Effectiveness of the South African expanded program of immunization against hepatitis B in children infected with human immunodeficiency virus-1 living in a resource-limited setting of Kwazulu-Natal.

Prevalence of Human-Immunodeficiency-Virus/Hepatitis-B-virus (HIV/HBV) coinfection and HBV vaccination response in children are unknown in Kwazulu-Natal. This study included 183 HIV-infected and 108 HIV-uninfected children aged between 5 and 15 years screened for HBV infection and vaccination. HBV infection occurred in 2.1% and 0% of HIV-infected and uninfected children respectively. Serological response to immunization was shown in 15.8% and 61.1% of HIV-infected and uninfected children, respectively (P < 0.001). Even if prevalence of HBV infection was low in these cohorts, HIV-infected children will stay at risk of infection if the vaccine schedule is not adapted. J. Med. Virol. 89:182-185, 2017. © 2016 Wiley Periodicals, Inc.

Differentiated umbilical cord matrix stem cells as a new in vitro model to study early events during hepatitis B virus infection.

UNLABELLED: The role of cell differentiation state on hepatitis B virus (HBV) replication has been well demonstrated, whereas how it determines cell susceptibility to HBV entry is far less understood. We previously showed that umbilical cord matrix stem cells (UCMSC) can be differentiated towards hepatocyte-like cells in vitro. In this study we infected undifferentiated (UD-) and differentiated (D-) UCMSCs with HBV and studied the infection kinetics, comparing them to primary human hepatocytes (PHHs). UD-UCMSCs, although permissive to viral binding, had a very limited uptake capacity, whereas D-UCMSCs showed binding and uptake capabilities similar to PHHs. Likewise, asialoglycoprotein receptor (ASGPR) was up-regulated in UCMSCs upon differentiation. In D-UCMSCs, a dose-dependent inhibition of HBV binding and uptake was observed when ASGPR was saturated with known specific ligands. Subsequent viral replication was shown in D-UCMSCs but not in UD-UCMSCs. Susceptibility of UCMSCs to viral replication correlated with the degree of differentiation. Replication efficiency was low compared to PHHs, but was confirmed by (1) a dose-dependent inhibition by specific antiviral treatment using tenofovir; (2) the increase of viral RNAs along time; (3) de novo synthesis of viral proteins; and (4) secretion of infectious viral progeny. CONCLUSION: UCMSCs become supportive of the entire HBV life cycle upon in vitro hepatic differentiation. Despite low replication efficiency, D-UCMSCs proved to be fully capable of HBV uptake. Overall, UCMSCs are a unique human, easily available, nontransformed, in vitro model of HBV infection that could prove useful to study early infection events and the role of the cell differentiation state on such events.

Phylogeographical footprint of colonial history in the global dispersal of human immunodeficiency virus type 2 group A.

Human immunodeficiency virus type 2 (HIV-2) emerged in West Africa and has spread further to countries that share socio-historical ties with this region. However, viral origins and dispersal patterns at a global scale remain poorly understood. Here, we adopt a Bayesian phylogeographic approach to investigate the spatial dynamics of HIV-2 group A (HIV-2A) using a collection of 320 partial pol and 248 partial env sequences sampled throughout 19 countries worldwide. We extend phylogenetic diffusion models that simultaneously draw information from multiple loci to estimate location states throughout distinct phylogenies and explicitly attempt to incorporate human migratory fluxes. Our study highlights that Guinea-Bissau, together with Côte d'Ivoire and Senegal, have acted as the main viral sources in the early stages of the epidemic. We show that convenience sampling can obfuscate the estimation of the spatial root of HIV-2A. We explicitly attempt to circumvent this by incorporating rate priors that reflect the ratio of human flow from and to West Africa. We recover four main routes of HIV-2A dispersal that are laid out along colonial ties: Guinea-Bissau and Cape Verde to Portugal, Côte d'Ivoire and Senegal to France. Within Europe, we find strong support for epidemiological linkage from Portugal to Luxembourg and to the UK. We demonstrate that probabilistic models can uncover global patterns of HIV-2A dispersal providing sampling bias is taken into account and we provide a scenario for the international spread of this virus.

HIV-1 low-level viraemia assessed with 3 commercial real-time PCR assays show high variability.

BACKGROUND: Current real-time PCR-based HIV-1 viral load (VL) assays allow the detection of residual viraemia in antiretroviral-treated patients. The clinical outcome of HIV1 patients experiencing low-level replication (<50 cop/mL) in comparison with fully suppressed patients is currently debated. We analysed variability of 3 VL assays <50 cop/mL, and evaluated the reproducibility of viral blips <100 cop/mL. METHODS: Three commercial VL assays were tested: Versant HIV-1 RNA 1.0 kPCR (Siemens), Abbott Realtime HIV-1, and Cobas Ampliprep/Cobas Taqman HIV-1 v2.0 (Roche). Ten replicates of a reference sample at 4 low target dilutions were tested to evaluate assay variability. Prospective collection of 181 clinical samples with detectable VL <50 cop/mL was used to evaluate intra-and inter-assay variability by triplicate testing. Samples from 26 patients experiencing a viral blip were retested. RESULTS: All assays showed substantial variability at low VL level: the coefficient of variation at 100, 50, 25 and 12 cop/mL ranged respectively from 32 to 44%, 35 to 68%, 41 to 83% and 33 to 77%. In the intra-assay evaluation of repeatability, 52.5 to 57.5% of detectable VL <50 cop/mL tested in triplicate showed at least one fully undetected result. Variability was similar in the inter-assay arm. The VL blips could only be reproduced in 19% of cases. CONCLUSIONS: The most recent versions of widespread commercial VL assays showed substantial variability at low levels and residual viraemia could not be consistently reproduced. Patient outcome studies comparing residual VL to full suppression are therefore biased when using commercial assays.

Immunogenicity of an adjuvanted 2009 pandemic influenza A (H1N1) vaccine in haemodialysed patients.

BACKGROUND: The 2009 pandemic of influenza A (H1N1) prompted an urgent worldwide vaccination campaign, especially of high-risk subjects, such as maintenance haemodialysis (HD) patients. Still the immunogenicity of the pandemic A (H1N1) vaccine in HD patients is unknown. METHODS: We prospectively studied the immunogenicity of a monovalent adjuvanted influenza A/California/2009 (H1N1) vaccine (Pandemrix, GSK Biologicals, Rixensart, Belgium) in HD patients and controls. Antibody level was measured using a seroneutralization assay before (D(0)) and 30 days after (D(30)) a single 3.75 µg vaccine dose. Specimens were tested in quadruplicates. Geometric mean (GM) antibody titers were determined in each subject at D(0) and D(30). Seroconversion was defined as an increase in GM titers by a factor 4 or more. RESULTS: Fifty-three adult HD patients [aged 71 ± 10, 58.5% males, on HD for a median of 38 (3 - 146) months] and 32 control subjects (aged 47.3 ± 14, 31.3% males) were analyzed. Baseline GM titers were similar in HD patients and controls [7.9 (6.6 - 9.6) vs 10 (6 - 17); p = 0.69]. Seroconversion was observed in 30 (93.8%) controls and 34 (64.2%) HD patients (p = 0.002). In addition, GM titers at D(30) were significantly higher in controls than in HD patients [373 (217 - 640) vs 75.5 (42.5 - 134); p = 0.001]. HD patients were significantly older than controls (p < 0.001) and more likely to be males (p = 0.02). However, by multivariate analysis, HD status [OR 0.13 (0.02-0.78), p = 0.03], but neither age [OR 0.99 (0.96 - 1.03); p = 0.7] nor male gender [OR 1.31 (0.45 - 3.85); p = 0.63] was independently associated with seroconversion. The vaccine was generally well tolerated by HD patients. CONCLUSIONS: Only 64% of chronic HD patients developed seroconversion after a single dose of adjuvanted influenza A (H1N1) vaccine, a much lower rate than in controls (94%). These results underscore the substantial immunodeficiency associated with End-Stage Renal Disease. The persistence of protective antibodies as well as the effect of a booster dose remain to be investigated in HD patients.

Polymorphisms of HIV-2 integrase and selection of resistance to raltegravir.

BACKGROUND: Human Immunodeficiency Virus type 2 is naturally resistant to some antiretroviral drugs, restricting therapeutic options for patients infected with HIV-2. Regimens including integrase inhibitors (INI) seem to be effective, but little data on HIV-2 integrase (IN) polymorphisms and resistance pathways are available. MATERIALS AND METHODS: The integrase coding sequence from 45 HIV-2-infected, INI-naïve, patients was sequenced and aligned against the ROD (group A) or EHO (group B) reference strains and polymorphic or conserved positions were analyzed.To select for raltegravir (RAL)-resistant variants in vitro, the ROD strain was cultured under increasing sub-optimal RAL concentrations for successive rounds. The phenotype of the selected variants was assessed using an MTT assay. RESULTS: We describe integrase gene polymorphisms in HIV-2 clinical isolates from 45 patients. Sixty-seven percent of the integrase residues were conserved. The HHCC Zinc coordination motif, the catalytic triad DDE motif, and AA involved in IN-DNA binding and correct positioning were highly conserved and unchanged with respect to HIV-1 whereas the connecting residues of the N-terminal domain, the dimer interface and C-terminal LEDGF binding domain were highly conserved but differed from HIV-1. The N155 H INI resistance-associated mutation (RAM) was detected in the virus population from one ARV-treated, INI-naïve patient, and the 72I and 201I polymorphisms were detected in samples from 36 and 38 patients respectively. No other known INI RAM was detected.Under RAL selective pressure in vitro, a ROD variant carrying the Q91R+I175M mutations was selected. The Q91R and I175M mutations emerged simultaneously and conferred phenotypic resistance (13-fold increase in IC50). The Q91R+I175M combination was absent from all clinical isolates. Three-dimensional modeling indicated that residue 91 lies on the enzyme surface, at the entry of a pocket containing the DDE catalytic triad and that adding a positive charge (Gln to Arg) might compromise IN-RAL affinity. CONCLUSIONS: HIV-2 polymorphisms from 45 INI-naïve patients are described. Conserved regions as well as frequencies of HIV-2 IN polymorphisms were comparable to HIV-1. Two new mutations (Q91R and I175M) that conferred high resistance to RAL were selected in vitro, which might affect therapeutic outcome.

Drug resistance in HIV-infected children living in rural South Africa: Implications of an antiretroviral therapy initiated during the first year of life.

INTRODUCTION: Management of antiretroviral-drug resistance in HIV-infected children is a global health concern. We compared the long-term virological outcomes of two cohorts of children living in a rural setting of South Africa. The first cohort initiated treatment before one year and the second after two years of age. The aim of this study was to describe the long-term consequences of early treatment initiation in terms of viral load and drug-resistance. METHODS: This retrospective study was conducted at the Edendale Hospital located in a peri-urban area of KwaZulu-Natal. Children were included during their planned appointment. Drug resistance was assessed genotypically on proviral DNA. RESULTS: From the 161 children included in this study, 93 samples were successfully genotyped. Both cohorts had comparable viral loads, but children treated early more often presented NRTI or NNRTI mutations, while there was no difference for PI mutations rates. CONCLUSIONS: Treatment was highly effective when comparing virological outcomes in both early- and late-treated cohorts. The persistence of NNRTI mutations could lead to treatment failures in children older than 3 years initiating their therapy with a NNRTI, or for those switching from a PI to NNRTI based regimen. The accumulation of NRTI mutations may lead to a functional PI monotherapy and consequently to viral escape. To promote access to HIV genotyping in resource-limited settings is challenging but essential to avoid inappropriate therapy switches in case of virological failure, and to adapt national treatment guidelines in line with the epidemiology of resistance.

Prevalence and epidemiology of HIV type 1 drug resistance among newly diagnosed therapy-naive patients in Belgium from 2003 to 2006.

This study is the first prospective study to assess the prevalence, epidemiology, and risk factors of HIV-1 drug resistance in newly diagnosed HIV-infected patients in Belgium. In January 2003 it was initiated as part of the pan-European SPREAD program, and continued thereafter for four inclusion rounds until December 2006. Epidemiological, clinical, and behavioral data were collected using a standardized questionnaire and genotypic resistance testing was done on a sample taken within 6 months of diagnosis. Two hundred and eighty-five patients were included. The overall prevalence of transmitted HIV-1 drug resistance in Belgium was 9.5% (27/285, 95% CI: 6.6-13.4). Being infected in Belgium, which largely coincided with harboring a subtype B virus, was found to be significantly associated with transmission of drug resistance. The relatively high rate of baseline resistance might jeopardize the success of first line treatment as more than 1 out of 10 (30/285, 10.5%) viruses did not score as fully susceptible to one of the recommended first-line regimens, i.e., zidovudine, lamivudine, and efavirenz. Our results support the implementation of genotypic resistance testing as a standard of care in all treatment-naive patients in Belgium.

Transmitted drug resistance, selection of resistance mutations and moderate antiretroviral efficacy in HIV-2: analysis of the HIV-2 Belgium and Luxembourg database.

BACKGROUND: Guidelines established for the treatment of HIV-1 infection and genotype interpretation do not apply for HIV-2. Data about antiretroviral (ARV) drug efficacy and resistance mutations is scarce. METHODS: Clinical data about HIV-2 infected patients in Belgium and Luxembourg were collected and the effect of ARV therapy on plasma viral load and CD4 counts were analysed. Viral RNA encoding for protease (PR) and reverse transcriptase (RT) from ARV-naïve and treated patients were sequenced. RESULTS: Sixty-five HIV-2 infected patients were included in this cohort. Twenty patients were treated with 25 different ARV combinations in a total of 34 regimens and six months after the start of ARV therapy, only one third achieved viral load suppression. All of these successful regimens bar one contained protease inhibitors (PIs). Mean CD4 gains in the group of viral load suppressors and the group of patients treated with PI-containing regimens were respectively significantly higher than in the group of non-suppressors and the group of PI-sparing regimens. The most frequent mutations selected under therapy (compared to HIV-2 ROD) were V71I, L90M and I89V within PR. Within RT, they were M184V, Q151M, V111I and K65R. All of these mutations, except K65R and M184V, were also found in variable proportions in ARV-naïve patients. CONCLUSION: Despite a high rate of ARV treatment failure, better virological and immunological results were achieved with PI-containing regimens. The analysis of polymorphic positions and HIV-2 specific mutations selected during therapy showed for the first time that transmission of drug resistant viruses has occurred in Belgium and Luxembourg. The high heterogeneity in ARV combinations reflects a lack of guidelines for the treatment of HIV-2 infection.

[Emerging viral infections in Belgium and abroad]. / Les maladies virales émergentes en Belgique et ailleurs.

Viral diseases are a moving world with epidemic upsurges, the disappearance of some infections and the temporary or definitive appearance of others. Each year our country is stricken by flu epidemics, but at some points of time unexpected new flu viruses may cause a worldwide pandemic. Presently a number of viral diseases have disappeared or are disappearing from our country, as smallpox, poliomyelitis, rabies, measles or rubella. Many factors, some changing others more persistent, may explain the high appearance rate of new viral infections: increasing intercontinental travel, globalization of transportation, increased contacts of man with new ecological niches, great variability and adaptability of viruses, climate changes, increasing density of populations and urbanisation, changing human behaviour (more sexual freedom, use of intravenous drugs, etc...), changing social and economic conditions, which influence housing, nutrition, access to water, etc...). Viruses take all opportunities in our changing world and have many more surprises in store for us.

Modulation of the NF-κB signaling pathway by the HIV-2 envelope glycoprotein and its incomplete BST-2 antagonism.

The HIVs have evolved by selecting means to hijack numerous host cellular factors. HIVs exploit the transcription factor NF-κB to ensure efficient LTR-driven gene transcription. However, NF-κB is primarily known to act as a key regulator of the proinflammatory and antiviral responses. Interestingly, retroviruses activate NF-κB during early stages of infection to initiate proviral genome expression while suppressing it at later stages to restrain expression of antiviral genes. During HIV-1 infection, diverse viral proteins such as Env, Nef and Vpr have been proposed to activate NF-κB activity, whereas Vpu has been shown to inhibit NF-κB activation. It is still unclear how HIV-2 regulates NF-κB signaling pathway during its replication cycle. Here we confirm that human BST-2 and HIV-1 Env proteins can trigger potent activation of NF-κB. Importantly, we demonstrate for the first time that the HIV-2 Env induces NF-κB activation in HEΚ293T cells. Furthermore, the anti-BST-2 activity of the HIV-2 Env is not sufficient to completely inhibit NF-κB activity.

Genetic polymorphisms and resistance mutations of HIV type 2 in antiretroviral-naive patients in Burkina Faso.

Natural polymorphisms in the pol gene of HIV-2 may influence the susceptibility to antiretroviral drugs and the choice of treatment. We collected samples in centers for anonymous HIV testing in Ouagadougou, Burkina Faso, in patients supposedly naive for any antiretroviral treatment. Eighty-four samples were first tested as HIV-2 positive in Burkina Faso and then shipped to Brussels, Belgium, for confirmation of the serological status and plasma viral load. Fifty-two samples were confirmed as HIV-2 positive in Belgium. Twelve others were HIV-1 positive and 20 were dually reactive. Twenty-one of HIV-2 confirmed samples had an HIV-2 plasma viral load higher than 1000 copies/ml. These viruses were sequenced in the protease and reverse trancriptase genes and 17 sequences of the pol gene were obtained. Highly polymorphic positions were identified in protease and RT genes. Two samples harbored known resistance mutations: M184V RT mutation in one and Q151M with M184V in the other. Phylogenetic analysis showed that viruses in Burkina Faso did not cluster separately from published sequences from neighboring countries. The two resistant strains were unrelated. Our findings imply either that resistant viruses are circulating in Burkina Faso or that some individuals take unsupervised treatment. Both hypotheses present problems.

Specific and quantitative detection of human polyomaviruses BKV and JCV by LightCycler real-time PCR.

BACKGROUND: BK virus (BKV) and JC virus (JCV) are the only two known human polyomavirus that typically establish subclinical persistent infections. In immunocompromised hosts reactivation of the JCV infection is the cause of the central nervous system disease progressive multifocal leucoencephalopathy (PML), while BKV may cause renal nephropathy and haemorrhagic cystitis. OBJECTIVES: The goal of this study was to develop a specific quantification assay for each polyomavirus by LightCycler real-time polymerase chain reaction (PCR) based on SYBR Green I detection. STUDY DESIGN: DNA fragments of 138bp and 233bp from the "large T antigen" region of JCV and BKV, respectively, were amplified. The ability of the designed primer sets to separately quantify BKV DNA or JCV DNA and the PCR efficiency were assessed on reference DNA samples. Known amounts of cloned JCV DNA and BKV DNA from TEBU-BIO nv (Boechout, Belgium) were used to generate standard curves for the quantification assays. Species-specificity of the PCR was evaluated with cloned DNA and with DNA from patient samples. RESULTS: The assay allowed a specific quantification over a 7log dynamic range. Seventeen copies each of the viral genes were reproducibly and accurately detected. The primer sets generated specific DNA fragments for each virus confirmed by agarose gel analysis and by cycle sequencing. The similarities of the amplified gene sequences by BLAST analysis were 99% and 100% for BKV and JCV, respectively. There was no cross-reactivity within the dynamic range of the standard dilutions. CONCLUSIONS: We developed LightCycler real-time PCR assay based on SYBR Green I detection that provided rapid and specific quantification of polyomavirus load.

Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism.

BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2) relies on the envelope glycoprotein (Env) to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET) experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletion that is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involving the asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail.

Molecular testing of multiple HIV-1 transmissions in a criminal case.

OBJECTIVE: To test the a priori hypothesis of HIV-1 transmission from one suspect to six recipients in a criminal case. METHODS: Partial pol and/or env sequences were obtained for at least two samples of the suspect and the victims. Appropriate local controls were sampled based on epidemiological and subtype criteria. Phylogenetic testing was performed using different reconstruction methods. RESULTS: Phylogenetic analyses consistently inferred a monophyletic cluster for the suspect and victim samples in both genome regions. This was highly supported by parametric and non-parametric bootstrapping techniques. Moreover, the controls most closely related to the suspect-victim cluster had a similar geographical origin to the suspect. CONCLUSIONS: Taking into account the limitations on the conclusions that can be drawn from molecular investigations we could infer that our molecular data is consistent with a scenario of multiple HIV transmission between suspect and victims.

Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler real-time PCR.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART. In the present study, a relative quantification assay of HIV-1 proviral DNA by LightCycler real-time PCR based on SYBR Green I detection was developed in comparison to the number of purified CD4+ cells as estimated by the quantification of the beta-globin gene. METHODS: The ability of the designed gag primers to quantify HIV-1 Group M and the PCR efficiency were assessed on HIV-1 reference isolate subtypes A, B, C and D. The 8E5 cell line containing a single defective copy of HIV-1 proviral DNA was used as a standard for both the HIV-1 target gene and the beta-globin reference gene. The assay was applied on thirty consecutive patient samples received for RNA viral load determinations and on retrospective samples from fifteen patients undergoing 2 years of structured treatment interruption (STI). RESULTS: The lower limit of quantification was 50 HIV-1 DNA proviral copies per CD4+ cell sample. The dynamic range was from 50 to 106 HIV-1 DNA copies per CD4+ cell sample with intra- and inter-assay coefficients of variability ranging from 3.1% to 37.1%. The beta-globin reference gene was quantified down to a limit of 1.5 pg of DNA/microl (approximately 5 cells) with intra- and interassay coefficients of variability ranging from 1.8% to 21%. DNA proviral load varies widely among HIV-1 infected patients. Proviral load and plasma viral load rebound were high in STI patients who took longer to achieve an undetectable plasma viral load under therapy. A statistically significant correlation was observed between DNA proviral load and RNA steady state viral load in STI patients (p-value = 0.012). CONCLUSION: We have developed a fast, sensitive and specific relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells. The assay enables the monitoring of HIV-1 proviral load, which may be useful to monitor therapy efficacy especially in patients with undetectable plasma RNA viral load, and allows the exploration of viral reservoirs.

Prevalence of HIV co-receptor polymorphisms in HIV-infected patients and uninfected volunteers in Luxembourg.

PURPOSE: To describe the prevalence of nine HIV-1 co-receptor polymorphisms in Luxembourg (CCR5-Delta 32, CCR5-58755-A/G, CCR5m303, CCR5-59029-A/G, CCR2-64I, CCR5-59653-C/T, CX(3) CR1-V249I, CX(3) CR1-T280M, and SDF1-3'A) in 288 HIV-1-infected patients and in 158 uninfected, healthy volunteers. METHOD: The presence of mutations was detected using PCR-restriction fragment length polymorphism (RFLP)-based methods. RESULTS: We did not find any significant differences in genotype distributions and allele frequencies between infected and uninfected participants in Luxembourg. The distribution of genotypes agreed in all groups with Hardy-Weinberg predicted proportions. CCR5-Delta 32 was significantly associated with HIV-1-infected slow progressor patients. CONCLUSION: Overall, allele frequencies were comparable to frequencies reported in previous studies in Caucasian populations.

Quantitative real-time PCR on Lightcycler for the detection of human immunodeficiency virus type 2 (HIV-2).

Similar to HIV-1, the viral load in HIV-2 is a marker of the evolution of infection and the success of therapy. No approved or commercially distributed assay exists to determine the plasma viral load of HIV-2. We therefore developed a quantitative real-time PCR to determine the plasma RNA viral load of HIV-2, based on the reference strains ROD and EHO, which represent subtypes A and B of HIV-2, respectively. After testing several pairs of primers, a set was chosen that recognised the 5'-LTR region of a subtypes A and B consensus sequence. The quantification of the PCR reaction was done using SYBR-Green I as the fluorescent dye in a Lightcycler system and relying on an external standard curve. The method was then optimised with reference strains, an validated further using patient samples and an inter-laboratory comparison. Good specificity was obtained for both subtypes of HIV-2, and a linear range between 10 and 10(6) copies of viral RNA. The limit of quantitation was 250 copies per millilitre of plasma. The coefficient of variation ranged from 0.7 to 5.6%, depending on the concentration of the target sequences.

Stable hepatitis C virus RNA detection by RT-PCR during four days storage.

BACKGROUND: Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV) RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4 degrees C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples. METHODS: Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 - 25.4 degrees C) or at 4 degrees C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems). RESULTS: The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4 degrees C). CONCLUSIONS: We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4 degrees C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results.

Western blot seroindeterminate individuals for human T-lymphotropic virus I/II (HTLV-I/II) in Fortaleza (Brazil): a serological and molecular diagnostic and epidemiological approach.

How to handle Western blot (WB) seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2) constitutes a challenge for blood banks and families. We made a cross-sectional study of 191 enzyme linked immunoassay (EIA) reactive individuals from the hematological center (HEMOCE) of Fortaleza (Brazil), examining their serological (WB) and molecular (PCR) diagnosis, and demographic profiles, as well as a possible association of their condition with other infectious pathologies and risk factors. Ethical institutional approval and personal consent were obtained. Out of 191 EIA reactive individuals, 118 were WB seroindeterminate and 73 were seropositive for HTLV-1/2. In the PCR analysis of 41 WB seroindeterminate individuals, 9 (22%) were positive and 32 (78%) were negative for HTLV-1/2. The demographic analysis indicated a trend towards a predominance of males among the seroindeterminate individuals and females in the seropositive ones. The seroindeterminate individuals were younger than the seropositive ones. We did not find any association of these conditions with syphilis, Chagas disease or HIV or hepatitis, and with risk factors such as breast-feeding, blood transfusion, STD (syphilis) and IDU.