Paula Durante Andrade

Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies

Chagas disease, caused by Trypanosoma cruzi, is an important endemic illness in Latin America. Serologic tests for T. cruzi detection in blood are sensitive, but their specificity is unsatisfactory. Direct detection of parasites in blood, either by xenodiagnosis or hemoculture, is highly specific but of low sensitivity. Molecular assays such as the Polymerase chain reaction (PCR), which amplifies certain repetitive sequences of nuclear DNA has been used as a good alternative tool for T. cruzi detection in human blood. The present study aimed to test PCR diagnosis in chagasic chronic patients and doubtful serologic patients attended in GEDOCH (Chagas Disease Study Group/UNICAMP, Brazil). A 149 bp fragment originated from nuclear DNA was specifically detected in chronic chagasic patients. The results of these tests were compared with serologic diagnosis performed using standard techniques and xenodiagnosis. We found that 43 out of 50 patients previously serodiagnosed as chagasic were positive using the N-PCR method. Thirteen of 30 patients with doubtful serologic results were confirmed as positive by N-PCR. Our results suggest that the N-PCR may be a complementary tool to serology in the diagnosis of Chagas disease, and that it is usefull for parasite detection in patients with chronic disease and patients with doubtful serologic results.

Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.

High frequency of Human Cytomegalovirus DNA in the Liver of Infants with Extrahepatic Neonatal Cholestasis

BackgroundBiliary atresia (BA) is the most severe hepatic disorder in newborns and its etiopathogenesis remains unknown. Viral involvement has been proposed, including the human cytomegalovirus (HCMV). The aims of the study were to use the polymerase chain reaction (PCR) to screen the liver tissue of infants with extrahepatic cholestasis for HCMV and to correlate the results with serological antibodies against HCMV and histological findings.MethodsA retrospective study in a tertiary care setting included 35 patients (31 BA, 1 BA associated with a choledochal cyst, 2 congenital stenosis of the distal common bile duct and 1 hepatic cyst). HCMV serology was determined by ELISA. Liver and porta hepatis were examined histologically. Liver samples from infants and a control group were screened for HCMV DNA.ResultsTwelve patients had HCMV negative serology, 9 were positive for IgG antibodies and 14 were positive for IgG and IgM. Nine liver and seven porta hepatis samples were positive for HCMV DNA but none of the control group were positive (general frequency of positivity was 34.3% – 12/35). There was no correlation between HCMV positivity by PCR and the histological findings. The accuracy of serology for detecting HCMV antibodies was low.ConclusionThese results indicate an elevated frequency of HCMV in pediatric patients with extrahepatic neonatal cholestasis. They also show the low accuracy of serological tests for detecting active HCMV infection and the lack of correlation between HCMV positivity by PCR and the histopathological changes.

Surveillance of active human cytomegalovirus infection in hematopoietic stem cell transplantation (HLA sibling identical donor): search for optimal cutoff value by real-time PCR

BackgroundHuman cytomegalovirus (CMV) infection still causes significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, it is extremely important to diagnosis and monitor active CMV infection in HSCT patients, defining the CMV DNA levels of virus replication that warrant intervention with antiviral agents in order to accurately prevent CMV disease and further related complications.MethodsDuring the first 150 days after allogeneic HSTC, thirty patients were monitored weekly for active CMV infection by pp65 antigenemia, nested-PCR and real-time PCR assays. Receiver operating characteristic (ROC) plot analysis was performed to determine a threshold value of the CMV DNA load by real-time PCR.ResultsUsing ROC curves, the optimal cutoff value by real-time PCR was 418.4 copies/104 PBL (sensitivity, 71.4%; specificity, 89.7%). Twenty seven (90%) of the 30 analyzed patients had active CMV infection and two (6.7%) developed CMV disease. Eleven (40.7%) of these 27 patients had acute GVHD, 18 (66.7%) had opportunistic infection, 5 (18.5%) had chronic rejection and 11 (40.7%) died - one died of CMV disease associated with GVHD and bacterial infection.ConclusionsThe low incidence of CMV disease in HSCT recipients in our study attests to the efficacy of CMV surveillance based on clinical routine assay. The quantification of CMV DNA load using real-time PCR appears to be applicable to the clinical practice and an optimal cutoff value for guiding timely preemptive therapy should be clinically validated in future studies.

Simultaneous monitoring of CMV and human herpesvirus 6 infections and diseases in liver transplant patients: one-year follow-up

OBJECTIVE: The aim of this study was to simultaneously monitoring cytomegalovirus and human herpesvirus 6 active infections using nested-polymerase chain reaction and, together with clinical findings, follow the clinical status of patients undergoing liver transplant. INTRODUCTION: The human β-herpesviruses, including cytomegalovirus and human herpesvirus 6, are ubiquitous among human populations. Active infections of human herpesvirus 6 and cytomegalovirus are common after liver transplantation, possibly induced and facilitated by allograft rejection and immunosuppressive therapy. Both viruses affect the success of the transplant procedure. METHODS: Thirty patients submitted to liver transplant at the Liver Transplant Unit, at the Gastro Center, State University of Campinas, SP, Brazil, were studied prospectively from six months to one year, nested-polymerase chain reaction for cytomegalovirus and human herpesvirus 6 DNA detections. Two or more consecutive positive nested-polymerase chain reaction were considered indicative of active infection. RESULTS: Active infection by cytomegalovirus was detected in 13/30 (43.3%) patients, median time to first cytomegalovirus detection was 29 days after transplantation (range: 0-99 days). Active infection by human herpesvirus 6 was detected in 12/30 (40%) patients, median time to first human herpesvirus 6 detection was 23.5 days after transplantation (range: 0-273 days). The time-related appearance of each virus was not statistically different (p = 0.49). Rejection of the transplanted liver was observed in 16.7% (5/30) of the patients. The present analysis showed that human herpesvirus 6 and/or cytomegalovirus active infections were frequent in liver transplant recipients at our center. CONCLUSIONS: Few patients remain free of betaherpesviruses after liver transplantation. Most patients presenting active infection with more than one virus were infected sequentially and not concurrently. Nested-polymerase chain reaction can be considered of limited value for clinically monitoring cytomegalovirus and human herpesvirus 6.

Monitoring and Detection of Cytomegalovirus in Liver Transplant Recipients

Cytomegalovirus (CMV) is a β-herpesvirus. CMV infections are a common complication contributing to morbidity and mortality after liver transplantation. Among organ transplant recipients, CMV can reactivate from latency during the first 6 months. This prospective study performed from February 2008 to December 2009 examined liver transplant recipients during the first 6 months. Two methods were performed to detect CMV infections: antigenemia (AGM) and nested (PCR). Ninety-four patients, including 72 men (76.6%) and 22 women (23.4%) underwent liver transplantation during this period. We analyzed 575 samples including 465 for AGM and PCR. Forty-three (9.25%) showed positive AGM as detected 2 to 179 days posttransplantation with a mean of 50 days and a median of 35 days, and 93/465 (20%) showed positive PCR at 0 to 186 days posttransplantation with a mean of 31 days and a median of 38 days. Among the 43 antigenemia patients, 38 samples were positive for up to 5 cells 18 of which were PCR-positive. Five samples were positive with more than 5 cells, including 3 that were PCR-positive. Only 4.51% had AGM and were PCR-positive in the same sample. Despite only 9.25% (43/465) showing AGM, the current study suggested the utility of routine monitoring to detect early CMV infection among liver transplantation patients seeking to reduce morbidity and mortality.

Identification of Bacterial Infections and Clinical Manifestation Associated With Cytomegalovirus in Liver Transplantation Patients

INTRODUCTION Liver transplantation has become the most effective therapy for the treatment of patients with end-stage liver disease. With new immunosuppressive agents the incidence of acute rejection has been significantly reduced, but infection has become a serious problem. OBJECTIVE Our objective was to correlate cytomegalovirus (CMV) positivity of antigenemia and polymerase chain reaction (PCR) with clinical manifestations and bacterial infections among patients undergoing liver transplantation. METHODS This prospective study included patients monitored for 6 months for early detection of CMV infection. Sample collections were performed at the time of surgery and weekly until the second month followed by fortnightly in the third month, and monthly in the fourth to sixth month. CMV infection was defined by positive antigenemia (>3 cells) or 2 positive PCR tests associated or not with clinical symptoms. The methodology for the diagnosis of bacterial infection was through biochemical tests and the automated VITEK/bioMérieux (identification and antibiogram) using samples of urine and blood cultures. Chi-square test was used for dicotomic variables with significant differences when P < .05. RESULTS Sixteen patients (32%) had CMV infections, including 13 (81%) with concomitant infections. Thirty-four patients (68%) did not have CMV infections and 8 of these (24%) had bacterial infection. There was a high correlation with bacterial infections among CMV-positive patients. CONCLUSION Bacterial infections after liver transplantation were associated with CMV infection.

Co-infection and Clinical Impact of Human Herpesvirus 5 and 6 in Liver Transplantation

BACKGROUND Human herpesvirus (HHV) 5 and 6 remain latent after primary infection and can be reactivated after immunosuppression for organ transplantation. An association between HHV-5 and HHV-6 has been reported in liver transplant patients. The coinfection is associated with clinical manifestations and graft dysfunction. OBJECTIVE The aim of this study was to monitor herpesviruses in liver transplant recipients to better understand issues involving coinfection with HHV-5/6 and correlations with acute cellular rejection episodes and bacterial infections. METHODS Forty-five adult liver transplant patients of median age 47 years (range, 18-66), gave blood samples and liver biopsies in the first 6 months after their surgeries. Viremia was detected with the use of nested PCR and antigenemia; the Banff classification was used to detect allograft rejection. RESULTS IgG positive for HHV-5 was observed in 94% of subjects whose main indication (67%) for transplantation was hepatitis C. Twenty-three (51.1%) displayed cytomeg virus (CMV) infections and 12 (26.7%) HHV-6 infection. There were 6 patients (13.3%) with HHV-5/6 coinfections. Eighteen of the 23 patients had CMV disease, showing a strong correlation between a positive test and CMV disease; 6 displayed an acute cellular rejection episode in the same period (χ(2) = 6.62; P < .03). Four out of 6 patients who displayed coinfections (HHV-5/6) had concomitant bacterial infections; 3/6 experienced graft rejection episodes. During follow-up, 1 patient had HHV-6 infection diagnosed as encephalitis followed by fever on the 24th day after surgery. The median 32 days for HHV-6 detection by nested PCR positivity was shorter than 38 days for HHV-5. CONCLUSIONS HHV-5/6-infected patients displayed more allograft rejection episodes, coinfections, and concomitant bacterial infections, besides an higher risk for CMV disease.

Prevalence of HIV-1 subtypes in Brazilian children with perinatally acquired infection.

HIV-1 infection has increased among women in recent years. The HIV-1 env gene (structural gene) has the greatest variation in all the HIV gene regions. In this study, 58 samples from infants infected with HIV-1 via perinatal transmission were analyzed. All the 58 samples were submitted to Nested-polymerase chain reaction of the env gene region for posterior viral genotyping using EN 70 and EN 85 (first polymerase chain reaction) and EN 80 and EN 95 (second polymerase chain reaction) primers, with the product of the 682 base pair amplification. After Nested-polymerase chain reaction for genotyping, purification of the product, and direct sequencing in a MegaBace 1000 automatic sequencer, 56 genotypes were found in the 58 HIV-1-positive children of the study, where 47 (83.93%) were HIV-1 subtype B infected and 9 (16.07%) were HIV-1 subtype F1 infected. The results demonstrate the predominance of subtype B followed by subtype F in Southeast Brazil.

CARACTERIZAÇÃO MOLECULAR DOS SOROTIPOS DE ESTREPTOCOCOS DO GRUPO B POR REAÇÃO MULTIPLEX PCR

OBJETIVO: Os sorotipos (Ia, Ib e II ao IX) do estreptococo do grupo B (GBS) sao classificados baseado nas variacoes em seus polissacarideos capsulares; sua prevalencia difere entre diferentes areas geograficas. Nos examinamos a prevalencia de todos os sorotipos do estreptococo do grupo B em amostras de swabs vaginal e retal obtidas de 363 mulheres seguidas em um centro de referencia brasileiro, o Hospital da Mulher Professor Doutor Jose Aristodemo Pinotti; a susceptibilidade bacteriana a antibioticos foi tambem determinada. METODO A prevalencia de estreptococo do grupo B positivo foi avaliada por aglutinacao em latex e atraves de analise por multiplex PCR; susceptibilidade bacteriana a antibioticos, tais como clindamicina, eritromicina, levofloxacin, linezolide, penicilina e tetraciclina foi determinada pelo metodo de disco difusao. RESULTADOS: (a) Tanto a cultura padrao para estreptococo do grupo B quanto a analise por multiplex PCR testaram positivos para 83 swabs. A prevalencia para colonizacao por GBS foi 20%. O sorotipo Ia foi o mais prevalente (n= 43/83; 52%), seguido pelo sorotipo V (n= 14/83; 17%); De acordo com a origem anatomica, o sorotipo Ia positivou 27/59 (46%) e 16/24 (67%) das amostras vaginais e retais, respectivamente; o teste de PCR tambem identificou os sorotipos Ib, II, III, VI. O sorotipo VI e raramente descrito e nao reportado no Brasil ou na America Latina ate esta data. (b) O teste de aglutinacao em latex somente identificou 44 amostras positivas, todas das quais foram sorotipadas: 34 destas amostras (77%) tiveram os sorotipos coincidindo com aqueles identificados pela multiplex PCR. (c) Somente uma amostra (sorotipo Ia) mostrou resistencia a eritromicina e clindamicina. CONCLUSAO: Estudos regionais sobre a prevalencia dos sorotipos do estreptococo do grupo B sao essenciais para guiar medidas imunoprofilaticas (vacinas) e a implementacao de adequada antibiotico profilaxia. Neste estudo, a incidencia do sorotipo VI foi descrita pela primeira vez na populacao Brasileira, um novo e raro sorotipo do estreptococo do grupo B.