CLOSTRIDIUM DIFFICILE INFECTION

Transcription

1 GRIGORE T. POPA UNIVERSITY OF MEDICINE AND PHARMACY IASI MULTIDISCIPLINARY RESEARCHES ON THE DIAGNOSIS AND TREATMENT OF THE CHRONIC HEPATITIS, TUBERCULOUS MENINGITIS AND CLOSTRIDIUM DIFFICILE INFECTION - - MIHAELA-CĂTĂLINA LUCA, MD, PhD IASI 2017

2 CONTENTS Thesis summary... 2 Rezumatul tezei... 4 Section I Brief review of professional and academic contribution Researches in chronic viral hepatitis area Epidemiology and diagnosis of chronic viral hepatitis Extrahepatic manifestations of the chronic hepatitis B The treatment of the clinical forms of chronic hepatitis B and the responsivity to treatment Chronic hepatitis C treatment Researches in tuberculous meningitis area Epidemiology of tuberculous meningitis Clinical and paraclinical aspects of tuberculous meningitis Researches on tuberculous meningitis in different populational groups (sucklings and children) III. Researches in digestive infection with Clostridium difficile Epidemiology of Clostridium difficile infection Risk factors and diagnosis Treatment of infection with Clostridium difficile Section II Plans of Career Development and Future Evolution of Research Section III References

3 THESIS SUMMARY The habilitation thesis reflects my scientific-professional activity, from the postdoctoral period (from 1996 until now), in the field of infectious diseases. The theses is an overview of my activity in the field of infectious diseases, an activity performed in the Infectious Diseases Discipline of the "Grigore T Popa" University of Medicine and Pharmacy Iasi, at Iasi Clinical Universitary "St Parascheva" Infectious Diseases Hospital. The thesis describes as a priority the development of strategies for the diagnosis of tuberculous meningitis, a difficult disease to be early diagnosed. This topic has been a constant concern of my activity since the scientific period of the doctorate, and then continued after the doctoral thesis, within a grant. By studying the tuberculous meningitis occasioned by the doctoral work, but also as a result of of the expertise accumulated in the Clinical Hospital of Infectious Diseases Iasi, the premises of the bacillary meningitis study were created, both clinically, diagnostically and therapeutically. At the same time, my professional medical activity in the field of infectious diseases, with the integration of the university and postgraduate education component, added in time the experience of scientific research. This was done in the form of multicentre clinical trials - as a principal investigator, and later in research projects as director, responsible and member of grant teams. The paper illustrates the continuing interest in the development of the current state of knowledge in research areas characteristic of infectious diseases, taking into account the academic evolution in accordance with the curricular requirements and the standards of higher medical education at the level of the European Union. Over the last decades, the World Health Organization has demonstrated the increase in the number and severity of infectious diseases globally, amid growing numbers and aggressiveness of pathogens. The empowerment thesis is structured into 3 main sections, the broadest section being: Section I covers the scientific achievements of the post-doctoral period (after 1998), pursuing the following fields of research: - research on chronic viral hepatitis (epidemiology and diagnosis, extrahepatic manifestations of chronic hepatitis B, treatment of clinical forms of chronic hepatitis B and their responsiveness to treatment, and treatment of chronic hepatitis C virus). Treatment of HBV has made remarkable progress in 2

4 recent years, and currently the existing therapeutic options manage to rapidly and efficiently reduce viral load. - tuberculous meningitis researches (epidemiology, clinical and paraclinical aspects of tuberculous meningitis, researches of tuberculous meningitis in various population groups - infants and children), with the introduction of new diagnostic techniques in practice (Quantifeon test). Studies focused on the performance of an immunological assay that evaluates the patient's T-IFN γ- IFN production in response to some M. tuberculosis antigens (ESAT-6 and CFP-10). - research into Clostridium difficile infection domain (epidemiological aspects, risk factors, diagnosis and treatment). Section II is dedicated to professional, scientific and academic contributions, as well as to the presentation of research projects. The academic and professional career is reviewed, as well as the research projects presented in detail. Academic development plans include the development of teaching materials for students, the involvement of students and PhD students in infectious disease research activities, postgraduate courses, as well as national and international cooperation activities involving young doctors and doctoral students. Future research directions include: - extinction of genotyping methods in the algorithm of diagnosis and therapy of chronic hepatitis, - continuing clinical and therapeutic studies of meningitis. - continue research on digestive infections with Clostridium difficile, given the particular incidence and complications. Future research will reflect further studies and diagnosis and new therapies as well as continuing studies in other areas of interest. The future research directions will represent a continuation of the issues addressed so far with the expansion of the area of interest and other pathologies. Section III includes the bibliography that supports the presented data in the empowerment thesis and complements them with the state of the research. 3

5 Rezumatul tezei Teza de abilitare reflectă activitatea mea profesional - științifică desfășurată in perioada ulterioară tezei de doctorat (din 1996 pana în prezent), în domeniul bolilor infecțioase. Lucrarea se constituie ca o privire de ansamblu a activității mele în domeniul bolilor infecțioase, activitate desfașurată în Disciplina Boli Infecțioase a Universitații de Medicină și Farmacie Grigore T Popa Iași, în cadrul Spitalului Clinic Universitar de Boli Infecțioase "Sf. Parascheva" Iasi Teza descrie mai ales activități de dezvoltare a unor strategii pentru diagnosticul meningitei tuberculoase, afecțiune dificil de diagnosticat precoce. Această temă a reprezentat o preocupare constantă a activității mele încă din perioada doctoranturii, continuându-se și după susținerea tezei de doctorat, in cadrul unui grant, prin studiul meningitei tuberculoase, studiu care a continuat preocuparile din domeniul tezei de doctorat, avand ca support și expertiența acumulată in Spitalul Clinic de Boli Infecțioase Iași ; astefel s-au creat premizele studiului meningitelor bacilare, atât din punct de vedere clinic, diagnostic, cât și terapeutic. Totodată, activitatea mea profesională medicală în specialitatea boli infecțioase, cu integrarea componentei de învățământ universitar și postuniversitar, a adaugat în timp și experiența acumulată dataorită cercetări științifice. Aceasta s-a realizat sub formă de studii clinice multicentrice - ca investigator principal, iar mai apoi în cadrul unor proiecte de cercetare ca director, responsabil științific și membru in echipe de granturi. Lucrarea ilustrează interesul continuu pentru dezvoltarea stadiului actual al cunoașterii în arii de cercetare de interes pentru domeniul bolilor infecțioase, având în vedere evoluția academică, în acord cu cerințele curriculare și cu standardele invațamântului superior medical, la nivelul Uniunii Europene. In ultimele decenii, Organizația Mondială a Sănătății a demonstrat creșterea ponderii și gravității maladiilor infecțioase la nivel global, pe fondul creșterii diversității și agresivității agenților patogeni. Teza de abilitare este structurată în 3 mari secțiuni principale Sectiunea I cuprinde realizările științifice din perioada postdoctorală (după 1998), urmărind următoarele domenii de cercetare: - cercetări în domeniul hepatitelor cronice virale (epidemiologie și diagnostic, manifestări extrahepatice ale hepatitelor cronice B, terapia formelor clinice de hepatită cronică B și responsivitatea lor la tratament, precum și tratamentul hepatitei cronice cu virus C). Tratamentul infecţiei VHB a făcut progrese remarcabile în ultimii ani, şi, în prezent, opţiunile terapeutice existente reuşesc să reducă rapid şi eficient încărcătura virală ; 4

6 - cercetări în domeniul meningitei tuberculoase (epidemiologie, aspecte clinice și paraclinice ale meningitei tuberculoase, cercetări ale meningitei tuberculoase emeningitis în diferite grupuri populaționale- sugari și copii), cu introducerea de tehnici noi de diagnostic în practică (testul Quantiferon). Studiile s-au axat pe cercetarea performanţelor unui test imunologic care evaluează producţia de γ-ifn a limfocitelor T ale pacientului ca răspuns la unele antigene prezente la nivelul M. tuberculosis (ESAT-6 şi CFP-10); - cercetări în domeniul infecțiiilor asociate cu Clostridium difficile (aspecte epidemiologice, factori de risc, diagnostic și tratament). Sectiunea II este dedicată contribuțiilor profesionale, științifice și academice, precum și prezentarea proiectelor de cercetare. Este trecută in revistă cariera academică și profesională, precum și proiectele de cercetare, care sunt prezentate detaliat. Planurile de dezvoltare academică includ elaborarea unor materiale didactice pentru studenți, implicarea studenților și doctoranzilor în activități de cercetare țn domeniul bolilor infecțioase, redactarea de cursuri postuniversitare, precum și activitati de cooperare națională și internațională, cu implicarea tinerilor medici și doctoranzilor. Direcțiile viitoare de cercetare se referă la: - extiderea genotiparii in algoritmul de diagnostic si terapie al hepatitelor cronice, - continuarea studiilor clinice și terapeutice a meningitelor. - continuarea cercetărilor în domeniul infecțiilor digestive cu Clostridium difficile, având în vedere incidența deosebită și complicațiile. Cercetările viitoare vor reflecta continuarea studiilor și asupra diagnosticului și noilor terapii, precum și continurea studiilor și în alte domenii de interes. Directiile de cercetare viitoare vor reprezenta o continuare a problemelor abordate pînă in prezent cu extinderea zonei de interes și in alte patologii. Secţiunea III include bibliografia care susține datele prezentate în teza de abilitare și le completează cu stadiul cercetării. 5

7 BRIEF REVIEW OF PROFESSIONAL AND ACADEMIC CONTRIBUTION I started my university career in 1991 as assistant professor of Infectious Diseases at UMF "Grigore T. Popa" Iasi. From the same year, I worked as a secondary doctor at the Clinical Hospital of Infectious Diseases Iasi. Over 26 years, I had evolved into the infectious disease specialty, having successively obtained, by contest, the didactic degrees of the chief of works (2000) and the lecturer (2003). From the medical level, I obtained the titles of specialist physician (1994) and consultant physician (1998) as a result of clinical activity and exams. Since 2007 I coordinate the activity of Section III of the Clinical Hospital Infectiose Iasi as Head of Section Physician. The researches elaborated during this period were marked by the collaboration with a series of disciplines such as: microbiology, biochemistry, genetics, morphopathology, and epidemiology. Collaboration with these disciplines has allowed the introduction of new methods for the prevention, diagnosis, treatment and monitoring of some infectious diseases with major health impact on the population as: chronic hepatitis, tuberculous meningitis, bacterial meningitis, Clostridium difficile infections. The diagnosis and treatment of tuberculous meningitis has been a constant theme of my professional and academic activity, which has gained new valences with the finalization of the doctoral thesis in 1998, with the theme "Therapeutic Studies in Tuberculosis Medicine". Researches in this field continued within a Grant CNCSIS type A completed in The research results allowed me to establish partnerships with specialists from the country (Timisoara) and abroad (France, Denmark, Turkey, Hungary, Croatia, Serbia, Albania) for the microbiological diagnosis of tuberculous meningitis in a multidisciplinary study conducted in the region of South-Eastern Europe - the Haydarpasa-1 study. Research continued in a similar institutional component in the Haydarpasa-II study, where a predictive method of unfavorable tuberculous meningitis was developed using the Hamsi score. The results of these studies have been published in prestigious specialist journals: «Clinical Microbiology and Infection and Journal of Neurology». A priority research direction has been the management of infections in general, that of Clostridium difficile, in particular. The research undertaken in this direction focused on the analysis of the influence of risk factors on C. difficile infection, 6

8 operative diagnosis, and the development of effective treatments for this type of infection. These studies have been the subject of several scientific articles and chapters of infectious disease books. Also, the appreciation of these results allowed me to be part of a multidisciplinary team of specialists from the country and abroad with whom I conducted research on the management of infections. The results of the study were published in the prestigious Journal of Infection. Diagnosis and study of the effectiveness of chronic hepatitis B and C virus infections treatment have been a constant concern. These infections are a real challenge for the medical system due to the difficulty of developing an effective treatment, for their disease, as well as the many complications, some of which are serious, accompanying it. Research has been directed to the use of methods of genotyping the virus, with the support of my team's colleagues, monitoring the treatment for infected patients with various genotypes of viruses and the study of extrahepatic manifestations. The experience gained during the academic and medical activity determined me to undertake the elaboration of this empowerment thesis. 7

9 My research activity was focused, prioritary, on the following directions: 1. researches in chronic hepatitis field; 2. researches in tuberculous meningitis area; 3. researches in digestive infection with Clostridium difficile. Chapter 1. RESEARCHES IN CHRONIC HEPATITIS FIELD In chronic hepatitis area I made some studies regarding epidemiologic data, and diagnosis of B and C chronic hepatitis, the particularities of B hepatitic virus infection, the extrahepatic manifestations in chronic C hepatitis, the treatment of the B and C chronic hepatitis and the impact of chronic hepatitis on the quality of life, with many medico-social and psychological aspects Epidemiology and diagnosis Epidemiologic, România is one of the countries with a medium endemicity: the HBsAg carriers being: 2-7% and the proportion of anti-hbs antibodies of 20-60%. GLOBOCAN mentions Romania as ranking second in Europe in the incidence of hepatocellular carcinoma (HCC), with a mortality of 10.5 / 100,000 for men and 4 / 100,000 for women, infection caused by HBV being associated with an increased risk for this cancer. WHO (World Health Organization) specifies that worldwide there are more than 240 million individuals chronically infected with HBV [63]. Romania is part of the intermediate endemicity area, porting chronic HBsAg prevalence between 2-7%. According with HBsAg prevalence in general population, Romania belong to intermediate endemicity area [63, 202]. Although HBV vaccine, introduced in Romania since 1995 in new-born, significantly reduced the morbidity and mortality through HPV infection, chronic hepatitis, cirrhosis and liver cancer remain a public health problem as in many parts of the world; complications of chronic HBV infection remain the 7th leading cause of death from infectious diseases worldwide. A ECDC (European Centre of Diseases Control) report states that, in 2012, 14,745 cases of hepatitis HBV have been reported by the 27 EU countries, Romania being cited with 486 cases of acute hepatitis HBV [193]. Trends over time are difficult to interpret due to changes in case definitions and practices reported in some countries. It is important to distinguish between acute and chronic infection with HBV. 8

10 The infection with hepatotrope viruses, namely hepatitis B virus and hepatitis C virus afflicts 3 up to 5% of the world population representing the ninth most frequently occurring cause of death [166]. The concept of chronic viral hepatitis appeared during the Second World War, when liver chronic diseases are described at soldiers with jaundice episodes. During the 50 s, the terms of chronic persistent hepatitis (corresponding to a nonprogressive variation of the viral hepatitis, with prolonged evolution) and chronic active hepatitis are introduced. Although this new terminology can be found in the monograph The Chronic Hepatitis edited in 1966, the first classification authored by an international group of hepatologists, which tries to correlate the histopathologic aspects of the liver biopsy with the prognosis of the disease, is published in There were described many genotyping methods of VHB, including the direct secvention [178], PCR based restriction fragment length polymorphism [188], line probe assay [9] and enzyme-linked immunoassay [188]. Recently was discovered by Naito and co. a new method of genotyping based on the PCR amplification, using specific primers of type, that can identify VHB genotypes. The treatment of HCB made remarcable progresses in the last years, and in present, the therapeutical options can rapidly and efficient reduce the viral load. Identifying of the VHB infectant genotypes has a big importance in choosing the antiviral therapy. In hepatitis B, a number of parameters are evaluated prior to treatment was identified as a part of the predictors of sustained virologic response interferon alpha-based therapies. During treatment, the best predictor is the evolution of viraemia. Early viral suppression is usually associated with a favorable virological response with less risk of subsequent mutations which induce resistance to antiviral. Viremia became negative after only two to four weeks of starting treatment. 8-10% of patients the disappearance of HBsAg and seroconversion occurs with the appearance of the patient's antibodies we anti-hbs.in meth 8-10% disparition of HBsAg and the seroconversion, with apparition of HBs-Ab. Chronic HBV affects nearly 350 million patients worldwide and may further progress to cirrhosis and/or hepatocellular carcinoma (HCC) in15 40% of cases. HBV accounts for 30% of cirrhotic and 50% of HCC cases. Over the world, at least ten genotypes of HBV (A through J) have been identified on the basis of more than 8% difference in their genome sequences [196]. Higher rates of HCC have been found in persons infected with genotypes C and F (compared with genotypes B or D), and in those infected with certain subtypes of genotype A found in southern Africa although aflatoxin exposure may play a role in sub-saharan Africa. Over the past three decades, treatment outcomes for chronic hepatitis B (CHB) have improved, first with IFN alpha and with nucleotide analogues (NAs). Currently, seven antiviral agents (six NAs lamivudine, adefovir, entecavir, telbivudine, tenofovir, emtricitabine, as well as 9

11 standard and two formulations of PEG-IFN) are approved and widely licensed for the treatment of CHB [200]. Numerous studies have investigated the influence of HBV genotypes on clinical outcomes. Genotype C is associated with rapid fibrosis, high HCC development rate, recurrence, and metastasis compared to genotype B. It appears that genotype D may be associated with more severe disease compared to A, and that genotype F is linked to high mortality rates [74, 96]. Although HBV genotypes do not significantly influence response to nucleoside/ nucleotide antiviral therapy, responsiveness to IFN is clearly affected by genotype. HBeAg-positive patients infected with genotype B have a better response compared to those with genotype C, and genotype A responds better than genotype D, especially during short term therapy. Genotype A also responds well to PEG-IFN. The response to PEGIFN has been reported to be influenced by HBV genotypes in many studies. Some authors [49, 84] reported that HBeAg loss as a response to PEG-IFN monotherapy or PEG-IFN plus lamivudine (LAM) was influenced by HBV genotype, and that genotypes A and B had a better response compared to C and D, after 52 weeks of treatment. Genotyping is still used as a research tool in the UK and isn t currently used to guide treatment with interferon based therapies. However, with such a high proportion of the CHB population carrying genotype there is a question as to whether genotyping should now be incorporated. It is possible that the use of routine genotyping could help identify those patients who are at higher risk of liver disease progression so that IFN-based therapies can be targeted earlier. In some studies, DNA/HBV detection with accurate methods is an imperative requirement in daily routine diagnostic of chronic HBV patients. Fast and accurate DNA/ HBV detection can be followed by genotyping of HBV, in order to correct choose the antiviral therapy [178]. The aim of presented study was to compare two molecular techniques for detection and genotyping of DNA/ HBV: a screening method using Real Time PCR (Amplisens HBV-FRT) technology and INNO LiPA HPV respectively, which involve classical PCR followed by hybridization on strips. We have tested 19 samples using two molecular biology techniques: first we performed detection of DNA/HBV with a Real Time PCR technique and second we tested the same extracted DNA from each patient, in order to detect the genotype of HBV. The DNA/HBV was purified from 200μL blood from each patient, using Invisorb Spin Virus DNA Mini Kit. The kit is using the classical buffers for binding, washing, ellution, and use also proteinase K, RNA-ase free water; for testing with Amplisens HBV-FRT a negative control (PCE) and Positive Control-1-HBV were necessary too. The DNA purification involved one step of incubation at 56 C for 15 min in Biosan thermobloc, and 4 steps of centrifugation (Beckman Coulter microcentrifuge) and washing steps. The DNA/HBV was eluted in 100μL ellution buffer and it was used immediately for PCR amplifications. 10

12 The Real Time PCR detection used Amplisens HBV-FRT, AccuPower PyroHotStart Taq PCR PreMix and MX3005P thermocycler, Stratagene. All the Real Time PCR reagents were unfrozen, mixed and centrifuges from the PCR kit. The reaction mix contained: 10 μl PCR-mix-1-FL HBV + 5μL PCR-mix-2-FRT + 0.5μL Polymerase TaqF/sample. After mixing of these reagents were added the DNA samples (10 μl each) and also 10μL of each of the controls: Positive Control-1-HBV, C- purified from Negative Control, C+ from DNA calibrator PIC2 HBV and NCA (water for negative control of amplification). The DNA isolated from the patients were pippeted in the plate in row A from 1 to 12 and in row B from 1 to 7. The negative controls were in row H, from 9 to 12. The thermal profile can be seen in table 1.I and in figure 1.1. The HBV genotyping was performed using INNO-LiPA HBV Genotyping kit Innogenetics N.V., Ghent, Belgium and AccuPower PyroHotStart Taq PCR PreMix. This method involved a first step of outer amplification which supposed that in the Eppendorf tubes with premixed AccuPower PyroHotStart Taq PCR PreMix was added 38μL water + 2μL Outer Primer Mix + 10μL ADN or water for negative control. The PCR amplification was realized in Gene Amp 9700 termocycler and has used the amplification protocol detailed in table 1.II. Determinations were made in Grigore T. Popa University of Medicine and Pharmacy Iasi by dr. Ramona Ursu. The second amplification was the nested one, in which the total reaction volume was 50μL: 46 μl water + 2μL Nested Primer Mix + 2μL amplicons or water for negative control. In this step, amplification was similar with the outer amplification protocol, with single difference of 35 cycles instead of 40 cycles. The products of nested amplification were then hybridized on the strip, using TwinCubator device, using the steps from the kit protocol. Table 1.I. The steps of thermal Real Time PCR amplification [179] 11

13 Fig The thermal profile using Amplisens HBV-FRT kit [178] Table 1.II. The amplification protocol for outer amplification [178] The analysis of Real Time amplification had used the channels FAM and JOE, and the exactly distribution of each of them for samples and control type can be seen in table 1.III. Table 1.3. The analysis of results [178] In figure 1.2 can be seen the amplification plots of Real Time PCR amplification for unknown samples and for positive controls. 12

14 Fig The amplification plots for the unknown samples and for the positive controls The Ct values for the patients (JOE/HEX) varied from to The Ct values for internal extraction control (FAM) varied from to The results of INNO LiPA Genotyping method: from 20 samples, one was the negative control, and the samples from 19 patients had the next results: 8 were negative, 9 had genotype D and 2 were detected having genotype A of HBV. In figure 1.3 can be observed the negative control strip, a strip with genotype D and a strip with genotype A. The results of both methods can be observed in table 1.IV. All the results obtained by both methods were validated with the help of positive and negative controls from the kits. Both testing were performed in 2 working days, which easily can be seen like an advantage in daily routine testing. The Real Time PCR amplification is less than 2 h, shorter than the classical ELISA methods (3.5 h) used for HBsAg detection, for example. The INNO LiPA assay is take longer because needs two PCR amplification, but is having the advantage of genotype detection, which can be used to optimize the antiviral therapy. The possible explanation for the negative results is that these patients are receving antiviral therapy and is possible that in the moment of testing, the DNA not to be detectable because of virus replication supression. INNO-LiPA is a reverse hybridization method that has been developed by Innogenetics and is commercially available as INNO- LiPA. The overall success rate for the detection of all eight HBV genotypes by this method was 98% using 100 clinical specimens with the majority having viral titers ranging from 105 to 107 IU/mL. Like advantages of this method we can mention that is easier and cheaper than sequencing, overall success rate for the detection of all HBV genotypes was recently found to be 98% and it is highly specific [74]. Like disadvantage, is relatively expensive compared to PCR-based and serological techniques. Amplisens HBV-FRT assay is recognized being a sensitive and specific assay. 13

15 Fig The reading card of INNO LiPA kit with a strip positive for D genotype, the negative control and a strip positive for A genotype of HBV [179] Table 1.IV The correlation of the results obtained by the two PCR methods [178] Although HBV genotyping before anti-viral therapy is not recommended by current guidelines from three regional liver associations, the American Association for 14

16 the Study of Liver Disease (AASLD), the European Association for the Study of Liver (EASL), and Asian Pacific Association for the study of liver (APASL), the impact of HBV genotype on therapeutic response to both interferon-based and nucleotide analogues has been increasingly recognized [6, 74, 96,108, 130, 160]. In conclusion, in the North-East region of Romania, the D genotype is the most frequent and even in a small patients group, was proposed a possible diagnosis algorithm which can be used for management of chronic HBV cases: DNA/HBV detection by Real Time PCR followed by HBV genotyping. The algorithm has the advantage of being cost and time efficient for optimal management of HBV positive patients. Further studies are needed on larger groups of patients to confirm these preliminary results. According to EASL (European Association for the Study of the Liver) clinical practice guidelines, pre-treatment assessment of liver disease involves assessing biochemical markers (AST, ALT), detection of DNA / HBV, particularly by Real Time PCR quantification (known method for sensitivity, specificity, accuracy and range limitations results), testing for other causes of chronic hepatitis (HDV coinfections, HCV and / or HIV), liver biopsy (recommended for determining the degree of necrosis and fibrosis) [31, 63, 194, 195]. The aim of this study was to assess the HBV genotype prevalence in a pilot study from North-eastern population of our country, and second, we aimed to correlate the type of HBV genotypes with the response to antiviral therapy [63,145]. Experimental part consist of a prospective cohort study of 20 chronic B hepatitis patients was conducted at the Sf. Parascheva University Hospital of Iasi for Infectious Diseases in the interval July October 2014 [63], regarding the genotypes circulated in Northeast region of Romania The serological markers (HBsAg, HBeAg and anti-hbe Ab) were tested with the ARCHITECT i1000 SR, Immunoassay (Abbott Diagnostics Analyzer) and the DNA / HBV were detected using COBAS TaqMan 48 Real-Time PCR System for Quantitation of Hepatitis B Virus (Roche Diagnostics). Blood samples collected in DTA tubes were then sent to the Microbiology Laboratory of the Gr. T. Popa University of Medicine and Pharmacy Iasi for HBV genotyping. The HBV genotyping: DNA/HBV purification: DNA/HBV was purified using the kit Instruction for the Invisorb Spin Virus DNA Mini Kit, STRATEC Molecular GmbH, D-The purity and concentration of DNA were analyzed with Nano Drop Pearl nan photometer. INNO LiPA PCR amplification purposed two steps: (outer and nested amplification) each of them using dedicated primers. For both amplifications (outer and nested) we used Gene Amp PCR System 9700, Applied Biosystem thermocycler. Test procedure for LiPA involved the next steps: denaturing, hybridizing the samples, washing the strips, developing the color using twincubator hybridization bath. Statistical analysis: Data were analyzed using SPSS version 20.0 (SPSS, Chicago, IL, 15

17 USA). The level of statistical significance (p-value, the probability of maximal error) was considered 0.05 (5%), a probability (confidence interval) of 95% showing that the decision was fair. Thus, statistical significance was defined as p <0.05 (95% CI). The HBV genotyping was a blind testing, without knowing clinical and microbiological data for the patients. Were genotyped the DNA/HBV from 20 patients aged years old (mean 37.75), 8 (40%) women, 10 (50%) being from urban areas. 13 cases (65%) were positive for HbeAg, and 14 (70%) presented anti HbeAb. The mean of ALAT values was ( IU/mL). Fibrosis grade 1 and 2 was detected each for 7 (35%) of patients, F3 for 5 (25%) and F3 F4 for 1 (5%) patient. 15(75%) of the patients are under Pegasys / Pegintron therapy. The mean of DNA / HBV was , 3000 IU/mL (2500, ,00 IU/ ml). The D genotype of HBV was detected for 18 (90%) of the patients. One had only genotype A (5%) and another one had double co-infection with both D and A genotypes (5%).Overestimation of incidence of hepatitis B virus mixed-genotype infections by use of the new line probe INNO LiPA geno-typing assay. Qutub MO et al, 2006, developed a standardized, single-round INNO LiPA PCR amplification protocols and automated sample processing by the MagNA Pure LC instrument were evaluated, with improved efficiency and suitability for routine laboratory use. One alternative of INNO LiPA genotyping method is TRUGENE HBV genotyping method, which was found to have better sensitivity, by Bassaras M et al [11]. Mercier Met al. in 2011 found that INNO-LiPA overestimates mixed infections as a result of erroneous genotype H detection, in comparison with sequencing of PCRamplified DNA /HBV samples [129]. The high prevalence of D genotype of HBV in the tested patients explain the lack of response to interferon therapy, as none of the patients had <2 x 10 copies /ml DNA / HBV. The only one patient positive for A genotype / HBV had positive HBe antibodies and a low value of viral load of DNA/HBV (5900 IU/mL), which is in accord with the known favorable response in case of this genotype from the literature. Was used F test ANOVA to test for differences among the mean of viremia (DNA/HBV) and the genotype of HBV, the presence / absence of HBeAg and antihbe, Ab, the severity of fibrosis, and the presence / absence of interferon therapy. There was a statistically significant difference between groups as determined by oneway ANOVA. There were also tested the differences between ALT values and the same previous analyzed parameters, but we did not found any statistical significant difference. The mathematical models showed that for our treated patients with interferon, the high level of viremia associated with a high level of ALT (16.1%) are predictors for D genotype of HBV, but these results cannot be extrapolated to general population (p > 0.05) (fig. 1.4). 16

18 Table 1.V. Genotypes VHB frecquency detected by different authors INNO Lipamethod [114] Author/year Country VHB genotype Prevalence Bokharaei-Salim F et al., 2014 Azerbaijan D A A+ D 93.2% 5.8% 0.97% Sayan M et al., 2014 Turkey E First case Constantinescu I et al., 2014 Romania D A+ D 60.5%) 31.4%) Chamni N et al., 2014 Tailand C 81.3% Vutien P et al., 2014 California B C Baxa DM et al., 2013 USA A C D E Baha W et al., 2012 Maroc D A Scotto G et al., 2010 South Italy E D B C A 67.5% 24.2% 43.7% 4.34% 4.34% 4.34% 90.45% 5.9% 45.13% 18.1% 15.3% 13.2% 4.9% Was used ROC curve for calculating the HBV DNA level differentiating HBeAg-negative and the cut-off value was set at copies/ml with 75% sensitivity and 80% specificity (fig. 1.5). Fig.1.4. Mathematical model DNA/HBV ALT values for prediction of D genotype of HBV [114] 17

19 Fig.1.5. Receiver Operator Curve (ROC) for calculating the DNA/HBV level differentiating HBeAg negative [114] 55% (11/20) of our patients were infected with Mediterranean HBV strains, being HbeAg negative, anti-hbe Ab positive with high levels of DNA/ HBV. The D genotype of HBV detected in 90% is known to be associated with precore mutation and non-a HBV genotypes. The most important strength of our study is that we are assessing for the first time the HBV genotype prevalence in patients from Northeastern Romania. The weakness of our study is that the number of tested patients is low, but our results are correlating with other studies performed in our country, which stated that the most prevalent genotype is D. In the close future is intended to extend this HBV genotyping in more patients and also to assess the antiviral therapy efficiency according with the HBV genotypes. In conclusion, in this pilot study regarding the genotypes circulated in Northeast region of Romania, the most predominant genotype detected was D (90 %). It would be useful for the chronic B hepatitis patients to adapt antiviral therapy according with the HBV genotype, in order to obtain like endpoints HBeAg seroconversion, HBsAg loss, DNA/HBV undetectability. As risk factors with statistical significance for the unfavourable evolution were identified: high viral load, the type genotype, HBeAg, the severity of fibrosis and IFN therapy. An important diagnosis element of chronic hepatitis are the operational scores [166]. Starting from the quantification of the specific lesions for chronic hepatitis B and C, our study focused on the correspondence between the necroinflammatory activity and the fibrosis stage ascertained through the Ishak scoring system, (ii) the classification overlaps and differences of Ishak vs. METAVIR score [101, 134]. 18

20 The histopathologic criterion, which differentiated the two forms, is the periportal necrosis occurring in fragments, called piecemeal necrosis. Soon, it became clear that the parenchymal lesions, especially the bridging necrosis constitute an important predictive factor in the progression of the disease (the evolution towards cirrhosis), and thus the chronic lobular hepatitis was defined. Later, the pathologic aspect was broadened, taking into account several other options, such as nonspecific reactive hepatitis a less aggressive form than chronic persistent and chronic lobular hepatitis, and chronic septal hepatitis a form of chronic active hepatitis with remission features. After more than 30 years from the initial classification, important knowledge regarding the etiology, the pathogenesis, the clinical profile and the pathology of the chronic hepatitis bring about new aspects, the old terminology becoming a confusion source, by using the same diagnosis, such as chronic active hepatitis, for diseases with different origins (viral, autoimmune, metabolic). These reasons imposed the formulation, by the International Liver Research Association, of a new classification based on etiology, because of the substantial differences between the clinical aspects, the prognosis and the treatment of the different types of chronic hepatitis. In the pathologic exam of liver biopsy, the implicit subjective component was clearly diminished by the introduction and development of certain scoring systems: Scheuer, Ludwig, Knodell, Ishak, and METAVIR [15, 80, 97, 159]. The scoring systems have unavoidably strengths and weaknesses in their main objective the semiquantitative classification of the necroinflammatory activity (NIA) and the staging of fibrosis. In time, the research groups have published relevant results supporting or contesting the diagnostic and prognostic importance of these scoring systems. Our research is based on the morphologic image resulted from the semiquantitative evaluation of a significant group of liver biopsies performed in chronic hepatitis B and C. Starting from the quantification of the specific lesions, we focused on (i) the correspondence between the NIA and the fibrosis stage ascertained through the Ishak scoring system, (ii) the classification overlaps and differences of Ishak vs. METAVIR score [166]. This analysis created the necessary support for the critical interpretation of the different classification systems, applicable in the assessment of the chronic hepatitis. The study group consisted of 953 cases of chronic viral hepatitis (202 cases with chronic hepatitis B and 751 cases with chronic hepatitis C), diagnosed in the Pathology Laboratory of the Sf. Parascheva Clinical Hospital of Infectious Diseases, Iassy, between 2008 and The liver biopsy was performed on all the patients included in the study group through the percutaneous method, using the special Hepafix kit (B. Braun Melsungen AG, Germany), after echographic guidance [166]. 19

21 The liver biopsies met the minimal criterion necessary for the evaluation namely a minimum of three portobiliary spaces. The study group had, for each specimen, between 3 and 12 portobiliary spaces, with an average of 6 8. The evaluation of the lesions was performed in semiquantitative manner. For the biopsies of the patients with chronic hepatitis B and C we applied the Ishak score which quantified individually the NIA (score 1 18) and the fibrosis (score 1 6) [80]. At the same time, the biopsies of the patients with chronic hepatitis C were evaluated also through the application of the METAVIR scoring system. As results, the pathologic picture of the investigated liver biopsies was characterized by the presence of the specific basic lesions: inflammatory infiltrate, piecemeal necrosis, bridging necrosis and fibrosis, together with associated lesions developed in the hepatic parenchyma. In our group, the morphology of the chronic hepatitis ranged from mild to severe forms, some with a tendency towards cirrhosis. In the mild forms, the inflammatory infiltrate (formed by lymphocytes, macrophages, few plasma cells and rare neutrophils and eosinophils) was restricted to the portobiliary space, the hepatic architecture being preserved. Gradually, the severity was signaled through: (i) the development of piecemeal necrosis, where the chronic inflammatory infiltrate goes beyond the location in the portobiliary space and invades the adjacent parenchyma, accompanied by necrotic hepatocytes from the limiting plates, and the presence of a lobular inflammation with focal hepatocytic necrosis; (ii) the development of bridging necrosis (porto-portal, portocentral, and central-central). We noticed frequent aspects typical for the hepatocyte regeneration, such as binucleated young cells with basophil cytoplasm, rosette-like dispositions of the hepatocytes mimicking the glandular pattern, or local segmental thickenings of the hepatocyte plaques [166]. Various degrees of Kupffer cells hyperplasia was observed in both types of chronic hepatitis B and C. Hepatocytic steatosis was a common morphologic element, more often macrovesicular but also microvesicular, with an unsystematic topography most of the times. For the 202 cases diagnosed with hepatitis B, the values conferred by the semiquantitative evaluation after the application of the Ishak scoring system for the NIA and fibrosis are presented in table 1.VI. Thirty-nine cases from the total of 202 presented also chronic hepatitis D with the following NIA profile: seven mild cases, 27 moderate and five severe. In the investigated group, the steatosis was associated with the NIA lesions as follows: 20

22 Tabel 1.VI. Distribution of the cases with hepatitis B based on NIA and fibrosis on NIA and fibrosis Ishak scoring system [166] For the mild NIA: from the total of 48 cases, in 34 it was absent, in 11 it was present in 10% of the whole specimen and in three cases it was present in 20% of the whole specimen; For the moderate NIA: from the total of 148 cases, in 99 it was absent, in 18 it was present in 10% of the whole specimen, in 27 cases it was present in 20% of the whole specimen and in 4 cases it was present in 30% of the entire specimen; For the severe NIA: from the total of six cases, in four it was absent, in one case it was present in 10% of the entire specimen and in one case it was present in 20% of the entire specimen. For the 751 cases diagnosed with hepatitis C, the values conferred by the semiquantitative evaluation after the application of the Ishak scoring system (for the NIA and fibrosis) as well as that of the METAVIR scoring system are presented in table 1.VII. From the total of 751 cases, 16 cases presented also hepatitis B, with the following NIA profile: mild in two cases and moderate in 14 cases. The liver biopsy was and still remains a major diagnosis tool in the evaluation of chronic hepatitis patients, because it allows the assessment of the morphological background of the disease and consequently of the severity and expansion degree of the morphological changes in the hepatic parenchyma. 21

23 Tabel 1.VII. Distribution of the cases with the Ishak (NIA, fibrosis) and the METAVIR scoring system [166] In the last decade, the advances in the development of the biologic and imaging techniques lead to the introduction of new methods, less invasive than liver biopsy. These methods are based on the follow-up of the seric biochemical markers and the imagistic evaluation of the liver through elastography, in order to measure its rigidity, with a correspondence in fibrosis. However, the experts still rely on liver biopsy not necessarily for the confirmation of the clinic diagnosis but rather for the estimation of the prognosis and the choice (or not) of the antiviral therapy. The pathologic picture of the chronic hepatitis may vary considerably from case to case, but in essence these variations are due to the relationship between the three possible components: inflammatory reaction, fibrosis (or cirrhosis) and hepatocellular changes. The morphologic features are generally similar to all forms of chronic, viral or other types of hepatitis. Nevertheless, there are some distinct elements, which we will discuss in correlation with our study. In chronic hepatitis B, the hepatocytes with ground glass cytoplasm were evident, appearance caused by a massive production of AgHBs in the hepatocytes, which determines the proteic coating of the HBV to be accumulated in excess within the RER cisternae. The morphologic image for the hepatitis C was characterized by the presence of lymphoid aggregates in the portobiliary spaces which is considered to be a specific signature and by lesions of the biliary pathways. Regarding the evolution of the disease, we must stress the caution with which the piecemeal and the bridging necrosis must be interpreted. Although these lesions do not imply an unavoidable progression of the disease, the loss of the hepatocytes continuity leads to the formation of fibrous septae, which, associated with the hepatocytic regeneration, triggers the development of cirrhosis. 22

24 This study is focused on a morphologic analysis performed on almost 1000 liver biopsies investigated during a three years interval. The semiquantitative evaluation of the microscopic specimens was achieved based on a previous rigorous instruction; the scores granted being periodically ascertained through an interobserver s diagnostic appraisal. Within this context, our results create the premises for a three-directional discussion: (i) the significance of the analysis on the correlation between the NIA and fibrosis, (ii) the motivation of the choice and application of a certain scoring system and (iii) the minimal condition necessary for the assessment of a liver biopsy [105]. The correlation between the NIA and the fibrosis is essentially the main element which permits a prognostic evaluation and, at the same time, an adequate therapeutic decision, fact that the moderate NIA (score 7 10) involves simultaneously an extremely variable development of fibrosis from the limited, restricted form (score 1) to the expanded form, characterized by fibrous bridges (score 4). On the contrary, for the mild and severe NIA, the correlation with a specific degree of fibrosis respects (in a certain manner) a better-defined pattern. Thus, the mild form (score 1 6) associates fibrosis score 1 (mild) while the severe form (score 11 16) associates fibrosis score 3 and 4 (severe). Conversely, in the chronic hepatitis C, the analysis of the correspondence between the NIA and the fibrosis revealed a different pattern, with a wider variability. Firstly, the mild NIA (score 1 6) correlated with mild fibrosis (score 1), moderate (score 2) but also severe (score 3). We believe it is necessary to underline this variability recorded in the expression of fibrosis. Thus, fibrosis score 1 was present in the case of NIA score 2 but also in the case of a NIA score 6. At the same time, the NIA score 6 was associated with mild fibrosis (score 1), moderate (score 2) but also severe (score 3). Comparing the values of the scores, the development pattern of the fibrosis is similar with the image present in moderate NIA (score 7 10) in hepatitis B. For the moderate NIA (score 7 10), the expansion degree of the fibrosis ranges from score 1 to score 6 similar to the results obtained in the mild NIA for the hepatitis B with the remark that the overwhelming majority of the cases (606 out of 672) presented fibrosis score 3 which is the first step on the scale of severe fibrosis quantification. Moreover, the results obtained for the severe NIA (score 11 18) were similar to the ones in hepatitis B cases, associated with severe fibrosis with the remark that in this class of the classification the severity degree was higher (score 4 and 5). The comparison between hepatitis B and C based on the NIA and fibrosis offers, through the numeric values of the Ishak scoring system, accurate proofs, which support the aggressivity of hepatitis C, because it develops fibrosis more quickly, even on the background of mild NIA. Also, our results reveal the fact that the NIA and the fibrosis are not processes which progress in a consistent pattern. The extreme diversity 23

25 of the fibrosis degree reflects, the biologic individuality that defines the response to the viral aggression [122, 136, 137]. The application of the METAVIR scoring system forth the cases with chronic hepatitis C confirmed the abovementioned observation, namely that there is not a direct correlation between necroinflammation and fibrosis. These data showed that a moderate NIA might be accompanied by fibrosis scores from 1 to 6. However, our findings revealed the occurrence of overlaps in the classification of some cases. Thus, score A3F3 resulted both for moderate NIA (score 7 10) with fibrosis F4, and for severe NIA (score 11 12) with fibrosis F4/F5. Another example is the score A2F1 resulted for moderate NIA (score 7 10) with fibrosis F1 and F2, respectively. Hence, we must draw the attention on the possibility of an identical score for lesions with different intensity which can be designated as an important limit of the METAVIR scoring system. Our activity centered on the liver biopsy required the accumulation of abilities specific for this type of pathology. In time, the experience increased mainly through the comparative application of several scoring systems, in order to create our own opinion with respect to the informational advantages offered by each of them. Although the Scheuer classification [105], the METAVIR system and the Batts and Ludwig visual interpretation provide simple and easy to apply criteria, from our point of view the information obtained is not refined [105]. Most interpretation issues regard the extremely different aspects of fibrosis. In these cases, the specialists recommend the Ishak staging system, because it makes available several classification steps and hence it increases the staging accuracy of the lesions. The final purpose of the scoring systems used in the interpretation of the liver biopsy, by the evaluation of the degree and activity stage of the inflammatory disease and fibrosis, is to convey clear and precise information to the clinician. Consequently, the choice and the usage of an operational scoring system must be founded on: (1) the existence of a consensus between pathologists and clinicians on the pathologic criteria quantified in the selected scoring system; (2) the concise and consistent formulation, in the pathology report, of the information about classification and staging; the possibility to apply the obtained information in diagnosis and therapy. Based on our experience in the semiquantitative evaluation of liver biopsies, both for hepatitis B and C, we use the Ishak scoring system. Our choice is supported by the fact that the wide range of numeric values attributed for the evaluation of NIA and fibrosis provides for more precise criteria for the appraisal of the degree of damage to the hepatic parenchyma at the time of the diagnosis. Supplementary to the Ishak scoring system, for the hepatitis C we perform at the same time a METAVIR scoring system assessment, because it allows an assessment of the entire histologic activity, with the addition of the interface hepatitis and of the associated lobular necrosis components. 24

26 The controversies in the literature on the histologic grading and the staging systems may be explained by the possible sampling errors. We believe that the liver biopsy is a representative sample for the hepatic parenchyma. However, we must take into account the fact that the liver biopsy, regardless of its basic length or width, is only a finite segment from an organ with an immense potential diversity in the expression of disease. The pathologists obviously prefer larger samples for the interpretation [47], and comparative data indicate the fact that 20 mm long samples are necessary, which have to include at least 11 portobiliary spaces in order to have an optimal specimen for grading and staging [37]. In the study group, each microscopic specimen had between 3 and 12 portobiliary spaces, with an average of 6 to 8 a value that can support a correct diagnostic evaluation. The diagnosis based on the semiquantitative assessment of the lesions, according to the parameters included in the scoring system, relies on the examination of the microscopic specimens in special stains: trichrome Szekely, Gordon Sweet silver impregnation, PAS [156]. A last comment must be made with respect to the expertise in the field which corresponds to the recommendations in the literature the main investigator having 10 years experience in liver pathology fact that increases the coherence and the accuracy of the diagnosis and decreases the issues associated with the dimensions of the sample [156]. The qualitative and semi quantitative appraisal of the liver biopsy allows an adequate evaluation of the severeness of the liver injury. However, a certain degree of subjectivity unavoidably intervenes in the use of the operational scores for the quantification of the lesions. The significant decrease of the subjective factor in the assessment implies a solid experience in the field which leads to the choice of a particular score system or the comparative application of several systems in complete consensus with the clinician. Changes in the viral subtypes, in hygienic measures and the effects of the national vaccination program are factors that can influence the epidemiological and clinical features of acute hepatitis B. The clinical and epidemiological profile of acute hepatitis B has significantly changed in the last 15 years, the decrease in the annual number of cases being paralleled by an increase of severe or cholestatic forms of disease [95, 147]. This research aims to analyze how a set of epidemiological and clinical characteristics of acute hepatitis B have changed over the last 15 years in a Regional Infectious Diseases Hospital [179]. Were retrospectively studied and compared two groups of patients diagnosed with acute hepatitis B in the Infectious Diseases Hospital of Iasi between 1997 and 2001 (group cases) and between 2007 and 2011 (group cases). The AHB diagnosis was set further to the detection of a severe hepatic cytolysis syndrome (ALT>1000 UI/l) and of the presence of IgM anti- HBc antibodies. Patients with IgM/IgG antibodies against hepatitis D virus were excluded. 25

27 The annual number of AHB cases was found to have decreased almost constantly over the last 15 years, from 168 in 1997, to a minimum of 20 in 2008 (Figure 1.6). The average number of hospitalized patients was in the first group, and 4.1 times lower (28.4) in the 2 nd group, the difference being statistically significant (p = ). The average age of the patients in the 1 st group was 25.7 years (95%CI: ), which is significantly lower (p < ) than in the 2 nd group 35.1 years (95% CI: ). As far as the sex ratio is concerned, the males prevailed in both groups, with a more pronounced predominance in the second group (average M/F ratio 1.4 vs. 2), which is close to the statistical significance threshold (p = 0.053). Most of the patients in both groups came from urban areas, yet significantly (p = 0.003) more patients coming from rural areas were found in the second than in the first group (average R/U ratio 0.49 vs. 0.8). HBV transmission risk factors were more commonly identified in the patients included in the 1 st group (30.6%) as compared to the second group (16.2%) (p = 0.04). Figure 1.6. Annual number of AHB cases over the studied period [179] Among these, in the second group, the following were found: unprotected sexual intercourse with partner suffering from chronic hepatitis B, complex dental surgery, surgical procedures, recent tattoos/ piercing, iv drug use. The dominant most common symptoms during the pre-jaundice period were of digestive nature in both groups (34.6% vs. 32.4%), and no significant differences between the frequencies of the other types of onset were reported (ANOVA F=0.77, p = 0.4) (Figure 1.7). The pre-jaundice period duration ranged from 2 to 20 days in the 1st group, with an average of 5.72 days (CI 95% ), whereas the average of the second group was 6.92 days (CI 95% ); the difference was not significant. 26

28 The maximum values of the hepatic cytolysis syndrome during the disease acme (quantified by ALT measurements) were similar in both groups: the 1 st group (95% CI: ), and the 2 nd group UI/l (95% CI: ). Figure 1.7. Types of clinical symptoms in the pre-jaundice period [179] The difference was not statistically significant (p = 0.29). The average level of the maximum total serum bilirubin value was higher in the 2nd group mg/l (95%CI: ) than in the 1 st one mg/l (95% CI: ), whereas the difference had statistical significance (p = ). The anicteric forms of the disease were more frequently diagnosed in the second group of patients 12.7% against 7.2% in the 1 st group, and the difference was close to the statistical significance threshold (p = 0.051). A comparative approach of the percentages of severe and fulminant forms in the two groups (Table 1.VIII) enabled us to note significantly higher occurrences in the 2nd than in the 1st group: 57% vs. 34.7% (p < ). As for disease duration, the forms prolonged by cytolysis occurred in similar percentages in the two groups: 14.9% vs. 15.5%; not statistically significant. The cholestatic forms of the disease were significantly more common in the 2 nd (19.7%) than in the 1 st (8.7%) group, the difference having statistical significance The frequency of AgHBs determination concomitantly with the IgM antihbc antibodies increased and reached 72.5% in the second group against only 28.1% in the first one. The number of acute AgHBs negative forms remained constant, being 3.6% (6/164) in the 1 st group against 3.9 (4/103) in the 2 nd, p =

29 Table 1.VIII. Clinical forms distribution depending on severity, in the two groups [179] Clinical Forms Groups Mild (no.) % Moderate (no.) % Severe (no.) % Fulminant (no.) % 1 st group nd group The percentage of patients that were administered antiviral therapy (lamivudine) during the acute stage was higher in the 2nd group (28.9%) than in the 1st one (3.4%), p < deaths were reported in the 1 st group (0.5% mortality rate) and 2 in the 2 nd (1.4% mortality rate) due to AHB (fulminant forms), the difference not having statistical significance (p = 0.4). The incidence rates of this infection has decreased because of improved antiseptic measures, to a stricter control of transfusion products and vertical transmission, and to the large-scale implementation of an anti-hepatitis B virus vaccination program, considerably in most European countries in the last years. From the viewpoint of AgHBs carriers prevalence, Romania is considered a moderately endemic country, as this percentage varied, between 2.15% and 7.91% (mean value %) in Whereas in 1989 the acute hepatitis B incidence was reported to be 43 cases/105 inhabitants, in 2004 it went down to 8.5 cases/105 inhabitants (1), and in 2010 it reached a level of 2.4 cases/105 inhabitants. Therefore, the 4.1 times decrease in the number of AHB cases diagnosed at the Infectious Hospital of Iaşi, which was noticed between the two 5-year time intervals under survey (namely and ), is in agreement with infectious diseases the national decreasing trend. The considerably higher average age of the patients examined between 2007 and 2011 (35.1 vs. 25.7, p<0.0001) may be accounted for by the effects of the free compulsory newborn vaccination campaign started in 1995, which was later extended to include school children (1999), and 18-year olds (2004). The masculine sex prevalence among acute hepatitis B patients has been noted by other authors as well (6, 7), and in the study it tended to be more pronounced in the second group of patients (p = 0.053). Although, just as in literature, the patients of urban extraction prevailed in both study groups, was found significantly (p = 0.003) more rural patients involved in the second interval, possibly due to incomplete vaccine coverage or to failing preventive health education. 28

30 Due to the high concentration levels in the blood of patients suffering from chronic virus B infection, the sexual or blood-related virus transmission risk is high. The infection s transmission route could not be identified in most of the patients (especially in the second group). The most common risk factor for VBH transmission in the 2 nd group was tattoos or piercings performed with non-sterile equipment, followed by unprotected sexual intercourse with partners that later proved to be chronic virus carriers. Nevertheless, acute infection also occurred in patients having undergone dental or surgical procedures that apparently observed all hygienic measures. The beginning of the disease is usually the noisiest from the standpoint of clinical symptoms, the spectrum and frequency of which seem not to have significantly changed between the two time intervals under survey. The clinical manifestations of the pre-jaundice period were dominated mostly by dyspeptic or neuro-vegetative symptoms. Pseudoinfluenzal and pseudo-rheumatismal symptoms were sometimes reported, and even a few cases (3 in the 1st group and 1 in the 2nd) with eruptive onset (Gianotti-Crosti syndrome). The hepatic cytolysis syndrome is pronounced during acute hepatitis B, and it sometimes reaches impressive values (the maximum value recorded in our group was 9200 UI/l), but is not usually indicative of the severity of the disease; the mean value of the maximum ALT recorded was similar in both groups ( vs UI/l). The total serum bilirubin value is generally correlated with the severity of the disease and it was significantly higher in the 2 nd than in the 1 st patients group (175.6 vs mg/l, p=0.0003). In addition to higher maximum values, the 2nd group patients also exhibited a slower decrease of these values, as the forms prolonged by cholestasis were significantly more frequent (19.7 vs. 8.7%, p = ). The drop in the number of cases reported over the last few years was accompanied by an increase in their severity, which is supported by the significant rise in the number of severe and fulminant clinical forms between the 1st and 2nd group. The changes affecting the clinical spectrum of acute hepatitis B, materialized in more frequently occurring severe or cholestasis-prolonged forms in our region, over the last 15 years, are difficult to explain and they may suggest the involvement of new viral subtypes, as genotype or prec/c mutations [182,195]. The more severe clinical forms and improved lamivudine accessibility determined doctors to use this therapy significantly more frequently these last few years (28.9 vs. 3.4% of the patients). However, AHB mortality rates have not decreased between the two time intervals we researched, as they amounted to 0.5% in 1997 through 2001 and to 1.4 between 2007 and The small number of deaths made the difference not to have any statistical significance. The last 15 years have brought about considerable changes in the acute virus B hepatitis features in our region. Epidemiologically we have seen a 4.1 times decrease in the annual number of cases, a significant increase in the average age of the patients and in the number of patients from rural areas. From a clinical point of view we found 29

31 a significant increase in the frequency of severe and cholestasis prolonged forms. Therapeutically there was a more frequent use of antiviral therapy (lamivudine), which did not necessarily result into lower mortality rates Extrahepatic manifestations of the chronic hepatitis B There are numerous extrahepatic syndromes associated with HBV infection: vascular, renal, and cutaneous manifestations, essential mixed cryoglobulinemia, and neurological phenomena. Their recognition may contribute to early diagnosis of chronic HBV infection and effective treatment. In our study group the extrahepatic manifestations have often complicated the course of chronic hepatitis B taking a wide range of clinical forms and involving many organs or systems (skin, joints, nervous, renal, cardiovascular, and hematologic systems). Although in the literature the incidence of chronic hepatitis B- associated PAN is of 1%-5%, in our study is was of 5%. The importance of diagnosing the extrahepatic manifestations is related to therapy, as corticotherapy can negatively influence the course of chronic HBV [115]. The current importance of acute hepatitis B virus (HBV) infection is due to its wide-spread around the world, high potential for chronicity and possible progression to liver cirrhosis or hepatocellular carcinoma. Hence the interest of researchers to supplement the data accumulated so far regarding the pathogenesis, clinical and laboratory aspects of chronic forms is accompanied by a persistent production of immune complexes (which fix and activate the complement) causing extrahepatic lesions. Autoimmune phenomena, with the presence of circulating nonspecific antimitochondrial and anti-albumin) have been also reported [115]. There are numerous extrahepatic syndromes associated with HBV infection: vascular (vasculitis, panarteritis nodosa), renal cutaneous (papular dermatitis), essential mixed cryoglobulinemia and neurological phenomena [46, 54]. The cases of chronic HBV infection admitted to the Iasi "St. Paraschieva Infectious Diseases in Iasi in the interval were retrospectively studied. The 180 patients included in the study were diagnosed by physical examination, liver function, serological, virological and histological tests. Our aim was to determine the prevalence and course of extrahepatic manifestations during the disease in view of adjusting the regimens, on the one hand for treating these complications and, on the other hand, for rethinking the strategy for monitoring and treating the patients with chronic HBV infection (as some therapeutic interventions to manage these complications can negatively influence the course of the underlying disease, as it is the case with panarteritis nodosa (PAN). The 180 patients included in the study accounted for 8.7% of all chronic hepatitis cases admitted during the study period. The mean age of the study patients was 41 years (range years), most patients being male (65%) and living in areas 30

32 (70%). The extrahepatic manifestations encountered during the course of chronic hepatitis C are listed in table 1.IX. Tabel 1.IX. Extrahepatic manifestations associated chronic hepatitis B [115] System Manifestations No. % Locomotor Arthralgia Arthritis Renal Glomerulonephritis Gianotti- Crosti syndrome Nonspecific exanthem Cutaneous Raynaud syndrome Panarteritis nodosa Essential mixed cryoglobulinemia Henoch- Schonlein purpura Myocarditis Pericarditis 0 0 Cardiovascular Hypotension Bradycardia Tachycardie Gastritis/Duodenitis Digestive Catarrhal pancreatitis Byliary dyskinesia Peripheral neuritis Nervous Polyneuritis Myelitis, extrapyramidal syndromes Endocrine Disorders of glucose metabolism Hypothyroidism Bone marrow aplasia Hematologic Thrombocytopenia Hemolytic anemia

33 During the course of chronic HBV infection cutaneous manifestations were present in 62% of patients. Rash was encountered in about 25% of patients. Gianotti-Crosti syndrome or "papular acrodermatitis of childhood", which is a "papular acrodermatitis of childhood", which is a self-limiting papular or papulovesicular rash, was present in the acute phase of infection in 6 cases, especially among women (15% cases) and young adults, being located in most cases on the distal portion of the limbs and accompanied by pruritus. The recovery occurred spontaneously after days and the associated manifestations included fever, diarrhea, and lymphadenopathy. Cryoglobulinemia was present in 38 (21.1%) cases and Henoch-Schonlein pur pura in 22 (12.22%) cases. Essential mixed cryoglobulinemia is described in association with the HBsAg carrier status; in our study group it manifested by: purpura (14 cases), Raynaud syndrome - 46 (25.6%) cases and arthralgia - 34 (18.9%) cases, triggered by exposure to cold. Fatigue was present in all cases, and renal involvement was found in 29 (16%) cases, manifested by glomerulonephritis. Three of the patients also had chronic kidney failure. Panarteritis nodosa (PAN) is a rare event, but severe in chronic hepatitis B; in our study its prevalence was of 5% (7.27). Cutaneous PAN was found in 9 (5%) cases, with the following manifestations: livedo reticularis and painful subcutaneous nodules, ulcerated nodules and distal gangrene manifested by fever, polyarthralgia, myalgia, nettle rash, hypertension, neuritis, soft tissue nodular lesions, livedo reticularis, and in severe forms: cutaneous ulcers, distal gangrene acute colecystitis, vascular occlusions. Other encountered cutaneous extrahepatic manifestations were: nonspecific rash maculopapular - 45 (25%) cases, livedo reticularis - 3(1.7%) cases, erythema nodosum - 46 (25.6%) cases, stellar angioma -14 (7.77%) cases and palmar erythrosis - 6 (3.33%) cases; acanthosis nigrans was present in 2(1.1%) cases. Late cutaneous porphyria: dysmetabolic condition associated with uroporphyrindecarboxylase deficiency has been associated with the presence of anti-hbc antibodies in blood, being reported in one patient. The manifestations at the level of the locomotor system were represented by arthralgia - 34(18, 9%) cases and arthritis 3(1,7%) cases and were clinically characterized polyarthralgias and arthrites accompanied by joint swelling, the joint involvement being symmetrical, gener-alized, and affecting especially the small joints of the hand and foot. The renal extrahepatic manifestations consisted in glomerulonephritis, seen in 5 (2.77%) cases, nephrotic syndrome, proteinuria and hematuria. Immune complex deposits in patients with viral hepatitis have been detected in the kidneys in the absence of kidney disease and the most in chronic hepatitis B was the nephrotic syndrome. Proteinuria and hematuria were reported in 2 (1,1%) cases and were accompanied by fever. Neurological manifestations related to the action of HBV or to the induced immune response were present in 20 (11.1%) cases. These were classified 32

34 as early and late, occurring several we eks after the onset, and included: peripheral neuritis (which occurred 4-21 days after - 11 (6.1%) cases, Guillain-Barre syndrome - 8 (4.4%) cases and extrapyramidal syndrome - 2 (1.1%) cases. The endocrine events were represented by disorders of glucose metabolism - 35(9.4%) cases and hypothyroidism - 15(8,3%) cases. The rec-orded hematologic manifestations in order of their frequency were: thrombocytopenia -95(52.7%) cases, hemolytic anemia - 34 (18.8%) cases, and phenomena of aplastic anemia in 2 (1,1%) cases. Cardiovascular orded hematologic manifestations in order of their frequency were: thrombocytopenia - 95(52.7%) cases, hemolytic anemia - 34 (18.8%) cases, and phenomena of aplastic anemia in 2 (1,1%) cases. Cardiovascular manifestations during chronic hepatitis B were seen in 43 (23.8%) cases, being repr esented by: myocarditis - 1 (0.6%) cases, phenomena of hypotension in 28 (15.6%) cases, bradycardia - 5 (2.2%) cases and tachycardia in 8 (4.4%) cases. Also recorded were digestive manifestations in 49 cases: gastritis 18 (24.4%) cases and biliary dyskinesia in 31 (17.2%) cases. The pathogenesis of extrahepatic manifestations in chronic hepatitis B is generally immune mediated. Panarteritis nodosa (PAN) is rare, with a prevalence of 1-5%, but a very severe complication of chronic HBV affecting the small and mediumsized vessels. It is more common among patients in North America and Europe but rare in Asians. In our study its prevalence was of 5%, thus concords the values reported in the literature. The incidence of HBV infection in patients with PAN ranges from 30% to 70%. In the US and Europe, where HBV infection is late the association with PAN is prevalent compared to Asia where HBV transmission is predominantly perinatal and the association of PAN has not been reported. Guillevin et al. [73] show that the clinical manifestations of HBV associated PAN were similar to those of typical PAN except for: gastrointestinal complications (perforation and bleeding, present in 46.3% of cases), malignant hypertension (29.6% cases), renal infarction and orhi-epididymitis (26%). Untreated, the prognosis of HBV- associated PAN is poor, with 30-50% mortality, consequence of vasculitis [54]. In glomerulonephritis, the HBsAg-anti-HBs circulating immune complexes were identified in the glomerular basement membrane. All three major antigens of HBV (HBsAg, HBeAg and HBcAg) were located in the glomerular capillary wall or mesangium. Deposits of immune complexes in patients with viral hepatitis were detected in the kidney even in the absence of kidney disease. The most common form of glomerulonephritis in chronic hepatitis B was the nephrotic syndrome. Healing is frequently associated with HBsAg seroconversion and only a small proportion of patients progresses to kidney failure [54]. " Arthritis-dermatitis" syndrome is common among patients with chronic hepatitis B, being present in about 1/3 of patients [71, 73]. Joint and skin manifestations are varied and remit spontaneously [75]. The essential mixed cryoglobulinemia remains controversial. There are 3 types of cryoglobulins; Type II 33

35 (monoclonal IgM and polyclonal IgG) and type III (polyclonal IgM and monoclonal IgG) cryoglobulins are classified as essential and are found in patients with chronic HBV infection. The prevalence of cryoglobulinemia ranges from 0% to 15% [163, 164]. Purpura and arthralgies are manifestations. Cryo-globulinemia is frequently associated with Raynaud's phenomenon, arthritis and Sicca syndrome, which can cause renal, neurological and vascular complications [7, 164] The treatment of the clinical forms of chronic hepatitis B and the responsivity to treatment Treatment of VHB infection has remarcable progresses in the last years, and now, the therapeutical options can rapidly and efficient reduce the viral load. The VHB infection has important medical and socio-economics implications. That s why the problem of an etiological treatment is very important. For the treatment of CBH the FDA (Food and Drugs Administration) has approved two different antivirals: conventional interferon or pegylat alfa and nucleoside analogues (NA) (lamivudine, telbivudine, emtricitabine, entecavir) and nucleotide (adefovir and tenofovir). As predictors of response to interferon therapy, international criteria were established, based on the amount of DNA / HBV (<2 x 10 UI/ ml) determined by RT PCR, useful for initiation and continuation of viral therapy, increased transaminases and HBV genotypes: genotypes A and B proved to be associated with a good response in the appearance of anti-hbe Ab, secondary sero-conversion and loss of HBsAg compared with genotypes D and C. The 10 types of VHB have different geographical distributions and population shifts occur due to intravenous drug use, sexual behavior and migration situation. For making therapeutic decisions based on clinical evidence, studies have been conducted, which compared the effectiveness of antiviral medication - NA versus interferon. Clinical decisions, individualized for each patient should be taken immediately based on response rate to therapy and on the basis of adverse reactions. Management of antiviral therapy should be based on virological monitoring to facilitate early evaluation of partial response to treatment, and treatment failure. Our study has 38 patients diagnosed with chronic hepatitis B. We followed the clinical and paraclinical evolution under the treatment with Lamivudine. The medical area that use blooding techniques has a higher risk from the epidemiologic point of view for the B hepatitis infection, the infection spreading, stomathology being the place where such an infection can be contracted, the dentist and the patient being both affected in the same measure, the risk of infection with VHB virus. In the Infectious Diseases Hospital of Iasi was realized a retrospective study on patients with chronic B virus hepatitis treated with Lamivudine (Zefix). These patients were clinic and paraclinic followed after a common protocol, with evaluation at 1, 3, 6 and 12 months of therapy. Patients had these periodic evaluations hospitalized, and we had also the 34

36 epidemiologic context of apparition of the viral hepatitis B infection. The studied group was represented by 38 patients with chronic B hepatitis: HBsAg positive, HbeAg positive (35 patients), other 3 patients were diagnosed with chronic hepatitis of mixt etiology B and C: HBsAg positive and anti-hbc Ab positive. From all the patients, a half (126.31%) presented in their records stomathologic treatments, 3 of them were diagnosed with acute viral hepatitis at days after stomathologic treatment. Were introduced Lamivudine treatment (100 mg/day) and the clinic and paraclinic evaluation: the biologic liver tests and serologic markers. From the clinical point of view, under therapy, the general status of the patients got better, and the serological markers, were followed the seroconversion HbeAg in HBeAb. After 6 months of treatment, the results were spectaculous in 37 of those 38 patients, seroconversion was present in 97.36% cases. The liver tests had the same favorable impact under Lamivudine, transaminasis presented important decreassed values in the first 3 months of treatment, then had normal values. Bilirubinemia had not very high values at the begining of the treatment (20-40 mg/l), and the values decreased in the first month of treatment; after 3 months the values were erau normal in all the patients.we followed also the evolution of the factors that are in coagulation, through determination of Quick time; evolution being favorable in the first months (table 1.X). Thrombocites number grew in the course of treatment (table 1.XI). The studied patients don t have adverse reactions, and the renal functional probes were unmodified. The imagistic evaluation of the liver after 6 months of treatment showed ameliorated aspects of echography. 20 patients received hepatotrophic therapy and 2 patients received endovenous coagulation factors. Table 1. X. Quik time values at treated patients with Lamivudine Lenght of the therapy Values of Quick test 1 month 20 40% 3 months 50 65% 6 months > 66% Table 1. XI. No. of thrombocites in patients treated with Lamivudine Lenght of the therapy No. thrombocite/mm 3 1 month 40,000 58,000 3 months 60,000 80,000 6 months >100,000 35

37 We may say that the Lamivudine treatment had a favorable clinic and paraclinic effect in the studied patients. The Hbe Ag-HBe Ab seroconversion was present in all patients in the first 6 months of treatment. The transaminases values were normalized after 3-6 months of treatment. We saw a favorable effect on the markers, that reflected the coagulation factors. The clinic and paraclinic evolution of the patients under Lamivudine therapy recomands this agent like a hope for the patients with VHB infection future Chronic hepatitis C treatment Chronic hepatitis C is another important public health problem. In Romania, the big number of the hepatitis C virus infected patients represents an important public health problem, with a severe evolution of the disease to cirrhosis or liver cancer. We can say that the routes of transmission are: surgical and stomatology treatments with insufficient sterilized instruments, together with the sexual one another transmission route of VHC. The principal aim of the antiviral treatment is prevention of the severe complications, realized by obtaining of sustained viral response (SVR), which means undetectable (RNA-VHC) at the finish of treatment and after 6 months after the end of treatment [117]. Both types of Pegylate Interferon used in treatment (Pegasys or Pegintron) associated with Ribavirine have no differences in therapy results in our study. The development of new diagnosis methods and new therapeutically strategies determine better response rates to treatment, remaining the problem of no responders patients. In Romania an important particularity represents the preponderance of the genotype 1 (95.5%) in population, which is resistant to treatment and more predisposed to rebound after the treatment. The therapy protocol of patients presumes stopping therapy at no responders and continuing therapy to 42 weeks at the responders patients. Chronic hepatitis C is an important cause of morbidity and mortality due o hepatic disease. The retrospectively studied the evolution of 30 patients with chronic hepatitis C, treated in the Infectious Diseases Hospital of Iasi, with Peginterferon and Ribavirin for 48 weeks. As results, 18 patients were treated with Peginterferon and Ribavirin, and 12 with Peginterferon and Ribavirin. Most of them (73.3%) were adults, aged between 30 and 50 years, with a sex ratio M/F - 13/17. Most of them had risk factors for the transmission of HCV: 28 of them suffered surgery and 2 of them had infected sexual partners. 3 patients achieve a rapid virusologic response and 6 patients were relapsers. All the other 70% of patients had a sustained virologic response. The side effects were present in all patients, with a moderate intensity. 36

38 In conclusion the success (SVR) rate of antiviral therapy was higher than expected (especially for genotype 1 HCV). The patients with a viral relapse could be soon treated with the new protease inhibitors and hope for a cure. Chronic hepatitis C represents a major cause of morbidity and mortality through liver disease. The evolution of the chronic infection with hepatitis C virus is variable and sometimes very severe, leading to liver cirrhosis and hepatocellular carcinoma, which are the most frequent indications for liver transplant. Before the beginning of therapy, we determined: viral load, ALAT and a complete blood count; the degree of liver fibrosis was determined through liver biopsy or noninvasive tests (fibroscan). The viral load was repeated at 12 weeks of treatment and six months after the end of the therapy. We took into consideration the adverse haematological reactions of the therapy, represented by: anemia, leukopenia and/or thrombocytopenia. In the 30 patients study, the success (SVR) rate of antiviral therapy was higher than expected (especially for genotype 1 HCV). The patients with a viral relapse could be soon treated with the new protease inhibitors and hope for a cure. From the history of the patients we identified some risk factors for the transmission of HCV. The route of transmission could have been parenteral in 28 cases (93.3%) surgical treatments were involved in 11 cases (36.66%) and stomatological treatment in 19 cases (63.33%); two patients had HCV infected sexual partners (Table 1.XII). The value of viral load at the moment of initiating therapy varied between ui/ml and ui/ml; and the medium values of ALAT was of 125ui/l (between 60 to 200ui/l). Table 1.XII. Risk factors for HVC infections [117] The liver biopsy was done in 80% of the patients and 20% with fibro scan; 52% of these patients being classified with F2 medium fibrosis 37

39 Chapter 2. RESEARCHES IN TUBERCULOUS MENINGITIS AREA Tuberculous meningitis, an affection difficult to diagnose precocious precoce, was a permanent preocupation, being a study subject in many articles, where we realized a comparative analize of the evolutive particularities in children and adults, the results of the study confirming some data from literature, and also to underline the frecquency of the atypical manifestations in every age category, with an impact on the diagnosis and of the precocious therapeutical decision Epidemiology Tuberculous meningitis is still a very serious disease in Romania, with high morbidity and mortality, especially among children and elders. Early diagnosis should be considered to improve prognosis. This study indicates that a new and rapid diagnosis method is highly needed and improves the prognosis of tuberculous meningitis, shorting the time needed for an early and accurate treatment initiation. In Romania, the morbidity through tuberculosis is high (especially in the children age) having one of the most important incidence in Europe. The tuberculous meningitis still remain a public health problem the disease being classic considerate a children disease with a maximum recurrence in the first 3 years of life [21, 40]. The protection through BCG vaccination moves the incidence of this disease from the little child to the big one and young adult. The necessity of a epidemiologic, clinic and therapeutical researche în tuberculous meningitis is relevant because of the increased number of new cases of bacillary meningitis in the recrudescence conditions of tuberculosis in general, and because of the increasing morbidity through bacterian meningitis. In this aim were realised a lot of retro- prospective studies on cases of tuberculous meningitis admitted in the Clinical Hospital of Infectious Diseases Iași. A first study was done on a number of 82 patients children, with the diagnosis of tuberculous meningitis between [38]. The aim of this study was to evidentiate the clinic, epidemiologic and therapeutic aspects on 82 cases of tuberculous meningitis in children during 4 years. The diagnosis was based on clinical criterion and confirmed through isolation of M. tuberculosis in culture of cerebrospinal fluid in 2g% cases and by direct examination of cases. The treatment was done with quadruple association of antituberculous drugs with a favorable evolution and a lethality of 9.15%. The study showed a increased incidence, registrating in medium 2-4 new cases per year in children (29.94%), comparatively with cu adults % [98]. 38

40 Along with the rise of morbidity from tuberculosis, in general, we have witnessed an increase by 16% of bacillary meningitis at the Infectious Diseases Hospital of Iasi since the last decade; the annual incidence was influenced by the tuberculosis epidemic and the administration of anti-tuberculosis treatment previous to the bacteriological examinations. Out of the 82 cases of tuberculous meningitis studied, in the previous years the maximum incidence of cases was of 33 cases (40,24%) in 2000 and 15 cases (18,29%) in the first 8 months of The maximum incidence of cases was in the age group of infants and small children (with 9 cases, i.e. 23%), followed by the age group of the older child (with 15 cases, i.e. 18,29%) (table 2.I). The disease was mainly present in the male sex where 59 cases were recorded (72%). 22 patients in a coma were hospitalized (26%). 8 of them (0,75%) had problems remaining conscious and paralyses at the level the cranial nerves. Year Total Table 2.I. Repartition of tb meningitis cases on groups of age [38] Groups of age (years) 0-1 >1-4 >4-9 >9-14 >14-18 Nr % Nr % Nr % Nr % Nr % Total In 22 cases diseases associated with tuberculous meningitis were present: protein-calorie malnutrition, epilepsy, tuberculous granuloma, pulmonary tuberculosis, internal hydrocephaly. 81.7% of the patients had antecedents of tuberculosis and 28% of tuberculosis contact, as well as other pre-existent pathological states (flu, measles) that weakened the resistance to infection. Generalized tuberculosis preceded meningitis in 11.9% of the cases. 39

41 Diagnosis. The laboratory diagnostic of meningitis was carried out by examining the cerebrospinal fluid that had a clear aspect with predominant lymphocytes in sediment and pleocytosis varying between elements/mmc in 80% of the cases and over 500 elements/mmc. The certain diagnosis was obtained by emphasizing M. tuberculosis in the direct exam of the cerebrospinal fluid in 14.63% of the cases, and in 28% of the cases in culture; Koch s bacillus turned out to be multiresistant in 10 strains (12.1 %). The etiological treatment was administered in the first 48 hours of hospitalization and after days for the remaining patients. Treatment included: isoniazid, rifampicin, streptomycin, pyrazinamide or ethambutol in quadruple association, 7/7 scheme for two consecutive months in the hospital. The precocity in the institution of therapy was noticed in most cases at the simple suspicion of diagnostic much before the bacteriological confirmation of the disease. In severe forms of the disease with neuropsychiatric phenomena and coma (17% of the cases), dexamethasone was employed as pathogenic therapy. The efficiency of the therapy was shown by the favourable evolution of meningitis (90.25%) as neurological sequelae and complications were recorded. The sequelae were the following: hydrocephaly, hemiplegia and cerebral tuberculomas (in 2 cases) and cecity by optic nerve atrophy (1 case). In 9 cases, complications were signalled: neuropsychiatric dysfunctions (12%), motor dysfunctions (4%) and side effects of the anti-tuberculosis treatment (rifampicin hepatopathy and vision problems after ethambutol). Mortality caused by this disease in children was of 9.75% with an increase in the last two years, especially in the male sex and in the age group of 1-4 years. To conclude, in the previous years the tuberculous infection, in general tuberculosis and meningitis, in particular, increased significantly mainly in the age group of children in their first three years of age (despite BCG vaccination) Clinical and paraclinical aspects of tuberculous meningitis The etiological diagnostic of tuberculous meningitis by isolation in culture of Koch s bacillus has a long elaboration period, hence the drafting and implementation of new efficient methods such as the Quantiferon TB gold dosage in the cephalorachidian liquid. The aim of this study is presentation of tuberculous meningitis in the department of Iasi Infectious Diseases Hospital, where patients from all North-East Romania are attended to [119]. There were retrospectively studied the cases of tuberculous meningitis during regarding clinical and laboratory methods of diagnosis and used the classic 40

42 laboratory methods together with new ones, the quantification of gamma-interferon in blood, in presence of M. tuberculosis specific antigens [78]. A total of 58 patients (41 male [71%] and 17 female [29%]) were included. The mean age was 31 years (age range: 10 months 80 years) with 12 patients (21%) over 50 years old and 15 under 14 years old (26%). 15 (26%) patients had a history of tuberculosis. The clinical symptoms and signs on the admittance were compatible with tuberculous meningitis: consciousness alteration in 18 cases (31%), focal neurological signs in 11 cases (19%), paraplegia and/or hemiplegia in 7 cases (12%) and extraneurological tuberculosis was associated in 32 cases (55%). The patients fulfilled some of following diagnosis criteria: CSF (cerebrospinal fluid) pleocytosis (with 300 elements/ml for 17 cases); high CSF protein level (45 cases, 78%); low CSF glucose level (32, 55%); favorable response to antituberculous therapy and steroids (51 cases, 88%); and a mortality of 12% (7 cases). Microbiological diagnosis was done through culture in CSF with positive results in 18 cases: 6 cases (10%) were with negative CSF cultures and 12 (21%) had positive M. tuberculosis CSF cultures and positive Quantiferon TB Gold results. Also, in 3 cases (5%) we had multidrug resistant strains of M. tuberculosis. We used quadruple association of antituberculous drugs for treatment, with favorable response in 48 cases (83%) and with a mortality of 12% (7 cases). Sequels were present in 18 cases (31%), with: hydrocephalus in 10 cases (56%), arachnoiditis in 3 cases (16%) and tuberculomas in 5 cases (28%). This study indicates that a new and rapid diagnosis method is highly needed and improves the prognosis of tuberculous meningitis, shorting the time needed for an early and accurate treatment initiation. The study of tuberculous infection worldwide and in Romania shows a reemergence of this disease mainly because of the HIV pandemic and the problem of selecting multiresistant strains of Koch s bacillus. Classic microbiological diagnostic methods are intricate, slow and reduced in sensibility [9, 52]. A great part of the new diagnostic methods in molecular biology incur great costs which prevents their use at a large scale. Moreover, despite the precise data they provide, they take too much time from the clinician s viewpoint. Due to the good price-performance ratio, the Quantiferon TB Gold test [78] comes with a series of incontestable advantages: its average price is affordable even for Romanians, it provides much greater accuracy than classic methods (such as tuberculin skin test reaction); it is a rapid method which gives results in approximately 1-2 days, thus allowing the early onset of specific treatment. This leads to an important amelioration of the prognostic, decrease in mortality and treatment costs per patient by avoiding the implementation of a treatment in the absence of diagnostic certainty. 41

43 Tuberculosis affects annually 8-9 million people all over the globe, being responsible for 2-3 million deaths. [1, 33] The main research directions to fight against this disease are the following: the development of new methods for an early diagnostic; the discovery of an efficient treatment against multiresistant strains of Koch s bacillus and obtaining a more advanced and efficient vaccine than the current one. The meningian localization of tuberculous infection renders the etiologic diagnostic even more difficult due to non-specific manifestations, limited access to the place of infection and the lack of the practicing physician s proper degree of suspicion. National and international experience showed that current diagnostic methods did not significantly contribute to a lower incidence in pulmonary or extra-pulmonary tuberculosis [152,190,195]. The main advantage of diagnostic immunological tests consists in their capacity to account for the presence of a previous sensibility or infection without the emphasis of Koch s bacillus as necessary. This study focused on tackling the performance of an immunological test that evaluates the γ-ifn production of T-lymphocytes as response to some antigens present at the level of M. tuberculosis (ESAT-6 and CFP-10), not at the level of M. Bovis (BCG) [120]. Retrospectively, there were studied consultation sheets of patients diagnosed with tuberculous meningitis in 2006 at the Infectious Diseases Hospital of Iasi and prospectively, patients hospitalised between January 2007 December 2008 (55 cases) [120]. The following variables were analysed: demographic, epidemiological (contacts with other patients, provenance from hot zones, antecedents of tuberculosis), clinical (type of onset, symptomatology, state at hospitalization, clinical signs, evolution, sequelae), paraclinical, imagistic: thoracic radiography, cranial CT, haematological, biochemical, tuberculin skin test reaction (2 ui ppd), cytological test, biochemical and microbiological LCR study. It was harvested at hospitalisation and afterwards at variable intervals according to the clinical evolution. It was studied from the viewpoint of the macroscopic (cellularity), biochemical (albuminorrachia, glycorrachia, chloridorrachia) and bacteriological aspect (Lowenstein-Jensen medium slants). - Quantiferon TB Gold test -IFN blood dosage: via IT system (heparin tubes method): 1 negative control, 1 TB antigen and 1 mitogen for positive control according to the package leaflet. -IFN dosage in LCR: using the QuantiFeron TB gold kit and a separate antigen stimulus package (Ag specific ESAT6 and CFP10, negative and positive control. Immediately after LCR harvesting, this is transferred to a dry sterile capped microtube and homogenized through flipping; with a sterile pipette 1 ml LCR was poured in minimum 2 wells of a plaque of cell cultures to accomplish at least the negative ESAT control tandem; when a 42

44 larger quantity of LCR was available, 1 ml was added for the CFP10 reaction. Since it was possible to establish patients immune-competence by using mitogen in serum test, the compound was no longer used in the case of LCR. In adding antigenic stimuli, the ratio mentioned on the package leaflet was respected (3 drops/ml). After stirring it for 60 seconds, the culture plate was incubated for 24 hours at 37 C and then the work procedure of the Quantiferon TB Gold was applied, according to the package leaflet. The results were formulated as follows: negative, positive and indeterminate, considering the cut-off value of 0.35 UI/ml above the concentration of negative control. The control group consisted of 68 patients with other diseases (x with viral meningitis, z with bacterial meningitis and y without inflammation of meningitis) and without known contact or personal or heredocolateral antecedents of tuberculosis infection. In all these patients QTFG and LCR blood tests were carried out, as well as clinical and hematological evaluation. - Therapeutic: first intention treatment and antituberculous treatment schemes used - Evaluation of specific indicators (total number of hospitalization days for patients with tuberculous meningitis, average number of hospitalization days / patient / year) The statistical analysis was carried out using Microsoft Excel + Analyse-it software, as well as the t Student test independently and χ 2 test. The significance threshold considered was p=0.05. As results, demographic variables and the day of disease at the moment of QTFG testing were similar to patients with tuberculous meningitis at the control group. (table 2.II) Table 2. II. Demographic variables in the momentof QTFG testing [120] Parametres TB meningitis Control group Significance difference level Medium age (years) 32,81 (95% CI: 26,6-38,9) (95%CI: 28,0-42,1) p 1 =0.62 M/F Report p 2 =0.92 Urban/Rural p 2 >0.5 Days of illness when testing QTFG p 1 >0.5 1 test t Student, 2 test χ 2 M. tuberculosis was the third cause (21.9%) of acute meningitis during the period studied ( ) after other bacteria (51.7%) and viruses (26.4%). In spite 43

45 of the fact that the average number of cases during the period studied 32.7 was lower than the one between : 35.2 cases/year, the difference did not have statistical significance (t=0.44, p=0.67). A significant decrease of the case number showed in viral meningitis (75 vs 39.2 cases/ year, t=4,43, p=0.0043). There was a predominance of males %, and most patients from rural areas (ratio U / R = 0.89). The patients' age ranged between 1 and 82 being the average of (95% CI: 26.6 to 38.9). Almost one quarter of the patients were children under 14 years and 11.3% were represented by the elderly (> 60 years) bacteriană tuberculo tuberculous tuberculoasă virală bacteriană bacterial Figure 2.1. The annual number of cases of acute meningitis, according to aetiology [120] years years years Figure 2.2. The distribution of patients by age [120] Nine patients (16.4%) were severely imunocompromissed, being diagnosed also with AIDS. The number of CD4 lymphocytes at the moment of QTFG testing varied 44

46 between 26 and 321/mm 3 (average 92.1/mm 3 ). Other 13 patients were found to suffer from diseases associated with an immune-depressive effect that probably contributed to the grafting of tuberculous infection at the level of meningitis: 7 patients with chronic alcoholism, 2 with diabetes, 1 with hepatic cirrhosis, 1 with malnutrition, 2 with extreme ages. In 19 patients (34.5%), it resulted from the anamnesis that they had a long contact with other people they knew who also suffered from pulmonary tuberculosis (most frequently (73.8%) 12 family members). The presence of another simultaneous localization of the tuberculous infection was found in 58.2% of patients, most frequently in the first stage of the trial ( : 71.1%). The lung was included the most often, in 38.9% of cases (72.2% of identified localizations). miliar % pleural % pulmonar 72,2% pneumonie varf % articular 5.556% ganglionar 5.556% alte pneumonii % Figure 2.3. Primary outbreak of tuberculosis [120] As clinical manifestations, 16 patients (29.2%) had an altered state of conscience, 10 at hospitalization (6 being transferred from other services or hospitals), and other 6 entered a coma during hospitalization. The average duration of the coma was of 3.6 days (extreme periods: 1 and 14 days). From the patients (or relatives) that allowed the gathering of anamnestic data, it resulted that the disease onset was an insidious one in only 69.6% of the cases. The duration of premonitory symptoms was between 5 and 15 days. The patients displayed a wide range of signs and symptoms at internation and the most constant ones were the following: rebellious cephalalgia 92.7% of the cases, fever/ subfebrility 76.7% or extreme vomiting 63.6%. The meningeal syndrome of contracture was also highly present at hospitalization (72.7% of the patients). In the remaining patients, it was only drafted, due to the difficulty of its interpreting in a clinical context. Generalized tonic-clonic convulsions were present in 16 patients (out of which 13 were in meningeal coma). Other 5 patients had localized clonic convulsions at member (2c) and face level (1c). In only 54.5% of the cases leucocytosis was found at 45

47 hospitalization, the average number of white cells at that moment being of 9045 / mm 3 (CI95%: ). The great majority of patients (78.2%) had an increased percentage of polymorphonuclear cells in the leukocyte formula (average: 73.9%, CI95: ). The VSH level at hospitalization (available only in 43 patients) varied between 12 mm/h and 116 mm/h, its average being of 47.3 mm/h (CI95%: ). cough tuse other neurologic manifestations alte manifestări neurologice coma comă convulsions convulsii weight loss scădere ponderală fever febră vomiting varsaturi headache cefalee sudoration transpiraţii asthenia astenie Figure 2.4. Signs and symptoms on admission [120] In all patients, a lumbar puncture was performed. The LCR harvested was analysed from a cytological, biochemical and microbiological viewpoint and by applying the QTFG test. The LCR constants in patients with tuberculous meningitis are summarized in the table 2.III. The direct exam in Zhiel-Nielsen coloration method highlighted acid-alcohol resistant bacilli in 3 patients (5.4%c). The culture on the Lowenstein Jensen medium led to the bacteriologic confirmation of the diagnostic in 13 cases (23.6%c). The average interval necessary to grow Mycobacterium tuberculosis was of 24.2 days. In the rest of the cases, the diagnostic was supported by the clinical chart, cellularity and biochemical constants of LCR, the existence of other outbreaks of tuberculous infection and the favourable development under tuberculostatic treatment. Table 2.III. Analysis of CSF cytological and biochemical [120] 46

48 CSF Average 95% CI of Mean No. cells % limfocytes Albuminorachy Glicorahy Report glicorahy/glicemia Clorurorahy Testing patients using QTFG was carried out between 4 and 26 days from the onset of symptoms, with an average of 13.6 days (CI 95%: ). The results of Quantiferon TB Gold blood test in patients with tuberculous meningitis are shown in table 2.IV. Excluding the indeterminate results, the QTFG blood test sensibility was of 68.6% and the specificity of 93.9%. Considering that 4 tests were false positive, is results that the positive predictive value of the QTFG serum test in patients with tuberculous meningitis was of 89.7%.The predictive negative value was lower, of 70.4%. The results of Quantiferon TB Gold test in LCR of patients with tuberculous meningitis are shown in table 2.V. Table 2. IV. QuantiFERON-TB Gold blood test Result TB meningitis Witnesses non-tb No. cases % No. cases % total Positive Undeterminate Negative total Excluding patients with non-interpretable tests, QTFG test sensibility in the LCR of tuberculous patients was slightly lower as compared to the one obtained in serum 62.7%. Moreover, the specificity of the test in LCR was comparable to the one obtained in serum 95.3%. The positive predictive value calculated was of 91.4% and the negative predictive value of 76.2%. Comparing the average age of QTFG 47

49 patients positive in blood to the negative ones, no significant differenced were noticed: 33.9 vs 32.0 years, t=0.31, p=0.75. A similar situation can be found in the case of results from LCR: 31.9 vs 33.48, t=0.25, p=0.8. Out of the 13 children (with ages under 14), 4 were false negative in LCR and 3 in serum. Thus, it becomes clear that QTFG test sensibility was not significantly influenced by the patient s age; it was generally higher in adults than in children: blood 64.3 vs 61.5, p=0.88, LCR 59.5 vs 53.8, p=0.96. Table 2.V. Results QuantiFERON TB Gold test in CSF Quantiferon TB Gold în-lcr TB meningitis Witnesses non-tb Result No. cases % Nr. cases % total Positive Undeterminate Negative total Patients with positive results and those woth false negative results in blood had a similar number of leucocytes (8960,5 vs 9169,2, p=0.84) and lymphocytes (2471,2 vs 2396,1, p=0.86) in periphery. It was also found that patients with positive tests in LCR had more lymphocytes in LCR at the moment of testing as compared to those with false negative results: 272,4 vs 202,6/ mm 3, without a significant statistical difference (t=1.09, p=0.28). There were no statistically significant differences between biochemical constants in LCR based on the result of the Quantiferon test in LCR table 2.VII. First intention tuberculostatic treatment was implemented along with the diagnostic which was probably that of tuberculous meningitis (anamnestic, clinical data, LCR characteristics, imagistic explorations). Table 2.VI. QTFG test results (blood and CSF) according to the age group Age Blood LCR positive Negative + undeterminate positive Negative + unedeterminate 48

50 Children Adults Table 2.VII. Average CSF biochemical variables based on the QuantiFERON test result Biochemical variables Quantiferon positive Quantiferon faulse negative Independent t test Albuminorahy (g/l) t=-1.09, p=0.27 Glicorahy (g/l) t=0.26, p=0.79 Clorurorahy (g/l) t=-0.12, p=0.82 The most employed initial scheme was the association between Rifampicin (10 mg/kgc pr child, mg/day per adult) + Isoniazid (5 mg/kgc/day per child and 300 mg/day per adult), Pyrazinamid (25 mg/kgc/day per child and g/day per adult) + Ethambutol (25 mg/kgc/day per child, 1 1.5g/day per adult), in its daily administration 83.6% of the cases. In 9 patients, Ciprofloxacin was used instead of Rifampicin due to the simultaneous severe side effects at hepatic level. In patients that had problems of their conscious state, dexamethasone was administered (approximately 0.23 mg/kgc/day) for an average duration of 6.02 days. Of the side effects attributed to therapy, the following occurred: peripheral neuropathy (34.5%), mild cytolytic hepatitis (67.3%), and digestive intolerance (41.8%). The following complications were cited: hydrocephalia 8 cases, the development of intracranial tuberculomas 5 cases, paresis of cranial nerves 5 cases, hemiparesis 6 cases. In 27.3% of the cases, it was necessary to change therapy due to the side effects or unsatisfactory development. Death occurred in 12.7% of the cases, as 7 out of the 55 patients died during hospitalization. Among the patients for whom anti-tuberculosis therapy was initiated before QTFG testing, a greater number of false negative or indeterminate results was noted, as compared to the patients without treatment but without any statistical significance. Another prospectively study analyses the different diagnosis methods for tuberculous meningitis available in Infectious Disease Hospital Iasi, Romania. Retrospectively were studied the cases of tuberculous meningitis admitted from January 1st till October 2007, in Infectious Disease Hospital Iasi, regarding epidemiological, clinical data and laboratory methods of diagnosis: cerebrospinal fluid examination and culture. tuberculin skin test, radiological assays and quantification of 49

51 gamma-interferon in blood and cerebrospinal fluid using Quantiferon TB Gold assay (QTF) [121]. The patients were divided in 2 groups: one of 17 patients with TB meningitis and one control group of 28 patients with either bacterial (3c), lymphocytic meningitis (17c) or without central nervous system infection (7c). TB meningitis was more frequent in males, with a mean ratio of 2.5. The age of patients varied between 2 and 78 years, with a mean of 10.5 years. Many of the patients were children (35.6%) and 40% were older than 36 years. In 71.1% (32c) of the cases the meningeal infection was secondary disseminated from a primary site, most frequent a pulmonary one (68.7%) and bacteriological confirmation by culture in 6 cases (13.3%) with a medium of 21.4 days of cultivation time. For the other cases the diagnosis are established by clinical data, cell count and biochemical analysis of CSF, the presence of extrameningeal tuberculous infection and positive evolution under antituberculous therapy. TST was performed to all the patients except the AIDS patients; it had a sensitivity of 71.4% and a specificity of 62.5%. In whole blood the sensitivity of QTF was In CSF the specificity was higher than in blood (96.1 vs 88.4%), having a positive prediction value over 90%. Antituberculous therapy prior to QTF testing had a negative impact, 5 of 6 cases having negative QTF results. QTF (serum or CSF) had a better sensibility and specificity than TSL, CSF direct exam or culture and could be used to establish an early diagnosis of tuberculous meningitis. To diagnose the tuberculous meningitis and to evaluate the benefit of this early method of diagnosis of tuberculous meningitis, gamma-interferon levels comparatively in blood and cerebrospinal fluid (CSF), we also study the whole-blood and cerebrospinal fluid interferon-gamma release assay We have studied 50 patients with tuberculous meningitis admitted in the Clinic of Infectious Diseases Iaşi between , from the epidemiological, clinical, diagnosis and therapeutical points of view [122]. Tuberculous meningitis was more frequent in males, with a mean ration of 2.5. The age of patients varied between 2 and 78 years of age, with a mean value of 30.5 years. 35.6% of patients were children. In 32 cases the meningeal infection was secondary, disseminated from a primary site, most frequently from the lungs (68,7%). The bacteriologic confirmation was through culture in 13.3%, with an average of 23.4 days of incubation. In whole blood the sensitivity Quantiferon TB Gold assay (QTF) was 78.57%. In CSF the specificity was higher than in blood (96.1 vs. 88.4%), having a positive prediction value over 90%. Antituberculous therapy prior to QTF testing had a negative impact, 50 to 6 cases having negative QTF results. The performance indicators of TB Gold Quantiferon Test in serum or CSF are high, being more sensitive and rapid than the direct exam of CSF or BK culture. 50

52 The first study of Haydarpasa-1 [61] showed the implementation of new immunodiagnostic tests based on the Mycobacterium tuberculosis specific antigen, early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), that showed promising results in the diagnosis of tuberculosis infection. However, there are only few studies in the published literature on performance tests in cerebrospinal fluid [94, 143, 169]. We investigated whether a rapid diagnosis of tuberculous meningitis could be established by interferon-γ blood and cerebrospinal fluid (CSF) tests in children. In the same time, there were studies that provide data on the diagnosis of tuberculous meningitis in the largest case series ever reported. Tuberculosis remains a major global health problem and is second to human immunodeficiency virus infection as an infectious cause of death. In 2011, the global tuberculosis prevalence was 13 million, and the incidence was 8.7 million, while mortality due to tuberculosis was 1.4 million. Tuberculous meningitis, one of the extrapulmonary tuberculous diseases, occurs in <1% of all cases, and it is the most severe form of tuberculosis [152]. Tuberculous meningitis is seen in all age groups but recent data from Germany indicated that individuals aged 15 years and over accounted for 88% of all patients[56]. We know that the mortality rate for ranges tuberculous meningitis between 20% -69% worldwide with up to half of survivors experiencing irreversible sequelae (e.g. paraplegia, blindness, motor, cognitive deficits). Cerebrospinal fluid (CSF) examination is the mainstay in the diagnosis of TBM. Definitive diagnosis depends on detection of tuberculous bacilli in the CSF either by smear examination or by culture. However, it comprises many challenges because quick, reliable and affordable diagnostic tests are not always available. The sensitivity of CSF smear microscopy is low (10 60%) and depends on the capacity of laboratories and technicians experience. Added to that, the sensitivity of CSF culture is as low as 25% and availability of results after 2 6 weeks of incubation causes delays in making the proper diagnosis and initiating treatment. Hence, the diagnostic algorithm of tuberculous meningitis should be reevaluated with combinations of older and newer diagnostic modalities. Prognosis of the disease is largely inter-related to early diagnosis leading to initiation of proper treatment. This was a retrospective, multicentric and multinational study-1, made by Haydarpasa-in a cohort study that involved patients hospitalized for tuberculous meningitis between 2000 and 2012, involved patients with microbiologically confirmed tuberculous meningitis in many countries (Albania, Croatia, Denmark, Egypt, France, Hungary, Iraq, Italy, Macedonia, Romania, Serbia, Slovenia, Syria and Turkey) [59, 61]. In this multinational cohort were investigated the laboratory implications of the largest microbiologically confirmed tuberculous meningitis case series ever reported 51

53 and the main aim for this study is to provide data for the optimization of diagnostic approaches. As results, a positive culture, PCR or Ehrlich Ziehl Nielsen staining (EZNs) from the cerebrospinal fluid (CSF) was mandatory for inclusion of meningitis patients. A total of 506 TBM patients were included. The sensitivities of the tests were as follows: interferon-c release assay (Quantiferon TB gold in tube) 90.2%, automated culture systems (ACS) 81.8%, Lowenstein Jensen medium (L-J) 72.7%, adenosine deaminase (ADA) 29.9% and EZNs 27.3%. CSF-ACS was superior to CSF L-J culture and CSF-PCR (p <0.05 for both). Accordingly, CSF L-J culture was superior to CSF- PCR (p <0.05). Combination of L-J and ACS was superior to using these tests alone (p <0.05). There were poor and inverse agreements between EZNs and L-J culture (j = 0.189); ACS and L-J culture (j = 0.172) (p <0.05 for both). Fair and inverse agreement was detected for CSF-ADA and CSF-PCR (j = 0.299, p <0.05). Diagnostic accuracy of TBM was increased when both ACS and L-J cultures were used together. Non-culture tests contributed to tuberculous meningitis diagnosis to a degree. However, due to the delays in the diagnosis with any of the cultures, combined use of non-culture tests appears to contribute early diagnosis. Hence, the diagnostic approach to tuberculous meningitis should be individualized according to the technical capacities of medical institutions particularly in those with poor resources. Inclusion criteria were approved in the protocol by the Institutional Review Board of Istanbul Fatih Sultan Mehmet Training and Research Hospital, being: age over 14 years and clinical evidence of meningitis (fever, nuchal rigidity and CSF abnormalities) and microbiological confirmation of TB meningitis. At least one of positive CSF culture, PCR analysis and Ehrlich Ziehl Nielsen staining (EZNs) was mandatory for the inclusion of the patient into the study [131, 171, 172]. The diagnosis of TB meningitis was made by the clinicians at the participating centres. In addition to CSF and routine laboratory analyses, a neurological scale of Glasgow coma score (GCS), which aims to score the conscious state of the patient, was also recorded by the clinicians for each patient on admittance. A GCS of 13 was accepted as mild, 9 12 as moderate, and 8 as poor. Although three different types of interferon-c (IFN-c) release assay (IGRA) (QuantiFERON-TB Test, QuantiFERON-TB Gold Test and QuantiFERON-TB Gold Test In-Tube, all products marketed by Cellestis Ltd., Carnegie, Vic., Australia) were used, due to consistent specificity of >99% in low-risk individuals and a sensitivity as high as 92% in individuals with active disease, the results of QuantiFERON-TB Gold In-Tube test were included solely in our study. This in vitro diagnostic test used a peptide cocktail simulating esat-6, cfp-10 and tb 7.7(p4) antigens associated with M. tuberculosis infection to stimulate cells for IFN-c in heparinized whole blood drawn directly into specialized blood collection tubes (Quantiferon-TB Gold In-Tube Package Insert, Cellestis, 2006) 52

54 The results were calculated and interpreted according to the manufacturer s instructions. CSF adenosine deaminase (ADA) activity was evaluated with different marketed test kits by the Giusti method, and kinetic determination and spectrophotometric method in the participating centres. All estimations were performed according to the manufacturer s guidelines and CSF ADA activity was quantified as Unit/liter. CSF culture results: overall, 412 (81.4%) patients were culture positive. In 319 of 439 (72.6%) patients L-J culture yielded the pathogen and in 157 out of 192 (81.8%) patients ACS yielded the pathogen from the CSF cultures. In 66 (13%) cases, the microorganism was isolated in both L-J and ACS. Neither L-J nor ACS was performed in 29 (5.7%) cases. When automated systems were investigated in detail, the bacterium was isolated with MGIT-960 in 144 patients, BACTEC-9000 MB in five patients and MB/BacT Alert in eight patients. In two cases the microorganism was recovered in both manual MGIT and L-J. In two cases the microorganism was recovered from Middlebrook 7H12 together with MGIT960. Diagnostic tests for TBM other than CSF cultures: CSF-PCR (n = 206), IGRA (n = 41), CSF-EZNs (n = 469), and CSF-ADA (n = 137). (a) Sensitivities of the diagnostic tests in the diagnosis of TBM: IGRA (QuantiFERON-TB Gold Test In-Tube) (90.2%) was the most sensitive method followed by CSF-ACS (81.8%) in this study. When L-J was combined with IGRA with or without EZN, the cumulative sensitivity was 100%. In addition, concordant use of IGRA and EZN with or without PCR had 100% sensitivity. (b) The contribution of non-culture tests to diagnosis in case of culture negativity: When L-J and ACS were individually combined with other diagnostic tests, the contributions of other tests to TBM diagnosis in the L-J arm was seemingly high (12.8% for EZN, 24.6% for PCR and 10.9% for ADA). But the combined use of ACS and L-J did not benefit much from the non-culture tests (0.7% for EZN, 2.8% for PCR and 14.2% for ADA. The sensitivities of the microbiological diagnostic tests in microbiologically confirmed tuberculosis meningitis (n = 506). (c) The comparisons of diagnostic test results according to L-J culture results: CSF-EZN tended to be more positive when L-J culture was negative (p <0.0001). We could not establish any other association for other tests, which are presented in table 2.VIII. Table 2. VIII. Comparison of diagnostic test results according to Löwenstein-Jensen medium culture positivity [69] 53

55 Test methods Results CSF L-j culture * Positive Negative p value OR (95% CI) (n = 319) (n = 120) CSF-PCR IGRA CSF-ADA Pos 47 (48.5) 41 (59.4) Neg 50 (51.5) 28 (40.6) Pos 27 (87.1) 3 (100) Neg 4 (12.9) 0 (0.0) Pos 12 (18.2) 11 (31.4) Neg 54 (81.8) 24 (68.6) ( ) ( ) ( ) CSF-EZNs Pos 53 (17.7) 53 (46.5) Neg 247 (82.3) 61 (53.5) < ( ) * Data expressed as n (%); Pos, positive; Neg, negative; OR, Odds ratio; CI, confidence interval. CSF, cerebrospinal fluid; L-j, Löwenstein-Jensen medium; EZNs, Ehrlich-Ziehl-Neelsen staining; IGRA, interferon-γ release assay; ADA, adenosine deaminase (d) The comparisons of diagnostic test results according to ACS culture results: CSF-PCR tended to be negative when ACS culture was negative (p <0.0001). We could not establish any other association for other tests presented in table 2.IX. (e) The efficacies of diagnostic tests in predicting culture positivity: The most sensitive test was IGRA indicating culture positivity for L-J and ACS (73% and 82%). However, PCR was the most likely test indicating ACS positivity. (f) The efficacies of microbiological tests according to GCS: Both EZNs and PCR were significantly more positive when the GCS was >13 (p and p 0.006, respectively). (g) The agreements between the diagnostic tests: There were poor and inverse agreements between EZNs staining and L-J culture (j = ; p <0.0001); ACS culture and L-J culture (j = ; p 0.021); and IGRA and L-J culture (j = ; p 0.05). Accordingly, fair and inverse agreement was detected for CSF-ADA and CSF- PCR (j = ; p 0.003). Table 2.IX. Comparison of diagnostic test results according to automated culture system positivity [69] 54

56 Test methods Results Automatic automated culture system culture media * p value OR (95% CI) Positive (n = 157) Negative (n = 35) CSF-PCR Pos 39 (47.6) 2 (5.7) Neg 43 (42.4) 33 (94.3) < ( ) IGRA Pos 2 (100) 0 (0) ND Neg 0 (0.0) 0 (0) ND NA NA CSF-ADA Pos 18 (50) 2 (100) Neg 18 (50) 0 (0) ( ) CSF-EZNs Pos 40 (25.9) 9 (26.5) Neg 114 (74.1) 25 (73.5) ( ) * Data expressed as n (%); OR, Odds ratio; CI, confidence interval; Pos, positive; Neg, negative; NA, not applicable; ND, not determined. CSF, cerebrospinal fluid; L-j, Löwenstein-Jensen medium; ACS, automated culture system; EZNs, Ehrlich-Ziehl-Neelsen staining; IGRA, interferon-γ release assay; ADA, adenosine deaminase When the diagnostic tests for TB meningitis were compared with each other, the results were as follows: CSF-ACS culture (n = 157/192, 81.8%) was superior to CSF L-J culture (n = 319/439, 72.7%) (v2: 5.98, p 0.015, OR (95% CI): 0.59 ( )). Accordingly, CSF-ACS culture (n = 157/192, 81.8%) was better than CSF- PCR test (n = 118/206, 57.3%) (v2: 27.91, p <0.0001, OR (95% CI): 3.35 ( )), and CSF L-J culture (n = 319/439, 72.7%) was superior to CSF-PCR test (n = 118/206, 57.3%) (v2: 15.19, p <0.0001, OR (95% CI): 1.98 ( )). On the other hand, the comparisons of IGRA with microbiological diagnostic tests were as follows: CSF L-J culture (n = 319/439, 72.7%) was found to be worse than IGRA test (n = 37/41, 90.2%) (p 0.014, OR (95% CI): 3.48 ( )). Similarly, CSF-ACS culture (n = 157/192, 81.8%) was inferior to IGRA test (n = 37/41, 90.2%) (p 0.007, OR (95% CI): 3.83 ( )). Accordingly, the IGRA test (n = 37/41, 90.2%) was significantly more effective than the CSF-PCR test (n = 118/ 206, 57.3%) in indicating TB meningitis (p <0.0001, OR (95% CI): 6.89( )). Untreated tuberculous meningitis is associated with high mortality and neurological sequelae. Early diagnosis is the critical step for the start of treatment of the disease [57, 152]. The diagnosis of CNS tuberculosis is still a complex issue

57 because of the poor sensitivity and frequently delayed results of conventional tests, and lack of standardization or applicability problems of newer techniques. The detection of acid-fast bacilli in the CSF by EZNs or isolation of the microorganism by culture has long been accepted as the gold standard for the diagnosis of TBM. Many authors reported finding acid-fast bacilli in <20% of TB meningitis patients and culture positivity rates were reported to range around 25 75%. The ACS has been shown to be more efficient than L-J culture in the diagnosis of TB meningitis. Our findings confirm that CSF-ACS was superior to CSF L-J culture. Nevertheless, the agreement between these two culture types was poor. Concordant use of L-J and ACS provided significant benefit over using L-J or ACS alone. Hence, the diagnostic approach in CNS tuberculosis should be similar to that of other mycobacterial infections in which the use of solid medium along with a liquid medium is believed to maximize recovery. Unfortunately; only 13% of the treating clinicians in this study preferred this approach while in 5.7% of the cases no culture of any kind was performed. In addition, CSF-ACS and L-J cultures were found to be significantly better than molecular methods (81.8%, 72.7% and 57.3%, respectively). The efficacy of molecular methods was previously reported to fall behind culture methods. This seemed to be the case in TBM, too. Hence, we consider ACS as the gold standard in the confirmation of diagnosis of TBM and if only one type of culture is possible in the hospital, ACS should be preferred. Amplification of mycobacterial DNA by the use of molecular assays, such as nucleic acid amplification techniques, PCR (including real-time and nested PCR) in particular, potentially allows rapid diagnosis of TB meningitis molecular tests can also pick up dead bacteria and may further contribute to diagnosis. Hence, this feature may warrant molecular tests to be used in combination with other diagnostic tests. However, the sensitivity of PCR lies between EZNs and culture according to our data. In a meta-analysis on the role of nucleic acid amplification techniques in TB meningitis, the pooled specificity was 98% but the sensitivity was 56%. In our study, molecular tests detected 57.3% of the patients. Besides, a positive PCR result was not likely to indicate L-J culture positivity and no agreement was detected between molecular test methods and L-J or ACS cultures. Consequently, molecular methods cannot replace culture methods as the gold standard of diagnosis and should be used in combination with other diagnostic tools to diagnose TB meningitis. On the other hand, since they are less affected by the use of antibiotics, in comparison to cultures and EZNs, molecular methods may provide additional advantages in identifying the disease. The foremost diagnostic modality in many countries of high tuberculosis endemicity with poor resources is EZNs because of its low cost. According to our data, EZNs provided positive results in one-quarter of the patients and it had the lowest sensitivity in this study. In addition, EZNs was not likely to suggest L-J or ACS 56

58 culture positivity, and the agreement between L-J culture and EZNs was poor and inverse. Hence, a surprising number of specimens were EZNs positive and culture negative, a finding that is in contrast to those known for the pulmonary form of the disease. The probable reason for this situation may be the low bacterial count in CSF samples compared with sputum. The low bacterial count may have occurred by the immune reaction of host or by use of various drugs. However, there was not an inverse agreement between EZNs and ACS as in L-J. This is probably because of the high sensitivity of the ACS compared with L-J medium. Interestingly, EZNs or molecular testing was significantly more positive when GCS was mild. This is probably due to mild inflammation in less severe forms of TB meningitis, which may contribute to higher yields for EZNs and PCR. Assays based on the detection of IFN-c from lymphocytes after the administration of M. tuberculosis antigens have been introduced into clinical practice in recent years. In some case series IFN-c assays were found be useful in the diagnosis of TB meningitis. On the other hand, there are reports indicating the failure of these assays in the diagnosis of TB meningitis patients. Comparatively study from Japan, found 50% of patients with culture-positive TBM yielded negative IGRA results In our study, IGRA (QuantiFERON_-TB Gold Test In-Tube) was positive in 73% of L-J and 82% of ACS culture-positive patients and in 92% of the microbiologically confirmed cases. Moreover, we found that although the IGRA test was superior to CSF L-J and ACS cultures, it was not likely to predict culture positivity as in molecular tests, ADA and EZNs. On the other hand, this situation may provide an advantage particularly in favour of IGRA because this most sensitive test tended to be positive in culture-negative patients. Accordingly, the agreement between IGRA and L-J culture positivity was poor and inverse. The probable reason for this situation may be that the strong immune reaction, in which IFN-c is also a part, may contribute to eradication of the bacterium from CSF leading to culture negativity. Molecular tests provided positive results in slightly more than half of the patients, although they were inferior to IGRA. But we could not disclose agreement between IGRA and CSF-PCR in this study. Hence, these non-culture tests appear not to be confirmatory tests, but rather supplementary tests. Hence, these two tests have different kinetics in diagnosis and they should be used separately. Adenosine deaminase is most commonly present in human lymphoid tissue and in active T lymphocytes. Hence, it increases largely by the induction of T- cell-mediated immune responses. A meta-analysis reported that the sensitivity and specificity of ADA in TB meningitis were 79% and 91%, respectively. But, publication bias was questioned by a recent report to result in the overestimation of diagnostic accuracy in that meta-analysis. According to our data, although its 57

59 specificity and sensitivity for L-J culture positivity were 69% and as low as 18%, respectively, ADA was not very likely to indicate the isolation of the pathogen both in L-J culture and by ACS. In addition, other CNS disorders may produce positive ADA results and this may limit its use. However, we detected a poor and inverse agreement between ADA and PCR. The probable reason may be that the activated T lymphocytes may contribute to lower efficacy of PCR in ADA-positive samples. The strength of this study is that it is by far the largest microbiologically confirmed TB meningitis case series. Although its retrospective design is a limitation, it is nearly impossible to provide such a large prospective cohort sample. Another limitation was that there were numerous molecular tests used in the participating centers. We combined all of them as one block for statistical comparisons and were supposed to neglect differences between their sensitivities. In conclusion, in the diagnosis of TB meningitis, ACS has the highest sensitivity and should be the reference standard followed by L-J culture. Diagnostic accuracy is increased when both tests are used together. In addition, tests like EZNs, IGRA, ADA and PCR only contributed slightly to the TB meningitis diagnosis. However, as the major problem in diagnosis of TB meningitis appears to be the long time period for the recovery of the microbe, up to 2 6 weeks, combined use of EZNs, PCR, ADA and IGRA may circumvent the delays and appear to help early diagnosis. Hence, the data indicate that diagnosis of TB meningitis should be made by combination of diagnostic tests in two steps. In the first step, non-culture tests like EZNs, PCR, ADA and IGRA should be performed in combination according to their availability in the institution for the rapid diagnostic clues. In the second coexistent step, both ACS and L-J cultures should be performed together for the confirmation of TBM. If only one type of culture is feasible in the hospital, that should be ACS in the diagnosis of TB meningitis. The aim of the second study of Hamsi (Haydarpasa-II study) was scoring in the prediction of unfavorable outcomes from tuberculous meningitis. Hamsi scoring in the prediction of unfavorable outcomes from tuberculous meningitis used the results of Haydarpasa-II study [65]. Haydarpasa-II is a multicenter retrospective cohort study. It included 43 centers from 14 countries (Albania, Croatia, Denmark, Egypt, France, Hungary, Iraq, Italy, Macedonia, Romania, Serbia, Slovenia, Syria, and Turkey) which provided data from TBM patients hospitalized between 2000 and Haydarpas a-1 Study, which used the same database and evaluated the interrelations of microbiological tests, was published elsewhere [65]. Adult (age over 14) TB meningitis patients with microbiological confirmation were included in the study. At least one of the following tests on the CSF was mandatory for microbiological confirmation; a positive Ehrlich-Ziehl-Neelsen stain, 58

60 positive Mycobacterium tuberculosis (Mtb) culture, or positive Mtb-PCR [4]. All the TBM cases included in this study were followed and treated in accordance with the centers own policies. All epidemiological, clinical, laboratory, radiological and outcome data were retrieved from patient charts by the participant centers Unfavorable outcome. This term represents an outcome of either sequela or mortality. Sequelae were detected after the end of antibiotic treatment and defined as any dysfunction related to CNS that persisted during the follow-up period. These included motor deficit, cranial nerve palsy, ataxia, persisting unconscious state, seizures, difficulty with speech, hydrocephalus, diabetes insipidus, anal sphincter dysfunction, and coma. Stage of the disease The stage of the patients was determined according to traditional MRC system. A total of 12 % (63 out of 507 patients) of observations in four (basal meningitis, hydrocephalus, abscess, vasculitis) variables were missing. These variables depended on CT or MRI examinations of CNS at admission. Since missing observations belong to patients without these tests at admission, missing observations for all these four variables belong to the same patients and 89 % were from three countries. These variables were significant candidates for the initial model. Logistic regression protocol drops missing observations in a list-wise fashion. Dropping 12 % off all other covariates might lead to biased estimates and so we decided to input these observations. First, the missingness pattern was inspected and indicator variables were generated to further assess if the data are missing completely at random. Depending on the relationship between missing values and other covariates including outcome, we concluded to input missing observations. Multiple Imputation (MI) using monotone-missing patterns was applied. Number of imputations was determined according to the Monte Carlo error estimates those ideally have to be less than 10 % of standard errors of related coefficients. Eventually, we built up a multiple imputed data set and tested models with both complete cases and imputed data set. Binary logistic regression model was constructed via a bootstrap resampling procedure described in detail elsewhere. Briefly, each of 12 imputed data sets were replaced by resampling 200 times. The full model including all potential variables was tested across bootstrapped samples by means of logistic regression. In each bootstrap replication, significant variables were selected with backward elimination at a selection point of a = Since data vary between replicates, bootstrap inclusion frequencies of variables also vary, where weak predictors are found with less inclusion frequencies. Those variables that have more than 30 % inclusion frequency at least at half of the imputed data sets were included in the final model. The final model was tested with logistic regression analysis across imputed data sets and estimates were combined 59

61 according to Rubin s rules. To test the significance of continuous predictors, multivariable fractional polynomial model selection procedure was applied. Global heuristic shrinkage factor was obtained by the average of heuristic shrinkage factors obtained from imputed data sets. The model was internally validated by bootstrapping [21]. According to the predictive powers of terms from the final model, scores were assigned to build a severity scale. Finally, this severity scale was tested on complete cases and imputed data set individually. Data for 507 patients were submitted from in Haydarpasa-2 study. Unfavorable outcome was detected in 165 of 507 patients. Eighty-six patients died and case-fatality rate for this cohort was 17 %. A total of 119 sequealae were detected in 79 (16 %) patients. The squealae included motor deficit in 37 patients (7 %), cranial nerve palsy in 30 (6 %), ataxia in 11 (2 %), altered consciousness in 10 (2 %), seizures in 10 (2 %), difficulty with speech in nine (2 %), hydrocephalus in five (1 %), diabetes insipidus in four (1 %), anal sphincter dysfunction in two (0.3 %), coma in one (0.1 %). Continuous variables such as age and elapsed time were non-normally distributed, and only age was found to be significant (p<0.001). Among the other significant variables, neurological deficit represented motor deficit and/or cranial nerve palsy. Motor deficit and cranial nerve palsy were tested one by one and all together in the model. However, once the composite term neurological deficit was entered, motor deficit and central nerve palsy were both dropped from the model. After reviewing all the data submitted, we detected that six variables were missing among 63 patients. These included the detection of basal meningitis, hydrocephalus, tuberculoma, abscess, vasculitis, and cerebritis. All of these variables are diagnosed by CT scan or MRI which means that missing data occurred when neuroimaging had not been performed. Missing data were mostly from three countries; 55.6 % (35/63) from Romania, 19 % (12/63) from Iraq and 14.3 % (9/63) from Turkey. More importantly, missing and non-missing data were concomitantly submitted from same centers thus indicating that a CT scan or MRI had been applied selectively. There was a significant relationship between missing data for these variables and favorable outcome which likely indicates that in these centers physicians probably do not request these tests for patients with mild disease. Therefore, the missingness might be missing at random (MAR) pattern, though we could not be certain that the missing data occurred missing not at random (MNAR). We imputed the missing variables and estimates were carried out on this data set and on the complete data set as well. The 13 variables, indicated with asterisks in the right column, were included in the full model. The global heuristic shrinkage factor was found to be 0.89 which indicated that over fitting of the model is not severe and might be omitted. Selection frequencies of variables in the final model. Selection frequencies of variables those not 60

62 included in the final model were as follows; sphincter dysfunction, difficulty with speech, ataxia, and chronic renal disease were selected zero and abscess was selected two times during bootstrap sampling. Among the selected variables, only age was a continuous variable. Estimates from the logistic regression failed to show a significant association between age and the outcome variable in its continuous form. Multivariable fractional polynomial model also failed to show any significant association between transformed forms of age and the outcome. However, it was not significant if the patient also presented with nausea and vomiting. We, therefore, assigned three points to this altered consciousness without nausea and vomiting and two points when nausea and vomiting accompany altered consciousness. Various ranks were assigned to predictors and those developed scales were tested in terms of predictive accuracy. Final scale giving higher area under the curve. Next, we tested the predictive accuracy of clinical staging and compared to the severity index. The severity scale and the clinical staging with corresponding percentages of observed unfavorable outcome and adjusted predicted probabilities with standard errors are presented. In terms of predictive accuracy of clinical outcomes, the clinical staging system was inferior to the severity scale. This difference was significant (Fig. 2.5). When they compared the observed unfavorable outcome percentages by ranks, in the severity scale the relationship was almost linear. In contrast, the relationship between the clinical the severity scale the relationship was almost linear. In contrast, the relationship between the clinical stages and outcome did not give a linear association. Medium and advanced levels of clinical stages presented similar outcome percentages which also discourage the use of clinical staging as a predictive scale (Fig. 2.5). In the database, observed mortality rates for the scores 1 6 were 3.4, 8.2, 20.6, 31, 30 and 40.1 %, respectively. On the other hand, mortality rates were 1.3 % for the early MRC stage, 18.1 % for the medium stage, and 22.4 % for the advanced stage. 61

63 Fig Comparison of ROC curves In this study, the largest retrospective cohort in the literature consisting of microbiologically confirmed cases, a severity index with acceptable predictive accuracy was developed by considering clinical findings, comorbidities, and CNS imaging findings all together. The final model was highly stable as it provided similar estimates among complete cases and those with incomplete data sets and it was successfully validated by bootstrap estimation. Internal validation with bootstrapping has been reported to provide more reliable results compared to other methods such as split-sample analysis and cross-validation procedure Haydarpasa Meningitis Severity Index, HAMSI, developed by the aid of this logistic model gives a linear prediction for unfavorable outcome, which reflects the strength of the base model as well. This linear association between the ranks of the Severity Index and the outcome makes it more applicable in routine medical practice. Traditionally, severity of TBM patients has been assessed by clinical staging developed in 1948 by the MRC, which solely depends on clinical assessment. Clinical parameters in staging systems are relatively subjective and somewhat vague by definition. The MRC categorizes patients as early, medium and advanced stages. Early stage indicates TBM patients with mainly non-specific symptoms, with little or no clinical signs of meningitis, no pareses, in good general condition, and fully conscious. Advanced-stage patients are extremely ill, deeply stuporous or comatosed, or with gross pareses while mediumstage patients are in a condition between the advanced and the early stages. Signs of meningeal irritation with or without slight clouding of consciousness with focal neurological signs such as cranial nerve palsies or hemiparesis are included in the medium stage. On the other hand, the HAMSI 62

64 scoring handles consciousness as fully conscious or altered mental status, which is a more straightforward classification. This simplified categorization of mental status might avoid the confusion arising among the medium-stage patients in the MRC clinical staging system. Although correlations between the unfavorable outcome and the stages of MRC system were noted in various studies, it did not provide satisfactory prediction to forecast outcome in other studies Accordingly, we could not show a reasonable association between the MRC scoring stage and unfavorable outcome in this study. On the other hand, integration of objective and measurable parameters into clinical parameters may increase the stability and the predictive accuracy of the prediction models. At this critical point, the question can TBM clinical staging system developed by the British MRC in 1948 be improved arises. In this context, we first tried to implant objective parameters such as radiological imaging and, comorbid conditions. Unfortunately, adding these parameters did not improve the predictive accuracy of the MRC clinical staging system. Consequently, MRC system is somewhat ill-defined or subjective by its nature. In addition, since when MRC defined clinical staging system at a time when sophisticated neuroimaging was not available and it was derived on a different population with less knowledge than now in the twenty-first century, it provided fairly rough estimates of unfavorable outcome, really no longer applicable today. There have been other efforts to develop an improved severity index for TBM. In a recent relatively small study in which microbiological confirmation was established in less than half of the patients, impaired conscious, motor deficits, cerebral infarcts, and cisternal effacement were found to increase mortality. The authors recommended a new staging system based on their findings. The first three parameters, impaired conscious, motor deficits, and infarcts, were shown to increase unfavorable outcome in this study, too. They also found that diabetes mellitus and hydrocephalus also affected prognosis. According to our data, another significant point was that there was no relationship between the elapsed time from the onset of symptoms prior to treatment and outcome. This might be due to delayed diagnosis in mild cases. Another possibility is that, disease progression varies greatly among TBM patients. For example, patients at the early stages according to MRC system might not necessarily present to the hospital early. This point was underlined in the original study, too. One of the limitations of this study was that GCS could not be provided due to missing data in hospital records. 63

65 Fig Adjusted predictions and confidence intervals of scales If this study was prospectively planned and the data were collected, we would be able to apply GCS as measure of conscious. However, in a study of this type focusing on a quite rare disease, collecting data prospectively and reaching adequate case numbers would be practically impossible. In conclusion, altered consciousness, diabetes mellitus, immunosuppression, neurological deficits, hydrocephalus, and vasculitis predicted the unfavorable outcome in HAMSI scoring and the cumulative score provided a linear estimation of prognosis. On the other hand, if a patient with altered consciousness can complain about nausea and vomiting, then this seemed to be a more favorable parameter on the outcome of the disease. This might represent a slight distortion of conscious compared to those patients who cannot complain about nausea due to deeper mental changes. Consequently, we provided a strong model in the prediction of TBM outcome and accordingly, HAMSI scoring should be validated subsequently in other cohorts Researches on tuberculous meningitis in different populational groups (sucklings and children) Tuberculous meningitis in sucklings To study the tuberculous meningitis in sucklings, from the clinical, laboratory findings and therapy point of view. Being retrospective study of tuberculous meningitis of 23 infants under 1 year of age, admitted in the Infectious Diseases Hospital of Iasi-Romania, in the period [118]. There were identified in medium 29% new cases in children per year, comparatively with adults (14.8%). From these 23 cases, 60% were boys and 26% of the group of age 1 4 months. The onset was insidious in 72, 84%, with meningeal 64

66 syndrome in 91% cases and conscience troubles in 8% cases. The CSF was clear in 95% cases, with high values of proteins and low values of glucose and chlorus. M. tuberculosis was isolated in culture in 28% cases and 14.66% by directly exam. The therapy was then with quadruple association of antituberculous drugs, with favorable evolution 65% cases, 8 deaths being recorded. The factors of bad prognoses still remain: the little age, the hydrocephalus (5 cases), malnutrition (3 cases), and coma from the beginning (2 cases). We may say that tuberculous meningitis still remain a disease with a high incidence and severe evolution in sucklings, despite the progress of diagnostic methods and therapy. Another study determined the clinical, bacteriological and therapeutic profile of this type of infection in newborns and sucklings [118]. There were eighty less than one-year-old infants (15 newborns and 65 infants older than 1 month) with bacterial meningitis, admitted in our hospital between January 2000 and December 2003 were retrospectively studied. Most of the children were boys (67.5%), had significant co-morbidities (75%); the mean duration from the onset of the disease to hospital admission was 2.75 days and 32.25% of them already received antibiotics. Symptoms and signs were unspecific and in 23.75% cases the diagnosis was suspected only after the onset of generalized seizures. Only 60.1% of the CSF specimens were purulent. Other septic foci were present in 32.5% cases. The etiology of the disease could be established in only 48.75% of cases. In newborns, from 10 cases with known etiology, we isolated gram positive cocci: group B. Streptocococcus in 3 cases, S. aureus 2 cases, gram negative bacilli 3 cases and gram negative cocci: Neisseria meningitis 2 cases. After 1 month of age, from 29 patients with known etiology, the main pathogen was N. meningitis (12 cases) followed by H. influenzae (6 cases) and S. aureus (4 cases). Most organisms were sensitive to beta-lactam antibiotics (76.92%). The empirical etiological treatment used an association of two antibiotics (ampicillin 11.63% cases, a third generation cephalosporin in 39.54% cases, fluoroquinolone 12.7% cases, Vancomycin 4.65%). Dexamethazone was used, for a mean duration of 5.62 days, in 32.5% of cases. The mortality rate was 21.25% (33% in newborns). During the studied period and for this age group, bacterial meningitis was an elusive disease, still with a poor prognosis, despite vigorous etiological and supportive care. Quantiferon TB gold test in the diagnosis of tuberculous meningitis in children was approved as gold standard diagnosis test, because of the high sensibility and specificity. This was obiectivated in another retrospectively study, on 40 children with tuberculous meningitis, admitted in the Infectious Diseses Hospital of Iasi between October 2006 December 2009 [24]. In Romania, the morbidity through tuberculosis is high the nervous localization 65

67 (especially at children age) having one of the most important incidence in Europe. The tuberculous meningitis still remain a public health problem the disease being classic considerate a children disease with a maximum frequency in the first 3 years of life. The protection through BCG vaccination moves the incidence of this disease from the little child to the big one and young adult [131]. The aim of this study was to evidentiate the clinic-epidemiologic and therapeutic aspects on 82 cases of tuberculous meningitis in children during 4 years. The diagnosis was based on clinical criterions and confirmed through isolation of M.tuberculosis in culture of cerebrospinal fluid in 28% cases, and by direct exam in 12% of cases. The treatment was done with quadruple association of antituberculous drugs with a favorable evolution and a lethality of 9,75%. ' Another study analysed the performance of an interferon-gamma release assay in the diagnosis of tuberculous meningitis in children [24]. Tuberculous meningitis is a serious illness that, if not diagnosed and managed early, leads to a high rate of mortality and permanent disabilities. Despite the advances that have been achieved in the diagnosis of TBM and anti-tuberculosis and pathogenic therapies, the prognosis remains poor in many cases, and the mortality remains at approximately 20-30%. In 2012 WHO reported 8.6 million new cases of tuberculosis and 1.3 millions of deaths. In Romania, more than new tuberculosis cases or reinfections arise annually, and the incidence of new cases of children who develop serious forms of tuberculosis is high [190]. Studies have clearly demonstrated that the timing of TB meningitis treatment is the most critical factor in determining the ultimate outcome, which underscores the importance of early diagnosis. Clinical features and cerebrospinal fluid (CSF) findings are helpful in the diagnosis of TBM, but these features cannot be used to differentiate TB meningitis from other infectious and non-infectious disorders. The diagnosis of TBM is based on the analysis of the CSF collected by lumbar puncture. Diagnostic markers of TBM in the CSF typically include elevated protein levels, low glucose levels and predominant lymphocytic pleocytosis. More than half of TB meningitis cases cannot be confirmed microbiologically, and these patients are treated based only on clinical suspicion. Culturing Mycobacterium tuberculosis from the CSF requires a minimum of 2-3 weeks; therefore, the majority of patients with TBM begin treatment before their diagnoses are confirmed. Polymerase chain reaction analyses of CSF samples are not available in our hospital. Brain CT scans and MRIs may show features that are strongly suggestive of TB meningitis, but these techniques cannot diagnose the condition. In recent years, important progress has been made in the development of in vitro T-cell-based IFNgamma release assays (IGRAs). The QuantiFERON- TB Gold (QFT-G) and ELISPOT 66

68 assays are examples. These assays measure the amount of IFN-gamma that is released after blood is incubated with TB-specific antigens, early secretory antigenic target 6 (ESAT 6) and culture filtrate protein 10 (CFP 10), that stimulate IFN-gamma release from the sensitized T cells of the patients. A small number of studies have investigated the use of ELISPOT assays of mononuclear cells from the site of infection (including CSF) compared to PBMCs to aid in the diagnosis of active tuberculosis with conflicting results. The performance of the QFT-G test for CSF has not yet been studied. Further studies are also needed to establish the sensitivities and specificities of interferon-gamma release assays in the detection of active tuberculosis, particularly in children and immunocompromised patients. There were investigated whether rapid diagnoses of tuberculous meningitis in children could be established with the interferon-gamma blood and cerebrospinal fluid test QuantiFERON-TB Gold in the context of routine clinical practice and assessed the agreement of the results of this test with those of classical laboratory methods [25]. The study enrolled 125 children <17 years of age who were admitted with a diagnosis of meningoencephalitis on a period of 5 years, in the New St. John Emergency County Hospital Suceava and Clinic of Infections Disease Iasi in this study, the patients being separated into two groups: 1) 25 cases of definite TB meningitis and 38 cases of probable TB meningitis; 2) 62 non-tb patients (controls) [24]. Patients were classified as having confirmed TB meningitis (definite cases) if Mycobacterium tuberculosis was cultured from CSF and/or was detected in acid fast staining (AFS). Probable cases were not confirmed by culture and/or AFS, but with clinical, radiological or laboratory features that were suggestive of TBM and with clinical response to antituberculous treatment. The clinical features include fever, headache, vomiting, seizures, altered mental status or neurological deficit, neck stiffness and a history of illness longer than six days. Radiological evaluation may support the diagnosis of TBM in cases that are suspected active pulmonary TB due to chest X ray or changes in brain computed tomography (CT) such as hydrocephalus, basal meningeal enhancement, tuberculoma, cerebral infarcts and edema. The group with no TB cases was defined by evidence of others diseases and clinical improvement without antituberculous treatment, respectively on antimicrobial, antifungal or antiviral treatment. In the second group (i.e., control group), were enrolled patients with non-tb meningitis, partially treated pyogenic meningitis, viral meningitis (i.e., lymphocytic predominant pleocytosis with normal glucose and proteins level in the CSF), cerebral toxoplasmosis (typical scan findings, IgG serology positive finding and a response to empiric treatment), cryptococcal meningitis (positive culture and the presence of cryptococcal antigens) and HIV aseptic meningitis. 67

69 Neurological manifestations in the control group were present in cases of cerebral toxoplasmosis, cryptococcal meningitis or HIV aseptic meningitis and were clinically improved without antituberculous therapy. Complete physical examinations of the patients were performed on admission. Patient demographic information, clinical, microbiological and radiological data have been collected and correlated to the QFT-G results. Background information, including past history of tuberculosis or tuberculin skin test or BCG vaccination, contact grading, immune suppressed status were investigated. The sensitivity and the specificity were calculated for the chosen cut-off value. Indeterminate assay results were excluded from statistical analyses of the sensitivity and specificity. As results, of the total of 125 subjects who were enrolled in the study, 63 children were diagnosed with TB meningitis (25 cases fulfill the criteria for definite TBM and 38 fulfill the criteria for possible TB meningitis), and 62 were in the control group. The demographic characteristics of the patients showed: the male/female ratios were 1.33 in the TB meningitis group and 1.58 in the control group. The mean age of the first group was years, and the range was eight months to 17 years. The majority of the TB meningitis patients had lived in areas of poor socioeconomic status (76.2% versus 56.5%) and had experienced malnutrition (66.1% vs. 37.1%). In the TB meningitis group, 14.9% of the patients were immunocompromised: seven patients had AIDS, one had acute lymphatic leukemia and was receiving chemotherapy and one had diabetes. In controls group, nine subjects had AIDS and one had leukemia. All HIV patients included in this study had CD4 <200/mm³. In the TB meningitis group, 35.5% of the children had a known household contact (p=0.239) and 12.7% of the children had histories of TB (p=0.319). Only 58.7% of the children with TB meningitis had received BCG vaccinations (p=0.213). In the control group 71% of cases have been BCG-vaccinated. The symptoms observed in the physical examination upon admission that suggested TB meningitis were anorexia (95.2%), low fever or prolonged fever syndrome (71.4% of subjects with TBM), neck stiffness (88%), headache (76.2%), altered mental status (69.8%), coma (61.9%), cranial nerve palsies (12.7%) and hemiparesis (12.7%) (table 2.X). Classic diagnosis methods. High numbers of leukocytes in the blood were present upon admission in 41.3 % of the patients with TBM and 54% of the TB meningitis patients exhibited increased neutrophils. In the non-tb subjects group, 50% had leukocytosis in blood and 66.1% had increased neutrophils. All subjects with TB meningitis had high erythrocyte sedimentation rates (p=0.001) and only 48.7% of non TB meningitis. Chest radiographies were suggestive of pulmonary TB in 60.3% of the children (miliary or lobar forms or pleurisy) (p=0.001) in TBM group and normal of all cases of 68

70 non TBM. Brain CTs revealed hydrocephalus in 25.8% of subjects (p=0.001) and tuberculomas in 14.5% (p=0.006) in the first group. In the second group, CT scan showed multiple ring lesions and cerebral edema in 4.8% toxoplasmosis cases, hydrocephalus in 6.9% cases of cryptococcal meningitis and cerebral atrophy in 3.2% subjects. Table 2.X. Signs and symptoms of patients involved in the study Symptoms TBM (n=25) Probable TBM (n=38) Control group (u=62) p*/** Anorexia 24 (96.0%) 36 (94.7%) 42 (67.7%) / Confusion 17 (68.0%) 27 (71.1%) 21 (33.9%) / Coma 13 (52.0%) 26 (68.4%) 15 (24.2%) / Low grade fever 18 (72.0%) 27 (71.1%) 44 (71.0%) / Headacke 19 (76.0%) 29 (76.3%) 55 (88.7%) / Photophobia 6 (24.0%) 10 (26.3%) 17 (27.4%) / Neck stiffness 22 (88.0%) 34 (89.5%) 50 (80.6%) / CN palsies 3 (12.0%) 5 (13.2%) 5 (8.1%) / Hemi/Para paresis 3 (12.0%) 5 (13.2%) 5 (8.1%) / Cough 10 (40.0%) 14 (36.8%) 29 (46.8%) / Abdominal pain 5 (20.0%) 7 (18.4%) 10 (16.1%) / Vomiting 17 (68.0%) 30 (78.9%) 49 (79.0Vo) / * Chi-squared test (with Yates correction) was used to compare discrete variables between TBM and Probable TBM groups. ** Chi-squared test (with Yates correction) was used to compare discrete variables between TBM and Control groups. TBM - tuberculous meningitis: CN - cranial nerve There were performed TSTs for all patients (n=125). The TSTs were positive in 49.2% of the patients with TBM and in 19.9% of controls (p=0.001). According to the defined criteria, the CSF analyses indicated possible TB meningitis in 63.4% cases as suggested by moderate pleocytosis (i.e. >20 cells/mm³), 69

71 high CSF protein levels (i.e., >0.5 g/ dl) and low CSF glucose levels (i.e., <45 mg/ dl) (table 2.XI). The chloride levels in the CSF were decreased to 44.4% of TBM patients and 77.7% had elevated lymphocyte counts in their CSF. All TBM patients were negative for acid- fast staining, only 25 (39.6%) had positive M. tuberculosis cultures in CSF and two subjects had positive sputum culture. In one patient, we isolated a MDR strain. The mean time interval necessary for the M. tuberculosis cultures was 25.6 days (range days). In the controls group, all subjects had moderate CSF protein levels (0.8 2 g/dl), normal glucose and chloride levels. All controls had negative AFS and cultures from CSF. QuantiFERON-TB Gold results. Of the 25 cases of culture-confirmed TBM (i.e., the gold standard), 21 had QFT-G positive results, and four had indeterminate results. In group of probable TBM, out of 38 cases, 24 had positive, 13 negative and one indeterminate QFT-G results (table 2.XI). The percentage of QFT-G positive results for CSF and QFT-IT for blood was similar in TB meningitis patients. The 11.3% of patients with false positive QFT-IT results for blood had a history of active tuberculosis and one child with false positive QFT-G result for CSF had previously TBM. Were obtained 68.25% concordant positive QFT-G results (positive QFT for blood and CSF) of the first group and 80.64% concordant negative results (negative QFT for blood and CSF) in the group of controls (table 2.XI). In the TB meningitis group, we observed 9.52% discordant results (positive for CSF/negative for blood or negative for CSF/positive for blood), and we observed 12.90% discordant results in controls. In TB meningitis patients, we observed 12.5% indeterminate or false negative QFT-G results in patients with low number of cells in CSF and 7, 5% in group of controls. The time interval required to obtain QFT-G results was 48 hours. The mean time interval necessary for the diagnosis of TB meningitis by QFT-G positive results was 4.31 days. All 63 patients with definite or probable TB meningitis received antituberculous therapy with four first line drugs (rifampicin, isoniazid, pyrazinamide, ethambutol), and 90% of the patients received dexamethasone therapy. One patient with MDR strain received treatment with moxifloxacin, clarithromycin, rifampicin and pyrazinamide. Prognosis was unfavorable in 12.7% of patients with TB meningitis and 3.2% in the non-tb group and severe disabilities were observed significantly more frequently in patients with MTB than in the control group (30.9% vs.11.7%) (p =0.02). 70

72 Results Table 2.XI. QuantiFERON-TB Gold test for 63 TB meningitis patients and 62 controls TB Meningitis Probable TBM Non-TB controls No. cases % No. cases % No. cases % QFT-IT in blood (p values for F Test < 0.001) Positive Negative Indeterminate Total QFT-G in CSF (p values for F Test < 0.001) Positive Negative Indeterminate Total CSF - cerebrospinal fluid; QFT-IT - QuantiFERON TB Gold in Tube; QFT-G - QuantiFERON TB Gold; QFT-G and QFT-IT was manufactured by Cellestis Ltd. (Carnegie. Australia) In the control group, 40.3% of the patients had viral meningoencephalitis; 45.2% had bacterial meningitis, 4.8% had toxoplasma encephalitis, 6.4% had cryptococcal meningitis and 3.2% had HIV aseptic meningitis. All of the controls recovered without antituberculous therapy. The present report evaluates the performance of the QuantiFERON-TB Gold in Tube using whole blood and the performance of the QuantiFERON-TB Gold assay using CSF for the diagnosis of tuberculous meningitis among children in Romania. The diagnosis of TBM remains a challenge in children, as bacteriological confirmation is the exception, because of the paucibacillary nature of the disease. In this study, anorexia, altered mental status and coma were the clinical manifestations suggestive of the TBM diagnosis (p = 0.001). However, the initial presentation of the patients with TBM that could have been mistaken for other illnesses included cough and fever (38% of the children; these children received antibiotics for respiratory infection) and abdominal pain, vomiting, fever with suspected gastroenteritis or acute abdominal surgery (19%). On admission 11.1% of the patients exhibited no meningeal signs, and these signs appeared later in the evolution of the clinical courses of these patients. 71

73 Tuberculous meningitis is a severe illness in children, and 61.9% of the TB meningitis group was admitted to the hospital with coma, 69.8% with confusion and 25.4% with neurological signs. In other studies, coma has been found in 30-60% of TMB cases, seizures in 50% of children and cranial nerves palsies in 30-50% of patients. Other authors (Kumar et al. - in 1999 [100], Youssef et al. - in 2006 [191], Thwaites et al. - in 2002 [175] and Moghtaderi et al. - in 2009 [133]) analyzed the clinical and laboratory features that were predictive of tuberculous meningitis and found that history of illness >5-6 days, focal neurological deficits, abnormal movements were significantly more common in TB meningitis patients than other meningoencephalites. However, in this study, the onsets were frequently atypical (57% of patients) and manifested as digestive disorders or prolonged fever, and it was too late to seek care from infectious disease specialist in these cases. More than 60% of the cases of TB meningitis in this study had active pulmonary tuberculosis on chest X-ray, and 17.7% of the children had miliary tuberculosis, these results are similar to those of Donald et al. [20] who reported a strong association between TB meningitis and disseminated tuberculosis. According to literature, TST has a low sensitivity in children with disseminated tuberculosis or TBM, but QFT may be an alternative. In this report, brain CTs were highly suggestive of TBM (basal enhancement, hydrocephalus or tuberculoma) in 40% of the cases, similar results to those obtained by Andronikou et al. [1] who reported 41% sensitivity and 100% specificity for brain CTs in the detection of childhood TBM [21]. Another study, of Kumar et al. [100], reported 89% sensitivity and 100% specificity for CT findings in the diagnosis of TBM [16]. However, other conditions, such as fungal meningitis, toxoplasmosis, neurosarcoidosis and carcinomatous infiltration, can result in similar CT images. Abnormal CSF profile (including lymphocytic pleocytosis, decreased glucose, increased protein and low chloride levels in the CSF) may suggest TB meningitis, and some studies have found sensitivities and specificities that are comparable to those of PCR and superior to those of the culture [22]. A portion of the TBM patients (22.2%) exhibited polymorph nuclear reaction in the CSF and blood at admission, and these patients were initially treated with antibiotics for pyogenic meningitis. The outcomes were unfavorable, and the patients conditions worsened. Some studies indicated that cell-mediated immune response may decrease in cases of active TB, when untreated. The results of QFT-G from CSF were more frequently false negative in this group and probably will be better to repeat the test after few days. The AFS smears were negative in all of the cases and did not help us make rapid diagnoses. The CSF cultures were positive for M. tuberculosis in a small 72

74 number of cases (39.6%) and because of the long time required for cultivation, were not useful for the rapid diagnosis of TB meningitis The CSF specimens used for the culture Mycobacterium were obtained upon the admissions of the patients prior to the initiation of antituberculous treatment. In the culture-confirmed TB cases (i.e., the definite cases), the sensitivity of the QFT-G tests of the CSF was 84% (the sensitivity was 100% when we excluded the indeterminate results), and the sensitivity of the QFT-IT tests of the blood was 80% (95.2% when indeterminate results were excluded). The estimated sensitivity and specificity of QFT-G in CSF were higher than QFT-IT in blood (table 2.XII). In previous study, it has been reported that the sensitivity of QFT-IT with whole blood was 59-89% and specificity 70-98% [8, 11, 24]. We found only one false positive QFT-G results with CSF and this was correlated with history of TB. The false negative and indeterminate QFT-G results (18.6%) for CSF occurred frequently in patients with low numbers of cells in CSF. TB-specific interferon gamma producing T cells might be present in lower numbers in children comparative with adults, resulting in lower levels of IFN-gamma, the target cytokine measured with QFT-G [25-27]. Table 2.XII. Sensitivity and specificity of utilizing methods Method Sensitivity (%) Specificity (%) Accuracy (%) Area under the curve Asymptotic 95% Cl Culture from CSF TST QFT-IT in blood QFT- G in CSF CSF - cerebrospinal fluid; QFT-IT - QuantiFERON TB Gold in Tube; QFT-G - QuantiFERON TB Gold; TST - tuberculin skin test Due to its clinical, epidemiological and CSF features, the QFT-G test of the CSF enabled the rapid diagnosis of TB meningitis in 71.4% of patients, and 34.9% of these cases were later confirmed by positive cultures. A positive QFT-IT blood test cannot distinguish between latent and active TB, but a positive QFT-G of the CSF may explain the etiology of tuberculous meningitis. Jafari et al. showed that IGRA response assayed in mononuclear cells from the site of infection can provide superior discrimination between active TB and latent TB [28]. In the remaining patients (28.6%), the diagnoses of TB meningitis were supported either by positive sputum 73

75 cultures for M. tuberculosis (three cases) or clinical manifestations, exposure to TB, biochemical characteristics and the cytologies of the CSF, chest XRand CT scan results and favorable responses to antituberculous treatment. All of the cases were also negative for Gram and India ink stains and bacterial and fungal cultures. The estimated sensitivity and specificity of the QFT-G test of the CSF were superior to those of the TST (80.4% vs. 49.2% and 98.2% vs. 88.6%, respectively) (table V), and the sensitivity and specificity were higher for the tests of the CSF than those of the serum (figure 2.7). Fig Sensitivity and specificity of QuantiFERON in blood and CSF The QFT-G might represent a cost-effective alternative to the TST, particularly for BCG-vaccinated persons born in countries in which TB is prevalent, such as Romania. In our study, we had children those parents refused BCG vaccination, especially in rural areas. The national promotion of BCG vaccination has made the interpretation of TST results difficult. The TST has a low specificity in the BCGvaccinated population due to the cross-reactivity of the diagnostic antigens that are used in the TST for BCG vaccine strains. The greater specificity of the QFT-G and the requirement of only a single visit are compelling advantages, as TST requires a second visit for reading [29, 30]. The sensitivities of the QFT-G test of the CSF (84%) and the QFT-IT test of the blood (80%) for the culture confirmed TBM cases were similar to the sensitivity of the QFT-IT of the blood (80%) that was reported by Kampmann et al. for 209 children with active or latent TB, but the sensitivities of the TST estimated in our study were lower (56% vs. 83%). The indeterminate QFT-G results from CSF and blood were a little higher in group of TBM than in controls (11.1% vs. 9.7% and 8% vs.6.4%) and were lower 74

76 indicated for QFT-IT in blood by Kobashi [32]. The negative and indeterminate QFT- G results from the CSF were more frequent among infants and children <5 years old (16.66%) than in children >5 years (9.8%). Kampmann et al. [31] suggested that IFN-γ producing T-cells might be present in lower numbers in children and those IFN-γ release assays of young children are more likely to yield false-negatives. Thirty per cent of the QFT-G indeterminate results in the CSF (n=4/13) and 33% of the QFT-IT indeterminate results in the blood (n=3/9) exhibited inadequate responses to mitogen, and these patients were immunocompromised (HIV positive with CD4 cell counts < 100/mm³). The lymphocytopenia that appeared in HIV patients caused a decrease in the production of IFN-gamma and induced indeterminate QFT results in blood and CSF due to a lower mitogen level. These results are similar to those of the report of Ferrara et al. regarding QFT of the blood [33]. Kobashi et al. also noted higher percentages of indeterminate QFT results when the blood of immunocompromised patients was tested [98]. Results from some studies indicated that frequencies of positive immune responses to IGRA are higher in immunosuppressed patients (i.e. with HIV infection) than to the TST [29,30]. The present report was indicated that 71.4% of patients with coinfection TB-HIV had positive QFT-G results with CSF, so this test may be used for diagnosis of TB meningitis even in immunocompromised persons. In this study, as in other studies [161, 192], negative or indeterminate QFT-G in the CSF were also influenced by previous TB treatment testing. In the 22.2% of patients who had undergone antituberculous therapy for longer than 14 days prior to the QFT-G tests, the numbers of negative or indeterminate results were higher (85.7%). In 77.8% patients without antituberculous treatment prior to the QFT-G tests, we observed 12.2% indeterminate results. Therefore, it is better that QFT-G test to be performed before treatment anti TB. In 2006, Quan et al. [151] assessed TBM diagnoses with different assays and found a sensitivity of 84% and a specificity of 91.8% for the enzyme-linked immunospot assay (ELISPOT) of the CSF; these values were higher than those of culture (sensitivity 16%) and PCR. Our study revealed that the sensitivity and specificity of the QFT-G of the CSF were similar to those of the ELISPOT in Quan s study. In other studies in 2010, Patel et al. [145] found only a 58% sensitivity and 94% specificity for the ELISPOT of the CSF and Kim et al. reported 59% sensitivity and 89% specificity of the ELISPOT assay in mononuclear cells of the CSF So, in this report the sensitivity of the QFT-G for the CSF seems to be similar or higher than that of the ELISPOT in some studies (80.4% vs. 84%, 58% and 59%, respectively). However, the diagnosis of TBM requires a combination of epidemiological, medical history, clinical, radiological and laboratory findings to be taken into account 75

77 when interpreting the QuantiFERON TB Gold test results. This study had some limitations, because were not not compared the diagnostic value of QFT-G from CSF to PCR for M tuberculosis in CSF because that technique was not available in that hospital. Secondly, were included in the report a limited number of infants and immunosuppressed subjects. Furthers studies are needed to investigate if QFT-G for CSF is dependent of the very young age (< 2 years) or immunocompromised status of patients and to compare the sensitivity and specificity of QFT to those of PCR and ELISPOT for CSF are of significant interest. In immunocompromised patients with higher likelihood of indeterminate results, the ELISPOT is an alternative to the QFT-G for CSF. Other studies are also requires to estimate the period between exposure to TB, the onset of TBM and the occurrence of positive QFT test in CSF. An initially negative QFT-G result in CSF does not exclude TBM and will be useful to repeat the test in cases of presumed TBM. Making dynamics QFT tests could be useful for determining the effectiveness of anti TB drugs. In conclusion, although cultures for M. tuberculosis remain the gold standard in the diagnosis of TB the new immunological interferon gamma releasing tests seem to be promising for the management of TB meningitis. In present study, the estimated sensitivity of QFT-G for CSF was comparable to that of the QFT-IT for whole blood and higher than TST and culture and the estimated specificity of QFT-G for CSF seems to be higher than other methods. A positive QFT-G test in the CSF may be a solid argument for a diagnosis of TBM meningitis, at least in cases with negative cultures. Even in a paucibacillary population like children, the determination of gamma interferon in the serum and CSF are useful diagnostic markers of tuberculosis that may improve the management of TB meningitis by enabling rapid diagnosis and early initiation of treatment. About the adverse reactions of the antituberculous therapy, all the therapeutical agents are hepatotoxic, but the most important hepatotoxic anti T.B. agent is rifampicine The study of Rifampicine hepatopathy in patients with tuberculous meningitis is presented in the following study [113, 161]. Aspects related to the hepatotoxicity of tuberculous meningitis in the use of rifampicin have been described in the paper that shows the clinical-biological aspects of tuberculous meningitis treated with antituberculosis. The study retrospectively analyzed 32 cases of rifampicin hepatopathy in the 82 cases of bacillary meningitis hospitalized in Infectious Diseases Hospital of Iasi between 1999 and The liver function monitorizing was realized by following of seric transaminasis, the incidence of this adverse reaction being 39%, with oscillations between 4 and 13 cases per year, in a ratio of 2.5 and 13 cases/year, compared to the number of patients treated with antituberculous drugs. 76

78 The majority of the cases were met at the group of adult age (71.8%), and clinic the liver affection from the cours of Rifampicin treatment was manifested through: nausea (37.5%), vomiting (31.25%), abdominal pains (59.3%), jaundice (31.25%) and fever (18.75%). In 87,5% cases, rifampicine hepatopathy was characterized by increassing values of transaminasis until 20 the normal value, the normalisation of these parameters being realized in days from intreruption of this therapy. This study is an alarm sign on the liver function monitorization importance during the treatment with antituberculous drugs, observing a progresive increassing, from year to year of the cases of rifampicinic hepathopathy. Chapter III. RESEARCHES IN DIGESTIVE INFECTION WITH CLOSTRIDIUM DIFFICILE Over the past decades, both incidence and severity of Clostridium difficile infection (CDI) have increased worldwide. Today, CDI is recognized as the leading cause of infectious nosocomial diarrhea in developed countries, associated with increased morbidity, mortality, and healthcare costs. In addition to prior broadspectrum antibiotic therapy, other potential risk factors such as advanced age, prolonged hospitalization, multiple comorbidities, gastrointestinal surgery, chemotherapy, immunosuppression, hypoalbuminemia, have been identified to explain the increased incidence of CDI. Outline: the outbreak was first in 2002 and then it continued in other areas of the world along with the increase of contagiousness, the incidence and severity of infections produced by Clostridium difficile as consequence of the emergence of a variant of this bacteria (ribotype 027) that produces times a higher quantity of toxin as compared to the classic variants [110]. One of the aggressive strains of the bacteria produces much more toxins than other strains. The new strain is more resistant to many of the available drugs and was discovered by people that have not been to hospital or followed drug treatment. This has been the cause of several outbreaks since 2000 to present day. The bacterium synthesizes an additional toxin and has an increased capacity to form spores. The infection affects younger age groups, even pregnant women, as well as the much more frequent occurrence of community cases. In 2011 the increase of the ICD incidence in Romania was found and it was correlated to the highest ratio ever recorded in EU member states (for the cases determined by ribotype 027, approximately 70% of the total cases), the incidence of the disease rising year after year. 77

79 3.1. Epidemiology C. difficile infection is now considered a serious public healthcare problem, and with the increasing incidence worldwide, this infection appears to become dominant in the overall picture of nosocomial infections in Europe and the US. In Europe, CDI rate is 4.1/10,000 patients per day, with a mortality of 22% at 90 days. The economic impact of CDI is in Europe approximately $ 3 billion/year (CDI patient care in hospitals) and in US riched 1.3 billion/year. Moreover, ICD is considered to have exceeded the number of cases of nosocomial infection caused by methicillin-resistant staphylococcus, reaching an incidence rate of approximately 9% of all infections associated with healthcare services; CDI represented 20%-30% of the cases of associated diahoreea with antibiotics and is the most frequent known cause of infectious diarrhea in healthcare centers. In the US in 2010 there were registered 346,800 hospitalized cases with C. difficile infection, with 30,000 deaths, because CDI is 21% more common than Methicillin resistant Staphylococcus aureus (MRSA). C.difficile is a Gram positive, anaerobic, sporulated ubiquitous bacterium, present in: faecal flora (60-70% in neonates, 10-30% in hospitalized patients, 3% in healthy adults), genitourinary tract (18-71%), manure of various animals and birds, in lakes, in pools, sand, dust (Pakistan), hospital units and healthcare centres [8-10]. It takes two forms: vegetative inactivated by atmospheric oxygen, spores - resistant to disinfectants and antiseptics. In our country, the epidemiological phenomenon of CDI infection is controversial in the absence of conclusive epidemiological studies. With high probability, the number of cases of this infection has increased over the years, but in the absence of sensitive diagnostic tests, these cases could not be properly recorded. At the same time, there was an increase in the incidence of CDI in patients who were previously considered to be at low or no risk for this infection. This is the case of patients with IBD which may exhibit an unfavorable prognosis reported by many recently studies. Broad spectrum antibiotic therapy has been and remains the main risk factor for the acquisition of CDI. From an epidemiological viewpoint, the latest reports of World Health Organization have showed an increase of the incidence for the number of Clostridium difficile infections, mainly in the last decade [110, 168]. The majority of infections reported occurred in patients over 50. The increased number of elderly patients in EU countries suggests the fact that C. difficile infections will grow. Patients that are prone to being susceptible to Clostridium difficile infections are surgical ones, with chronic renal disease and those with cancer. Clostridium carrier ratios are from 0-3% in Europe to 15 % in Japan. These differences reflect variations in the sensibility of the culture method and selection of subjects that did not previously receive antibodies. 78

80 The number of asymptomatic carriers among healthy volunteers rose by 46% after antibodies were administered to them. The percentages of carriers reflect the following: 1-4% of the patients hospitalized with general problems, and 8-6% with neoplastic diseases. The incidence rate in pediatric patients, i.e. newborns is much higher reaching a percentage of 35-36%. Neonatal colonization is almost in all cases asymptomatic despite the Clostridium difficile strains that produce toxins. The absence of symptomatology is accounted for by the newborns immature intestinal flora and the lack of development in receptors for the intestines toxin. After 1-2 years, the rate of colonization drops, yet susceptibility for the symptomatology of the infection with Clostridium difficile grows. Nosocomial contamination has been suspected for a long time, yet highlighted along with the development of type and phenotype methods. This varies according to the populations studied, the use of antibiotics and the presence or absence of an increased incidence of infection with Clostridium difficile or diarrhea associated with it [88]. For instance, neoplastic patients have a contamination percentage of 2-6% in the absence of a previous outbreak and of 2-5% during it. Colonisation drawing on medium factors is the result of spore ingestion that outlives external conditions and resist from months to years. They survive stomach acidity and turn into vegetative organisms when they reach colon level. The contamination with this bacterium is the most common one in hospital and the patients infected are the most important reservoir for the bacterium [23]. The bacterium was also isolated from the hands of medical personnel as symptomatic infections were manifest in them, as well. Recent epidemiologic reports claim that Clostridium difficile replaced methicillin-resistant Staphylococcus aureus as the commonest cause of intra-hospital infection. Many reports from USA, Canada and Europe show an increase in the incidence of infection with Clostridium difficile by 2-4 times in the last decade, especially in elderly patients with prolonged hospitalizations. For instance, in Quebec, the incidence of Clostridium difficile infections increased by four times between with a 6,9% mortality. The European study group of Clostridium difficile (ESGCD) reported an incidence of infection with intra-hospital Clostridium difficile of 4,1 per patients hospitalized a day. Children and women in peripartum previously considered to be low risk populations show an increased incidence. Approximately new cases of colitis associated to Clostridium difficile are annually reported in the USA, and healthcare costs are high. Molecular epidemiology of C. difficile is extremely varied. Strain 027 proved to be the most widespread one and frequently occurs in North America, whereas strain 001 is the most identified one in Europe. Rare cases of infection with stream 027 were encountered in hospitals from Japan, Hong Kong and Australia. 79

81 The epidemiology of infection with Clostridium difficile has changed in recent years: the incidence of the disease rose (8 times in Quebec and 4 times in Germany), the affection is the most severe one (higher rates of septic shock), perforation and colectomy up to 18%). ICD is the most reluctant to standard therapy (clinical failure with metronidazole of 25% in 2004, as opposed to 9% in 2002) and recurrence ratios that doubled in people over 65; we witness the emergence and spread of new clones (ribotype 027 and 078 that prodice binary toxin) [110]. The toxinotype PCR 078 is an instance of emergent type of Clostridium difficile with transmission from animals to people and an increased virulence in people. Lately there has been an increase of costs incurred by the care of hospitalized patients and their death during hospitalization and the following period (3-5% of the total number of hospitalizations, 3 out of 29 with this diagnostic) due to the insufficient knowledge of the problem, as well as diagnostic capacity, the absence of diagnostic and treatment protocols, flaws in the organization of nosocomial infections control. Studies conducted in various regions of the world indicated that out of the antibiotic prescriptions, a proportion of at least 30-50% is not justified. Reducing the number of useless prescriptions of antibiotics is the main means to lower ICD incidence (both of first episodes and relapse). Etiologically speaking, Clostridium difficile is an anaerobic Gram positive bacillus, a spore that determines pseudomembranous colitis and antibiotics-induced diarrhoea; it is a bacterium that causes various symptoms which vary from diarrhoea to colon inflammation and may endanger the patient s life. Clostridium difficile colitis affects the elderly that have been hospitalized for a long time or follow long-term drug treatments and usually occur after the use of antibiotics. Despite common knowledge, this is not a commensal bacterium of the digestive tract. The bacterium comes from an exogeneous source and in certain given conditions may produce the disease. The spectrum of consequences in asymptomatic carriers may comprise dehydration, metabolic changes, intestinal perforation and haemorrhage. Mortality is high in aged population and of 1,5% in hospitalised cases with diarrhoea caused by Clostridium difficile. Clostridium difficile induced colitis is a result of disequilibrium of the colon s bacterial flora, of the colonization with Clostridium difficile and toxin production. Antibiotic treatment leads to the alteration of intestinal flora. In recent years infections with this bacterium have become more and more frequent, severe and difficult to treat. Every year tens of thousands of people get sick. In its mild form, the disease may be ameliorated if antibiotics administration is stopped. Symptoms vary according to the type of antibiotic used. Disease relapses occur most often in 7-14 days after terminating therapy (which rather suggests a relapse than a reinfection). Relapse is recorded in 15-35% of the patients with a previous episode and in 33-65% of those with 2 episodes. 80

82 Clostridium difficile may be encountered in soil, air, water and human and animal excrements. There is a pretty low percentage of healthy people in which the bacteria are present inside the thick intestine. However, Clostridium difficile is most often found in hospital units and healthcare centers (visited by most of the patients that carry this bacterium) and is transmitted via excrements. The bacterium forms resistant spores that may live in a chamber for a few weeks up to months. Two mechanisms are necessary for the diarrhoea associated to Clostridium difficile to occur: the destruction of normal gastro-intestinal flora, diminishing the resistance of colonisation by favouring the onset of Clostridium difficile bacterium and its takeover from an exogenous source. Other factors include host susceptibility, virulence and nature of the Clostridium difficile strain and the period of exposure to antimicrobial, respectively. At the normal population, there is more than 500 species of bacteria in the colon. A gram of excrements contains bacteria that resists colonisation and interfere with the multiplication of Clostridium difficile. Lactobacillus and group D enterococci have a greater antagonist activity, and the eradication or reduction of such bacteria by antibiotics creates a medium which is likely to develop Clostridium difficile. General population has great variation of bacteria in intestinal microflora and aged population is at great risk to develop a Clostridium difficile infection because protective bacterian diversity is affected by antibiotics, which allows the development of Clostridium difficile. There are 400 Clostridium difficile strains. The infection is produced by oral transmission/ via excrements and the bacterium multiplies in the colon. Only the toxin produced by strains produced the disease. Toxins are endocyted by all epithelial cells and destroyed by the actin cytoskeleton causing cellular death [8, 16, 23]. There are two types of toxins necessary to produce diarrhoea associated with Clostridium difficile. Toxin A 308 KDa enterotoxin destroys cellular adherence in colon mucosae at basal membrane, thus affecting the tip of intestinal villi. This causes a fluid secretion, mucosae destruction, diarrhoea and inflammation. Toxin B KDa cytotoxin enters the cell by endocytosis and induces cellular apoptosis. B toxin is 1000 times more potent in its cytotoxic effect than toxin A. Both toxins stimulate monocytes and macrophages that free interleukin 8, hence the tissue infiltration with neutrophils. Pathogen strains of Clostridium difficile produce two distinct toxins: an enterotoxin with toxic action which is lethal and responsible for clinical symptoms and a cytotoxin that exerts a cytopathic effect and mentions the laboratory diagnostic. Whereas toxin A is produced by almost all Clostridium difficile strains involved in the onset of the disease, it has been recently demonstrated that some strains secrete only toxin B. The colitis induced by Clostridium difficile results from disequilibrium of the bacterium flora in the colon, the Clostridium difficile colonization and toxin production. 81

83 Antibiotic treatment leads to alteration of the intestinal flora. Furthermore, Clostridium difficile forms thermoresistant spores that may last in the environment for a few months up to a few years. When contamination is prevalent, intra-hospital epidemic of diarrhea may occur. Clostridia colonisation occurs after the ingestion of acid-resistant spores. The spreading of clinical manifestations depends on the immune system to Clostridium difficile. In patients with low levels of IgG antitoxin A the disease is more severe than in patients with high levels that usually recover spontaneously. Some Clostridium difficile strains are non-toxicogenic and do not contain genes for toxin A and B. It is widely acknowledged that non-toxigene strains are non-pathogenic. Moreover, it was reported that elderly patients hospitalized with diarrhoea have non-toxigene Clostridium difficile strains which suggests that other pathogenic mechanisms should be studied, as well. It was suggested that broad-spectrum antibiotics cause changes in the normal intestinal flora which causes disequilibrium in the control mechanism of bacterial population in the colon, thus favouring the proliferation and colonisation with Clostridium difficile. The nature of changes in the intestinal flora that favours the colonisation with Clostridium difficile has not been established yet. The source of Clostridium difficile may be endogeneous if the patient is a carrier, or most often exogenous, nosocomial taken over from the environment. The most frequently involved antibiotics are the broad-spectrum ones as they have a major impact on normal intestinal flora, especially the ones administered orally. Here we can include penicillin, those that contain beta lactamase inhibitors, cephalosporins and clindamycin. Infection with Clostridium difficile may also be induced by antibiotics to which the bacterium is resistant, i.e. broad-spectrum penicillin. Along all antibiotics involved in producing infections with Clostridium difficile associated with diarrhoea, with a lower effect, there are aminoglycosides administered intravenously. Combinations of antibiotics have a low risk of colonisation with Clostridium difficile and induction of an infection with it. The relative risk of an antibiotic that produces a colonisation with Clostridium difficile depends on the duration and frequency of antibiotics administration. Antibiotics that can generate pseudomembranous colitis are cephalosporin (especially second and third generation ones). i.e. clindamycin, ampicillin, amoxicillin and fluorochinolone. The risk is greater in the case of prolonged treatments and associations with antibiotics. Colonisation with Clostridium difficile generates a broad spectrum of clinical conditions, from a state of asymptomatic carrier, mild selflimiting diarrhoea to pseudomembranous colitis and fulminant colitis. Even if the use of antibiotics is the most important favouring factor for the infection with Clostridium difficile, there are also other factors that play a significant role such as a susceptible host treated with chemotherapy and immunosuppressive agents, patients with 82

84 leukaemia in the units of intensive therapy. Pseudomembranous colitis may occasionally occur without exposure to antibiotics. If a contaminated surface will be reached by Clostridium difficile and then an aliment will be consumed, one may contact the disease. Healthy people will not get sick as the intestine contains millions of nonpathogenic bacteria that have the role to protect the organism from infections. If antibiotics are used for the treatment of an infection, they may destroy some of the non-pathogenic bacteria, along with the ones that cause the disease. Without their action, Clostridium difficile may rapidly proliferate. Clostridium difficile forms thermo-resistant spores that may persist in the environment from a few months to several years. When contamination is prevalent, hospital-acquired diarrhoea epidemics may occur. Colonisation with clostridia occurs after the ingestion of acid-resistant spores. Antibiotics that may generate colitis are: cephalosporin, clindamycin, ampicillin, amoxicillin, fluoroquinolone. Associations of antibiotics and prolonged treatment determine a high risk of colitis. Colonisation with Clostridium difficile generates a broad spectrum of clinical conditions, from the state of mild self-limiting diarrhoea to pseudo-membranous colitis and fulminating colitis. Host-related pathogenic factors. There are many host-related pathogenic factors: immuno-depression, adherence factors, chemotactism, S-stratum, enzymes (hyaluronidase, collagenase), toxins: toxin A (enterotoxic activity), toxin B (cytotoxic activity), binary toxin (ADP ribosyl actin-specific transferase). The effects of Clostridium difficile toxins on enterocytes determine ruptures of the cellular cytoskeleton, i.e. ruptures of the F-actin, cell form distortions, and enlargement of intercellular junction space, PN attraction and attraction of other local inflammatory cells. Risk factors of the infection with Clostridium difficile are highlighted, both primary and secondary: male sex, age over 65 or under 1 with associated comorbidities, prolonged hospitalisation and therapy with antibiotics. The most important secondary risk factors that are usually host-related are the following: associated comorbidities, i.e. irritable intestine, age > 64, duration of hospitalisation, antimicrobial agents, immunodeficiency and HIV, gastro-intestinal surgery, anti-acids, anti-depressives, malnutrition, low serical albumin (<2,5g/dL), neoplasic diseases, chemotherapy, cystic fibrosis and diabetes. The administration of broad spectrum antibiotics that interrupts the normal growth of microbial flora and determines the proliferation of Clostridium difficile remains the most acknowledged risk factor. Any type of antibiotic, especially clindamycin, cephalosporins, fluoroquinolones, ampicillin or amoxicillin, macrolides, tetracycline may cause the infection with Clostridium difficile. Exposure to metronidazole or vancomicin used as initial therapy in C. difficile infection may determine the Clostridium difficile infection. 83

85 The consumption of contaminated foods is another risk factor for the infections in the community. From a clinical viewpoint, Clostridium difficile is the cause of 25-30% of antibiotic associated diarrhoea. It is defined as inexplicable diarrhoea that occurred between 2 hours and 2 months after antibiotics administration associated with abdominal pain and cramps. Diarrhoea is the elimination of 3 or more non-formed excrements for at least 2 consecutive days. The diagnostic is established when toxin A and toxin B, respectively are identified in the excrement. The disease has a variety of clinical forms such as: status of asymptomatic carrier, mild-self-limiting diarrhoea, pseudomembranous colitis and fulminating colitis [88, 119]. It is characterized by: diarrhoea (which is rarely sanguinolent); colic-related abdominal pain anorexia and fever (30-50%). Other symptoms include: nausea, general state of sickness, hypo-albumin, anorexia, anasarca, occult intestinal bleeding. Clinical manifestations include abdominal cramps, dehydration, hypoalbuminuria (<30mg/L), watery diarrhoea, increase of inflammatory cells, serial proteins and mucus Risk factors and diagnosis The clinical suspicion of CDI is supported by the presence of diarrhea at hospital admission or during hospitalization. C. difficile causes various symptoms, ranging from asymptomatic to mild or severe diarrhea that can endanger the patient's life. Diarrhea is the most important clinical symptom to which fever and abdominal pain can be added. There are cases of severe evolution with colon dilatation when the entire organism is affected by substances freed at this level (toxic megacolon) with a death rate of 50-60%. For a person to develop ICD, two conditions are necessary: 1. Most of the intestine colonisation of the respective host occurs in the hospital environment and is facilitated by ill patients with this infection or other carriers of the germ, medical personnel included (mainly nosocomial infection). 2. The significant multiplication of C.difficile that produces large quantities of toxin determines the disease. This phenomenon is the consequence of flora destruction further to antibiotics administration, the excessive use of antibiotics leading to the frequency of the occurrence in such cases. 3. Exposure to an environment with a high risk of generating and transmitting various infections such as the healthcare environment, along with favourable conditions, may determine the development of CDI. The diagnostic is achieved by sigmoidoscopy and yellow plaques are observed in the colorectal mucous. Relapses occur in 10-25% of the patients treated. The diagnostics of colitis induced by C. difficile needs to be suspected in any patient with diarrhoea that received antibiotic 84

86 treatment in the previous two months and/ or diarrhoea out broke after minimum 72 hours with hospitalisation. Extra-intestinal manifestations are rare (bacteraemia, splenic abscess, osteomyelitis, reactive arthritis or tenosynovitis). Rare associations of colitis with clostridia comprise the following: consumption of antineoplastic-methotrexate agents, haemolytic-uremic syndrome, neoplasia, intestinal ischemia, renal failure, necrotizing enterocolitis, Hirschsprung disease, intestinal inflammatory disease, non-surgical intestinal procedures, naso-gastric tube procedures included. Laboratory diagnostic: has the purpose of signalling the presence of toxins (directly from excrements of cultures). The diagnosis methods may be classified as follows: a) Cytotoxicity tests with the highest sensibility ( gold standard ). b) Cultivation c) Detection of toxins A and B by immune-enzymatic methods directly from excrement manifestations or the supernatant of broth cultures. Detection of C. Difficult toxins A and B was performed by immunochromatographic assay. Thus, a predominance of the strains producing both toxins (69%) was identified, followed by the toxin B producing strains (24.28%), and a low percentage of those producing toxin A (6.88% %). Biological parameters were represented by elevated C-reactive protein (CRP), along with relatively low serum albumin levels. d) Detection of toxins A and B by molecular methods (PCR) to detect genes that codify C.difficile toxins (tcdb, tcdc or binary toxin). The sensibility and specificity of molecular toxins is high and offers a quick diagnostic. e) Detection of enzyme GDH (dehydrogenase glutamate) has high sensibility, yet low specificity; therefore, it can be used as test screening and cannot distinguish between toxigenic and non-toxigenic strains. The algorithm of etiologic diagnostic (ATLAS score) is presented in table 3.I. Etiological diagnosis brings arguments in favour of the initial approach. A diagnostic in 2-3 stages is recommended, applicable only in laboratories that have the possibility to carry out rapid detection tests and cultivation: a. A rapid detection test for the presence of toxins/ genes codifying for toxins is required in the following cases: In case of positivity, the ICD diagnostic is confirmed; In case of negativity, there is also a second stage: b. Stool sample cultivation to isolate anaerobic germs: Negativity of cultures makes improbable the ICD diagnostic; 85

87 Table 3.I. ATLAS score Parametre 0 points 1 point 2 points Age < 60 years years 80 years Temperature 37.5 C C 38.6 C Leucocite number (cel/ mm) < > Albumin (g/dl) > Sistemic antibiotic simultaneously to therapy ICD(>1 zi) NU DA In case of colony isolation identified as C. difficile, a third stage is required: c. A toxin/ detection test is conducted to codify toxins of bacterial culture: ICD is confirmed, if positive If negative, it is very likely for colonisation to occur and for the present disease to have a different etiology. In the case of ICD diagnosis, in the first stage a culture may be carried out from the sample of excrements, and not for diagnostic purposes, but to have available the bacterium for future tests. Retesting: the control of bacteriological efficiency in ICD therapy is not recommended (C. difficile may persist in colon for a few months after symptomatology remission); repetition of testing with the same method in a new stool test for the same diarrhoea episode is not recommended when the first result came out negative (it does not increase significantly the chance of a positive diagnostic). 1. Cultivation is the method with the highest sensibility and epidemiological importance. 2. C. difficile cultivation from the stool sample followed by toxin production (or the presence of genes that codify these toxins) is the safest diagnostic method; in some cases, the final result is obtained after several days. 3. Immunoenzymatic methods to detect toxin A and B from the stool sample are rapid, yet with low sensibility. There are several types of C.difficile infection: - nosocomial ICD in which symptoms occur more than 48 hours before hospitalisation, during hospitalisation and 28 days after release from hospital; - communitary ICD in which symptoms occur more than 8 weeks from hospital release; 86

88 - undeterminable ICD in which symptoms occur in a period of 4-8 weeks from hospital release. In patients with severe disease, radiological studies may help to determine complications: toxic dilatation and perforation. They may be indicated for other possible diagnostics, as well. The most serious complications are the following: toxic megacolon, colon perforation and peritonitis, recurrent colitis (in 20% of the patients). Most patients develop diarrhoea immediately after the initiation of antibiotic treatment. However, no sooner than 10 weeks from the completion of antibiotherapy, than 25-40% of the patients become symptomatic. Increased incidence in the previous years is associated with a hyper virulent strain that leads to multiple organ failure and increased mortality. The increased recurrence of C. difficile infections is a result of relapses of infections. Approximately of 33-75% of the cases are determined by infections with another strain. The disequilibrium with the intestinal microbial flora and in low immune response against C. difficile plays an important role in recurrence development. The results of the current studies confirm a real increase in the incidence of ICD. The incidence of CDI in 2011 was around 0.57%, and it reached 15.28% in Antibiotic therapy remains the main risk factor for the acquisition of CDI both in the hospital and community setting. Regarding antibiotic treatment used in the community setting, the results indicates that previous antibiotic exposure is a risk factor for community-acquired CDI. The clinical study Updates in the assesment of risk factors in Clostridium difficile infection in patients with infectious diseases intendeed to know the risk factors of the C. difficile infection in patients with infectious diseases [116]. The aim of this study is derivative from the need to knowledge the aspects of the emergence of this disease in the last period in our geographyc region. The study aims were to assess the clinical and epidemiological features of C. difficile infection by establishing the influence of risk factors involved in the occurrence of C. difficile infection in the patients enrolled in the study, to determine correlations between risk factors and clinical-evolutionary aspects of the patients establishing correlations between prognostic risk factors, comorbidities and disease severity [116]. It has been conducted a retrospective study between January 2014 and March 2015, 166 patients being included, using the following inclusion criteria: presence of diarrheal syndrome, positive test for Clostridium difficile toxin, suggestive aspects of pseudo-membranous colitis observed during colonoscopy, response to specific antibiotics for pseudo-membranous colitis and exclusion criteria: diarrheal syndrome of other type of etiology, the response to symptomatic treatment and/or empirical antibiotics for suspected bacterial etiology diarrhea. 87

89 All patients with flare activity were tested for diagnosis using immunechromatography assay for toxins A and B. The results were statistically processed using: student t test, χ2 test, and the relative risk (RR). We have used the RR score for calculating the major risk in diabetic patients. The relative risk (RR), its standard error and 95% confidence interval are calculated according to Bhangu and Altman. The following risk factors of CDI were assessed: incidence, age, sex, comorbidities, cause, and antibiotics. Fig Yearly distribution of cases with Clostridium difficile pseudomembranous colitis by gender As results, comparative analysis of pseudo-membranous colitis distribution, by years of study, revealed the following: in the first 12 months were diagnosed 109 cases, in a relatively equal distribution according to the gender (female 54 cases, male 55 cases); in the first 3 months of 2015 there has been a marked increase in the cases of pseudo-membranous colitis, 57 patients (Figure 3.1). It should be noted a slightly higher percentage of female cases in the period January-May 2014 (female 32 cases, male 25 cases), the yearly distribution of pseudomembranous colitis with Clostridium difficile percentage having no significant gender difference (p = 0.478). Yearly distribution of cases with C.difficile pseudomembranous colitis by gender Patients age ranged between 35 and 60 years (Figure 3.2). 88

90 Figure 3.2. Yearly distribution of cases with C. difficile. The distribution of patients according to sex with a predominance of females depicted in figure 3.3. Figure 3.3. The distribution of patients according to sex Depending on patient s previous medical history, the greatest risk was registered for diabetic patients (RR = 2.63) and for digestive symptoms patients (RR = 1.79) (Table 3.II). Table 3.II. Personal pathological antecedents - comorbidities 89

91 The smallest risk was noted for patients with renal diseases as comorbidities. In hospital, the major risk factor is represented by exposure to antimicrobial agents, especially to Clindamycin and β-lactamines. There were also identified other potential risk factors for CDI: the administration of protons pomp inhibitors, the presence of intestinal inflammatory diseases or irritable bowel syndrome. During the hospitalization, the prevalence in the first 7 days was 1% to 20%, increasing to 50% for at least 4 weeks. We also assessed early symptoms of infection with C. difficile, likewise debut in the community - patients having a history of surgery; associate debut in hospital health care; undefined onset- patients hospitalized 4-12 weeks prior to the early symptoms; community debut - without hospitalization in the last 3 months. There has been a growing trend in the number of cases between December 2014 and March 2015, particularly among immunocompromised patients (cancers, diabetes, liver cirrhosis, chronic renal failure). 48% of cases occurred in patients after recent surgery and 57% came from medical services. Causes of developing CDI are shown in Figure 3.4. Figure 3.4. Causes of developing CDI In terms of the abdominal discomfort, patients were suffering pains in more than 90% of cases, either diffuse or colicky pain. It is well known that the main risk factor is represented by the exposure to antimicrobial agents, especially Clindamycin and β- lactamines. Continuing the study using groups of antibiotics, it revealed that the highest risk is represented by the use of fluoroquinolones followed by cephalosporin (Figure 3.5). 90

92 Figure 3.5. The study group of antibiotics as a cause of CDI Unfortunately relapses were recorded in 28.67% of the cases. The treatment was carried out as follows: Metronidazole (66.26%), Vancomycin (5.42%) and Vancomycin+Metronidazole (28.31%) Treatment The treatment was described in the following researches. The treatment impact during the study period was successfully for 135 patients and relapsed for 31 patients. During the antibiotic treatment of CDIs the antibiotic resistance and physiological factors of the bacteria (spore formation, protective effects of the pseudo-membrane) may cause difficulties. The emergence of a new and highly toxic strain of Clostridium difficile resistant to fluoroquinolone antibiotics (such as Ciprofloxacin and Levofloxacin), which determine geographically dispersed outbreaks in North America, was reported in The United States Centers for Disease Control and Prevention in Atlanta warned about the emergence of an epidemic strain with increased virulence, antibiotic resistance, or both. Like prognosis, after a first treatment with Metronidazole or Vancomycin, Clostridium difficile recurs in about 20% of the cases, increasing 40% and 60% with subsequent recurrences. As conclusions, the analysis of the cases with Clostridium difficile infection is a state on unjustified and irrational use of antibiotics and also a warning represented by nosocomial infections. We found an increasing trend of the number of cases between December 2014 and March 2015 particularly among immunocompromised patients, 48% of cases occurred in patients after recent surgery and 57% came from medical services. The treatment was carried out with: Metronidazole, Vancomycin and Vancomycin + Metronidazole. Relapses were present in 28,67% cases, and treatment was realized with: Metronidazol (66,26%), Vancomicine (5,42%) şi 91

93 Vancomicine+Metronidazol (28,31%). Fidaxomicin was used in 5 cases, and feeces transplant in 8 cases. Figure 3.6. Prognosis of C. difficile infection The treatment with antibiotics, known as a risk factor in development of Clostridium difficile pseudomembranous colitis, has been documented in our study investigating the antibiotics groups, revealing that the highest risk is represented by the use of fluoroquinolones and cephalosporins. This study is warning once more on the unjustified and irrational use of antibiotics, as well as on the danger represented by nosocomial infections. Treatment of Clostridium difficile infection comprises the following: a) General principles: elimination of the emergence factor (terminating antibiotic administration), initiation of etiologic therapy and measures that limit interhuman transmitting, as rapidly as possible after the formulation of diagnostic suspicion. b) Etiologic treatment: diarrhoea requires rehydration and electrolytic recalibration. Oral therapy with efficient antimicrobial agents against clostridia is the most important option. In aged patients and the ones with severe forms, empirical antibiotics are the first option. 92

94 Table. 3.III. Therapeutic Guide (IDSA) in Clostridium difficile infection (after Infect Control Hosp Epidemiol 2010;31 (5):431-55) Severity Clinical picture Treatment S/Q First episode (Mild/Mod) WBC <15,000 OR scr< 1.5 x baseline Metronidazole 500 mg PO TID x days AI First episode (Severe) WBC >15,000 OR scr> 1.5 x baseline Vancomycin 125 mg PO QID x days Bl First episode (Severe/Complicated) Hypotension, shock, ileus, megacolon Vancomycin 500 mg PO/NG QID CIII PLUS Metronidazole 500 mg IV Q8H First Recurrence... Same as first episode All Second Recurrence... Vancomycin in a tapered or pulsed regimen S/Q = Strength of recommendation (A-C)/Quality of Evidence (I-1II) BIII In case of multiple recurrences, a prolonged cure of antibiotics of 4-6 weeks may be administered, followed by three-week vancomycin. The first episode is reserved to low or medium severity ICD forms: Metronidazole is the first line agent with a response rate of 86-92%. The recommended dosage is 500 mg every 8 hours, orally; days; yet no more than 3-4 days after symptoms remission due to the risk of neurotoxicity. Vancomycin is the most efficient antibiotic in the highly severe cases of the disease. The response rate to treatment is of %. The recommended dose is of: 125 mg every 6 hours, orally, for days. Then 125 mg every 12 hours for 7 days; then 125 mg daily for 7 days, and finally 125 mg every 2-3 days for days. In toxic megacolon or ileus 500 mg of vancomicyn is administered every 6-12 hours by therapeutic enema (in ml saline liquid) and metronidazole 500 mg every 8 hours. In the case of relapses, the same options and doses are used as in the first relapse: Fidaxomicin is a new antibiotic drug with a macrocyclic structure, the first compound in his class proposed for certification by European Medicines Agency in the treatment of infections with Clostridium difficile [19, 41]. Clinical studies carried out insofar showed that it is as efficient as vancomycin in the treatment of infections with Clostridium difficile; however, the advantage 93

95 consists in the selective destruction of the bacteria by fixadomicin with minimum damage of the other bacterial species in the stomach. In addition, the action mechanism in fixadomicin reduces the chances of the action to be counterattacked by the bacterium resistance to other antibiotics. The basic rule to prescribe antibiotics is monotherapy, and associations of antibiotics are justified in the context of megacolon or ileus: (Metronidazole iv + vancomycin po/rectal). The antibiotic chosen needs to be adapted to the severity of the disease: metronidazol is not administered in severe forms and neither is vancomycin in the mild forms. The simultaneous administration of any other antibiotic systemically is forbidden as it increases duration up to symptomatology remission and the risk of unfavourable evolution (relapses, death). Systemic therapy is indicated only in the case of associations with other infections (most often enteral germs) or salvation treatment with tigecycline (with increased attention to the compromising of his activity as reserve antibiotic for the infections determined by Acinetobacter, Klebsiella, and MRSA). Transplant of fecal microbiota is an alternative therapy for recurrent cases of Clostridium difficile infections. This method uses intestinal microorganisms from a healthy donor to reestablish a normal microbial flora in the patient. Recent studies pointed out a 92% efficiency of the treatment realized by means of a colonoscopy, enema or tube procedures. The administration of faecal matters collected from healthy people via colonoscopy at the level of all colon segments or the infusion on nasogastric tube gave the best results in recurrence treatment. The basic rule in the prescription of antibiotics is monotherapy, and associations of antibiotics are justified in conditions of megacolon or paralytic ileus (Metronidazole iv + vancomycin po/rectal). The simultaneous administration of any other antibiotic systemically is forbidden as it increases duration up to symptomatology remission and the risk of unfavourable evolution (relapses, death). New therapies: the use of two neutralizing monoclonal antibodies acting against toxin A and B simultaneously administered with standard antibiotherapy, thus revealing the significant decrease of recurrence rate in patients that received antibodies, as opposed to the placebo group (8% vs 32%) (Lowy). I.v. immunoglobulins determine a favourable clinical response to a subgroup of patients with many ICD recurrences. [12], with a dose of 300 mg/kg or the same dose in 5 daily perfusions. c) Surgical intervention is indicated in case of: colon perforations, an aggravation of the patient s condition despite adequate medical treatment, toxic megacolon or severe ileus with shock ICD during recovery after a colon intervention. Subtotal colectomy and ileostomy are the types of interventions preferred. 94

96 The carrier status does not require any treatment. The mild forms of infection are spontaneously dealt with in 2-3 days after termination of antibiotic treatment. Due to the lethal potential of colitis, curative therapy requires immediate initiation. The death risk is 2-3 times reduced, from 35%-80% to 12-35% in case of early intervention, especially before the very severe damage of the colon. f) Recurrence prevention by colon recolonisation with detritivore flora through prebiotics or transplant of enteral flora from healthy donors. The outcome for most infected patients in that they remain asymptomatic carriers of the bacteria and they will almost never develop colitis. 20% of the patients treated will relapse in the infection by spore germination or reinfection. Response to treatment with vancomycin is usually favourable. The efficiency of treatment was analyzed in the study of C.difficile infection treatment in elderly was done in a clinical trial A Phase IIIb/IV Randomized, Controlled, Open-Label, Parallel Group Study to Compare the Efficacy of Vancomycin Therapy to Extended Duration Fidaxomicin Therapy in the Sustained Clinical Cure of Clostridium difficile Infection in an Older Population. Clostridium difficile infection (CDI) is caused by an overgrowth of C. difficile in the colon. Most cases are associated with previous antibiotic use which eradicates or disrupts the gut flora, allowing C. difficile to proliferate. Proliferating C. difficile produces harmful toxins that cause a variety of complications including pseudomembranous colitis, toxic mega colon, perforation of the colon and sepsis [Kuijper et al, 2006]. The clinical burden of CDI is compounded by the fact that as many as 25% of patients willsuffer a recurrence of infection within 30 days following treatment [Louie et al, 2011;Lowy et al, 2010; Bouza et al, 2008]. This issue is particularly problematic for patient groups at high risk of recurrence or those for whom the impact of recurrence would be most dramatic such as patients who are immunocompromised, receiving concomitant antibiotics [Hu et al, 2009; Pépin et al, 2005; Kyne et al, 2001], who have had CDI previously [Pépin etal, 2005; McFarland et al, 2002], who have renal impairment [Bauer et al, 2011] or those aged 65 years or over. Metronidazole and vancomycin have been the mainstays of treatment for CDI for several decades. The 2014 European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines have recognized the robust phase 3 randomized controlled trial evidence supporting the use of fidaxomicin in the treatment of CDI resulting in a moderate recommendation for the use of fidaxomicin [Debast et al, 2014]. Fidaxomicin is a macrocyclic antibacterial agent with selective bactericidal activity against C. difficile and minimal effect on the colonic microflora. Fidaxomicin inhibits RNA synthesis by bacterial RNA polymerase at a distinct site from that of rifamycins [Sergio et al, 1975]. Fidaxomicin in the lumen of the lower gastrointestinal (GI) tract with low systemic exposure. 95

97 In the EU, fidaxomicin treatment, as tablets, was approved under the trade name DIFICLIR in December 2011 [41]. Fidaxomicin tablets were approved in the US and Canada under the trade name of DIFICID in May 2011 and June 2012, respectively. In all 3 regions, fidaxomicin tablets were approved for use in adults ( 18 years of age) for treatment of CDI. Of those patients enrolled in the phase 3 registration studies, more than half the patients were 60 years of age or older [Debast et al, 2014]. Advanced age is a major risk factor for the development of CDI [Bauer et al, 2011; Bignardi et al, 1998] and is also correlated with increased severity of the disease and other adverse outcomes, including death [Louie et al, 2013]. Regression modeling of results from the 2 double-blind randomized multicenter phase 3 studies demonstrated progressive deterioration in CDI treatment outcomes. These occurred with advancing age (by decade) in those aged 40 and older; the clinical cure rate decreased by 17% (odds ratio [OR] = 0.83 (95% CI: ), (P = 0.008); recurrence rate increased by 17% OR = 1.17 (95% CI: ), (P = 0.007); and sustained clinical cure rate reduced by 13% (OR = 0.87 (95% CI: ), (P < 0.001). Fidaxomicin treatment was associated with a 60% lower risk of recurrence (P < 0.001) and a greater sustained clinical cure, compared to vancomycin therapy, after adjusting for age, concomitant antibiotics and C. difficile strain [Louie et al, 2013]. The challenges for therapeutic success posed by this population present the ideal scenario for demonstrating clinical effectiveness of treatment interventions and an opportunity to address a significant ongoing unmet medical need in this disease area [19]. The present study assessed the comparative effectiveness and safety of extended pulsed fidaxomicin (EPFX) vs standard vancomycin treatments in patients 60 years of age and older. This phase 3b/4 study evaluated if EPFX therapy was superior to the standard vancomycin therapy in an older population We followed the Fidaxomicina versus Vancomicina therapy beneffits in older patients, with comorbidities. This study assessed the comparative effectiveness and safety of extended pulsed fidaxomicin (EPFX) vs standard vancomycin treatments in patients 60 years of age and older. This phase 3b/4 study evaluated if EPFX therapy was superior to the standard vancomycin therapy in an older population. This was a phase 3b/4 study that evaluated whether EPFX therapy was superior to the standard vancomycin therapy in an older patient population. The study was conducted in accordance with the protocol, Good Clinical Practice (GCP), International Committee on Harmonisation (ICH) guidelines, applicable regulations and guidelines governing clinical study conduct and the ethical principles that have their origin in the Declaration of Helsinki. 96

98 This was a multicenter, randomized (1:1), parallel-group, open-label study with patients aged 60 years with confirmed presence of Clostridium difficile toxin A or B in stool, respectively. The study drug was either vancomycin 125 mg oral capsules taken 4 times daily for 10 days or fidaxomicin 200 mg oral tablets taken twice daily for the first 5 days (day 1 to day 5). The primary objective of this study was to evaluate the efficacy of EPFX in the treatment of CDI in male and female patients aged 60 years and older compared with standard vancomycin therapy and the primary endpoint of this study was: Rate of sustained clinical cure of CDI at 30 days after end of treatment (EoT) (day 40 or day 55). The secondary objectives were to compare EPFX and standard vancomycin treatment on sustained clinical cure, clinical response and the rate of relapse and recurrence of CDI. In addition, the Health-Related Quality of Life (HRQoL) and safety were assessed. Main secondary efficacy variables were: sustained clinical cure of CDI at day 40, day 55 and day 90; clinical response of CDI at day 12; rate of relapse at day 90 as determined by whole genome sequencing of C. difficile isolates from patients who have documented recurrence after test of cure (TOC). Other secondary efficacy variables were: Clinical response of CDI at 2 days after EoT (day 12 or day 27 for standard vancomycin and EPFX, respectively); Time to resolution of diarrhea (TTROD) in hours; Rate of recurrence of CDI at day 40, day 55 and day 90; Time to recurrence after EoT (only for patients with clinical response); Disease-free survival after day 10 (only for patients with clinical response). Study Design and Plan Description Overall: This was a phase 3b/4 randomized, controlled, parallel-group, openlabel study in which 364 female and male patients of at least 60 years of age with CDI confirmed positive for presence of C. difficile toxin A or B in stool (within 48 hours prior to randomization) were to be enrolled. Patients randomized to the extended pulsed fidaxomicin (EPFX) arm took 200 mg fidaxomicin from day 1 to day 5 twice daily, followed by a 1-day gap (day 6) before starting alternate day dosing of 1 tablet of fidaxomicin 200 mg daily from day 7 to day 25. Patients randomized to the standard vancomycin arm took 125 mg vancomycin from day 1 to day 10, 4 times daily. Patients visited the clinic at screening (visit 1), at day 12 (visit 2) and 27 (visit 3) for the assessment of TOC and clinical 97

99 response as determined by the investigator. Safety was followed from the time of written informed consent until end of study (EoS) visit 6 (day 90) for all patients. Patients with increased unformed bowel moments (UBMs) following clinical response at TOC (UBMs greater than the frequency recorded on day 10 for the standard vancomycin arm or day 25 for the EPFX arm), had to attend an unscheduled visit for the assessment of recurrence of CDI. The study also included 2 sub-studies to analyze microbiome profiles and/or pharmacokinetics of the concentration of fidaxomicin in blood (plasma) and stool. Both assessments were carried out on day 5, and visits 2, 3, 4 and 5 (see also Table 3.IV). Screening. Informed consent was obtained before performing any study-related procedures. Patients were asked to give informed consent to participate in 1 or both sub-studies. Patients underwent eligibility screening, including safety laboratory assessments, physical examination, estimation of the number of UBMs, stool collection, vital signs, completing the EQ-5D-5L questionnaires and recording of the CDI clinical assessment and severity score. Randomization was based on a 1:1 ratio: Fidaxomicin (200 mg) oral tablets twice daily from day 1 to day 5, followed by once daily (200 mg) every other day from day 6 to day 25; Vancomycin (125 mg) oral capsules 4 times daily from day 1 to day 10. If a patient signed informed consent for participation, they were randomized in 1 or both substudies. A total of 35 patients participated in the pharmacokinetic (plasma) sub-study and 65 patients participated in the microbiome sub-study. Table 3.IV. Dosing regimens Randomization was stratified by: baseline CDI severity (severe/non-severe, where severe was defined as leukocyte count > 15 x 109/L or rise in serum creatinine [> 50% above the patient s 98

100 normal levels] or albumin < 30 g/l), presence or absence of cancer (defined as diagnosis of cancer within the past 6 months and/or on therapy for the treatment of cancer), age ( 75 years or < 75 years), and the number of previous recurrences (0, 1 or 2) in the 3 months prior to the study Treatment period: in the case of treatment failure or any confirmed recurrence after assessment of clinical response from day 12 (visit 2), stool samples were collected for laboratory testing. All samples underwent culture, ribotyping and susceptibility testing. In order to distinguish relapse and re-infection in patients with a recurrence of CDI after TOC, whole genome sequencing of isolates was to be carried out on samples from both day 1 and confirmed recurrence. From visit 1 (day 1) to TOC visit 2 (day 12) for patients in the standard vancomycin arm, or visit 3 (day 27) for patients in the EPFX arm, patients completed a Subject Diary to record the total number of UBMs passed each day, along with the time of the last UBM for each day, or the daily volume of stool (for patients with rectal collection devices), and intake of the number of tablets/capsules of study drug taken each day. At baseline, visit 2, 3, 4 and 5 and at unscheduled visits for assessment of recurrence of CDI, patients completed the EQ-5D-5L questionnaires. If a patient was discharged from the hospital and was not participating in the sub-study, they could complete the questionnaires for visit 4 and visit 5 at home. The duration of the treatment period was from the start of study drug dosing to the end of study drug dosing. Follow-up Period: following completion of the treatment period, safety, efficacy and health economics outcomes were followed-up until EoS visit 6 (day 90). Patients were to be followed up at least every 2 weeks to check for CDI recurrence, AEs and concomitant medications. The study flow chart of the main study is provided in figure 3.7. Fidaxomicin is believed to reduce recurrence of CDI primarily through minimal collateral damage to colonic microflora allowing recovery to start during therapy. The current summary of product characteristics (SmPC) approved dosing regimen for fidaxomicin (200 mg every 12 hours for 10 days) was not optimized for recovery of microflora but chosen based on existing practice for vancomycin and metronidazole. Hence, the study dosing of fidaxomicin (Dificlir ) to be administered was not in line with the current approved SmPC for Dificlir and has been selected for the purpose of maximizing the attributes of this product in treating CDI. Patients received 200 mg twice daily for 5 days from day 1 to day 5. From day 6 to day 25, patients received 200 mg once daily on alternate days. This extension of the dose allowed a 99

101 longer period for the protective microflora to recover and extended the period of time over which C. difficile cells were suppressed, and to kill any dormant spores that germinate. Legend: EoT: end of treatment; EoS: end of study; F: treatment failure; Fidax: fidaxomicin (extended pulsed fidaxomicin arm); R: recurrence of CDI; Rand: randomization; Screen: screening; TOC: test of cure; Vanc: standard vancomycin arm Figure 3.9. Study flow chart The extension of the dose also targeted the early recurrence period documented in the phase 3 studies the first 15 days after the end of therapy during which the majority of recurrences occurred. By covering this period, this revised dosing regimen could potentially reduce the recurrence rate further. The total dose of fidaxomicin received over 25 days was equal to that received in the dosing regimen approved in the SmPC and had been designed to be simple enough for patients to follow. This dosing regimen was tested in an in vitro gut model [Chilton et al, 2014] and simulation of CDI demonstrated rapid reduction of C. difficile viable counts and toxin levels to below the level of detection within 3 days, which was sustained out to 21 days post the last treatment intervention (the end of the study). The impact on the gut microflora was limited, supporting the rationale for this study. The dosing of vancomycin (Vancocin ) to be administered in this study was in line with the SmPC for Vancocin. Patients received 125 mg 4 times daily for 10 days from day 1 to day 10. Conclusions were: the interim analysis revealed a significant treatment difference in the rates of sustained clinical cure in favor for EPFX-treated patients 100