Respiratory torque teno virus load at emergency department visit predicts intensive care unit admission of SARS-CoV-2 infected patients.

Accurate prediction of COVID-19 severity remains a challenge. Torque teno virus (TTV), recognized as a surrogate marker of functional immunity in solid organ transplant recipients, holds the potential for assessing infection outcomes. We investigated whether quantifying TTV in nasopharyngeal samples upon emergency department (ED) admission could serve as an early predictor of COVID-19 severity. Retrospective single-center study in the ED of Saint-Louis Hospital in Paris, France. TTV DNA was quantified in nasopharyngeal swab samples collected for SARS-CoV-2 testing. Among 295 SARS-CoV-2 infected patients, 92 returned home, 160 were admitted to medical wards, and 43 to the intensive care unit (ICU). Elevated TTV loads were observed in ICU patients (median: 3.02 log copies/mL, interquartile range [IQR]: 2.215-3.825), exceeding those in discharged (2.215, [0; 2.962]) or hospitalized patients (2.24, [0; 3.29]) (p = 0.006). Multivariate analysis identified diabetes, obesity, hepatitis, fever, dyspnea, oxygen requirement, and TTV load as predictors of ICU admission. A 2.91 log10 copies/mL TTV threshold independently predicted ICU admission. Nasopharyngeal TTV quantification in SARS-CoV-2 infected patients is linked to the likelihood of ICU admission and might reflect respiratory immunosuppression.

BK virus genotypes and humoral response in kidney transplant recipients with BKV associated nephropathy.

BACKGROUND: Among kidney transplant recipients (KTR) with BK virus associated nephropathy (BKVN), BKV genotypes' evolution and anti-BKV humoral response are not well established. We aim to analyze BKV replication and genetic evolution following transplantation, and characterize concomitant anti-BKV-VP1 humoral response. METHODS: We retrospectively analyzed 32 cases of biopsy-proven BKVN. Stored plasma and kidney biopsies were tested for BKV viral load, and VP1 sequencing performed on positive samples. BKV-VP1 genotype-specific neutralizing antibodies (NAbs) titers were determined at transplantation and BKVN. RESULTS: At the time of BKVN diagnosis, BKV viral load was 8.2 log10 IU/106 cells and 5.4 log10 IU/mL in kidney and plasma, respectively. VP1 sequencing identified the same BKV-subtype in both compartments in 31/32 cases. At the time of transplantation, 8/20 (40%) of biopsies tested positive for BKV detection, whereas concomitant BKV viremia was negative. VP1 sequencing identified a different subtype compared to BKVN in 5/6 of these samples. This was confirmed following transplantation: 8 patients had a BKV+ biopsy before BKV viremia, and VP1 sequencing identified a different subtype compared to BKVN in all of them. After the onset of BKV viremia and prior to BKVN diagnosis, the BKV subtype in BKV+ plasma and kidney biopsy was the same as the one isolated at BKVN. BKV-VP1 NAbs titers were significantly higher at the time of BKVN compared to transplantation (p = .0031), with similar titers across genotypes. CONCLUSION: Altogether, our data suggest that among some KTR with BKVN, the BKV genotype from the donor may not be responsible for BKVN pathogenesis.

Prevention of SARS-CoV-2 transmission during a large, live, indoor gathering (SPRING): a non-inferiority, randomised, controlled trial.

BACKGROUND: Mass indoor gatherings were banned in early 2020 to prevent the spread of SARS-CoV-2. We aimed to assess, under controlled conditions, whether infection rates among attendees at a large, indoor gathering event would be similar to those in non-attendees, given implementation of a comprehensive prevention strategy including antigen-screening within 3 days, medical mask wearing, and optimised ventilation. METHODS: The non-inferiority, prospective, open-label, randomised, controlled SPRING trial was done on attendees at a live indoor concert held in the Accor Arena on May 29, 2021 in Paris, France. Participants, aged 18-45 years, recruited via a dedicated website, had no comorbidities, COVID-19 symptoms, or recent case contact, and had had a negative rapid antigen diagnostic test within 3 days before the concert. Participants were randomly allocated in a 2:1 ratio to the experimental group (attendees) or to the control group (non-attendees). The allocation sequence was computer-generated by means of permuted blocks of sizes three, six, or nine, with no stratification. The primary outcome measure was the number of patients who were SARS-CoV-2-positive by RT-PCR test on self-collected saliva 7 days post-gathering in the per-protocol population (non-inferiority margin <0·35%). This trial is registered with ClinicalTrials.gov, NCT04872075. FINDINGS: Between May 11 and 25, 2021, 18 845 individuals registered on the dedicated website, and 10 953 were randomly selected for a pre-enrolment on-site visit. Among 6968 who kept the appointment and were screened, 6678 participants were randomly assigned (4451 were assigned to be attendees and 2227 to be non-attendees; median age 28 years; 59% women); 88% (3917) of attendees and 87% (1947) of non-attendees complied with follow-up requirements. The day 7 RT-PCR was positive for eight of the 3917 attendees (observed incidence, 0·20%; 95% CI 0·09-0·40) and three of the 1947 non-attendees (0·15%; 0·03-0·45; absolute difference, 95% CI -0·26% to 0·28%), findings that met the non-inferiority criterion for the primary endpoint. INTERPRETATION: Participation in a large, indoor, live gathering without physical distancing was not associated with increased SARS-CoV-2-transmission risk, provided a comprehensive preventive intervention was implemented. FUNDING: French Ministry of Health. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Evaluation of a saliva molecular point of care for the detection of SARS-CoV-2 in ambulatory care.

Rapid identification of SARS-CoV-2-infected individuals is a cornerstone for the control of virus spread. The sensitivity of SARS-CoV-2 RNA detection by RT-PCR is similar in saliva and nasopharyngeal swabs. Rapid molecular point-of-care tests in saliva could facilitate, broaden and speed up the diagnosis. We conducted a prospective study in two community COVID-19 screening centers to evaluate the performances of a CE-marked RT-LAMP assay (EasyCoV) designed for the detection of SARS-CoV2 RNA from fresh saliva samples, compared to nasopharyngeal RT-PCR, to saliva RT-PCR and to nasopharyngeal antigen testing. Overall, 117 of the 1718 participants (7%) tested positive with nasopharyngeal RT-PCR. Compared to nasopharyngeal RT-PCR, the sensitivity and specificity of the RT-LAMP assay in saliva were 34% and 97%, respectively. The Ct values of nasopharyngeal RT-PCR were significantly lower in the 40 true positive subjects with saliva RT-LAMP (Ct 25.9) than in the 48 false negative subjects with saliva RT-LAMP (Ct 28.4) (p = 0.028). Considering six alternate criteria for reference tests, including saliva RT-PCR and nasopharyngeal antigen, the sensitivity of saliva RT-LAMP ranged between 27 and 44%. The detection of SARS-CoV-2 in crude saliva samples with an RT-LAMP assay had a lower sensitivity than nasopharyngeal RT-PCR, saliva RT-PCR and nasopharyngeal antigen testing.Registration number: NCT04578509.

Accuracy of saliva and nasopharyngeal sampling for detection of SARS-CoV-2 in community screening: a multicentric cohort study.

Nasopharyngeal sampling for nucleic acid amplification testing (NAAT) is the standard diagnostic test of coronavirus disease 2019. Our objectives were to assess, in real-life conditions, the diagnostic accuracy of a nasopharyngeal point-of-care antigen (Ag) test and of saliva NAAT for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in ambulatory care. This was a prospective cohort study from 19 October through 18 December 2020 in two community COVID-19 screening centers in Paris, France. Two nasopharyngeal swabs and one saliva sample were simultaneously collected. Diagnostic accuracies of nasopharyngeal Ag testing and of three saliva NAAT methods were assessed as compared to nasopharyngeal NAAT. A total of 1452 ambulatory children and adults were included. Overall, 129/1443 (9%) participants tested positive on nasopharyngeal NAAT (102/564 [18%] in symptomatic and 27/879 [3%] in asymptomatic participants). Sensitivity was 94%, 23%, 96%, and 94% for the three different protocols of saliva NAAT and for the nasopharyngeal Ag test, respectively. Estimates of specificity were above 95% for all methods. Diagnostic accuracy was similar in symptomatic and asymptomatic individuals. Diagnostic accuracy of nasopharyngeal Ag testing and of saliva NAAT is similar to that of nasopharyngeal NAAT, subject to compliance with specific protocols for saliva. Registration number: NCT04578509.

Deep sequencing analysis of M184V/I mutation at the switch and at the time of virological failure of boosted protease inhibitor plus lamivudine or boosted protease inhibitor maintenance strategy (substudy of the ANRS-MOBIDIP trial).

BACKGROUND: The ANRS12286/MOBIDIP trial showed that boosted protease inhibitor (bPI) plus lamivudine dual therapy was superior to bPI monotherapy as maintenance treatment in subjects with a history of M184V mutation. OBJECTIVES: We aimed to deep analyse the detection of M184V/I variants at time of switch and at the time of virological failure (VF). METHODS: Ultra-deep sequencing (UDS) was performed on proviral HIV-DNA at inclusion among 265 patients enrolled in the ANRS 12026/MOBIDIP trial, and on plasma from 31 patients experiencing VF. The proportion of M184V/I variants was described and the association between the M184V/I mutation at 1% of threshold and VF was explored with logistic regression models. RESULTS: M184V and I mutations were detected in HIV-DNA for 173/252 (69%) and 31/252 (12%) of participants, respectively. Longer duration of first-line treatment, higher plasma viral load at first-line treatment failure and higher baseline HIV-DNA load were associated with the archived M184V. M184I mutation was always associated with a STOP codon, suggesting defective virus. The 48 week estimated probability of remaining free from VF was comparable with or without the M184V/I mutation for dual therapy. At failure, M184V and major PI mutations were detected in 1/17 and 5/15 patients in the bPI arm and in 2/2 and 0/3 in the bPI+lamivudine arm, respectively. CONCLUSIONS: Using UDS evidenced that archiving of M184V in HIV-DNA is heterogeneous despite past historical M184V in 96% of cases. The antiviral efficacy of lamivudine-based dual therapy regimens is mainly due to the residual lamivudine activity.

Multicenter comparison of the Cobas 6800 system with the RealStar RT-PCR kit for the detection of SARS-CoV-2.

BACKGROUND: RT-PCR testing is crucial in the diagnostic of SARS-CoV-2 infection. The use of reliable and comparable PCR assays is a cornerstone to allow use of different PCR assays depending on the local equipment. In this work, we provide a comparison of the Cobas® (Roche) and the RealStar® assay (Altona). METHODS: Assessment of the two assays was performed prospectively in three reference Parisians hospitals, using 170 clinical samples. They were tested with the Cobas® assay, selected to obtain a distribution of cycle threshold (Ct) as large as possible, and tested with the RealStar assay with three largely available extraction platforms: QIAsymphony (Qiagen), MagNAPure (Roche) and NucliSENS-easyMag (BioMérieux). RESULTS: Overall, the agreement (positive for at least one gene) was 76 %. This rate differed considerably depending on the Cobas Ct values for gene E: below 35 (n = 91), the concordance was 99 %. Regarding the positive Ct values, linear regression analysis showed a coefficient of determination (R2) of 0.88 and the Deming regression line revealed a strong correlation with a slope of 1.023 and an intercept of -3.9. Bland-Altman analysis showed that the mean difference (Cobas® minus RealStar®) was + 3.3 Ct, with a SD of + 2.3 Ct. CONCLUSIONS: In this comparison, both RealStar® and Cobas® assays provided comparable qualitative results and a high correlation when both tests were positive. Discrepancies exist after 35 Ct and varied depending on the extraction system used for the RealStar® assay, probably due to a low viral load close to the detection limit of both assays.

Difficulties of Identifying the Early HIV Antibody Seroconversion Period Depending on the Confirmatory Assay.

BACKGROUND: Identification of HIV infection at the early stage is valuable for patient management, for prevention, and for research purposes. In practice, identification of a recent HIV infection at diagnosis proves challenging after HIV antibody seroconversion but can be suspected using Western blots (WBs) or immunoblots (IBs) as confirmatory assays. METHODS: Five commercially available confirmatory assays were compared using 43 samples from recently infected individuals. This included 2 WBs (New LAV Blot I, Biorad, and HIV Blot 2.2, MP Biomedicals), 2 IBs (INNO-LIA HIV I/II, Fujirebio, and RecomLine HIV-1 & HIV-2, Mikrogen Diagnostik), and 1 immunochromatographic single-use assay (Geenius HIV1/2 supplemental assay, Biorad). RESULTS: Following the manufacturer's recommendations for interpretation, the 2 WBs led to indeterminate results for 30% and 42% of the samples, suggesting recent infection, compared with 2%-7% for the 3 other assays. When interpreted based on the Fiebig classification, concordant stages were observed in 42% of samples, and only 49% were classified as early seroconversion by all 5 assays. For the remaining specimens, the distinction with chronic infection was highly variable depending on the assay (5%-100%). CONCLUSIONS: Clinical laboratories must consider this variability, which must be kept in mind both for initial diagnosis and for multicenter studies for which inclusion criteria refer to serological profiles by confirmatory assays.

Characterization of HIV-1 diversity in various compartments at the time of primary infection by ultradeep sequencing.

We used next-generation sequencing to evaluate the quantity and genetic diversity of the HIV envelope gene in various compartments in eight patients with acute infection. Plasma (PL) and seminal fluid (SF) were available for all patients, whole blood (WB) for seven, non-spermatozoid cells (NSC) for four, and saliva (SAL) for three. Median HIV-1 RNA was 6.2 log10 copies/mL [IQR: 5.5-6.95] in PL, 4.9 log10 copies/mL [IQR: 4.25-5.29] in SF, and 4.9 log10 copies/mL [IQR: 4.46-5.09] in SAL. Median HIV-1 DNA was 4.1 log10 copies/106 PBMCs [IQR: 3.15-4.15] in WB and 2.6 log10 copies /106 Cells [IQR: 2.23-2.75] in NSC. The median overall diversity per patient varied from 0.0005 to 0.0232, suggesting very low diversity, confirmed by the clonal aspect of most of the phylogenetic trees. One single haplotype was present in all compartments for five patients in the earliest stage of infection. Evidence of higher diversity was established for two patients in PL and WB, suggesting compartmentalization. Our study shows low diversity of the env gene in the first stages of infection followed by the rapid establishment of cellular reservoirs of the virus. Such clonality could be exploited in the search for early patient-specific therapeutic solutions.

Proliferative memory SAMHD1low CD4+ T cells harbour high levels of HIV-1 with compartmentalized viral populations.

We previously reported the presence of memory CD4+ T cells that express low levels of SAMHD1 (SAMHD1low) in peripheral blood and lymph nodes from both HIV-1 infected and uninfected individuals. These cells are enriched in Th17 and Tfh subsets, two populations known to be preferentially targeted by HIV-1. Here we investigated whether SAMHD1low CD4+ T-cells harbour replication-competent virus and compartimentalized HIV-1 genomes. We sorted memory CD4+CD45RO+SAMHD1low, CD4+CD45RO+SAMHD1+ and naive CD4+CD45RO-SAMHD1+ cells from HIV-1-infected patients on anti-retroviral therapy (c-ART) and performed HIV-1 DNA quantification, ultra-deep-sequencing of partial env (C2/V3) sequences and phenotypic characterization of the cells. We show that SAMHD1low cells include novel Th17 CCR6+ subsets that lack CXCR3 and CCR4 (CCR6+DN). There is a decrease of the % of Th17 in SAMHD1low compartment in infected compared to uninfected individuals (41% vs 55%, p<0.05), whereas the % of CCR6+DN increases (7.95% vs 3.8%, p<0.05). Moreover, in HIV-1 infected patients, memory SAMHD1low cells harbour high levels of HIV-1 DNA compared to memory SAMHD1+ cells (4.5 vs 3.8 log/106cells, respectively, p<0.001), while naïve SAMHD1+ showed significantly lower levels (3.1 log/106cells, p<0.0001). Importantly, we show that SAMHD1low cells contain p24-producing cells. Moreover, phylogenetic analyses revealed well-segregated HIV-1 DNA populations with compartmentalization between SAMHD1low and SAMHD1+ memory cells, and limited viral exchange. As expected, the % of Ki67+ cells was significantly higher in SAMHD1low compared to SAMHD1+ cells. There was positive association between levels of HIV-1 DNA and Ki67+ in memory SAMHD1low cells, but not in memory and naïve SAMHD1+ CD4+ T-cells. Altogether, these data suggest that proliferative memory SAMHD1low cells contribute to viral persistence.

In-depth Sampling of High-risk Populations to Characterize HIV Transmission Epidemics Among Young MSM Using PrEP in France and Quebec.

BACKGROUND: A better understanding of HIV transmission dynamics among populations at high risk is important for development of prevention strategies. We determined HIV transmission networks from infected individuals enrolled in the pre-exposure prophylaxis (PrEP) IPERGAY trial in combination with the ANRS PRIMO and Montreal PHI cohorts to identify and characterize active clusters of transmission in this high-risk population. METHODS: Genotypic resistance tests were performed on plasma samples from 31 IPERGAY participants. Reverse transcriptase sequences were analyzed in combination with unique HIV pol sequences from 1351 individuals enrolled in the PRIMO ANRS cohort (1999-2014) and 511 individuals enrolled in the Montreal PHI cohort (1996-2016). Network analyses were performed to infer putative relationships between all participants. RESULTS: Overall, 1893 participants were included. Transmission network analyses revealed that 14 individuals (45.2%) from the IPERGAY trial were involved in 13 clusters sampled over a median period (interquartile range) of 2 (0.3-7.8) years, including 7 dyads and 6 larger clusters ranging from 4 to 28 individuals. When comparing characteristics between clustering individuals enrolled in the PRIMO cohort (n = 377) and in IPERGAY (n = 14), we found that IPERGAY participants had a higher viral load (5.93 vs 5.20 log10 copies/mL, P = .032) and reported a higher number of partners in the last 2 months (P < .01). CONCLUSIONS: These results demonstrate high rates of HIV transmission clustering among young high-risk MSM enrolled in the IPERGAY trial. In-depth sampling of high-risk populations may help to uncover unobserved transmission intermediaries and improve prevention efforts that could be targeted to the most active clusters.

Anti-gp41 antibody levels reflect HIV viral suppression and cellular reservoir in long-term antiretroviral-treated trial participants.

BACKGROUND: A major challenge to HIV cure strategies is the quantification of persistent reactivation-prone virus in people living with HIV. OBJECTIVES: To determine whether anti-gp41 antibody levels correlate with viral suppression and HIV-1 DNA levels in patients on ART. METHODS: Participants with plasma HIV-1 RNA below 50 copies/mL for >12 months were included from three ANRS cohorts (COPANA, MONOI and APROCO). Antibody levels to gp41 were measured by a low-sensitivity enzyme-linked immunoassay. Correlations with patient and virus characteristics, plasma HIV-1 RNA load (standard and ultrasensitive tests) and cell-associated HIV-1 DNA were assessed. RESULTS: Median age was 41 years and 77.5% of the 683 participants were men. Median CD4+ T cell count was 582 cells/mm3 and median viral suppression duration was 6.6 years (IQR 2.0-9.5). The overall median anti-gp41 antibody titre was 1.3 (IQR 0.6-1.9); median HIV-1 DNA level was 2.6 (IQR 2.1-3.0) log10 copies/106 leucocytes; and HIV-1 RNA was undetectable in 56% of samples. A lower titre of anti-gp41 antibodies correlated with male gender, longer viral suppression and lower HIV-1 DNA burden. Sustained undetectable HIV-1 RNA was associated with lower anti-gp41 levels [median 1.1 (IQR 0.5-1.6) versus 1.4 (IQR 0.7-1.9), P = 0.009]. CONCLUSIONS: Anti-gp41 levels decreased with the duration of antiviral suppression on ART. Lower titres were associated with lower HIV-1 DNA levels and longer duration of viral suppression, reflecting minimal antigen stimulation. Anti-gp41 antibody titration may be a useful biomarker reflecting long-term HIV-1 suppression on ART.

Viral rebound in semen after antiretroviral treatment interruption in an HIV therapeutic vaccine double-blind trial.

OBJECTIVES: This study aimed to determine the timing and level of HIV rebound in blood and seminal plasma and to characterize the HIV rebounding populations after antiretroviral treatment interruption (ATI) in HIV-1-infected participants enrolled in a therapeutic vaccine trial. DESIGN: A 12-week (W) ATI period was proposed at W36 to patients enrolled in the VRI02/ANRS149-LIGHT trial. Paired blood and semen samples were collected before (W32 or W36) and during ATI (W38, W40, W42, W44, and W48). METHODS: HIV-RNA and HIV-DNA were quantified sequentially from blood and semen samples. Ultradeep sequencing (UDS; Roche/454) of partial env HIV-DNA/RNA (C2V3) was performed in both compartments. RESULTS: HIV-RNA rebounded in blood plasma and seminal plasma of all ten participants after ATI [median peak of 5.12 log10 cp/ml (range: 4.61-6.35) and 4.26 log10 cp/ml (3.20-4.67), respectively]. HIV-RNA rebound was detected in blood plasma as soon as W38 in 8/10 patients, and in seminal plasma between W38 and W40 in 8/10 patients. Phylogenetic approaches showed intermingled HIV-RNA populations from plasma and semen during ATI, suggesting a lack of viral compartmentalization between blood and semen. CONCLUSION: Our data demonstrate rapid and high HIV rebound in semen after ATI, raising concerns about high risk of HIV sexual transmission during HIV cure trials.

Ultra-deep sequencing improves the detection of drug resistance in cellular DNA from HIV-infected patients on ART with suppressed viraemia.

Background: Standard genotypic tests performed on HIV DNA from patients on suppressive ART, with previous resistance-associated mutations (RAMs) detected in their plasma, underestimate resistance. We thus compared ultra-deep sequencing (UDS) with bulk sequencing of DNA to detect RAMs previously identified in plasma. Methods: We sequenced the DNA of 169 highly treatment experienced patients with suppressed viraemia (ANRS 138-EASIER trial). Protease (PR) and reverse transcriptase (RT) genes from HIV DNA were sequenced by bulk sequencing and UDS, comparing 1% and 20% as thresholds of detection for UDS. Results: Patients were highly treatment experienced (13.6 years). UDS of DNA was successful for the RT and PR genes in 133 (79%) and 137 (81%) patients, respectively. The detection of RAMs was similar by bulk sequencing and UDS with a 20% cut-off. However, the detection of RAMs by UDS with a 1% cut-off was significantly higher than that of bulk sequencing for RT codons D67N (65.4% versus 52.3%), M184V (66.2% versus 52.3%), L210W (48.9% versus 36.4%) and T215Y (57.9% versus 42.1%) and PR codons M46I (46% versus 26%), I54L (12.4% versus 3.9%), V82A (44.5% versus 29.9%) and L90M (57.7% versus 42.5%). Conclusions: Genotypic resistance testing of cellular HIV DNA of well-controlled patients should use UDS technology with a sensitivity threshold of 1% to improve the detection of the resistant reservoir.

Drug resistance among patients who acquired HIV infection in a preexposure prophylaxis trial.

BACKGROUND: The IPERGAY ANRS trial showed that on-demand preexposure prophylaxis (PrEP) with tenofovir (TDF) and emtricitabine (FTC) was highly effective in preventing HIV infection among highly exposed MSM. Here, we analyzed drug resistance-associated mutations (RAMs) among all participants who acquired HIV infection during this trial. METHODS: Resistance was analyzed on frozen plasma at the time of HIV diagnosis among participants enrolled in the double-blind and open-label phases of the ANRS IPERGAY trial. Reverse transcriptase sequencing was performed, using population-based and ultradeep sequencing (454 GS Flex). Adherence was measured by pill counting and by plasma tenofovir and FTC assay. RESULTS: During the trial, 31 participants were diagnosed with HIV-1 infection (subtype B, 64.5%), using antigen/antibody immune assay in 29 cases and plasma HIV RNA assay in two. The median plasma HIV-1 RNA level was 5.52 log10 copies/ml. Drug resistance was tested in 12 participants before starting PrEP, in six assigned to TDF/FTC group and in 13 assigned to placebo group. Primary resistance to nucleoside reverse transcriptase inhibitors (zidovudine) and/or nonnucleoside reverse transcriptase inhibitors was detected in six participants (19%; 95% confidence interval 7-42). No major or minor TDF-resistant or FTC-resistant variants were detected. CONCLUSION: No TDF or FTC resistance-associated mutations were found among participants who acquired HIV in the ANRS IPERGAY trial.

Detectable HIV-RNA in semen of HIV controllers.

BACKGROUND: Whether spontaneous low levels of HIV-1 RNA in blood plasma correlate with low levels of HIV-1 RNA in seminal plasma has never been investigated in HIV controller (HIC) men so far. METHODS: HIC men enrolled in the ANRS CODEX cohort were eligible for the present study if they had no symptoms of sexually transmitted infections (STI). Two paired samples of blood and semen were collected four weeks apart. HIV-RNA was quantified in blood plasma (bpVL) and in seminal plasma (spVL), and cell-associated HIV-DNA was quantified in peripheral blood mononuclear cells (PBMC) and in non-sperm cells (NSC). Spearman rho tests were used to estimate correlations between bpVL and spVL. RESULTS: Ten men were enrolled. At Day 0 (D0), spVL was detectable in four patients: 458; 552; 256 copies/mL and PCR signal detectable below limit of quantification (LoQ, 40 copies/mL). At Day 28 (D28), spVL was detectable in the same four participants in whom spVL was detectable at D0 with 582; 802; 752 and 50 copies/mL, respectively. HIV-DNA was detectable below LoQ in NSC of one patient at D0 visit. No patient had detectable HIV-DNA in NSC at D28 visit. At D0, bpVL and spVL were highly positively correlated (Spearman rho: 0.94; p = 0.0001). Similar results were found at D28. CONCLUSION: We show that HIV-RNA can be detected in the semen of HIC men, with levels positively correlated with those measured concomitantly in blood plasma. HIC men should be aware of the risk of HIV genital shedding, especially if viral blips are reported.

Unravelling the dynamics of selection of multiresistant variants to integrase inhibitors in an HIV-1-infected child using ultra-deep sequencing.

Background: Ultra-deep sequencing (UDS) allows detection of minority resistant variants (MRVs) with a threshold of 1% and could be useful to identify variants harbouring single or multiple drug-resistance mutations (DRMs). Objectives: We analysed the integrase gene region longitudinally using UDS in an HIV-1-infected child rapidly failing a raltegravir-based regimen. Methods: Longitudinal plasma samples at baseline and weeks 4, 8, 13, 17 and 39 were obtained, as well as the mother's baseline plasma sample. Sanger sequencing and UDS were performed on the integrase gene using Roche 454 GS-Junior. A bioinformatic workflow was developed to identify the major DRMs, accessory mutations and the linkage between mutations. Results: In Sanger sequencing and UDS, no MRV in the integrase gene was detected at baseline in either the mother or the child. The major DRM N155H conferring resistance to raltegravir and elvitegravir was detected in 4% of the sequences by week 4 using UDS, whereas it was not detected by Sanger sequencing. The double mutant E92Q + N155H, conferring resistance to the entire integrase inhibitor class, including dolutegravir, emerged at week 8 (16%) and became rapidly dominant (57% by week 13). At the last timepoint under raltegravir (week 17), Y143R emerged, leading to different resistance mutation patterns: single mutants N155H (47%) and Y143R (24%) and double mutants E92Q + N155H (13%), Y143R + N155H (2%) and E92Q + Y143R (2%). The polymorphic substitution M50I was preferentially selected on resistant variants, especially on E92Q + N155H variants. Conclusions: This case study illustrates the usefulness of UDS in detecting early MRVs and determining the dynamics of selected HIV-1 variants in longitudinal analysis.

Time course of total HIV-1 DNA and 2-long-terminal repeat circles in patients with controlled plasma viremia switching to a raltegravir-containing regimen.

BACKGROUND: Early integration of HIV proviral DNA into the host cell genome prevents viral eradication, despite suppressive HAART. In vitro, integrase inhibitors reduce proviral DNA levels and rapidly increase 2-long-terminal repeat (LTR) circle levels. We examined the effect of raltegravir on the time course of HIV-1 DNA forms in patients with controlled viremia. PATIENTS AND METHODS: The EASIER-ANRS 138 randomized trial demonstrated that switching from enfuvirtide to raltegravir maintained virological suppression in treatment-experienced patients with viral load below 400 copies/ml. We analyzed total HIV-1 DNA and 2-LTR circle levels measured at weeks (W)0 and 24 in the first 30 patients enrolled in each arm, and at W48 in the raltegravir arm. RESULTS: At W0 the total DNA level was 3.6 log(10)/10(6) peripheral blood mononuclear cell (PBMC) in both groups, and 2-LTR circles were detected in six patients (median 89 copies/10(6) PBMC). At W24 the total DNA level was 3.6 log(10)/10(6) PBMC in both groups, and 2-LTR circles were detected in three new patients. At W48 the total HIV DNA level in the raltegravir group was 3.5 log(10)/10(6) PBMC, and 2-LTR circles were undetectable. No significant change in total HIV DNA occurred between W0 and W24 in either arm (P = 0.71) and no significant change was observed in the raltegravir arm at W48. DISCUSSION: In most patients on effective HAART, including regimens containing an integrase inhibitor, the viral reservoir, reflected by the HIV-1 DNA load, is stable and nondynamic during the 48 weeks of follow-up.