AND PCR. University of São Paulo, Bauru, Brazil 2 Graduate student, Stomatology Department, Bauru School of Dentistry,

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1 International Journal of Clinical Dentistry ISSN: Volume 7, Number 1 Nova Science Publishers, Inc. VIRAL OPPORTUNISTIC INFECTIONS IN ORGAN AND TISSUE TRANSPLANTED PATIENTS: COMPARISON BETWEEN CLINICAL EXAMINATION AND PCR Paulo Sérgio da Silva Santos, DDS, MSc, PhD 1, José Endrigo Tinoco-Araujo, MSc, PhDc 2, Ana Paula Bloise, DDS 3, and José Roberto Pereira Lauris, MSc, PhD 4 1 Professor, Stomatology Department, Bauru School of Dentistry, University of São Paulo, Bauru, Brazil 2 Graduate student, Stomatology Department, Bauru School of Dentistry, University of São Paulo, Bauru, Brazil 3 Paulista University, São Paulo, Brazil 4 Professor, Department of Paedodontics, Orthodontics and Public Health of Dentistry, Bauru School of Dentistry, University of São Paulo, Bauru, Brazil ABSTRACT Purpose: To evaluate correlation between clinical suspicion and laboratory diagnosis of viral opportunistic infections in transplanted patients. Methods: We assessed 29 patients with oral infections with a clinical aspect of viral lesions. We scraped the oral lesions, collected secretions using swab and sent samples to the molecular biology laboratory for PCR according to the clinical diagnostic hypotheses. Results: We found 44.8% of cases suspected of having HSV, 6.9% of CMV and in 48.3% there was doubts about the diagnosis. The PCR was positive for HSV in 34.5% of cases, for CMV in 6.9%, for both HSV and CMV in 10.3% and negative in 46.7% of the cases. The sensitivity of clinical examination was 100% for both HSV and CMV, but the possibility of having the infection by HSV or CMV was respectively 44% and 25%. We suggest that clinical examination is not sufficient to establish the final diagnosis of viral lesions in transplanted patients, making it necessary for exams of high reliability as the PCR. Corresponding author: Professor Paulo Sérgio da Silva Santos Faculdade de Odontologia de Bauru FOB/USP Departamento de Estomatologia, Al. Octávio Pinheiro Brizolla, 9-75, Bauru, SP, Brasil. CEP: Tel: paulosss@fob.usp.br

2 96 Paulo Sérgio da Silva Santos, José Endrigo Tinoco-Araujo, Ana Paula Bloise et al. INTRODUCTION Oral examination is methodical and requires that all evaluated areas point to the proper diagnosis of existing diseases. In this article we suggest that clinical examination alone is not enough for the diagnosis of opportunistic infections with oral manifestations and viral aspects in organ and tissue transplant recipients, making it necessary to perform complementary exams, with the request oriented by clinical examination and preferably molecular biology, for the beginning of appropriate antiviral therapy. The herpes simplex virus (HSV) and cytomegalovirus (CMV) are major causes of morbidity and mortality in transplant recipients, affecting 50 75% of these patients [1, 2]. HSV-1 is frequently reported in patients with compromised immunity. Differently from recurrences in immunocompetent patients, the oral lesions in immunocompromised patients are more extensive, aggressive, painful, and slow to heal. They are also important causes of morbidity and mortality after transplantation [2, 3]. There are few studies related to the identification of oral HSV and CMV infections by molecular biology in immunocompromised patients [4, 5]. In solid organs transplantation, CMV may lead acute rejection and chronic vascular disease, and is also associated with skin infections, esophagitis, chronic graft-versus-host disease after bone marrow transplantation (BMT), and pneumonia, which may result in opportunistic infection with severe morbidity and mortality [6, 7, 8]. HSV infection is less severe clinical problem in transplant patients, occasionally appearing as a reactivation of a latent infection but can lead to adverse conditions such as hepatitis, gingival overgrowth, periodontal disease, genital lesions, and painful ulcerative lesions on the edge of the tongue, lips, and mucosa [1, 9]. It is not uncommon to verify in renal transplanted patients oral lesions such as labial herpes caused by HSV-1 or HSV-2, hairy leukoplakia, nonspecific ulcers (CMV), and other injuries related to various types of HSV [10, 11]. Laboratory diagnosis by virus isolation is considered the gold standard. Polymerase chain reaction (PCR) and hybrid capture are new methods that can detect small quantities of virus genetic material more quickly and are approved by the literature as reliable methods for early antiviral treatment. PCR has been a useful tool for diagnosis, especially when conventional methods are flawed, such as lesions with reduced viral load, and immunosuppressed patients [3]. In order to compare the detection of HSV DNA by PCR and virus isolation in cell culture, Wald et al. [12] evaluated 36,000 samples of secretions from a group of 296 immunocompetent patients and found that the PCR test was the most efficient test for detecting infections, with positivity in 12.1%, demonstrating greater reliability of the PCR when compared with virus isolation (3% positive). The PCR test is relevant to the virus preservation when it is submitted to unfavorable temperature or transport conditions that may affect the quality of the sample [12]. The exfoliative cytology is an important test for the diagnosis of infections related to HSV. The exfoliative cytology and PCR tests are consistent for diagnosis of these infections, but this does not exclude the possibility of false negative results [2]. This study aims to evaluate the correlation between clinical diagnosis and PCR test of viral opportunistic infections in organ and tissue transplanted patients.

3 Viral Opportunistic Infections in Organ and Tissue Transplanted Patients 97 MATERIAL AND METHODS This study is in accordance with an Institutional Review Board, in accordance with the Helsinki Declaration of 1975 (as revised in 1983). This is a retrospective study in which, after medical request, 29 transplanted patients with oral lesions were assessed by the same dentist in a hospital bed for the investigation of opportunistic infections. Oral diseases with a clinical aspect of viral lesions triggered by HSV and/or CMV were submitted to scraping and chiseling with a metal spatula, and the secretions were collected using a swab. The samples were stored in dry tubes and sent immediately to the molecular biology laboratory for PCR according to the clinical diagnostic hypotheses. The clinical diagnosis criteria of HSV lesions were superficial ulcerations with fibrinous backgrounds with gray or white color, erythematous halo, coalescing, with brown crust, variable intensity of pain, persistent, not always associated with vesicles, and usually associated with regional lymphadenopathy. The clinical diagnosis criteria for CMV lesions were chronic ulcerations with variable pain intensity, usually solitary, frequently associated with regional lymphadenopathy. The oral assessment and clinical diagnosis of viral lesions of all patients was performed by a single professional experienced in oral diagnosis. The PCR test for HSV was performed when oral lesions had clinical features consistent with infection by herpes simplex virus infection, and the PCR test for CMV was performed when oral lesions had clinical features compatible with cytomegalovirus. When clinical signs and symptoms were not elucidative for the differential diagnosis between viral lesions, we requested PCR for both HSV and CMV. Five microliters of this DNA were used as a template for our in-house CMV PCR, HSV PCR, and internal control PCR (human beta-globin gene). The CMV primers were IE-F 5= AGC TGC ATG ATG TGA GCA AG 3= as the forward and IE-R 5= GAA GGC TGA GTT CTT GGT AA 3= as the reverse [13,14]. The size of the specific CMV immediate-early gene-amplified product was 147 bp. The HSV primers were HSV-J 5= TAC ATC GGC GTC ATC TGCGGG G 3= and HSV-K 5= AGT TCG TGC GCG GTG AGG ACA AAG T 3=.11 The amplified product was a 290-bp internal fragment of the HSV DNA polymerase gene, common to HSV-1 and HSV-2. For each sample, an internal quality-control PCR, detecting the human beta-globin gene, was performed using the same master mix with the primers PCO-3 5= ACA CAA CTG TGT TCA CTA GC 3= and PCO-4 5= CAA CTT CAT CCA CGT TCA CC 3= [15]. The size of the specific CMV-amplified product (147 bp), HSV (290 bp), and the internal control human beta-globin gene (110 bp) were assessed by comparison with a commercial 100-bp ladder. Internal controls were used in the PCR assays to exclude false-negative results due to inefficient specimen processing or the presence of compounds that inhibit the amplification enzyme. Inclusion of a control made it possible to distinguish between specimen preparation and PCR failures and truly negative results. In our study, if the amplified product of the internal control (human beta-globin gene) was not seen in the gel after staining, both PCRs (CMV, HSV, and beta-globin gene) were repeated twice: (1) once again and (2) once with a 1/10 dilution of the original DNA extraction. Therefore, positive or negative results for CMV and/or HSV PCRs were only defined if the internal control amplicon was observed in the gel electrophoresis of each sample.

4 98 Paulo Sérgio da Silva Santos, José Endrigo Tinoco-Araujo, Ana Paula Bloise et al. For statistical analysis, we used the SigmaStat software, applying sensitivity and specificity tests, the concordance test, and Spearman correlation coefficient, adopting a significance level of 5% (p<0.05). RESULTS AND DISCUSSION We assessed 29 patients aged 30.3 ± 12.4 years (14 men and 15 women), with different transplant procedures: hematopoietic stem cell (48%), kidney (45%), heart (3%) and liver (3%) transplantation. We found 44.8% (N=13/29) cases suspected of having HSV and 6.9% (N=2/29) cases suspected of having CMV. The cases in which there was doubt about the clinical diagnosis between viral lesions was 48.3% (N=14/29). According to this clinical diagnosis hypothesis, we requested respectively PCR for HSV and PCR for CMV. When there was doubt about the etiopathogenesis, we requested PCR for HSV and CMV. Table 1 shows correlation between clinical diagnosis and PCR test results. When we requested a PCR test for HSV, the test was positive in 34.5% of cases (N=10/29). When we requested a PCR test for CMV, the test was positive in 6.9% of cases (N=2/29). When we requested PCR tests both for HSV and CMV, 10.3% (N=3/29) of cases were positive for both HSV and CMV, totaling 13 cases of HSV and 5 of CMV. In 48.3% (N=14/29) of the cases the PCR test was negative. Table 1. Correlation between Clinical Examination and PCR Test (p=.01) PCR Test Negative HSV CMV HSV+CMV Clinical Exam N % N % N % N % p HSV CMV HSV+CMV TOTAL In statistical analyses we used the concordance test and found a relationship only in 20% of cases. Applying the Spearman correlation coefficient, we found a significant correlation between clinical and laboratory data (p=.01). Applying tests of sensitivity and specificity, we found that clinical examination was 100% sensitive in cases of HSV infection, and the predictive value indicated a 44% chance of having an HSV infection. For CMV infections, the sensitivity of clinical examination was also 100%, and the possibility of having infection was 25%. The development of molecular biology was one of the greatest achievements in biological sciences, and the discovery of polymerase chain reactions (PCRs) was beneficial to the sequencing of genetic and molecular analyses, as in the diagnosis of genetic diseases and detection of bacteria, viruses, and fungal infections. It allows in vitro synthesis of nucleic acids from the replication of a DNA segment, with excellent results. The PCR test is a standard test for diagnosis and a tool for research in dental sciences, applied in studies of periodontal disease, tooth decay, infections, and oral cancer [16].

5 Viral Opportunistic Infections in Organ and Tissue Transplanted Patients 99 Virus isolation is the most used test for the diagnosis of viral infections, but the PCR test is described as the most reliable and sensitive method, primarily indicated for immunosuppressed patients [3]. The sensitivity of the PCR test is four times greater than the cytological examination, and its reliability is unquestionable. The development of methods for nucleic acids amplification allowed the identification and quantification of mucosal pathogens [12] and can detect viruses in small samples without the risk of interference from unfavorable temperature or transport conditions that may cause failures of conventional methods. In this study we found that HSV opportunistic infections were more frequent, but most patients had lesions with atypical features, making it impossible to determine the etiologic agent just by clinical examination. Immunosuppression may justify atypical manifestations of opportunistic infections in transplanted patients because the immune response in these patients is not equal to the response of a immunocompetent patient who does not use immunosuppressive drugs continuously [2, 11, 17]. Considering that the appropriate treatment of opportunistic infection depends on the appropriate diagnosis of its cause, we believe it is important to carry out additional tests. The sensitivity and specificity tests showed that clinical examination is fully capable of diagnosing HSV and CMV infections; however, the predictive value showed that 44% of patients clinically diagnosed with HSV probably had the disease. For CMV, the positive predictive value indicated a 25% chance of having the disease. These results suggest that clinical examination is not sufficient to complete the viral diagnosis of suspicious lesions in transplanted patients due to unusual clinical manifestations that may present themselves. Therefore, it is necessary to request additional tests with an indication consistent with clinical features. The equilibrium between viral persistence and immune regulation of the host is maintained in healthy subjects, but may be modified in many immunological conditions related to organ transplantation [18, 19]. Oral infections are threats that can cause systemic infections and cause rejection of a transplanted organ. Among the sources of oral infection that can cause some impairment in this group of patients, the most common viral infections are related to CMV and HSV [20]. The HSV and CMV infections are related to morbidity and mortality in patients undergoing organ and tissue transplantation, affecting 50 75% of these patients [21]. The type and intensity of immunosuppression are related to the occurrence of infections by both viruses. In transplanted organs, CMV may cause acute rejection and vascular disease [22], dermatological infections, esophagitis, chronic graft-versus-host disease in BMT, and pneumonia. HSV may cause hepatitis and genital lesions in conditions of immunosuppression [23]. Changes related to oral opportunistic infections caused by viruses may be important for investigating systemic commitments, especially when related to immunosuppression in transplantation, and there is a risk of secondary infections. The number of transplanted patients is increasing, and the formation of multidisciplinary teams for treating these patients has become fundamental, a trend in which dental sciences should take part. In the clinical examination, the herpetic lesions (HSV) were more prevalent, followed by lesions without elucidative clinical features but with viral disease appearance. We suggest that clinical examination alone is not sufficient to establish the final diagnosis of viral lesions in organ and tissue transplanted patients, making it necessary for

6 100 Paulo Sérgio da Silva Santos, José Endrigo Tinoco-Araujo, Ana Paula Bloise et al. exams of high reliability, but clinical suspicion is essential for the appropriate application of the PCR test. CLINICAL SIGNIFICANCE Oral lesions in immunocompromised patients are more extensive, aggressive, painful, and slow to heal. There are few studies identifying oral HSV and CMV infections by molecular biology in immunocompromised patients. The herpes simplex virus (HSV) and cytomegalovirus (CMV) are major causes of morbidity and mortality in transplant recipients, affecting 50 75% of these patients. The oral lesions in immunocompromised patients are more extensive, aggressive, painful, and slow to heal. They are also important causes of morbidity and mortality after transplantation. We suggest that clinical examination alone is not enough for the diagnosis of opportunistic infections with oral manifestations and viral aspects in organ and tissue transplant recipients, making it necessary to perform complementary exams, with the request oriented by clinical examination and preferably molecular biology, for the beginning of appropriate antiviral therapy. REFERENCES [1] Lima RB, Santos PSS, Malafronte P, Muller H, Caiaffa-Filho HH, Sens YAS. Oral manifestation of cytomegalovirus associated with herpes simplex virus in renal transplant recipient. Transplant. Proc. 2008; 40: [2] Gomez RS, Carneiro MA, Souza LN, Victoria JMN, de Azevedo WM, De Marco L et al. Oral recurrent human herpes virus infection and bone marrow transplantation survival. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2001; 91: [3] Kang YN, Oh HK, Chang YC, Kim HC, Lee SL, Hwang M et al. Systemic herpes simplex virus infection following cadaveric renal transplantation: A case report. Transplant. Proc. 2006; 38: [4] Gheui EM, Ship JA. Systemic diseases and their treatments in the elderly: impact on oral health. J. Public Health Dent. 2000; 60: [5] Summers SA, Tilakaratne WM, Fortune F, Ashman N. Renal disease and the mouth. Am. J. Med. 2007; 120: [6] Nebbia G, Mattes FM, Ramaswamy M, Quaglia A, Verghese G, Griffiths PD et al. Primary herpes simplex virus type-2 infection as a cause of liver failure after liver transplantation. Transpl. Infect. Dis. 2006; 8: [7] Correia-Silva JF, Victoria JMN, Guimaraes ALS, Salomao UE, de Abreu MHNG, Bittencourt H et al. Cytomegalovirus shedding in the oral cavity of allogeneic haematopoietic stem cell transplant patients. Oral. Dis. 2007; 13: [8] Gelb B, Feng S. Management of liver transplant patient. Expert Rev Gastroenterol Hepatol 2009; 3: [9] Griffiths WJH, Wreghitt TG, Alexander GJ. Reactivation of herpes simplex virus after liver transplantation. Transplant. 2005; 80:

7 Viral Opportunistic Infections in Organ and Tissue Transplanted Patients 101 [10] Schander K, Jontell M, Johansson P, Norden G, Hakeberg M, Bratel J. Oral infections and their influence on medical rehabilitation in kidney transplant patients. Swed. Dent. J. 2009; 33: [11] Otero R, Martins C, Ferreira D, Benati F, Santos N, Castro G. Identification of Herpesvirus types 1-8 in oral cavity of children/adolescents with chronic renal failure. J. Oral. Pathol. Med. 2011; 40: [12] Wald A, Huang ML, Carrell D, Selke S, Corey L. Polymerase chain reaction for detection of herpes simplex virus (HSV) DNA on mucosal surfaces: Comparison with HSV isolation in cell culture. J. Infect. Dis. 2003; 188: [13] Espy M, Aslanzadeh J, Smith T. PCR Detection of Herpes simplex DNA sequences in fluid. In: Persing D, Smith T, Tenover F, editors. Diagnostic Molecular Microbiology Principles and Applications. Washington, DC: American Society for Microbiology; p [14] Epsy M, Smith T. PCR detection of cytomegalovirus DNA sequences in clinical specimens. In: Persing D, Smith T, Tenover F, editors. Diagnostic Molecular Microbiology Principles and Applications. Washington, DC: American Society for Microbiology; p [15] Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA-polymerase. Science 1988; 239: [16] Valones MAA, Guimaraes RL, Brandao LAC, Souza PRE, Carvalho AAT, Crovela S. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Braz. J. Microbiol. 2009; 40: [17] Lopez-Pintor RM, Hernandez G, de Arriba L, de Andres A. Comparison of oral lesion prevalence in renal transplant patients under immunosuppressive therapy and healthy controls. Oral. Dis. 2010; 16: [18] Razonable RR, Paya CV. The impact of human herpesvirus-6 and-7 infection on the outcome of liver transplantation. Liver. Transplant. 2002; 8: [19] Razonable RR, Eid AJ. Viral infections in transplant recipients. Minerva Med. 2009; 100: [20] Da Silva Santos PS, Bitu F, Coracin FL, Mancusi Sobrinho R, Lima RB. Oral complications associated with organ and tissue transplants: a literature review. J. Bras. Transplant. 2009; 12: [21] Rubin R. Cytomegalovirus in solid organ transplantation. Transplant. Infect. Dis. 2001; 3 Suppl. 2: 1-5. [22] Sagedal S, Nordal K, Hartmann A, Sund S, Scott H, Degré M et al. The impact of cytomegalovirus infection and disease on rejection episodes in renal allograft recipients. Am. J. Transplant. 2002; 2: [23] Singh N, Dummer J, Kusne S, Breinig M, Armstrong J, Makowka L et al. Infections with cytomegalovirus and other herpesviruses in 121 liver transplant recipients: transmission by donated organ and the effect of OKT3 antibodies. J. Infect. Dis. 1988; 158:

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Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed. Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons