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Background: Management of a novel respiratory virus causing severe pneumonitis included the use of antibiotics to prevent bacterial co-infections and secondary infections. However, the impact of this antibiotic use on the selection of resistant bacterial isolates needs to be evaluated. Methods: We conducted a single-center retrospective study from November 14, 2020 to December 31, 2021 to assess the prevalence of several members of the nasopharyngeal microbiota from PCR-positive SARS-CoV-2 subjects. The study population corresponded to 1030 nasopharyngeal swabs positive for SARS-CoV-2 at the university hospital of Rouen site in symptomatic patients aged 16 years and older. Real-time PCR was used to detect the presence of Haemophilus influenzae, Streptococcus pneumonia, Neisseria meningitidis and influenza A virus. An analysis of the ftsI gene was further used to analyze beta-lactam resistance in H. influenzae. Results: The results reveled less than expected carriage rate with 5% for H. influenzae, 1.2% for N. meningitidisand 3.7% for S. pneumoniae and an absence of influenza A. On the other hand, there was a significant difference (pftsI gene reveals 26% of predicted resistance to amoxicillin without resistance to third generation cephalosporins. Conclusions: COVID-19 pandemic has disrupted bacterial and viral epidemiology, leading to lower circulation of several respiratory pathogens.

HIV-1 is characterized by high genetic diversity and genetic recombination is one of the major evolution processes. Despite their great genetic divergence, HIV-1 group M, pandemic, and group O, endemic in Cameroon, can generate HIV-1/MO intergroup recombinants. The current description of 19 HIV-1/MO recombinant forms (URF_MO) raises the question of a possible benefit of the recombination and the modalities of their emergence.Therefore, the objectives of this work were to studyin vitrothe replicative potential of HIV-1/MO recombinant forms.The replicative potential was analyzed, based on a simple recombination pattern, [Ogag/pol-Menv], harboring a breakpoint in Vpr, due to a recombination hotspot in this region. For this, a chimeric infectious molecular clone (IMC) was synthesized from HIV-1/M subtype B and HIV-1/O subgroup T and recombinant viruses were obtained by transfection and co-culture. To compare the replicative potential of recombinant viruses with HIV-1/M and HIV-1/O parental viruses, two markers were monitored in culture supernatants: Reverse Transcriptase (RT) activity and P24 antigen concentration. The results showed a superiority of the group M parental virus compared to group O for both markers. In contrast, for the HIV-1/OM recombinant virus, RT activity data did not overlap with the concentration of P24 antigen, suggesting a hybrid behavior of the recombinant, in terms of enzyme activity and P24 production.These results highlighted many hypotheses about the impact of recombination on replicative potential and demonstrated once again the significant plasticity of HIV genomes and their infinite possibility of evolution.ImportanceHIV-1/M and HIV-1/O can generate HIV-1/MO intergroup recombinants. The current description of 19 URF_MO raises the question of a possible benefit of recombination in terms of emergence. The objectives of this work were to studyin vitrothe replicative potential of HIV-1/MO recombinant viruses. For this, a chimeric infectious molecular clone (IMC) generated from HIV-1/M subtype B and HIV-1/O subgroup T and recombinant viruses were obtained by transfection and co-culture. To compare the replicative potential of recombinant viruses with HIV-1/M and HIV-1/O parental viruses, RT activity and P24 antigen concentration were monitored in culture supernatants. A superiority of the group M parental virus compared to group O was observed for both markers whereas a hybrid behavior in terms of enzyme activity and P24 production was found for the recombinant virus. These results demonstrated once again the significant plasticity of HIV genomes and their infinite possibility of evolution.

Objectives HIV-1 group O (HIV-1/O) is one of the four HIV-1 groups and is endemic in Cameroon, representing 1% of HIV-1 infections in the population. Around 50% of the strains of this group naturally show a mutation (Y181C) providing them with resistance to NNRTIs and making therapeutic management more difficult. Today, the WHO recommends the use of integrase strand transfer inhibitors (INSTIs) as first-line treatment. Bictegravir and cabotegravir are the two most recent INSTIs. Because of the genetic polymorphism of HIV-1/O, studies are required to evaluate their phenotypic susceptibility to these two drugs. Patients and methods We performed a phenotypic study on a large panel including 41 HIV-1/O clinical isolates and other rare non-group M HIV-1 (2 HIV-1/N and 1 HIV-1/P) to evaluate in vitro susceptibility to bictegravir and cabotegravir. Results The results showed an overall susceptibility of non-group M strains to the two drugs compared with HIV-1 group M. There was no difference between the mean (min–max) IC50 of HIV-1/M [1.86 (0.93–4.12) and 5.24 (1.76–12.41) nM for bictegravir and cabotegravir, respectively] and HIV-1/non-M [2.17 (0.03–9.47) and 4.88 (0.02–15.64) nM for bictegravir and cabotegravir, respectively]. However, we found a significant difference between IC50 values for bictegravir and cabotegravir in the whole panel (P value Conclusions This study has shown encouraging results regarding the clinical use of these drugs in HIV-1/non-M-infected patients, which will need to be confirmed with clinical data.

Background Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity. Methods We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection. Results For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs. Conclusion Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

International audience; Objective: HIV-1 transmission leads to a genetic bottleneck, with one or a few variants of the donor quasispecies establishing an infection in the new host. We aimed to characterize this bottleneck in more detail, by comparing the properties of HIV envelope glycoproteins from acute and chronic infections within the particular context of a male-to-male transmission cluster. Design: We compared the genotypic and phenotypic properties of envelope glycoproteins from viral variants derived from five study participants from the same transmission cluster. Methods: We used single-genome amplification to generate a collection of full-length env sequences. We then constructed pseudotyped viruses expressing selected Env variants from the quasispecies infecting each study participant and compared their infectivities and sensitivities to various entry inhibitors. Results: The genotypic analyses confirmed the genetic bottleneck expected after HIV transmission, with a limited number of variants identified in four study participants during acute infection. However, the transmitted sequences harbored no evident common signature and belonged to various genetic lineages. The phenotypic analyses revealed no difference in infectivity, susceptibility to the CCR5 antagonist maraviroc, the fusion inhibitor enfurvitide or type-I interferon between viruses from participants with acute and chronic infections. The key property distinguishing transmitted viruses was a higher resistance to soluble CD4 þ , correlated with greater sensitivity to occupation of the CD4 þ receptor by the anti-CD4 þ antibodies LM52 and SK3. Conclusion: These results suggest that envelope glycoproteins from transmitted/ founder viruses bind CD4 þ less efficiently than those of viruses from chronic infections.

In February 2022, samples collected in northwest France showed discordant molecular results. After virological and epidemiological investigations, 17 cases of Deltacron XD recombinant severe acute respiratory syndrome coronavirus 2 were confirmed by sequencing or suspected due to epidemiological links, showing evidence of an extended transmission event and circulation of this form, with low clinical severity.

International audience; Three confirmed infections with the SARS-CoV-2 B.1.640 variant under monitoring were reported in Normandy, north-western France in late November 2021. Investigations led to the identification of two events linked to the same cluster. A total of 75 confirmed and probable B.1.640 cases were reported. All had completed the primary vaccination series. Sixty-two cases were older than 65 years. Fifty-six cases had symptoms and four were hospitalised. This investigation provides preliminary results concerning a variant with limited information currently available.

To eliminate HIV infection, there are several elements to take into account to limit transmission and break viral replication, such as epidemiological, preventive or therapeutic management. The UNAIDS goals of screening, treatment and efficacy should allow for this elimination if properly followed. For some infections, the difficulty is linked to the strong genetic divergence of the viruses, which can impact the virological and therapeutic management of patients. To completely eliminate HIV by 2030, we must therefore also be able to act on these atypical variants (HIV-1 non-group M) which are distinct from the group M pandemic viruses. While this diversity has had an impact on the efficacy of antiretroviral treatment in the past, recent data show that there is real hope of eliminating these forms, while maintaining vigilance and constant surveillance, so as not to allow more divergent and resistant forms to emerge. The aim of this work is therefore to share an update on the current knowledge on epidemiology, diagnosis and antiretroviral agent efficacy of HIV-1 non-M variants.

Serological assays were performed on 85 human immunodeficiency virus-controller samples . 6% presented a negative rapid screening test 7% presented an indeterminate Western blot. The enzyme immunoassay ratio decreased in controllers who had continual negative ultrasensitive HIV RNA results since inclusion.

International audience; Although integrase strand transfer inhibitors (INSTIs) are widely used in HIV-1 group M (HIV-1/M) infections, little is known about their efficacy against genetically divergent HIV-1 group O (HIV-1/O) strains. Previous phenotypic works have demonstrated the variable susceptibility of HIV-1/O strains, depending on INSTI drugs. Clinical data are very limited and obtained from a few patients.OBJECTIVES:To investigate the virological success rate of an INSTI-based combination of antiretroviral therapy (cART) in a large population of HIV-1/O-infected patients, and to describe resistance-associated mutations (RAM) at virological failure.METHODS:The virological response of 39 patients receiving INSTI-based cART during their follow-up was analysed i) at the last point of the first INSTI initiation and ii) at their most recent visit. RAM analysis was performed at virological failures. Resistance interpretation was based on the French National Agency of Research on AIDS and viral hepatitis (ANRS) rules.RESULTS:Virological success at both time points of analysis was high, with more than 87% of the patients with undetectable plasma viral load. Among the six patients with virological failure, three selected RAM described for HIV-1/M resistance, and two had already RAM, before INSTI initiation.CONCLUSION:Our results show that HIV-1/O infected patients receiving INSTI-based cART presented a high rate of virological success whatever their previous lines; we have also shown that resistance rules for HIV-1/M could be considered when failure occurs. These data are of importance especially in the context of WHO recommendations for a wider use of this class.

BACKGROUND The genetic divergence of HIV-1 group O is high relative to pandemic group M, which could impact detection and quantification of plasma RNA. Recent commercial kits for RNA quantification seem to show good performances in HIV-1/O, but discrepancies are still observed. Here, we compare the performances of 3 commercial assays for the RNA quantification of HIV-1/O. METHODS We studied the RNA quantification of 117 clinical samples using Abbott RealTime HIV-1, Cepheid Xpert HIV-1 Viral Load, or Roche Cobas TaqMan HIV-1 v2. First, we conducted a qualitative description, and second, we focused on a quantitative analysis of the results above 40 cp/mL. The degree of agreement between methods and the strength of the correlation of viral load determination were estimated using Bland-Altman plot and Passing-Bablok regression with the Spearman coefficient, respectively. RESULTS Our 2-by-2 analysis showed that the Abbott and Cepheid assays were very close in terms of correlation and dispersion of points, whereas Roche presented higher values in the highest range of quantification (>5 log10). The Cepheid assay combined better correlation with the consensus value and a lower dispersion of values, leading to an overall better performance of quantification. The quantification was still impacted by intragroup genetic diversity with, here, 1 strain (YBF26). CONCLUSIONS Using a new approach to compare the performances of RNA quantification between more than 2 techniques, we demonstrated that Cepheid could be the most suitable assay for HIV-1/O quantification, although the results from all assays remained strain dependent.

Genome-wide association studies suggest that HLA-C expression is a major determinant of viral load set points and CD4+ T cell counts in HIV-infected individuals. On the one hand, efficient HLA-C expression enables the killing of infected cells by cytotoxic T lymphocytes (CTLs). On the other hand, HLA-C sends inhibitory signals to natural killer (NK) cells and enhances the infectivity of newly produced HIV particles. HIV-1 group M viruses modulate HLA-C expression using the accessory protein Vpu, possibly to balance CTL- and NK cell-mediated immune responses. Here, we show that the second human immunodeficiency virus, HIV-2, can use its accessory protein Vif to evade HLA-C-mediated restriction. Furthermore, our mutational analyses provide insights into the underlying molecular mechanisms. In summary, our results reveal how the two human AIDS viruses modulate HLA-C, a key component of the antiviral immune response., HLA-C-mediated antigen presentation induces the killing of human immunodeficiency virus (HIV)-infected CD4+ T cells by cytotoxic T lymphocytes (CTLs). To evade killing, many HIV-1 group M strains decrease HLA-C surface levels using their accessory protein Vpu. However, some HIV-1 group M isolates lack this activity, possibly to prevent the activation of natural killer (NK) cells. Analyzing diverse primate lentiviruses, we found that Vpu-mediated HLA-C downregulation is not limited to pandemic group M but is also found in HIV-1 groups O and P as well as several simian immunodeficiency viruses (SIVs). We show that Vpu targets HLA-C primarily at the protein level, independently of its ability to suppress NF-κB-driven gene expression, and that in some viral lineages, HLA-C downregulation may come at the cost of efficient counteraction of the restriction factor tetherin. Remarkably, HIV-2, which does not carry a vpu gene, uses its accessory protein Vif to decrease HLA-C surface expression. This Vif activity requires intact binding sites for the Cullin5/Elongin ubiquitin ligase complex but is separable from its ability to counteract APOBEC3G. Similar to HIV-1 Vpu, the degree of HIV-2 Vif-mediated HLA-C downregulation varies considerably among different virus isolates. In agreement with opposing selection pressures in vivo, we show that the reduction of HLA-C surface levels by HIV-2 Vif is accompanied by increased NK cell-mediated killing. In summary, our results highlight the complex role of HLA-C in lentiviral infections and demonstrate that HIV-1 and HIV-2 have evolved at least two independent mechanisms to decrease HLA-C levels on infected cells.

International audience; ObjectivesTo determine natural phenotypic susceptibility of non-group M HIV-1 to integrase strand transfer inhibitors (INSTIs) in a large panel of 39 clinical strains from groups O, N and P and to identify genotypic polymorphisms according to susceptibility levels.MethodsSusceptibility to raltegravir, elvitegravir and dolutegravir was evaluated in 36 HIV-1/O, 2 HIV-1/N and 1 HIV-1/P strains plus an HIV-1/M reference strain. IC50 values were determined after 3 days, and fold changes (FCs) were calculated relative to the HIV-1/M strain. Genotypic polymorphism was determined by amplification of codons 19–263 of the integrase; the natural occurrence of resistance-associated mutations was analysed using the main resistance algorithms and the IAS-USA list. VESPA analysis of the strain sequences was used to determine a signature pattern associated with higher FC.ResultsSimilar IC50 results were observed for the three drugs. Based on the value for the HIV-1/M reference strain, the data showed FC values 10 for six strains (15%). Analysis of the non-M integrase sequences showed a high level of polymorphism without a major genotypic impact; it also revealed mutations that may be associated with the highest FC values obtained for elvitegravir.ConclusionsOur phenotypic data showed that non-M strains are globally susceptible to the three currently used INSTIs, but the impact of the high FC values observed for some strains with elvitegravir needs to be explored. Clinical data are now needed to confirm these phenotypic results.

HIV-1 group O (HIV-1/O) are rare variants that are mainly found in Cameroon, where they represent several thousands of cases. The reasons for their limited diffusion remains poorly understood: their emergence is estimated as ancient as that of pandemic HIV-1/M, and ancient cases of exportation on diverse continents have been identified for both groups. In France, more than a hundred cases have been identified so far that are mostly linked with Cameroon. HIV-1/O have developed a great level of genetic diversity and an atypical phylogenetic structure whose description remained ambiguous, due to the existence of several nomenclatures, which have only recently been unified. Their great genetic diversity and divergence from HIV-1/M probably impacts diverse properties, such as counteraction of Tetherin. Further studying the epidemiologic, evolutionary and virologic characteristics of these variants will allow for a better understanding of their limited spread and the future of this epidemic. In this respect, the recent description of diverse M/O intergroup recombinant forms underlines an evolutionary potential.

HIV-1 group O (HIV-1/O) are rare variants that are mainly found in Cameroon, where they have caused several thousand cases. The reasons for their limited diffusion remain poorly understood: their emergence is estimated to have been as long ago as that of the HIV-1/M pandemic, and old cases of exportation to different continents have been identified for both groups. In France, more than a hundred cases have been identified thus far, mostly linked to Cameroon. HIV-1/O have developed a high level of genetic diversity and an atypical phylogenetic structure, for which the description has remained ambiguous due to the existence of several nomenclatures. These have only recently been unified by taking the evolutionary history of the viruses into account. Their high genetic diversity and divergence from HIV-1/M likely affects diverse properties of the strains, such as their ability to counteract the action of tetherin. Further studies of the epidemiological, evolutionary, and virological characteristics of these variants will allow a better understanding of the reasons for their limited spread and the future of the epidemic. In this respect, the recent description of diverse M/O intergroup recombinants highlights their evolutionary potential.

International audience; Blood collected and dried on a paper card - dried blood spot (DBS) - knows a growing interest as a sampling method that can be performed outside care facilities by capillary puncture, and transported in a simple and safe manner by mail. The benefits of this method for blood collection and transport has recently led the World Health Organization to recommend DBS for HIV and hepatitis B and C diagnosis. The clinical utility of DBS sampling to improve diagnostics and care of HIV and hepatitis B and C infection in hard to reach populations, key populations and people living in low-income settings was highlighted. Literature about usefulness of DBS specimens in the therapeutic cascade of care - screening, confirmation, quantification of nucleic acids, and resistance genotyping -, was reviewed. DBS samples are suitable for testing antibodies, antigens, or nucleic acids using most laboratory methods. Good sensibility and specificity have been reported for infant HIV diagnosis and diagnosis of hepatitis B and C. The performance of HIV RNA testing on DBS to identified virological failure on antiretroviral therapy is also high but not optimal because of the dilution of dried blood in the elution buffer, reducing the analytical sensitivity, and because of the contamination by intracellular HIV DNA. Standardized protocols are needed for inter-laboratory comparisons, and manufacturers should pursue regulatory approval for in vitro diagnostics using DBS specimens. Despite these limitations, DBS sampling is a clinically relevant tool to improve access to infectious disease diagnosis worldwide.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains.

International audience; Background: Nucleic acid tests performed on blood samples collected on Dried Blood Spot (DBS) and detection of HCV core antigen (HCVcAg) are two approaches that may facilitate access to HCV diagnosis in low and middle incomes countries. In this study we evaluate HCV RNA and HCV antigen testing on DBS in HIV/HCV co-infected peoples who inject drugs in Vietnam.Method: One hundred and four HIV/HCV seropositive patients managed in outpatient care at the Haiphong Viet Tiep hospital were included in this study from February to March, 2014 (ANRS 12262 study).Results: Eighty-six subjects were tested positive for HCV RNA in serum, median (IQR): 6.9 log10 IU/ml (5.6–7.4 log10 IU/ml). Genotypes consisted of 57 G1 (69%), 3 G3 (4%), and 22 G6 (27%). HCV RNA was detected on DBS specimens in 79 out 86 subjects with chronic hepatitis C (sensitivity 92.5%; 95% CI: 85.1–96.9%). HCV RNA level on DBS and serum was moderately correlated (r = 0.24; p = 0.05) suggesting a degradation of HCV RNA due to transportation and storage conditions. HCVcAg was detected in 75/86 dB specimens (sensitivity: 87.2%; 95% CI: 78.3–93.4%), with a strong positive relationship between DBS HCVcAg and serum HCV RNA levels (r = 0.80; P

International audience; Objectives: We estimated the prevalence of transmitted-drug-resistance-associated mutations (TDRAMs) in antiretroviral-naive chronically HIV-1-infected patients.Patients and methods: TDRAMs were sought in samples from 660 diagnosed HIV-1-infected individuals in 2015/2016 in 33 HIV clinical centres. Weighted analyses, considering the number of patients followed in each centre, were used to derive representative estimates of the percentage of individuals with TDRAMs. Results were compared with those of the 2010/2011 survey (n = 661) using the same methodology.Results: At inclusion, median CD4 cell counts and plasma HIV-1 RNA were 394 and 350/mm3 (P = 0.056) and 4.6 and 4.6 log10 copies/mL (P = 0.360) in the 2010/2011 survey and the 2015/2016 survey, respectively. The frequency of non-B subtypes increased from 42.9% in 2010/2011 to 54.8% in 2015/2016 (P < 0.001), including 23.4% and 30.6% of CRF02_AG (P = 0.004). The prevalence of virus with protease or reverse-transcriptase TDRAMs was 9.0% (95% CI = 6.8-11.2) in 2010/2011 and 10.8% (95% CI = 8.4-13.2) in 2015/2016 (P = 0.269). No significant increase was observed in integrase inhibitor TDRAMs (6.7% versus 9.2%, P = 0.146). Multivariable analysis showed that men infected with the B subtype were the group with the highest risk of being infected with a resistant virus compared with others (adjusted OR = 2.2, 95% CI = 1.3-3.9).Conclusions: In France in 2015/2016, the overall prevalence of TDRAMs was 10.8% and stable compared with 9.0% in the 2010/2011 survey. Non-B subtypes dramatically increased after 2010. Men infected with B subtype were the group with the highest risk of being infected with a resistant virus, highlighting the need to re-emphasize safe sex messages.

Objective This study was aimed at evaluating the performance of three CE-marked rapid diagnostic tests (RDTs): Multisure-HCV, First Response® and Toyo®; for screening anti- HCV antibody using plasma samples. Results Overall, 200 plasma samples were used. Sensibility and specificity of these RDTs range from 71 to 99 and 78 to 100% respectively. Multisure scored a sensitivity at 99% (95% CI 97–100%) and First Response reached a specificity at 90% (95% CI 85–94.9%). Further studies should be conducted to establish an algorithm using these RTDs for the detection of HCV infection in Cameroon.

International audience; OBJECTIVES:HIV/1 group P (HIV-1/P) is the last HIV/1 group discovered and, to date, constitutes only two strains. To obtain new insight into this divergent group, we screened for new infections by developing specific tools, and analysed phenotypic and genotypic properties of the prototypic strain RBF168. In addition, the follow-up of the unique infected patient monitored so far has raised the knowledge of the natural history of this infection and its therapeutic management.DESIGN/METHODS:We developed an HIV-1/P specific seromolecular strategy and screened over 29 498 specimen samples. Infectivity and evolution of the gag-30 position, considered as marker of adaptation to human, were explored by successive passages of RBF168 strain onto human peripheral blood mononuclear cells. Natural history and immunovirological responses to combined antiretroviral therapy (cART) were analysed based on CD4+ cells and plasmatic viral load evolution.RESULTS:No new infection was detected. Infectivity of RBF168 was found lower, relative to other main HIV groups and the conservative methionine found in the gag-30 position revealed a lack of adaptation to human. The follow-up of the patient during the 5-year ART-free period, showed a relative stability of CD4+ cell count with a mean of 326 cells/μl. Initiation of cART led to rapid RNA undetectability with a significant increase of CD4+ cells, reaching 687 cells/μl after 8 years.CONCLUSION:Our results showed that HIV-1/P strains remain extremely rare and could be less adapted and pathogenic than other HIV strains. These data lead to the hypothesis that HIV-1/P infection could evolve towards, or even already corresponds to, a dead-end infection.

Major differences exist between HIV-1 and HIV-2 in terms of epidemiology, pathogenicity, sensitivity to antiretrovirals. Determining the type of HIV infecting a patient is essential for management. The aim of this study was to evaluate the ability of simple/rapid tests to differentiate between HIV-1 and/or HIV-2 infections. We analyzed 116 samples from patients infected with HIV-1 (n = 61), HIV-2 (n = 47), or HIV-1+HIV-2 (n = 8) at the chronic stage of infection. Each sample was tested with SD Bioline HIV-1/2 3.0, ImmunoFlow HIV1–HIV2, ImmunoFlow HIV1–HIV2 (WB), Genie III HIV-1/HIV-2, ImmunoComb HIV1&2 BiSpot. HIV-1, or HIV-2 single infection was identified with a sensitivity ranging from 90% to 100%. The ability to detect dual infection was less sensitive (12.5–100%). SD Bioline HIV-1/2 3.0, ImmunoFlow HIV1–HIV2, and Genie III were unable to detect HIV-1 group O infection in one, one and two cases, respectively. The specificity of detection of HIV-1, HIV-2, or HIV-1+HIV-2 antibodies differed greatly (36–100%). ImmunoComb BiSpot had the highest sensitivity values (99–100% for HIV-1, 98% for HIV-2, and 75–87.5% for dual infection) and specificity values (94–100% for HIV-1, 100% for HIV-2, and 97–100% for dual infection). In conclusion, this study showed that no single rapid test had a perfect sensitivity/specificity ratio, particularly in the case of the double infections. J. Med. Virol. 87:2061–2066, 2015. © 2015 Wiley Periodicals, Inc.

Background In 2014–2015, 242 individuals aged 2–89 years were newly diagnosed with human immunodeficiency virus type 1 (HIV-1) in Roka, a rural commune in Cambodia. A case-control study attributed the outbreak to unsafe injections. We aimed to reconstruct the likely transmission history of the outbreak. Methods We assessed in 209 (86.4%) HIV-infected cases the presence of hepatitis C virus (HCV) and hepatitis B virus (HBV). We identified recent infections using antibody (Ab) avidity testing for HIV and HCV. We performed amplification, sequencing, and evolutionary phylogenetic analyses of viral strains. Geographical coordinates and parenteral exposure through medical services provided by an unlicensed healthcare practitioner were obtained from 193 cases and 1499 controls during interviews. Results Cases were coinfected with HCV (78.5%) and HBV (12.9%). We identified 79 (37.8%) recent ( Conclusions Unsafe injections in Cambodia most likely led to an explosive iatrogenic spreading of HIV, associated with a long-standing and more genetically diverse HCV propagation.

HIV-2 infection is characterized by a very low replication rate in most cases and low progression. This necessitates an approach to patient monitoring that differs from that for HIV-1 infection. Here, a new highly specific and sensitive method for HIV-2 DNA quantification was developed. The new test is based on quantitative real-time PCR targeting the long terminal repeat (LTR) andgagregions and using an internal control. Analytical performance was determined in three laboratories, and clinical performance was determined on blood samples from 63 patients infected with HIV-2 group A (n= 35) or group B (n= 28). The specificity was 100%. The 95% limit of detection was three copies/PCR and the limit of quantification was six copies/PCR. The within-run coefficients of variation were between 1.03% at 3.78 log10copies/PCR and 27.02% at 0.78 log10copies/PCR. The between-run coefficient of variation was 5.10%. Both manual and automated nucleic acid extraction methods were validated. HIV-2 DNA loads were detectable in blood cells from all 63 patients. When HIV-2 DNA was quantifiable, median loads were significantly higher in antiretroviral-treated than in naive patients and were similar for groups A and B. HIV-2 DNA load was correlated with HIV-2 RNA load (r= 0.68; 95% confidence interval [CI], 0.4 to 0.8;P< 0.0001). Our data show that this new assay is highly sensitive and quantifies the two main HIV-2 groups, making it useful for the diagnosis of HIV-2 infection and for pathogenesis studies on HIV-2 reservoirs.

Simian immunodeficiency viruses (SIVs) use their Nef proteins to counteract the restriction factor tetherin. However, a deletion in human tetherin prevents antagonism by the Nef proteins of SIVcpz and SIVgor, which represent the ape precursors of human immunodeficiency virus type 1 (HIV-1). To promote virus release from infected cells, pandemic HIV-1 group M strains evolved Vpu as a tetherin antagonist, while the Nef protein of less widespread HIV-1 group O strains acquired the ability to target a region adjacent to this deletion. In this study, we identified an unusual HIV-1 group O strain (RBF206) that evolved Vpu as an effective antagonist of human tetherin. While both RBF206 Vpu and Nef exert anti-tetherin activity in transient-transfection assays, mainly Vpu promotes RBF206 release in infected CD4 + T cells. Although mutations distinct from the adaptive changes observed in group M Vpus (M-Vpus) were critical for the acquisition of its anti-tetherin activity, RBF206 O-Vpu potently suppresses NF-κB activation and reduces CD4 cell surface expression. Interestingly, RBF206 Vpu counteracts tetherin in a largely species-independent manner, degrading both the long and short isoforms of human tetherin. Downmodulation of CD4, but not counteraction of tetherin, by RBF206 Vpu was dependent on the cellular ubiquitin ligase machinery. Our data present the first example of an HIV-1 group O Vpu that efficiently antagonizes human tetherin and suggest that counteraction by O-Nefs may be suboptimal. IMPORTANCE Previous studies showed that HIV-1 groups M and O evolved two alternative strategies to counteract the human ortholog of the restriction factor tetherin. While HIV-1 group M switched from Nef to Vpu due to a deletion in the cytoplasmic domain of human tetherin, HIV-1 group O, which lacks Vpu-mediated anti-tetherin activity, acquired a Nef protein that is able to target a region adjacent to the deletion. Here we report an unusual exception, identifying a strain of HIV-1 group O (RBF206) whose Vpu protein evolved an effective antagonism of human tetherin. Interestingly, the adaptive changes in RBF206 Vpu are distinct from those found in M-Vpus and mediate efficient counteraction of both the long and short isoforms of this restriction factor. Our results further illustrate the enormous flexibility of HIV-1 in counteracting human defense mechanisms.

HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3′ processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3′ processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.

The presence of HIV-1 non-B subtypes in Western Europe is commonly attributed to migration of individuals from non-European countries, but the possible role of domestic infections with non-B subtypes is not well investigated. The French mandatory anonymous reporting system for HIV is linked to a virological surveillance using assays for recent infection (env region, followed by phylogenetic analysis and search for transmission clusters. Two hundred thirty-three recent HIV-1 infections with non-B variants were identified. They involved 5 subtypes and 7 circulating recombinant forms (CRFs). Ninety-two cases (39.5%) were due to heterosexual transmissions, of which 39 occurred in patients born in France. Eighty-five cases (36.5%) were identified in men having sex with men (MSM). Forty-three recent non-B infections (18.5%) segregated into 14 clusters, MSM being involved in 11 of them. Clustered transmission events included 2 to 7 cases per cluster. The largest cluster involved MSM infected by a CRF02_AG variant. In conclusion, we found that the spread of non-B subtypes in France occurs in individuals of French origin and that MSM are particularly involved in this dynamic.

To assess the virological response, genotypic resistance profiles, and antiretroviral plasma concentrations in HIV-2 antiretroviral-treated (antiretroviral therapy, ART) patients in Côte d'Ivoire.A cross-sectional survey was conducted among HIV-2 patients receiving ART. Plasma HIV-2 viral load was performed using the Agence Nationale de Recherche sur le SIDA et les hépatites virales (ANRS) assay. Protease and reverse transcriptase sequencing was performed using in-house methods and antiretroviral plasma concentrations were assessed using ultra performance liquid chromatography combined with tandem mass spectrometry.One hundred and forty-five HIV-2-treated patients were enrolled with a median CD4 cell count of 360 cells/μl (interquartile range, IQR = 215-528). Median duration of ART was 4 years (IQR = 2-7) and 74% of patients displayed viral load less than 50 copies/ml. Median plasma HIV-2 RNA among patients with viral load more than 50 copies/ml was 3016 copies/ml (IQR = 436-5156). Most patients (84%) received a lopinavir/ritonavir-based regimen. HIV-2 resistance mutations to nucleoside reverse transcriptase inhibitors and protease inhibitors were detected in 21 of 25 (84%) and 20 of 29 (69%) samples, respectively. The most prevalent nucleoside reverse transcriptase inhibitor resistance mutations were M184I/V (90%), Q151M (24%), and S215F/Y (24%). The most prevalent protease inhibitor resistance mutations were V47A (60%) and I54M (30%). Median CD4 cell counts were 434 cells/μl (292-573) and 204 cells/μl (122-281) in patients with viral load less than 50 copies/ml and those exhibiting virological failure (P 0.0001), respectively. The proportions of patients with adequate antiretroviral plasma concentrations were 81 and 93% in patients displaying virological failure and in those with viral load less than 50 copies/ml, respectively (P = 0.046), suggesting good treatment adherence.We observed adequate drug plasma concentrations and virological suppression in a high proportion of HIV-2-infected patients. However, in cases of virological failure, the limited HIV-2 therapeutic arsenal and cross-resistance dramatically reduced treatment options.

International audience

Dried blood spots (DBS) are widely proposed as a plasma surrogate for monitoring antiretroviral treatment efficacy based on the HIV-1 RNA level (viral load [VL]) in resource-limited settings. Interfering coamplification of cell-associated HIV-1 DNA during reverse transcription (RT)-PCR can be avoided by using nucleic acid sequence-based amplification (NASBA) technology, which is based on an RNA template and isothermic conditions. We analyzed VL values obtained with DBS and plasma samples by comparing isothermic NASBA (NucliSENS EasyQ HIV-1 V2.0; bioMérieux) with real-time RT-PCR (Cobas TaqMan HIV-1 V2.0; Roche). Samples from 197 HIV-1-infected patients were tested (non-B subtypes in 51% of the cases). Nucleic acid extractions were performed by use of NucliSENS EasyMAG (bioMérieux) and Cobas AmpliPrep (Roche) before the NASBA and RT-PCR quantifications, respectively. Both quantification assays have lower limits of detection of 20 (1.3) and 800 (2.9) log 10 copies/ml (log) in plasma and DBS, respectively. The mean (DBS minus plasma) differences were −0.39 and −0.46 log, respectively, for RT-PCR and NASBA. RT-PCR on DBS identified virological failure in 122 of 126 patients (sensitivity, 97%) and viral suppression in 58 of 70 patients (specificity, 83%), yielding 12 false-positive results (median, 3.2 log). NASBA on DBS identified virological failure in 85 of 96 patients (sensitivity, 89%) and viral suppression in 95 of 97 patients (specificity, 98%) and yielded 2 false-positive results (3.0 log for both). Both technologies detected HIV-1 RNA in DBS at a threshold of 800 copies/ml. This higher specificity of NASBA technology could avoid overestimation of poor compliance or the emergence of resistance when monitoring antiretroviral efficacy with the DBS method.

Several B/CRF02_AG Unique Recombinant Forms (URFs) have previously been identified in France. Here we show that one of them (URF5_B/02/G) is emerging in MSM, a high-risk population where HIV incidence and number of superinfections are increasing. We describe this new Circulating Recombinant Form, CRF56_cpx, estimate the time to its most recent common ancestor, investigate its origins and show that it probably shares common ancestors with strains from the East Mediterranean.

SUMMARY The AIDS pandemic that started in the early 1980s is due to human immunodeficiency virus type 1 (HIV-1) group M (HIV-M), but apart from this major group, many divergent variants have been described (HIV-1 groups N, O, and P and HIV-2). The four HIV-1 groups arose from independent cross-species transmission of the simian immunodeficiency viruses (SIVs) SIVcpz, infecting chimpanzees, and SIVgor, infecting gorillas. This, together with human adaptation, accounts for their genomic, phylogenetic, and virological specificities. Nevertheless, the natural course of non-M HIV infection seems similar to that of HIV-M. The virological monitoring of infected patients is now possible with commercial kits, but their therapeutic management remains complex. All non-M variants were principally described for patients linked to Cameroon, where HIV-O accounts for 1% of all HIV infections; only 15 cases of HIV-N infection and 2 HIV-P infections have been reported. Despite improvements in our knowledge, many fascinating questions remain concerning the origin, genetic evolution, and slow spread of these variants. Other variants may already exist or may arise in the future, calling for close surveillance. This review provides a comprehensive, up-to-date summary of the current knowledge on these pathogens, including the historical background of their discovery; the latest advances in the comprehension of their origin and spread; and clinical, therapeutic, and laboratory aspects that may be useful for the management and the treatment of patients infected with these divergent viruses.

To optimize standard treatment of chronic hepatitis C in responder patients who have achieved undetectable viral load, a prospective study was conducted to determine the factors and kinetics of virologic relapse. Responder patients were monitored 2, 4, 8, 12, 16, and 24 weeks after the end of treatment with pegylated interferon and ribavirin. Forty-seven of the 154 patients (30.5%) relapsed. Relapse was significantly associated with absence of rapid virologic response (RVR), retreatment, higher baseline viral load, older age, and lower weight-based dose of pegylated interferon. Relapse was more frequent in patients failing to achieve a RVR after receiving pegylated interferon alpha 2a

OBJECTIVE HIV-1 has been classified into four groups: M, N, O and P. The aim of this study was to revisit the cross-group neutralization using a highly diverse panel of primary isolates. DESIGN The panel of viruses included nine HIV-1 group O primary isolates, one recombinant M/O primary isolate, one group N primary isolates, one group P primary isolate, two group M (subtype B) primary isolates and the HIV-1 group M adapted strain MN. METHODS All the viruses were tested for neutralization in TZM-bl cells, using sera issued from patients infected by viruses of group M (n = 11), O (n = 12) and P (n = 1), and a panel of nine human monoclonal broadly neutralizing antibodies (HuMo bNAbs). RESULTS Although the primary isolates displayed a wide spectrum of sensitivity to neutralization by the human sera, cross-group neutralization was clearly observed. In contrast, the bNAbs did not show any cross-group neutralization, except PG9 and PG16. Interestingly, the group N prototype strain YBF30 was highly sensitive to neutralization by PG9 (IC50: 0.28 μg/ml) and PG16 (IC50

International audience; BACKGROUND: Due to the prevalence of HIV-1 group M and the endemicity of HIV-1 group O infections in Cameroon, patients may be infected with both viruses and/or with HIV-1/MO recombinant forms. Such atypical infections may be deleterious in terms of diagnosis and therapeutic management due to the high divergence of HIV-1/O. The aim of this study was to identify prospectively such atypical infections in Cameroon.RESULTS: Based on serological screening by env-V3 serotyping and a molecular strategy using group-specific (RT)-PCRs, we identified 10 Cameroonian patients harboring three different profiles of infection: (1) 4 HIV-1/M + O dual infections without evidence of recombinant; (2) 5 recombinants associated with one or both parental strains; and (3) 1 new recombinant form without parental strains.CONCLUSIONS: This work highlights the dynamic co-evolution of these two HIV groups in Cameroon that could lead to the emergence of a circulating recombinant form MO, and the need for accurate identification of such atypical infections for precise diagnosis, virological monitoring and therapeutic management with adapted tools.

Background In France, HIV infection diagnosis was modified by a decree, published in 2010, that requires the use of HIV Ag/Ab assays able to detect at least 2 IU/ml of p24Ag. This measure raises the concern of the capacity of these assays to equally detect all HIV variants. Objectives To assess the performance of HIV Ag/Ab assays for the detection of p24Ag from diverse HIV isolates. Study design Ten HIV Ag/Ab assays were compared using two p24Ag reference standards, 297 samples from 99 HIV-1 and HIV-2 cell-culture derived isolates including various subtypes and groups, and 9 native specimens from subjects with primary HIV infection. Results The p24Ag limit of detection (LOD) ranged from 0.505 IU/ml to 1.901 IU/ml and, from 11.9 pg/ml to 33.5 pg/ml when using WHO and French national standards, respectively. The overall percentage of positive samples ranged from 26.8% to 74.5%. Five assays failed to detect all dilutions of at least one group M subtype, three missed all group O and six all the group P samples. Three assays were able to detect 2–10 of the 30 HIV-2 samples. The distribution of LODs for each group M isolate showed a wide dispersion between the assays. Percentage of isolates detected at a p24Ag level less than 2 IU/ml varied from 22% to 98.7%. Conclusion This study demonstrated that, even though their analytical sensitivity fulfills the requirements, many of HIV Ag/Ab assays could fail to detect HIV primary infection due to HIV-1 non-B, non-M and HIV-2 strains.

Background: A new version of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 assay (CA/CTM v2.0) has been introduced to overcome the underquantification observed with the first version. Methods: We compared the Roche Cobas CA/CTM v2.0 and Abbott RealTime HIV-1 assays for HIV-1 group M and non-M viral load measurement. Results: We found a good correlation (r = 0.96) between the 2 techniques for the 260 HIV-1 group M plasma samples tested. The Roche Cobas assay gave significantly higher values than the Abbott assay, and 51 samples (20%) yielded differences greater than 0.5 log 10 copies per milliliter. Conversely, 2 samples were more than 0.5 log 10 copies per milliliter higher with the Abbott assay than with the Roche Cobas assay. Among the 84 samples with undetectable viral load in the Abbott assay (detection limit 40 copies/mL), 17 (20%) were detectable with the CA/CTM v2.0 assay (detection limit 20 copies/mL), with values ranging from 41 to 897 copies per milliliter. Extrapolation of the Abbott curves led to 10/17 (59%) of these samples being quantifiable. HIV-1 groups O and P were similarly quantified by the two techniques. Conclusion: The results of the Roche Cobas CA/CTM v2.0 and Abbott RealTime HIV-1 assays correlate well. The new version of the CA/CTM assay shows improved sensitivity. Nevertheless, the 2 assays differ by more than 0.5 log 10 copies per milliliter for some samples.

Screening for HIV infection has improved since the first immunoassays. Today, diagnosis of HIV infection can be performed with fourth-generation tests that track both the patient's antibodies and HIV antigen. The objective of this study was to evaluate the clinical sensitivity and specificity of the new DiaSorin Liaison XL Murex HIV Ab/Ag assay compared to another fourth-generation assay, the Abbott Architect HIV Ag/Ab Combo kit. This work was performed on a large panel of 900 samples, including negative samples ( n = 493) and HIV-positive ( n = 407) representatives of HIV-1 group M subtypes and circulating recombinant forms (CRFs), HIV-1 group O, and HIV-2 variants. The results highlight the high specificity (98.9%) and sensitivity (100%) of this new fourth-generation assay, which are consistent with its use for the screening and diagnosis of HIV infections with the current circulating strains.

Resume Introduction Le VIH-1 groupe O (VIH-O), principalement retrouve au Cameroun, presente une tres grande diversite genetique ayant des consequences sur le diagnostic et la prise en charge des patients, necessitant la mise au point d’outils specifiques. Objectif Presenter les moyens actuellement disponibles pour depister les infections a VIH-O et assurer leur traitement, ainsi que les premieres donnees de RES-O, reseau francais d’identification et de suivi de patients infectes par le VIH-O. Methodes Le diagnostic a ete confirme par serotypage de l’enveloppe, specifique de groupe. La charge virale a ete evaluee par une technique LTR-O, mise au point au laboratoire et comparee a la trousse non specifique RealTime HIV-1 (Abbott). Le sequencage des regions cibles des antiretroviraux (protease, transcriptase inverse [TI], integrase et Gp41) a ete realise par amorces specifiques. L’analyse des mutations de resistance a ete faite grâce a l’algorithme ANRS utilise pour les VIH-M. Resultats L’infection par un VIH-O a ete confirmee chez 117 patients. La charge virale montre une equivalence entre les deux techniques, avec une tendance a la sous-quantification de la technique Abbott superieure a 1 Log pour 5 % des prelevements. Parmi les souches etudiees, 70 a 100 % possedent au moins neuf mutations dans la protease, quatre dans la TI et une dans la Gp41, avec pour consequence une resistance genotypique naturelle a certaines molecules antiretrovirales. Discussion Le diagnostic et le suivi d’une infection par le VIH-O est possible. L’impact du polymorphisme naturel de ce variant sur la reponse au traitement est meconnu.

Objectives: To estimate the prevalence of transmitted drug resistance mutations and non-B subtype circulation in antiretroviral-naive chronically HIV-1-infected patients in France. Methods: Resistance mutations were sought in samples from 530 newly diagnosed HIV-1-infected patients from October 2006 to March 2007. Protease and reverse transcriptase mutations were identified from the 2007 Stanford Resistance Surveillance list. Results: Reverse transcriptase and protease resistance mutations were determined in 466 patients with duration of seropositivity

Resume A ce jour, plus de 40 virus de l’immunodeficience simienne (VIS), contre partie animale des VIH ont ete decrits. Les structures genomiques de ces VIS sont proches de celles des VIH. L’apparition des VIH serait le resultat de passages de VIS a l’homme. L’Afrique centrale se caracterise par une remarquable diversite VIH : tous les sous-types du groupe Majeur, la majorite des formes recombinantes, les variants des groupes M, O, N et P y sont retrouves. Cette situation temoigne de l’anciennete de l’epidemie dans cette region. Le VIH-1 du groupe Majeur (M) represente la majorite des isolats caracterises a ce jour (plus de 30 millions d’infections en 2010) avec 9 differents sous-types (A-K) et plus de 40 formes recombinantes. Le VIH-1 groupe O se caracterise par une tres grande diversite genetique. La decouverte des rares cas de VIH-1 groupe N a ete la premiere preuve d’une transmission inter-espece des VIS des chimpanzes a l’homme et de l’importance des mecanismes de recombinaison entre les souches lentivirales des primates. En 2009, un variant atypique du VIH-1 dit groupe P presentant un lien phylogenetique etroit avec les VIS des gorilles a ete mis en evidence chez une patiente camerounaise, vivant en France. Les preuves de ces transmissions inter-especes des virus de l’immunodeficience des primates a l’homme sont confortees par les similarites des genomes viraux, les parentes phylogenetiques et les prevalences des VIS chez leurs hotes naturels et par la superposition geographique entre VIS et VIH.

HIV-1 are now classified into four groups: M (major), O (outlier), N (non-M – non-O), and a new group P, the prototype of which is the divergent strain recently characterized in a Cameroonian patient [1]. Recombination between HIV strains is a very frequent phenomenon, greatly increasing genetic div

Background Low-cost in-house technologies for genotypic drug resistance testing use reagents with quality labels for research only. Here, we report on the results of PCR amplifications in negative-controls that were observed in two independent laboratories. Methods Positive PCR amplifications of protease and reverse transcriptase fragments for genotypic drug resistance testing of HIV on dried blood and/or plasma spots were observed on negative-control samples and were analysed in detail by PCR and sequence and phylogenetic analyses to identify the origin of the PCR contamination. Results Detailed analysis revealed that the RT-PCR enzymes were contaminated with an HIV-based vector commercialized by the same company. Conclusions These observations show the need to implement quality control steps that verify for the absence of HIV in new reagent batches because this can significantly compromise molecular diagnosis of HIV and genotypic drug resistance tests using in-house protocols.

Background Dried sample spots have molecular applications, but few data are available on conditions of HIV-1 RNA amplification. Objectives To determine the impact of (i) the sample type (plasma or serum), (ii) various storage periods, (iii) transfer at ambient temperature of the dried spots via postal mail, and (iv) two different methods of elution–extraction, to amplify partial pol and env genes with a view to both phylogenic and resistance analyses. Methods Fourteen samples (dried plasma spot and dried serum spot) from seven patients were stored at 20–25 °C for 2, 5 and 7 days. Two extraction buffers were tested on these samples, followed by reverse transcriptase-polymerase chain reaction (RT-PCR) on pol and env genes. Sixteen spots from eight other patients were sent by postal mail at ambient temperature between two laboratories, and were analysed at both sites. Results We observed no influence of the 2–7 day storage period, whatever the sample type and viral load, but the choice of the extraction procedure is a critical step. Analysis of the mailed spots resulted in a good agreement between the two laboratories. Conclusions This study shows that amplification of HIV-1 RNA on spots is possible in various conditions by different operators and using appropriate reagents. This finding could be particularly useful for large-scale molecular and resistance epidemiological studies in resource-rich and resource-limited countries alike.

INTRODUCTION The lesser pathogenicity of HIV-2 relative to HIV-1 is generally attributed to its slower replication. To compare the amounts of total HIV DNA during human HIV-1 and HIV-2 infection, we developed a quantitative real-time PCR method with a unique external quantification standard based on a single plasmid harboring both the HIV-1 and the HIV-2 LTR. METHODS Viral DNA load was compared between 40 HIV-1-infected and 42 HIV-2-infected antiretroviral-naive patients. RESULTS The difference between HIV-1 and HIV-2 proviral DNA load was highly significant in patients with CD4 cell counts > 500 cells/microl [HIV-1: n = 14; median, 2.5; interquartile range (IQR), 2.1-2.7; HIV-2: n = 22, median, 1.6; IQR, 1.0-2.0] and in patients with CD4 cell counts between 300 cells/microl and 500 cells/microl (HIV-1: n = 12; median, 2.7; IQR, 2.3-2.8; HIV-2: n = 11; median, 2.0; IQR, 1.0-2.4). Too few HIV-2-infected patients had CD4 cell counts < 300 cells/microl to detect a significant difference but DNA values were again lower in HIV-2-infected patients (HIV-1: n = 14; median, 2.9; IQR, 2.2-3.2; HIV-2: n = 9; median, 2.7; IQR, 2.2-3.3). CONCLUSIONS These differences are in line with the natural histories of the two infections and show that HIV-2 infection is a valid model for studying the pathophysiology of HIV infection in general.