Characterization of Torquetenovirus in amniotic fluid at the time of in utero fetal surgery: correlation with early premature delivery and respiratory distress.

Torquetenovirus (TTV) is a commensal virus present in many healthy individuals. Although considered to be non-pathogenic, its presence and titer have been shown to be indicative of altered immune status in individuals with chronic infections or following allogeneic transplantations. We evaluated if TTV was present in amniotic fluid (AF) at the time of in utero surgery to correct a fetal neurological defect, and whether its detection was predictive of adverse post-surgical parameters. AF was collected from 27 women by needle aspiration prior to a uterine incision. TTV titer in the AF was measured by isolation of viral DNA followed by gene amplification and analysis. The TTV genomes were further characterized and sequenced by metagenomics. Pregnancy outcome parameters were subsequently obtained by chart review. Three of the AFs (11.1%) were positive for TTV at 3.36, 4.16, and 4.19 log10 copies/mL. Analysis of their genomes revealed DNA sequences similar to previously identified TTV isolates. Mean gestational age at delivery was >2 weeks earlier (32.5 vs. 34.6 weeks) and the prevalence of respiratory distress was greater (100% vs. 20.8%) in the TTV-positive pregnancies. TTV detection in AF prior to intrauterine surgery may indicate elevated post-surgical risk for earlier delivery and newborn respiratory distress.

CCR5 genotypes and progression to HIV disease in perinatally infected children.

The CCR5 molecule, a chemokine receptor, is the most important co-receptor for macrophage-tropic HIV-1. A 32-bp deletion in the gene encoding CCR5 (CCR5-del32) confers nearly complete resistance to HIV-1 infection in homozygotes, and slows the rate of progression to AIDS in heterozygous adults. The aim of this study was to describe the CCR5 genotypes and the characteristics of HIV disease progression in perinatally infected children. From a total of 51 children analyzed for the CCR5-del32 mutation, 18 (35%) were considered to be rapid progressors, 28 (55%) were moderate progressors and 5 (10%) were slow progressors. A portion of the CCR5 gene was amplified by PCR from genomic DNA followed by agarose gel electrophoresis. Forty-nine children (96%) carried the homozygous wild type genotype for CCR5 while 2 (4%) carried the heterozygous wt/del32 genotype. In the population studied, the CCR5 genotype was unable to account for the differences in pattern of the disease progression among the three groups (rapid, moderate and slow progressors), and the allele frequency of CCR5-del32 was too low to allow statistical comparisons with adequate resolving power. Studies on larger populations may help to further elucidate the role of this allele and other host factors in the regulation of HIV-1 pathogenesis in children.

Analysis of HIV- type 1 protease and reverse transcriptase in Brazilian children failing highly active antiretroviral therapy (HAART).

The aim of this study was to evaluate the genotypic resistance profiles of HIV-1 in children failing highly active antiretroviral therapy (HAART). Forty-one children (median age = 67 months) receiving HAART were submitted to genotypic testing when virological failure was detected. cDNA was extracted from PBMCs and amplified by nested PCR for the reverse transcriptase and protease regions of the pol gene. Drug resistance genotypes were determined from DNA sequencing. According to the genotypic analysis, 12/36 (33.3%) and 6/36 (16.6%) children showed resistance and possible resistance, respectively, to ZDV; 5/36 (14%) and 4/36 (11.1%), respectively, showed resistance and possible resistance to ddI; 4/36 (11.1%) showed resistance to 3TC and D4T; and 3/36 (8.3%) showed resistance to Abacavir. A high percentage (54%) of children exhibited mutations conferring resistance to NNRTI class drugs. Respective rates of resistance and possible resistance to PIs were: RTV (12.2%, 7.3%); APV (2.4%, 12.1%); SQV(0%, 12.1%); IDV (14.6%, 4.9%), NFV (22%, 4.9%), LPV/RTV (2.4%, 12.1%). Overall, 37/41 (90%) children exhibited virus with mutations related to drug resistance, while 9% exhibited resistance to all three antiretroviral drug classes.

Seroreactivity to Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) latent nuclear antigen in AIDS-associated Kaposi's sarcoma patients depends on CD4+ T-cell count.

In AIDS/Kaposi's sarcoma (KS) patients, the sensitivity of immunofluorescence assays for detecting antibodies against latent nuclear antigen ranges from 52% to 93%. However, in classic and African KS, sensitivities above 90% have been reported systematically. This study evaluates whether CD4+ T-cell count affects seroreactivity to KSHV LANA and to lytic antigens in AIDS/KS patients. Kaposi's sarcoma-associated herpesvirus (KSHV) latent (IFA-LANA) and lytic (IFA-Lytic and ORF65/K8.1 EIA) antibodies were screened in 184 consecutive samples taken from 36 AIDS/KS patients grouped according to their CD4+ counts as follows: <100 (group A), 100-300 (group B), and >300 (group C) cells/mm(3). At enrollment, the immunofluorescence assay for the detection of antibodies against latent nuclear antigen (IFA-LANA) was positive in 3/11(27.2%) group A patients, in 10/11 (90.9%) group B patients, and in 14/14 (100%) group C patients (P < 0.01). Seropositivity to lytic antigens did not differ according to CD4+ T-cell count. Considering IFA-Lytic and ORF65/K8.1 EIA, seropositivity for lytic antigens was 100% in all three patient groups. In patients whose CD4+ count improved during follow-up, IFA-LANA seroconversion occurred; unstable counts resulted in a decrease in LANA antibody titers while the persistence of high counts resulted in unchanged, elevated antibody titers. In conclusion, LANA seroreactivity in AIDS/KS patients, as assessed by an immunofluorescence assay, depends on CD4+ T-cell count, rendering this evaluation important in the interpretation of seroepidemiological studies of KSHV infection in AIDS patients. To evaluate future serological tests based on latency-associated antigens, the selection of sera from KS patients with CD4+ cell count >300 cells/mm(3) as a positive gold standard is recommended.

CCR5 genotypes and progression to HIV disease in perinatally infected children

The CCR5 molecule, a chemokine receptor, is the most important co-receptor for macrophage-tropic HIV-1. A 32-bp deletion in the gene encoding CCR5 (CCR5-del32) confers nearly complete resistance to HIV-1 infection in homozygotes, and slows the rate of progression to AIDS in heterozygous adults. The aim of this study was to describe the CCR5 genotypes and the characteristics of HIV disease progression in perinatally infected children. From a total of 51 children analyzed for the CCR5-del32 mutation, 18 (35 percent) were considered to be rapid progressors, 28 (55 percent) were moderate progressors and 5 (10 percent) were slow progressors. A portion of the CCR5 gene was amplified by PCR from genomic DNA followed by agarose gel electrophoresis. Forty-nine children (96 percent) carried the homozygous wild type genotype for CCR5 while 2 (4 percent) carried the heterozygous wt/del32 genotype. In the population studied, the CCR5 genotype was unable to account for the differences in pattern of the disease progression among the three groups (rapid, moderate and slow progressors), and the allele frequency of CCR5-del32 was too low to allow statistical comparisons with adequate resolving power. Studies on larger populations may help to further elucidate the role of this allele and other host factors in the regulation of HIV-1 pathogenesis in children.

Analysis of HIV- type 1 protease and reverse transcriptase in Brazilian children failing highly active antiretroviral therapy (HAART)

O objetivo deste estudo foi avaliar o perfil de resistência genotípica do HIV-1 em crianças com falha terapêutica ao tratamento anti-retroviral (HAART). Quarenta e uma crianças (idade mediana = 67 meses) em uso de HAART foram submetidas ao teste de genotipagem no momento da detecção de falha ao tratamento. Foi realizada extração de cDNA de células periféricas mononucleares e amplificação do mesmo (regiões da transcriptase reversa e protease do gene pol) através de PCR-nested. O perfil genotípico foi determinado através do seqüenciamnto de nucleotídeos. De acordo com a análise genotípica, 12/36 (33,3%) e 6/36 (16,6%) crianças apresentaram, respectivamente, resistência e possível resistência ao AZT; 5/36 (14%) e 4/36 (11,1%), respectivamente, eram resistentes e possivelmente resistentes ao ddI; 4/36 %11,1%) apresentaram resistência ao 3TC e D4T, e 3/36 (8,3%) eram resistentes ao ABC. Uma alta porcentagem de crianças (54%) apresentou mutações relacionadas à resistência aos inibidores da trancriptase reversa não-análogos de nucleosídeos. As taxas de resistência e possível resistência aos inibidores da protease foram, respectivamente: RTV (12,2%; 7,3%); APV (2,4%; 12,1%); SQV (0%; 12,1%); IDV (14,6%; 4,9%); NFV (22%; 4,9%); LPV/RTV (2,4%; 12,1%). No total, 37/41 (90%) crianças apresentaram vírus com mutações relacionadas à resistência a alguma droga, sendo que 9% delas tinham vírus resistentes às três classes de drogas anti-retrovirais disponíveis.