Current status of herpesvirus identification in the oral cavity of HIV-infected children

INTRODUCTION: Some viruses of the Herpesviridae family are frequently the etiologic agents of oral lesions associated with HIV. The aim of this study was to identify the presence of herpes simplex virus types 1 and 2 (HSV-1, HSV-2), Varicella Zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus type 6, type 7 and type 8 (HHV-6, HHV-7 and HHV-8) in the oral cavity of HIV-infected children/adolescents and verify the association between viral subtypes and clinical factors. METHODS: The cells of oral mucosa were collected from 50 HIV infected children/adolescents, 3-13 years old (mean age 8.66). The majority (66%) of selected were girls, and they were all outpatients at the pediatric AIDS clinic of a public hospital in Rio de Janeiro. Nested-PCR was used to identify the viral types. RESULTS: Absence of immunosuppression was observed in 66% of the children. Highly active antiretroviral therapy (HAART) was used by 72.1% of selected and moderate viral load was observed in 56% of the children/adolescents. Viral types were found in 86% of the children and the subtypes were: HSV-1 (4%), HSV-2 (2%), VZV (4%), EBV (0%), HCMV (24%), HHV6 (18%), HHV-7 (68%), HHV8 (0%). CONCLUSIONS: The use of HAART has helped to reduce oral lesions, especially with herpes virus infections. The health professionals who work with these patients should be aware of such lesions because of their predictive value and the herpes virus can be found circulating in the oral cavity without causing lesions.

Human adenovirus detection among immunocompetent and immunocompromised patients presenting acute respiratory infection

INTRODUCTION: Human adenoviruses (HAdV) play an important role in the etiology of severe acute lower respiratory infection, especially in immunocompromised individuals. The aim of the present study was detect the HAdV through different methods: direct fluorescence assay (DFA) and nested-polymerase chain reaction (PCR-nested) from patients with acute respiratory infection (ARI) up to 7 days of symptoms onset.METHODS:Samples (n=643) were collected from different risk groups during from 2001 to 2010: 139 adults attended in an Emergency Room Patients (ERP); 205 health care workers (HCW); 69 from Renal Transplant Outpatients (RTO); 230 patients in hematopoietic stem cell transplantation (HSCT) program.RESULTS:Among all patients (n=643) adenovirus was detected on 13.2% by DFA and/or PCR: 6/139 (4.3%) adults from ERP, 7/205 (3.4%) from HCW samples, 4/69 (5.8%) from RTO and 68/230 (29.5%) from HSCT patients. Nested PCR showed higher detection (10%) compared to DFA test (3.8%) (p < 0.001). HSCT patients presented significantly higher prevalence of HAdV infection.CONCLUSIONS:Adenovirus detection through nested-PCR assay was higher. However the inclusion of molecular method in laboratorial routine diagnostic should be evaluated considering the reality of each specific health service.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%).

Molecular characterization of Torque teno virus and SEN virus co-infection with HIV in patients from Southern Iran

Introduction Torque teno virus (TTV) and SEN virus are circular single-stranded DNA viruses that cause blood-borne infections. The SEN virus (SEN-V) was originally detected in the serum of an injection drug user infected with human immunodeficiency virus (HIV). Recently TTV was discovered as a potential causative agent of non-A-E hepatitis. The aim of this study was to investigate the prevalence of the SEN-V-D/H and TTV in HIV patients and healthy blood donors in Iran. Methods One hundred and fifty HIV patients with a mean age of 50.46 ± 18.46 years and 150 healthy blood donors with a mean age of 48.16 ± 13.73 years were included in this study. TTV and SEN-V were detected by the PCR and were quantitatively assayed by competitive PCR (nested and semi-nested PCR). Restriction fragment length polymorphisms (RFLPs) were used to determine the heterogeneity of TTV. Results TTV and SEN-V were detected 96 (64%) and 84 (56%) of 150 HIV patients respectively. These rates were 34% (n=51) and 37.33% (n=56) in healthy blood donors (significant, p<0.05). PCR detected SEN-V/TTV DNA from 32 of the healthy blood donors (21.33%), while 65 (43.33%) of HIV patients were positive for SEN-V/TTV DNA. Of 150 HIV patients, 32.66% and 23.33% were positive for SEN-V-H and SEN-V-D, respectively and 18.66% (n=28) were co-infected with SEN-V-D/H. Conclusions The prevalence of SEN-VD/H and TTV is higher in HIV patients than in healthy blood donors in Southern Iran. Our results suggest that TTV and SEN-V might play a role in the development of liver disease in patients with immunodeficiency diseases.

Lack of evidence for human infection with Xenotropic murine leukemia virus-related virus in the Brazilian Amazon basin

Introduction This study confirmed the absence of natural infection with Xenotropic murine leukemia virus-related virus (XMRV) or XMRV-related disease in human populations of the Brazilian Amazon basin. We demonstrated that 803 individuals of both sexes, who were residents of Belem in the Brazilian State of Pará, were not infected with XMRV. Methods Individuals were divided into 4 subgroups: healthy individuals, individuals infected with human immunodeficiency virus, type 1 (HIV-1), individuals infected with human T-lymphotrophic virus, types 1 or 2 (HTLV-1/2), and individuals with prostate cancer. XMRV infection was investigated by nested PCR to detect the viral gag gene and by quantitative PCR to detect pol. Results There was no amplification of either gag or pol segments from XRMV in any of the samples examined. Conclusions This study supports the conclusions of the studies that eventually led to the retraction of the original study reporting the association between XMRV and human diseases.

Hepatitis B virus genotypes and mutations in the basal core promoter and pre-core/core in chronically infected patients in southern Brazil: a cross-sectional study of HBV genotypes and mutations in chronic carriers

Introduction In Brazil, little data exist regarding the distribution of genotypes in relation to basal core promoter (BCP) and precore/core mutations among chronic hepatitis B virus (HBV) carriers from different regions of the country. The aim of this study was to identify HBV genotypes and the frequency of mutations at the BCP and precore/core region among the prevalent genotypes in chronic carriers from southern Brazil. Methods Nested-polymerase chain reaction (nested-PCR) products amplified from the S-polymerase gene, BCP and precore/core region from 54 samples were sequenced and analyzed. Results Phylogenetic analysis of the S-polymerase gene sequences showed that 66.7% (36/54) of the patients were infected with genotype D (D1, D2, D3), 25.9% (14/54) with genotype A (A1, A2), 5.6% (3/54) with subgenotype C2, and 2% (1/54) with genotype E. A comparison of virological characteristics showed significant differences between genotypes A, C and D. The comparison between HBeAg status and the G1896A stop codon mutation in patients with genotype D revealed a relationship between HBV G1896A precore mutants and genotype D and hepatitis B e antigen (HBeAg) seroconversion. Genotype D had a higher prevalence of the G1896A mutation and the presence of a thymine at position 1858. Genotype A was associated with a higher prevalence of the G1862T mutation and the presence of a cytosine at position 1858. Conclusions HBV genotype D (D3) is predominant in HBV chronic carriers from southern Brazil. The presence of mutations in the BCP and precore/core region was correlated with the HBV genotype and HBeAg negative status.

Occult HBV infection status among chronic hepatitis C and hemodialysis patients in Northeastern Egypt: regional and national overview

INTRODUCTION: Occult hepatitis B infection (OBI) is considered to be one of the major risks for patients suffering from end-stage renal disease (ESRD) on regular hemodialysis (HD) and patients with chronic hepatitis C virus (HCV) infection. This study compared the prevalence of OBI among these two high-risk groups in the Suez Canal region, Northeastern Egypt, to obtain a better national overview of the magnitude of OBI in this region. METHODS: Serum samples were collected from 165 HD patients and 210 chronic HCV-infected patients. Anti-HCV antibody, hepatitis B surface antigen (HBsAg), total hepatitis B core (anti-HBc) antibody, and hepatitis B surface antibody (anti-HBs) were detected by enzyme-linked immunosorbent assay (ELISA). HCV RNA was detected using a quantitative real-time RT-PCR assay, and HBV was detected using a nested PCR. RESULTS: All patients were negative for HBsAg. A total of 49.1% and 25.2% of the patients in the HD and HCV groups, respectively, were anti-HBc-positive. In addition, more anti-HBs-positive patients were detected in the HD group compared to the HCV group (52.1% and 11.4%, respectively). Three cases were positive for HBV DNA in the HD group, while eighteen positive cases were detected in the HCV group. Both study groups showed significant differences in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level as well as anti-HBc, anti-HBs and HBV-DNA positivity. CONCLUSIONS: OBI was more prevalent among chronic HCV patients than HD patients in the Suez Canal region, Egypt, with rates of 8.5% and 1.8%, respectively. However, more precise assessment of this infection requires regular patient follow-up using HBV DNA detection methods.

HIV-1 drug resistance transmission networks in southwest Switzerland.

To determine viral subtypes and resistance mutations to antiretroviral treatment (ART) in untreated HIV-1 acutely infected subjects from Southwest Switzerland. Clinical samples were obtained from the HIV primary infection cohort from Lausanne. Briefly, pol gene was amplified by nested PCR and sequenced to generate a 1?kb sequence spanning protease and reverse transcriptase key protein regions. Nucleotide sequences were used to assess viral genotype and ART resistance mutations. Blood specimens and medical information were obtained from 30 patients. Main viral subtypes corresponded to clade B, CRF02_AG, and F1. Resistant mutations to PIs consisted of L10V and accessory mutations 16E and 60E present in all F1 clades. The NNRTI major resistant mutation 103N was detected in all F1 viruses and in other 2 clades. Additionally, we identified F1 sequences from other 6 HIV infected and untreated individuals from Southwest Switzerland, harboring nucleotide motifs and resistance mutations to ART as observed in the F1 strains from the cohort. These data reveal a high transmission rate (16.6%) for NNRTI resistant mutation 103N in a cohort of HIV acute infection. Three of the 5 resistant strains were F1 clades closely related to other F1 isolates from HIV-1 infection untreated patients also coming from Southwest Switzerland. Overall, we provide strong evidence towards an HIV-1 resistant transmission network in Southwest Switzerland. These findings have relevant implications for the local molecular mapping of HIV-1 and future ART surveillance studies in the region.

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott®), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95%) sera from patients in the study group and negative in 38 of the 39 (97%) sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Hepatitis C and hepatitis B virus infection in different hemodialysis units in Belo Horizonte, Minas Gerais, Brazil

The prevalence, virological and epidemilogical aspects of the hepatitis C virus (HCV) and the hepatitis B virus (HBV) infections vary among hemodialysis patients in different countries. Aiming at analyzing these aspects of HCV and HBV infections in hemodialysis patients in Belo Horizonte, MG, Brazil, we studied three hemodialysis units including 434 patients. Serology was used to detect anti-HCV and HBsAg. Reverse trancriptase nested polymerase chain reaction (RT-nested-PCR) of the 5'-noncoding region was used to detect circulating HCV RNA and restriction fragment length polymorphism analysis for genotyping. Seroprevalence varied from 26.5% to 11.1% for hepatitis C and from 5.9% to 0% for hepatitis B. Risk factors observed for HBV and/or HCV infections were the number of patients per dialysis unit, duration of treatment, number of clinics attended, number of blood units transfused, and lower level scholarity. Alanine aminotransferase levels were altered with a higher frequency in HBV or HCV seropositive patients. Half of ten patients, negative for anti-HCV, had detectable viremia by RT-nested-PCR, indicating that this technique should be used to confirm infections in this group of patients. The HCV genotype 1 was the most frequently observed, followed by the genotype 2, but no correlation was detected between genotype and clinical or epidemiological data.

Prevalence of Chlamydia trachomatis in human immunodeficiency virus-infected women in Cuba

To determine the prevalence rates and serovar distribution of Chlamydia trachomatis cervical infections in Cuban women, two different groups were selected. Group I consisted of 60 human immunodeficiency virus (HIV-1) seropositive women from different regions of Cuba and group II of 60 randomly selected women HIV seronegative and apparently healthy. C. trachomatis was detected in cervical scrapes by mean of nested polymerase chain reaction (PCR) specific for major out membrane protein. The overall prevalence rate of C. trachomatis in cervical scrapes determined by nested PCR was 10% in group I and the estimated prevalence was 6.6% for group II; 83.3% of HIV seropositive women with C. trachomatis infection reported history of pelvic inflammatory disease followed by cervicitis (50%). The control group C. trachomatis-infected women referred a history of cervicitis in 75% of cases. Other reports in the latter group included infertility and pelvic inflamatory disease in 50%. The present study is the first report of C. trachomatis prevalence in Cuba. It showed that there was not significantly difference in the prevalence rate of C. trachomatis between both groups.

Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positivity) by nested-PCR. The eight amplicons obtained by RT-PCR (P1, P3-P9), including one obtained by nested-PCR (P-2) and not obtained by RT-PCR, were sequenced and showed high homology (94.8% to 99.1%) with the N gene of Araraquara hantavirus. Although the serologic method ELISA is the most appropriate test for HCPS diagnosis, the use of nested RT-PCR for hantavirus in Brazil would contribute to the diagnosis of acute hantavirus disease detecting viral genomes in patient specimens as well as initial genomic characterization of circulating hantaviruses.

Diagnosis of Brazilian vesiculoviruses by reverse transcription-polymerase chain reaction

We describe a reverse transcription-polymerase chain reaction (RT-PCR) and a nested-PCR for diagnosis of Piry, Carajás, Cocal, and Alagoas vesiculoviruses from Brazil. The RNA extracts of viral and clinical samples were submitted to a RT-PCR using Vesiculovirus G primers that amplify part of the glycoprotein gene. The RT-PCR produced amplicons of expected size, 290 base pair, for the four studied viruses. The RT-PCR showed a high sensitivity being 151.3 times (2.18 log) more sensitive for the detection of Piry virus than the classical procedure for virus detection in tissue culture based on the viral cytophatic effect. Amplicons had nucleotides sequenced and were aligned in order to select internal primers for a nested-PCR to confirm the origin of Piry, Carajás, Cocal, and Alagoas Vesiculovirus. Ten blood and tarsal pad epithelial samples of infected Guinea-pigs had Vesiculovirus genome amplified by RT-nested-PCR.

Prevalence and risk factors of hepatitis C virus infection in hemodialysis patients from one center in Recife, Brazil

A hemodialysis population from a dialysis unit in the city of Recife, Northeastern Brazil, was screened to assess the prevalence of hepatitis C virus (HCV) infection and to investigate the associated risk factors. Hemodialysis patients (n = 250) were interviewed and serum samples tested for anti-HCV antibodies by enzyme-linked immunosorbent assay (ELISA). All samples were also tested for HCV RNA by reverse transcriptase nested polymerase chain reaction (RT-nested-PCR). Out of 250 patients, 21 (8.4%) were found to be seropositive by ELISA, and 19 (7.6%) patients were HCV RNA positive. HCV viraemia was present in 90.5% of the anti-HCV positive patients. The predominant genotype was HCV 1a (8/19), followed by 3a (7/19), and 1b (4/19). None of the anti-HCV negative patients were shown to be viraemic by the PCR. Univariate analysis of risk factors showed that time spent on hemodialysis, the number of blood transfusions and a blood transfusion before November 1993 were associated with HCV positivity. However, multivariate analysis revealed that blood transfusions before November 1993 were significantly associated with HCV infection in this population. Low prevalence levels were encountered in this center, however prospective studies are necessary to confirm these findings.

Ocurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.