PARACLINICAL DIAGNOSIS VALUE FOR ENDOCARDITIS INVOLVEMENT IN SEPSIS

Transcription

1 UNIVERSITY OF MEDICINE AND PHARMACY GRIGORE T. POPA IASI FACULTY OF MEDICINE DOCTORATE THESIS PARACLINICAL DIAGNOSIS VALUE FOR ENDOCARDITIS INVOLVEMENT IN SEPSIS - ABSTRACT- PhD Student, Isabela Ioana BEGEZSÁN (LOGHIN) Scientific coordinator, Prof. Univ. Dr. Carmen Mihaela DOROBĂŢ IASI 2013

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3 Contents General part I. Importance of infective endocarditis nationally and globally.. 6 I.1 Infective endocarditis - introductory notes I.2 Infective endocarditis - short history.. 6 II. Epidemiology III. Etiopathogenesis IV. Clinical picture of infective endocarditis 18 V. The diagnosis of infective endocarditis 22 V. 1 Bacteriological diagnosis blood cultures.. 25 V. 2 Diagnostic imaging echocardiography. 30 V. 3 Other modern techniques for bacterial detection. 35 VI. Prognosis of infective endocarditis.. 42 VII. Treatment and follow-up of infectious endocarditis VII. 1 Etiologic therapy VII. 2 Pathogen-specific therapy. 53 VII. 3 The role of cardiologist in infective endocarditis follow-up VII. 4 Role of cardiac surgery in the treatment and prognosis of infective endocarditis.. 55 Personal contribution VIII. Importance of the topic. Objectives IX. Materials and method.. 61 IX.1 Study 1 - retrospective study of infective endocarditis cases recorded in the interval IX.2 Study 2 - prospective study of the patients with endocardial involvement during severe sepsis recorded in the interval IX.2.1 Bacteriological diagnosis.. 63 IX.2.2 Diagnostic imaging IX.3 Study 3 - prospective experimental study on the use of molecular biology methods for detection of bacterial DNA IX.3.1 Platelet concentrates. 66 IX.3.2 Bacterial strains 67 IX.3.3 Inoculated plateled concentrates plating.. 67 IX.3.4 Bacterial detection by Bactiflow ALS. 67 IX.3.5 Isolation of bacterial DNA IX.3.6 Bacterial detection by 16s RNA PCR method. 69 IX.4 Study 4 - prospective study on the therapeutic efficacy of the use of PLEX ID - method for the detection of bacterial DNA, in patients with infective endocarditis.. 70 X. Results X.1 - Retrospective study of infective endocarditis cases recorded in the interval

4 X.1.1 Demographic and epidemiological data. 70 X.1.2 Clinical and evolutive features X.1.3 Positive diagnosis of infective endocarditis X.1.4 Prognostic considerations in the study group X.1.5 Therapeutic considerations in the study patients X.2 Study 2 prospective study of the patients with endocardial involvement during severe sepsis recorded in the interval X.2.1 Epidemiological characteristics X.2.2 Diseases associated with infective endocarditis X.2.3 Clinical and evolutive features 115 X.2.4 Positive diagnosis of infective endocarditis 123 X.2.5 Prognostic considerations in the study series X.2.6 Therapeutic consideration 144 X.3 Comparative discussions on the retrospective and prospective study of patients with infective endocarditis (Study 1 and Study 2, respectively) X.4 Study 3 - prospective experimental study on the use of molecular biology methods for detection of bacterial DNA (Bactiflow system and PCR). 156 X.5 Study 4 - prospective study on the therapeutic efficacy of the use of PLEX ID - method for the detection of bacterial DNA, in patients with infective endocarditis 166 X.6 The role of modern diagnostic methods in therapeutic decision 171 X.7 Role of adequate antibiotic therapy in determining patient prognosis - clinical considerations based on prognostic scoring system XI. Discussions 173 XII. Conclusions XIII. Final conclusions 178 XIV. Original elements of the thesis 180 XV. Perspective that opens the thesis XVI. References XVII. Annexes. 190 The doctorate thesis includes: pages (General part 159 pages, Personal contributions 123 pages) tables figures references - 3 annexes - 3 original papers (BDI B+ cotation) Note the abstract shows selective references and iconography, respecting numbering and contents of thesis. Key words INFECTIVE ENDOCARDITIS, PCR, ECHOCARDIOGRAPHY, BACTERIAN DNA, BLOOD CULTURE. Rector's Decision U.M.F. Grigore T. Popa Iasi of appointment of the doctoral committee no of Date of public defense of the thesis: Acknowledgements: The researcher was supported by the project POSDRU/88/1,5/S/

5 General part I. Importance of infective endocarditis nationally and globally I.1 Infective endocarditis - introductory notes The term "endocarditis" [Greek: "endon" = inside; "kardia" = heart; "ita" = inflammation] is described in the medical dictionary as the exudative and proliferative inflammatory lesion of the endocardium that can be located in the heart valves or inner lining of the cardiac chambers (1). Currently, this condition can be also described as a consequence of implanted intracardiac devices, one of the main risk factors for infection with Staphylococcus aureus in the bloodstream (3). I.2 Infective endocarditis - short history The history of infective endocarditis (IE) can be divided into several eras. Lazaire Riviere was the first to describe in 1723 the autopsy findings in a patient (although some sources suggest that the Frenchman first described some unusual "outgrowths" in 1646). In 1885, William Osler presented the first comprehensive description of endocarditis in English ("Synthetized work of others relating to endocarditis"). In 2000 and 2002 Durack Duke criteria have been modified and updated. In 2004, a research group of the British Society for Antimicrobial Chemotherapy published new perspectives regarding the guidelines for the treatment of endocardites caused by streptococci, enterococci and staphylococci, as well as by the HACEK group of microorganisms. II. Epidemiology Recent studies estimate that in the United States are recorded annually between 10,000 and 15,000 new IE cases (IE) (8). Currently, in Romania its incidence is still high, with an average of 3.4 cases per 100,000 persons/year in the past decade (2, 7). Today, the most affected age group is years. Unfortunately, the young adult age group (20-35 years) is increasingly affected (25-30%), taking into account the immunocompromised patients (especially AIDS) and the increasing number of intravenous drug and more recently of intravenous ethnobotanical users (6, 7). Ten percent of all IE cases are recorded in the pediatric population aged less than 10 years (11). In Romania, the male predominance among IE patients is a common finding of recent studies, the male/female ratio being approximately :1 (6). Overall mortality rate ranges between 22 to 24% compared to 40% in the underdeveloped areas. In countries with high economic growth, mortality is below 14-15% (7, 14). III. Etiopathogenesis 3

6 The great diversity of involved etiologic agents varies according to the type of affected valve (native or prosthetic) and infection type (nosocomial or community acquired). Most infective endocardites (>80%) are caused by: viridans streptococci (30-40%), Enterococcus species (5-18%), Staphylococcus spp (10-27%), Escherichia coli (1.5-13%), Pseudomonas aeruginosa, Klebsiella pneumoniae and the HACEK group gram-negative bacteria responsible for 1-4% of endocardites. Bartonella spp accounts for 3% of the etiologic agents of endocarditis. Candida and other fungi cause <10% of endocarditis cases, especially in intravenous drug users (4, 7, 15, 16). The risk factors for infective endocarditis in studies conducted over the past four decades were represented mainly by degenerative valvular lesions and cardiac surgery resulting in prosthetic valve or not, but over time some changes occurred being related to increasing intravenous drug use, intravascular prostheses, nosocomial exposure, hemodialysis and agerelated valvular sclerosis, much more important than rheumatic heart diseases, especially in developed countries (18). Also, beauty dermatological interventions, or at musculoskeletal level may be followed by unwanted endocardial infections in the absence of antibioprophylaxis (6). Another risky procedure, this time non-medical, is body piercing and tattoos, particularly tongue piercing (19). For all these, sterile conditions are recommended. (20, 21) The pathogenesis of endocarditis involves a complex sequencing and a series of converging events (37). The endothelial injuries caused by the turbulent blood flow in congenital or acquired heart disease cause the deposition of fibrin and platelets and subsequently lead to the formation of non-bacterial thrombotic endocarditis (6). IV. Clinical picture of infective endocarditis The clinical picture depends on the epidemiological profile, living conditions, absence or presence of a history of heart diseases or surgery followed by implantation of prosthetic material. In 90% of infective endocarditis cases fever is an important symptom and often is accompanied by chills, decreased appetite, and weight loss. A febrile syndrome lasting more than 21 days in a patient with heart disease requires that the suspected diagnosis of infective endocarditis to be confirmed or refuted (6). Afebrile cases are most common among elderly, cachectic, immunosuppressed, in cases of severe renal insufficiency or severe heart failure. As to the specific clinical signs, top list are the heart murmurs present in 85-90% of the patients with endocardial disease. Heart murmurs are absent only in onset infective endocarditis, right heart endocarditis, or parietal endocardial infections (15). New regurgitation murmurs or changes in preexisting heart murmurs are the result of heart valves changes caused by infection. V. The diagnosis of infective endocarditis Currently, the best criteria for the diagnosis of infective endocarditis are the Durack/Duke criteria. Bacteriological diagnosis identifies the etiologic agent in blood cultures (three samples), 4

7 and transthoracic and/or transesophageal echocardiography in dynamic allow confirmation of valve changes (28, 42). According to current guidelines, the diagnosis of infective endocarditis is made when endocardial microbial infection is demonstrated. In unclear cases, the diagnosis may be based on Duke criteria (44, 45). V. 1 Bacteriological diagnosis blood cultures Blood cultures are accepted as the gold standard for pathogen identification and quantitative susceptibility testing. However, in 31% of suspected endocarditis cases, blood cultures remain negative, as a result of prior antibiotic treatment, slow-growing, fast-growing or difficult to culture microorganisms, and the small amount of microorganisms per volume (46, 47, and 48). V. 2 Diagnostic imaging - echocardiography Transthoracic and transesophageal echocardiography (TTE/TEE) are imaging methods highly important in the diagnosis and management of infective endocarditis. The presence of three echocardiographic appearances determines the major diagnostic criteria: the presence of vegetations, intracardiac abscesses, new dehiscences of prosthetic valve (6). The sensitivity of TTE is between 40-63%; TEE is more sensitive that TTE, some studies reporting it to be about 100% (90-100%) (6, 56). V. 3 Other modern techniques for bacterial detection With the introduction of molecular biology and serology techniques in the diagnosis of IE, it is now possible to identify the etiologic agent in cases initially of unspecified etiology, fact that subsequently lead to the administration of optimal therapy, thereby improving the quality of care (62). PCR (polymerase chain reaction) allows rapid detection of germs difficult to diagnose by conventional means and identification of the genes involved in resistance to antibiotics. Currently, more and more authors claim the introduction of PCR in the main diagnostic criteria. The great advantage of the technique lies in the fact that PCR allows the diagnosis of some infection with the detection of bacterial and viral particles even when their number is small, even for species that are difficult to cultivate in vitro, or for highly antigenically variable pathogens with a long latency period (64). Another rapid bacterial detection method is flow cytometry by BactiFlow system that uses small aliquots of each solution and initiates an automatic fluorescence cell sorting analysis - FACS. BactiFlow is an affordable compact microbial analyzer, which provides real-time testing for non-filtered and filtered microbial samples. The results are provided in minutes to hours rather than days, creating a true competitive advantage (67, 68, and 69). 5

8 Fig. 7. BactiFlow system Fig. 8. PLEX ID identification of bacterial DNA PLEX - ID is a new revolutionary way to detect and identify different groups of bacteria as single microorganism or combination of organisms from isolates or directly from samples by nucleic acid amplification and subsequent base composition analysis (72). Generally, microbial screening takes six to eight hours since sampling to the result depending on extracted amount and rapidity of required amplification. Fig. 9. Bacterial detection and identification by PLEX ID PLEX-ID is the method for the detection and identification of bacteria and Candida species as well as of antibiotic resistance markers by nucleic acid amplification and subsequent mass spectrometry analysis. It is able to identify Staphylococcus spp, Bacillus spp, Gramnegative bacilli, etc (74). VI. Prognosis of infective endocarditis Without adequate treatment, infective endocarditis is often fatal, with a mortality rate of hospitalized patients ranging from 9.6 to 26%, with considerable patient-to-patient differences (75). Given the recent major changes in epidemiology and bacteriological profile with strains resistant to different antibiotics, the introduction of Carmeli score, especially in immunocompromised patients or with multiple severe comorbidities was a step forward (81, 82). Carmeli score, used to select the appropriate antibiotic therapy, introduces a new term - healthcare-associated infection, in addition to the two classic infection types: community and 6

9 nosocomial. This score uses three parameters: contact with the health system, history of antibiotic treatment, and patient characteristics, taking into account the highest score obtained (83). Since many severe endocarditis cases are monitored in cardiac surgery and intensive care units, useful could also be the APACHE II classification system used to assess the severity of the disease, and consequently the prognosis of patients diagnosed with infective endocarditis and the recommended procedures and therapy. VII. Treatment and follow-up of infectious endocarditis A correct treatment of this condition involves controlling the septic process and improving the noninfectious manifestations and/or sequels by antibiotic therapy and conservative pathogenic therapy aimed at combating the adverse effects of antibiotic therapy and with an adjuvant role. VII. 1 Etiologic therapy Antibiotic therapy is administered to sterilize the infective vegetations and other concomitant infection foci, and its optimization involves two stages: making the diagnosis, therapeutic options, administration rate and used dose, the duration of treatment being often longer than in other infections with the same germs due to the difficulty in sterilizing the vegetations (2, 7). VII. 2 Pathogen-specific therapy Pathogen-specific therapy should be guided by five major direction lines: 1. arrhythmias and hemodynamic decompensation, 2.anticoagulant treatment 3. antiplatelet therapy 4. fibrinolotic treatment 5. stimulating factors enabling correction of haematological disorders (2, 60). VII. 3 The role of cardiologist in infective endocarditis follow-up Heart medication is always administered in patients with infective endocarditis, and more than that, it is mandatory when cardiac complications do occurs. The main role of the cardiologist is to follow up the disease course, and in case of worsening of hemodynamic status to contact in due time the infectionist or surgeon. VII. 4 Role of cardiac surgery in the treatment and prognosis of infective endocarditis The surgical treatment of infective endocarditis is indicated in case of heart failure 7

10 NYHA class III-IV due to valvular dysfunction, myocardial or perivalvulare abscesses with no tendency to heal, mycotic aneurysm, purulent pericarditis, ruptured Valsalva sinus or ventricular septum, fungal infection (7). Personal contribution VIII. Importance of the topic. Objectives Nowadays, a rapid and accurate diagnosis of bacterial diseases (severe sepsis, infective endocarditis), and especially the identification of the etiologic agent are essential for instituting a targeted therapeutic scheme. In this respect, when faced with a patient suspected of having an infection, a good knowledge and availability of the laboratory investigation methods able to establish in the shortest time what kind of bacteria is involved, and then choose the appropriate antibiotic treatment are the key elements for success. Endocarditis with negative blood cultures is a challenge for the clinician. In particular, when faced with bacteria difficult to cultivate (such as Coxiella burnetii, Bartonela spp, Brucella melitensis, or Legionella pneumophila) it is more difficult to obtain positive results in bacterial identification. The following describes the main goals of the research carried out in four studies. The specific objectives of each study are presented below. Given that infective endocarditis remains a problem to consider at both adulthood and at an early age and that the involved factors are becoming more and more common, the deep insight into this disease was aimed at presenting the frequently encountered clinical and biological features of this disease essential for a rapid therapeutic and prophylactic intervention. The major need of using specific methods for the diagnosis and identification of bacteria involved in the etiology of infective endocarditis motivated us to compare the different methods for bacterial detection in terms of effectiveness, rapidity and specificity. Also approached are the molecular biology methods (PLEX ID), which greatly improved the sensitivity of bacteriological diagnosis, given the increasing variety of DNA types and primers for gene amplification (PCR). The goal was to establish and validate a diagnostic method based on Real-Time PCR for bacterial detection in various human platelet concentrate samples using generic primers for the 16S RNA region of bacterial genome. At the same time, we aimed at comparing the results of PCR method with a flow cytometry detection system based on the so-called digital flow cytometry - BactiFlow system as a method for bacterial detection. The main goal of all these was the possibility of establishing a first-line antibiotic therapy in the absence of a positive infection and antibiotic history with direct impact on the course and prognosis of this condition. IX. Materials and method IX.1 Study 1 - retrospective study of infective endocarditis cases recorded in the interval The study group included 70 patients admitted to the Iasi "Sf. Parascheva Infectious 8

11 Diseases Hospital in the interval diagnosed with infective endocarditis according to Duke criteria. The study protocol included the following data retrospectively analyzed: epidemiological features, clinical and evolutive features, positive diagnosis, treatment, course and prognosis. IX.2 Study 2 - prospective study of the patients with endocardial involvement during severe sepsis recorded in the interval We conducted a prospective study on a series of subjects suspected of sepsis with endocardial involvement. The study group consisted of 37 patients admitted to the Iasi Sf. Parascheva Infectious Diseases Hospital in the interval January 1, 2011 December 31, 2012 for making a definite diagnosis and specialized treatment. The study protocol included the following data: epidemiological features, clinical and evolutive features, positive diagnosis (according to Durack-Duke diagnostic and classification criteria), treatment, course, and prognosis. The patients included in the study were informed about the nature of the study and asked to sign the informed consent approved by the Research Ethics Committee. IX.3 Study 3 - prospective experimental study on the use of molecular biology methods for detection of bacterial DNA This prospective experimental study was conducted in the interval June September 2011 in the Department of Bacteriology of the Institute for Transfusion Medicine and Immunohematology Frankfurt, Germany, where we used methods of molecular biology, polymerase chain reaction (PCR) and digital flow cytometry (BactiFlow system) for the detection of bacteria and bacterial DNA in human platelet concentrate samples from apparently healthy anonymous donors to which to different types of bacterial strains were inoculated. Bacterial growth on plate was used for comparison IX.4 Study 4 - prospective study on the therapeutic efficacy of the use of PLEX ID - method for the detection of bacterial DNA, in patients with infective endocarditis The study included the post-antibiotic therapy testing of 13 patients diagnosed with infective endocarditis at the Bucharest "Prof. Dr. Matei Bals" National Institute for Infectious Diseases in 2012, in which the etiologic agent was identified by blood cultures, and at the end of therapy PLEX ID (Ibis Biosciences, Abbott Laboratories, Illinois, USA) for bacterial DNA detection of the previously isolated microorganism was performed. The protocol was followed as recommended by the manufacturer. X. Results X.1 - Retrospective study of infective endocarditis cases recorded in the 9

12 no of cases interval X.1.1 Demographic and epidemiological data The distribution of the study cases showed an increasing trend in the number of patients with infective endocarditis (y = 7.8 x - 2 in 2013 approximately 21 new cases being estimated y = 7,8x - 2 R 2 = 0, years Fig. 12. Year-distribution of infective endocarditis cases TABLE XXXV Risk-related profile of endocarditis patients Parameter Risk factors P cardiac Extracardiac Male 91,8% 40,4% 0,001 Age over 60 years 27,9% 14,9% 0,169 Urban 68,9% 42,6% 0,011 Cardiovascular family 37,7% 19,1% 0,043 history Nosocomial infections 47,5% 27,7% 0,036 X.1.2 Clinical and evolutive features Onset symptoms TABLE XXXVIII Signs of endocarditis according to infection source in study 1 patients Signs COMMUNITY (n=41) NOSOCOMIAL (n=29) Statistical significance RR IC95% N % N % χ 2 P Murmurs 6 14, ,6 12,97 0,001 2,89 1,68 4,99 10

13 Neurological 7 17,1 4 13,8 0,01 0,970 abnormalities 1,10 0,67 1,82 Embolic events 6 14,6 5 17,2 0,01 0,970 1,12 0,55 2,29 Eye disease 4 9,8 4 13,8 0,02 0,887 1,24 0,58 2,64 Hippocratic fingers 13 31, ,8 0,75 0,385 1,38 0,79 2,38 Hepato/splenomegaly 10 24, ,9 0,91 0,341 1,43 0,82 2,47 Roth spot 5 12,2 2 6,9 0,10 0,746 1,25 0,75 2,09 Janeway lesions 4 9,8 3 10,3 0,10 0,746 1,04 0,42 2,57 Osler nodules 3 7,3 3 10,3 0,10 0,990 1,23 0,52 2,89 Onycomycosis 13 31, ,8 0,75 0,385 1,38 0,79 2,38 Petechiae 4 9,8 6 20,7 0,89 0,347 1,57 0,86 2,85 Gate of entry In the study cases the most common entry gate was surgical (30%), followed by the urinary (20%) and dental (20%) ones; in 11.4% of the cases the gate of entry could be determined. X.1.3 Positive diagnosis of infective endocarditis Bacteriologic investigations In the study 1 patients positive blood cultures were recorded in 28 cases (40%). The number of detected strains was 28, which is one strain per patient. Etiologic agent TABLE XLV Etiologic picture in the study 1 patients with infective endocarditis % of total No strains strains % of total patients Total strains 28 Gram pozitive 25 89,3 35,7 Staphylococcus spp 9 32,1 12,9 Staphylococcus aureus 8 28,6 11,4 Staphylococcus haemoliticus 1 3,6 1,4 Streptococcus spp 8 28,6 11,4 Streptococcus bovis 1 3,6 1,4 Streptococcus mutis 1 3,6 1,4 Streptococcus mutans 1 3,6 1,4 Other streptococcus viridans 5 17,9 7,1 Enterococcus spp 8 28,6 11,4 Enterococcus faecalis 8 28,6 11,4 Gram negative 3 10,7 4,3 Achromobacter spp 1 3,6 1,4 Escherichia coli 1 3,6 1,4 Klebsiella ozaenae 1 3,6 1,4 Sensitivity of the etiologic agent involved in the etiology of infective endocarditis 11

14 The highest sensitivity to antibiotics of the etiologic agents was to tienam (100%), ertapenem (100%), vancomycin (95%), and teicoplanin (85%). Marked resistance was found to ceftazidime is noted marked (75%), ceftriaxone (60%), penicillin (60%), oxacillin (60%), chloramphenicol (60%) and co-trimoxazole (50%). Imaging In our study 1 cases vegetations over 10 mm in size were identified echocardiographically in 34.3% of the patients. In only one patient the echocardiographic findings were normal (1.4%) and in another patient echocardiography was not performed (1.4%). veg etations <10 mm 62,9% normal 1,4% not made 1,4% veg etations >10 mm 34,3% Fig. 36. Structure of study 1 patients according to echocardiographic findings 60 % vegetative lesions localization implant/prosthesis/pacemaker 24,3 pulmonary valve 0 tricuspid valve 5,7 aortic valve 30 mitral valve 51,4 Fig. 37. Distribution of vegetative lesions according to location - Study 1 X.1.4 Prognostic considerations in the study group Heart failure of different NYHA classes is the main complication, being present in about 35% of the study 1 patients. 34,3 % ,3 Heart failure Pericarditis Thromboembolis m 12

15 Fig. 38. Percentage of complications associated with endocarditis in study 1 subjects CARMELI SCORE Carmeli score-related characteristics of study 1 patients revealed a high risk for nosocomial infection in males (89.7%) aged about 54 years, residing in urban areas (62.1%), with prior antibiotic therapy (62.1%), positive blood cultures in 48.3% and surgical entry gate (58.6%). The average hospital stay was approximately 2.5 weeks (19 days), and presentation to the doctor was late (13 days). APACHE II SCORE APACHE II scores ranged between 7 and 20, with a mean value suggesting likely recovery highlighting permissive values. Individual values of APACHE II score correlated directly with patient age (r = 0.34), being significantly higher in elderly (p = 0.037). Apache II score was significantly higher in patients with surgical (17.63) and urinary entry (15, 67) while the lowest APACHE II score in patients in which the gate of entry was dental (10. 50) (p = 0.001). TABLE LXXXV Statistical indicators of APACHE II score by positive blood cultures Blood cultures N Average Std Deviation Std Error Confidence Interval Min Max p - 95%CI +95%CI Negative 17 13,59 2,830,686 12,13 15, Pozitive 20 15,05 4,097,916 13,13 16, Total 37 14,38 3,601,592 13,18 15, In our study 1 patients, the favorable outcome was the most common (42.9%), lethality being recorded in 5.7% and a poor prognosis in 10% of the patients investigated. Referred to cardiovascular surgery units were 41.4% of the endocarditis patients. X.1.5 Therapeutic considerations in the study patients Most commonly, the treatment of first choice was a combination of two antibiotics, of which aminopenicillin and aminoglycoside in 24.3% of the cases, the last generation treatment being used as first choice treatment in 18.6% of the all study l patients. TABLE LI Therapeutic schemes Antibiotiotics classes commonly used in the study patients Cephalosporins 3 rd generation + flouroquinolone/aminoglycosides Aminopenicilin/aminoglycosides + fluoroquinolone Oxazolidinone/carbapenems/lincosamides + quinolone Glycopeptide + fluoroquinolone/polymyxins 0,223 13

16 X.2 Study 2 prospective study of the patients with endocardial involvement during severe sepsis recorded in the interval X.2.1 Epidemiological characteristics Distribution/annual incidence In the study group, a slightly higher annual distribution of endocarditis cases was recorded in 2011 (52.8%) as compared with 2012 (47.2%) % % Fig. 40. Annual distribution of study 2 endocarditis patients TABLE LVIX Risk-factor related profile of endocarditis patient Parameter Risk factors P Cardiac Extracardiac Male 78,3% 82,6% 0,713 Age over 60 years 83,3% 85,0% 0,761 Rural 75,0% 85,0% 0,693 Cardiovascular family 100,0% 50,0% 0,046 history Nosocomial infections 95,7% 69,6% 0,021 X.2.2 Diseases associated with infective endocarditis Secondary cardiovascular involvement All patients in study 2 presented secondary cardiovascular involvement. 14

17 Total Male 60 years Rural with ATB positive blood culture Total Masculin 60 years Rural with ATB Positive blood culture % Aortic Bicuspid Arterial hypertension Pulmonary hypertension Ao stenosis PI MI TI AoI cronic heart failure Fig. 47. Series structure according to secondary cardiovascular events Gate of entry 40 % 20 Urinary Dental Cutaneous 0 surgicaly procedure Fig. 49. Series structure according to the gate of entry X.2.3 Clinical and evolutive features TABELUL LXVI 15

18 Epidemiological features of patients according to admission time EARLY LATE HOSPITALISATION HOSPITALISATION (n=12) (n=25) EPIDEMIOLOGICAL DATA NR. % NR. % GENDER Male 8 66, ,0 Female 4 33, ,0 AGE Average ± SD 60,75±18,25 57,48±13,80 BACKGROUNDS Urban 4 33, ,0 Rural 8 66, ,0 CARDIOVASCULAR COMORBIDITY Prezent 10 83, ,0 Absent 2 16,7 8 32,0 PREVIOUS ANTIBIOTICS TREATMENT With 6 50, ,0 Without 6 50,0 8 32,0 STATISTICAL SIGNIFICANCE 2 =0,15; GL=1 p=0,699 t=0,37; GL=35 p=0,548 2 =0,51; GL=1 p=0,475 2 =0,35; GL=1 p=0,557 2 =0,48; GL=1 p=0,487 Signs Murmurs New Modified Neurological abnormalities TABELUL LXVIII Signs of endocarditis in relation with infection source COMMUNITY NOSOCOMIAL Statistical RR (n=14) (n=23) significance N % N % χ 2 P IC95% 11 78, ,5 0,15 0,700 1,35 1,32 5, ,3 9 39, ,4 5,26 0, Embolic events 1 7,1 5 21,7 0,50 0,479 3,04 0,40 23,45 Eye disease 2 14,3 2 8,7 0,27 0,600 1,64 0,26 10,39 Hippocratic fingers 9 64,3 8 34,8 1,98 0,160 1,85 0,93 3,66 Hepato/splenomegaly 7 50, ,9 1,26 0,262 1,48 0,83 2,63 Roth spot 3 21,4 1 4,3 1,16 0,282 4,93 0,57 42,89 Janeway lesions 4 28,6 5 21,7 0,01 0,940 1,31 0,42 4,09 Osler nodules 2 14,3 2 8,7 0,27 0,600 1,64 0,26 10,39 Onycomycosis 8 57, ,5 0,22 0,640 1,31 0,69 2,52 Petechiae 5 35, ,8 0,14 0,705 1,34 0,59 3,05 X.2.4 Positive diagnosis of infective endocarditis Basic hematological and biochemical investigations Leukogram mainly revealed leukocytosis with increased polymorphonuclear (PMN) 16

19 counts. Bacteriological investigations In our study cases, positive blood cultures were recorded in 20 patients (54.1%). The number of detected strain was 22, which is an average of 1.1 strains per patient. The etiologic agent most commonly involved was Staphylococcus aureus: 27.3% of all strains and identified in 16.2% of patients, followed by Enterococcus faecalis: 18.2% of all strains and identified in 10.8% of the study subjects. It is worth mentioning that the involved gram negative bacteria were Klebsiella pneumoniae and Serratia marcescens % of all isolated strains, and identified in 10.8% of the investigated patients. Sensitivity of the involved etiological agent The highest sensitivity to antibiotics of the etiologic agents was to tienam (90%), levofloxacin (90%), colistin (85%), linezoid (85%), gentamicin (85%), teicoplanin (85%) and ertapenem (80%). Marked resistance was found to ceftazidime (75%), ceftriaxone (60%), penicillin (60%), oxacillin (50%) and co-trimoxazole (50%). TABLE LXXVI Percentage of positive blood cultures in study 2 patients of all etiologic agents identified in the laboratory in the interval Identified etiologic agent in the laboratory Pozitive blood cultures 2011 Pozitive blood cultures 2012 Number Study 2 % Number Study 2 % Staphylococcus aureus , ,4 Other coagulase negative staphylococcus Staphylococcus epidermidis , Staphylococcus haemolyticus Staphylococcus hominis Salmonella Neisseria Streptococcus pneumoniae Listeria Bacillus cereus ,2 Klebsiella pneumoniae , ,0 Escherichia coli Gemella Enterobacteriacee Pseudomonas aeruginosa Streptococcus alpha haemolytic ,0 Corinebacterii Proteus mirabilis Enterococcus spp , ,2 Acinetobacter baumannii Streptococcus pyogenes , Streptococcus faecalis Streptococcus galoliticus

20 Candida Serratia marcescens Imaging Vegetations over 10 mm in size were found echocardiographically in 43.2% of the patients. In only one patient echocardiographic findings were normal (2.7%) and in 3 patients echocardiography was not performed (8.1%). Echocardiographic location of vegetative lesions Vegetative injuries were typically found on the mitral valve (51.5%) and aortic valve (36.4%). Vegetations on implant/prosthesis/pacemaker were identified echocardiographically in 9.1% of all vegetative lesions. In 4 patients (10.8%) multiple vegetative lesions were found: two on the mitral and aortic valves, one on the tricuspid and pulmonary valves, and one on the mitral, aortic and pulmonary valves. Fig. 68. Mitral valve vegetation Fig. 69. Aortic valve vegetation 18

21 60 % vegetative lesions localization implant/prosthesis/pacemaker 9,1 pulmonary valve 6,1 tricuspid valve 15,2 aortic valve 36,4 mitral valve 51,5 Fig. 70. Distribution of vegetative lesions according to location X.2.5 Prognostic considerations in the study series Complications Heart failure accounted for about 57% of the complications found in our patients with infective endocarditis. 56,8 % ,6 8,1 Heart failure Pericarditis Thromboembolis m Fig. 73. Percentage of complications associated with endocarditis in study 2 patients CARMELI SCORE Carmeli score-related characteristics of study 2 patients revealed a high risk for nosocomial infection in males (56.5%), aged about 60 years, residing in rural areas (56.5.1%), with prior antibiotic therapy (62.1%), positive blood cultures in 47.8% and cutaneous entry gate (58.6%). The average hospital stay exceeded 3 weeks (20.61days), and presentation to the doctor was late (16.87 days). 19

22 age age APACHE II SCORE In study 2 patients, the favorable outcome was the most common (40.5%), lethality being recorded in 5.4% and a poor prognosis in 13.5% of the investigated patients. Referral of endocarditis patients for cardiological examination was of 21.6% and to cardiovascular surgery units of 18.9%. X.2.6 Therapeutic consideration Most commonly, the treatment of first choice was a combination of two antibiotics: aminopenicillin and aminoglycoside (35.1%), and last-generation treatment was used as first-line therapy in 18.1% of study 2 patients. TABLE LXXXVIII Therapeutic schemes Antibiotics classes commonly used in the study patients Glycopeptide + fluoroquinolone/polymyxins Aminopenicilin/aminoglycosides + fluoroquinolone Cephalosporins 3rd generation + flouroquinolon/aminoglycosides Oxazolidinone + quinolone Carbapenems + quinolone Lincosamides + quinolone X.3 Comparative discussions on the retrospective and prospective study of patients with infective endocarditis (Study 1 and Study 2, respectively) Compared to the retrospective study it was noticed that the average age of female patients in the prospective study was significantly higher (p <0.05), the same not being true for males (p> 0.05) ,77 52, ,74 63, Male Female Male Female study 1 study 2 Fig. 83. Comparative sex distribution of average age of endocarditis patients in the two studies Frequency distribution of subjects in the prospective study was predominantly rural (54.1%) 20

23 while in the retrospective study more patients came from urban areas (60%). urban 60,0% s tudy 1 urban 45,9% s tudy 2 rural 40,0% rural 54,1% Fig. 84. Comparative rural/urban distribution of endocarditis patients in the two studies Positive diagnosis There were no significant differences in the mean levels of hematologic/biochemical parameters between the two studies. TABLE XCVII Comparative statistical differences in the mean levels of hematologic and biochemical parameters between the two studies Parameter Study 1 Study 2 P White blood cells (/mmc) - admission - hospitalization - discharge ± ± ± ± ± ± 4785 >0,05 >0,05 >0,05 PMN (%) 68,99 ± 13,54 69,31 ± 16,46 >0,05 Lymfocites (%) 19,41 ± 9,99 18,79 ± 13,88 >0,05 ESR (mm/1h) - admission - discharge Fibrinogen (g/l) - admission - discharge C-reactive protein (mg/l) - admission - discharge 76,97 ± 38,32 49,44 ± 33,40 5,03 ± 1,61 4,46 ± 1,57 61, 01 ± 26,05 30,64 ± 22,82 78,68 ± 40,61 43,21 ± 28,06 4,62 ± 1,86 3,55 ± 1,02 63,92 ± 29,53 21,12 ± 16,24 >0,05 >0,05 >0,05 >0,05 >0,05 >0,05 Bacteriological investigations Frequency distribution of positive strains according to the etiological agent showed no significant differences between the two studies (p=0.378). 21

24 ,1 36,4 28,6 18,2 28,6 18,2 9,1 Study 1 Study 2 18,2 0 S taphylococc S treptococcu us spp E nterococcus s spp spp 0 B acillus cereus 10,7 B G N Fig. 87. Comparative distribution of strains according to the etiologic agent between the two studies In study 1, the 28 positive strains accounted for 9.6% of all strains isolated by blood cultures in the cases of sepsis reported in the interval , and in study 2, the 22 positive strains accounted for 10.9% of all isolated strains, frequency distributions without statistical significance (p>0.05) TABLE XCVIII Etiological picture in endocarditis patients versus all sepsis cases with positive blood cultures reported during the study interval Etiologic agent Total Positive blood cultures sepsis cases % of total Positive Positive blood Study 1 blood cultures Study 2 cultures sepsis sepsis cases cases % of total Positive blood cultures sepsis cases Gram pozitive , ,0 Staphylococcus spp , ,1 Streptococcus spp , ,2 Gram negative , ,2 E. coli , Total , ,9 Diagnostic imaging TABLE XCIX Visualization of vegetations over 10 mm in size according to echocardiography modes Transthoracic STUDY 1 STUDY 2 Echocardiography N % n % Vegetations >10 mm 23 95,8% % Emboligen risk 12 50,0% % 22

25 Cardiac insufficiency was significantly more often associated with endocarditis in study 2 patients (56.8% vs 34.3%). Over 20% of study 2 patients also presented pericarditis as compared with 10% in study 1 patients, inducing a twofold higher relative risk of endocarditis. Thromboembolism was present in 8.1% of study 2 patients, the relative risk being 1.89 times higher than in study 1 patients associating the same complication. Patients in the two studies had a similar favorable course, but study 2 patients were referred earlier for cardiologic examination that to cardiovascular surgery units. In study 1 patients the most frequently administered therapeutic scheme was aminopenicilin/aminoglycoside + fluoroquinolone (22.9%), while in study 2 patients the same scheme was also most frequently administered (35.1%) followed by 3 rd generation cephalosporin + fluoroquionolone/aminoglycoside in 32.4% of the subjects. Study Glycopeptide+fluoroquinolone/ polymyxine Aminopenicilin/aminoglycozides + fluoroquinolone Cephalosporins 3rd generation + fluoroquinolone/aminoglycozide Oxazolidinone + quinolone Study Carbapenems + quinolone Lincosamides + quinolone 0% 20% 40% 60% 80% 100% Fig. 89. Patients distribution according to therapeutic scheme In study 2 patients, 3 rd generation cephalospirins, aminoglicosides and antifungals were more frequently used. X.4 Study 3 - prospective experimental study on the use of molecular biology methods for detection of bacterial DNA This study dealt with bacterial detection and bacterial DNA identification in human platelet concentrate samples inoculated with 6 bacterial strains by molecular biology methods in view of determining their rapidity of identification. The first experiment day was day 0. We used 16 samples of mixed platelet concentrate obtained from apparently health human subjects containing at least 2x10 11 platelets/bag. On day 0 we cultivated on blood-agar plates 500 µl of each platelet concentrate. Twenty-four hours later the results were negative (no grown on any plate). TABLE CIII Day 0 before bacterial strains inoculation Bacterial strains Number of sample Plating CFU/ml BactiFlow ALS counts/ml PCR (Ct) 23

26 Bacillus cereus 1 -/0 Not done 0 2 -/0 Not done 0 Klebsiella pneumoniae 1 -/0 Not done 0 2 -/0 Not done 0 Serratia marcescens 1 -/0 Not done 0 2 -/0 Not done 0 Staphylococcus aureus 1 -/0 Not done 0 2 -/0 Not done 0 Staphylococcus epidermidis 1 -/0 Not done 0 2 -/0 Not done 0 Streptococcus pyogenes 1 -/0 Not done 0 2 -/0 Not done 0 Positive control 1 24,57 Also, we initiated a PCR using Klebsiella pneumoniae as positive control, and the results were again negative for our platelet concentrate samples. Bacterial detection by BactiFlow system was not initiated at this point of the experiment because samples negativity was evident. On the same day 0, we inoculated in 12 bags different UFC/ml platelet concentrate sample, obtaining in the end the same per bag concentration of the 6 bacterial strains: Bacillus cereus, Klebsiella pneumoniae, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes. The 3 platelet concentrate samples not inoculated with bacterial strains served as negative control. On day 1, following bacterial strains inoculation, we obtained on the blood-agar culture plate positive results for all inoculated samples, except for one Streptococcus pyogenes sample with no colony growth. We noticed that one Klebsiella pneumoniae and one Staphylococcus epidermidis sample presented a slower colony growth on the plate (264 UFC/ml and 1000 UFC/ml) compared to the other plates where colonies covered almost the entire plate surface. The negative control samples remained negative. Bacterial detection was done using BactiFlow system which showed the number of detected bacteria per ml. On day 1 after inoculation, one of the Streptococcus pyogenes sample remained negative (0 det./ml), all the other inoclutaed samples being positive (> 500 det./ml). Negative control remained negative for BactiFlow system (0< 500 det./ml). PCR results showed that bacterial DNA was not detected in a sample inoculated with Klebsiella pneumoniae and one with Streptococcus pyogenes.negative controls remained negative ((PCR Ct=undetermined/0). TABLE CV Day 1 following bacterial inoculation Bacterial strains Number of sample Plating CFU/ml BactiFlow ALS counts/ml PCR (Ct) Bacillus cereus 1 +/Full +/ ,17 2 +/Full +/ ,00 Klebsiella pneumoniae 1 +/264 +/ /Full +/ ,13 Serratia marcescens 1 +/Full +/ ,46 2 +/Full +/ ,97 24

27 Staphylococcus aureus 1 +/Full +/ ,89 2 +/Full +/ ,21 Staphylococcus epidermidis 1 +/ / ,60 2 +/Full +/ ,01 Streptococcus pyogenes 1 -/0 -/ /Full +/ ,94 1 -/0 -/40 0 Negative control 2 -/0 -/ /0 -/0 0 Positive control 1 25,05 On day 2, all methods used on day 1 were repeated. For the cultivation on plates with blood-agar growth medium of the platelet concentrate samples inoculated with the 6 bacterial strains, all samples were positive, except for the same sample with Streptococcus pyogenes on which no colony grown was seen. With the BactiFlow method, an increase in the number of detected bacteria for every tested sample, except for the same Streptococcus pyogenes sample which remained negative (119 det./ml). In one negative control sample an increase in the detected number was recorded (1268 det./ml). PCR results were positive for all inoculated samples, but remained negative for the same Streptococcus pyogenes sample, as it happened the previous day (day 1). Negative controls remained negative, without an increased fluorescent signal. On experiment day 3, the sample inoculated with Streptococcus pyogenes remained negative. All the other inoculated samples were positive, and negative controls stayed negative (with no growth). Bacterial detection by BactiFlow showed negative results for one of the samples inoculated with Streptococcus pyogenes (476 det./ml), and a slight increase in the number of detected bacteria for the second sample also inoculated with Streptococcus pyogenes. In one of the negative control samples an increase in detected number was noticed. PCR results were similar with those recorded the previous day: positive for the inoculated samples, but negative for the same sample inoculated with Streptococcus pyogenes. Negative controls remained negative and no amplification curve was described. Bacterial strains TABLE CVIII Day 6 after bacterial inoculation Number Plating BactiFlow ALS of samples CFU/ml counts/ml PCR(CT) Bacillus cereus 1 +/Full +/ ,25 2 +/Full +/ ,91 Klebsiella pneumoniae 1 +/Full +/ ,39 2 +/Full +/ ,49 Serratia marcescens 1 +/Full +/ ,67 2 +/Full +/ ,11 Staphylococcus aureus 1 +/Full +/ ,27 2 +/Full +/ ,00 25

28 Staphylococcus epidermidis 1 +/Full +/ ,41 2 +/Full +/ ,44 Streptococcus pyogenes 1 -/ /Full +/ ,07 1 -/0 -/ Negative control 2 -/0 -/ /0 -/79 0 Positive control 1 26,84 On the last experiment day, day 6, for the same sample inoculated with Streptococcus pyogenes no colony growth on the blood-agar growth medium was noticed. Negative controls remained negative, in agreement with PCR results describing no amplification curve for the sample inoculated with Streptococcus pyogenes. However, BactiFlow bacterial detection method showed an increase in the number of detected bacteria for the sample inoculated with Streptococcus pyogenes, which remained negative the previous experiment days. Fig. 92. BactiFlow results on experiment day 3 26

29 delta RN delta RN 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0-0,1-0, CT Fig. 94. PCR results on day 0 before bacterial inoculation positive control Amplification curve (red) represents the positive control 1,4 1,2 1 0,8 0,6 0,4 0,2 0-0,2-0, Fig. 95. PCR results amplification curves on day 1 following bacterial strain inoculation Caption of amplification curves on colors: DNA expression of Serratia marcescens in the inoculated platelet concentrate DNA expression a Bacillus cereus in the inoculated platelet concentrate DNA expression a Klebsiella pneumoniae in the inoculated platelet concentrate DNA expression a Staphylococcus epidermidis in the inoculated platelet concentrate DNA expression a Staphylococcus aureus in the inoculated platelet concentrate DNA expression a Streptococcus pyogenes in the inoculated platelet concentrate DNA expression of positive control Amplification curve negative control (aqua). Negative control: platelet concentrates (without bacterial strain inoculation). CT 27

30 CT delta RN 1,4 1,2 1 0,8 0,6 0,4 0,2 0-0,2-0, Fig. 98. PCR results amplification curves on day 6 after bacterial strain inoculation The end results of PCR analysis indicated the fact that except for Streptococcus pyogenes in the tested samples, all the other inoculated bacteria in the platelet concentrate samples were detected starting with day 2. CT days Bacillus cereus Bacillus cereus Klebsiella pneumoniae Klebsiella pneumoniae Serratia marcescens Serratia marcescens Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Staphylococcus epidermidis Streptococcus pyogenes Streptococcus pyogenes positive control Fig. 99. Final analysis of PCR results By BactiFlow method it was noticed that one of the samples inoculated with Streptococcus pyogenes Streptococcus pyogenes remained negative for bacterial detection since day 1 to the last experiment day. 28

31 CFU / ml But on experiment day 6 the results were considered negative despite the over 500 det./ml, a possible blood platelet clumping being suspicioned days Bacillus cereus Bacillus cereus Klebsiella pneumoniae Klebsiella pneumoniae Serratia marcescens Serratia marcescens Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Fig Final analysis of BactiFlow system Staphylococcus results epidermidis X.5 Study 4 - prospective study on the therapeutic efficacy of the use of PLEX ID - method for the detection of bacterial DNA, in patients with infective endocarditis Epidemiological features In 69.2% of the patients diagnosed with infective endocarditis in which following antibiotic therapy a test aimed at identifying traces of bacterial DNA by PLEX ID was performed were males, from urban areas, and with a mean age of 48 years. Upon admission most patients presented subfebrility. Mean white blood cell counts during hospital stay were 40% above the upper reference limit. ESR was high, between 4 and 140 mm/1h, average 71.1 mm/1h. The average of individual fibrinogen levels was above the upper reference limit. The mean levels of C reactive protein were very high (73 mg/l) TABLE CIX Descriptive data of patients with infective endocarditis tested by PLEX ID Characteristic Parameter Male 9 (69,2%) Age (years) 48,0 ± 22,2 (5 71) Urban 9 (69,2%) Fever on admission ( C) 37,2 ± 0,6 (36 38) Pathologic personal history - Chronic kidney disease - Diabetes - Anemia 4 (30,8%) 2 (15,4%) 7 (53,8%) 29