Seroprevalence of HBV, HCV and HIV co-infection in selected individuals from state of São Paulo, Brazil

Few studies are available on hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co-infection in populations living in small and medium-sized Brazilian cities. We evaluated the seroprevalence of these viruses in selected individuals from a clinic of infectology, who were referred to the University Regional Hospital of the West Region of state of São Paulo, Brazil. Among a total of 7,021 individuals seen in the clinic following receipt of preliminary ELISA results or having the suggested clinical signs of viral hepatitis or HIV, 1,228 were systematically screened. Isolated or associated HBsAg, HCV and HIV antibodies were found in 44.9% of the subjects. Anti-HIV antibodies were found in 24.7% of the patients, 20.3% of whom had an HIV monoinfection and 4.4% of whom were co-infected with hepatitis viruses (HCV: 4%; HBV: 0.4%). Anti-HCV antibodies were found in 14% of the patients and 5.9% had anti-HBsAg antibodies. HCV infection affected males more than females (p < 0.05) and individuals > 50-years old had an increased prevalence of anti-HCV compared to HIV (p = 0.0001) or HBV (p = 0.0063). HCV-RNA was detected in 73.5% of the samples with a predominance of genotype 1 (72.5%). A significant percentage (44.9%) of the selected individuals was positive for antibodies against HBV, HCV and/or HIV; these patients would otherwise have remained undiagnosed.

Sequencing of E2 and NS5A regions of HCV genotype 3a in Brazilian patients with chronic hepatitis

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

Evaluation of polymerase chain reaction in the routine diagnosis for tegumentary leishmaniasis in a referral centre

The present study investigated the diagnostic value of polymerase chain reaction (PCR) performed in parallel to conventional methods at an American tegumentary leishmaniasis (ATL) referral centre for diagnosis. Accuracy parameters for PCR were calculated using 130 patients with confirmed ATL (ATL group), 15 patients established with other diseases and 23 patients with a lesion suggestive of ATL, but without parasitological confirmation (NDEF group). PCR showed 92.3% sensitivity, 93.3% specificity, a 99.2% positive predictive value and a 13.84 positive likelihood ratio. In the NDEF group, PCR confirmed ATL in 13 of the 23 patients, seven of whom responded to leishmaniasis treatment and six who presented spontaneous healing of the lesion. PCR should be included in the routine diagnostic procedures for ATL, especially for cases found to be negative by conventional methods.

Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children

The diagnosis of visceral leishmaniasis (VL) generally requires the use of invasive tests for the collection of infected tissue (aspirates of bone marrow, spleen, liver or lymph nodes). This difficulty has led to the search for safer and less painful techniques to confirm the occurrence of the disease in children. Polymerase chain reaction (PCR) is a method that is advantageous in that it allows the use of peripheral blood samples for diagnosis. This paper reports the utilisation of PCR on peripheral blood samples to diagnose VL in 45 children in Mato Grosso do Sul, Brazil. This technique is compared with methods carried out using tissue collected by invasive procedures, including direct microscopy, culture and detection of Leishmania DNA by PCR in bone marrow aspirates. The results show that PCR of peripheral blood provides great sensitivity (95.6%) that is similar to that from the PCR of bone marrow aspirates (91.1%) and higher than that achieved with microscopy (80%) or culture (26.7%) methods. PCR of peripheral blood proved to be a suitable tool for the diagnosis of VL in children because it is highly sensitive and safe, with tissue collection being less invasive than in traditional tests.

Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction

Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97%) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33%) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5%. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.

Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients

This study was designed to assess the effect of GB virus (GBV)-C on the immune response to human immunodeficiency virus (HIV) in chronically HIV-infected and HIV- hepatitis C virus (HCV)-co-infected patients undergoing antiretroviral therapy. A cohort of 159 HIV-seropositive patients, of whom 52 were HCV-co-infected, was included. Epidemiological data were collected and virological and immunological markers, including the production of interferon gamma (IFN-γ) and interleukin (IL)-2 by CD4, CD8 and Tγδ cells and the expression of the activation marker, CD38, were assessed. A total of 65 patients (40.8%) presented markers of GBV-C infection. The presence of GBV-C did not influence HIV and HCV replication or TCD4 and TCD8 cell counts. Immune responses, defined by IFN-γ and IL-2 production and CD38 expression did not differ among the groups. Our results suggest that neither GBV-C viremia nor the presence of E2 antibodies influence HIV and HCV viral replication or CD4 T cell counts in chronically infected patients. Furthermore, GBV-C did not influence cytokine production or CD38-driven immune activation among these patients. Although our results do not exclude a protective effect of GBV-C in early HIV disease, they demonstrate that this effect may not be present in chronically infected patients, who represent the majority of patients in outpatient clinics.

Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction

This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed.

Genetic diversity of NS3 protease from Brazilian HCV isolates and possible implications for therapy with direct-acting antiviral drugs

The hepatitis C virus (HCV) NS3 protease has been one of the molecular targets of new therapeutic approaches. Its genomic sequence variability in Brazilian HCV isolates is poorly documented. To obtain more information on the magnitude of its genetic diversity, 114 Brazilian HCV samples were sequenced and analysed together with global reference sequences. Genetic distance (d) analyses revealed that subtype 1b had a higher degree of heterogeneity (d = 0.098) than subtypes 1a (d = 0.060) and 3a (d = 0.062). Brazilian isolates of subtype 1b were distributed in the phylogenetic tree among sequences from other countries, whereas most subtype 1a and 3a sequences clustered into a single branch. Additional characterisation of subtype 1a in clades 1 and 2 revealed that all but two Brazilian subtype 1a sequences formed a distinct and strongly supported (approximate likelihood-ratio test = 93) group of sequences inside clade 1. Moreover, this subcluster inside clade 1 presented an unusual phenotypic characteristic in relation to the presence of resistance mutations for macrocyclic inhibitors. In particular, the mutation Q80K was found in the majority of clade 1 sequences, but not in the Brazilian isolates. These data demonstrate that Brazilian HCV subtypes display a distinct pattern of genetic diversity and reinforce the importance of sequence information in future therapeutic approaches.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

Response to treatment in Brazilian patients with chronic hepatitis C is associated with a single-nucleotide polymorphism near the interleukin-28B gene

A single-nucleotide polymorphism (SNP) upstream of interleukin (IL)28B was recently identified as an important predictor of the outcome of chronic hepatitis C patients treated with pegylated interferon plus ribavirin (PEG-IFN/RBV). The aim of this study was to investigate the association between the IL28B gene polymorphism (rs12979860) and virological response in chronic hepatitis C patients. Brazilian patients (n = 263) who were infected with hepatitis C virus (HCV) genotype 1 and were receiving PEG-IFN/RBV were genotyped. Early virological response (EVR) (12 weeks), end-of-treatment response (EOTR) (48 weeks), sustained virological response (SVR) (72 weeks) and relapse were evaluated using conventional and quantitative polymerase chain reaction (PCR) assays. The frequency of the C allele in the population was 39%. Overall, 43% of patients experienced SVR. The IL28B CC genotype was significantly associated with higher treatment response rates and a lower relapse rate compared to the other genotypes [84% vs. 58% EVR, 92% vs. 63% EOTR, 76% vs. 38% SVR and 17% vs. 40% relapse rate in CC vs. other genotypes (CT and TT), respectively]. Thus, the IL28B genotype appears to be a strong predictor of SVR following PEG-IFN/RBV therapy in treatment-naïve Brazilian patients infected with HCV genotype 1. This study, together with similar research examining other SNPs, should help to define adequate protocols for the treatment of patients infected with HCV genotype 1, especially those with a poor prognosis.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids

We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR) assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.