Higher Levels of SARS-CoV-2 Genetic Variation in Immunocompromised Patients: A Retrospective Case-Control Study.

BACKGROUND: A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection lasts longer in immunocompromised hosts than in immunocompetent patients. Prolonged infection is associated with a higher probability of selection for novel SARS-CoV-2 mutations, particularly in the spike protein, a critical target for vaccines and therapeutics. METHODS: From December 2020 to September 2022, respiratory samples from 444 immunocompromised patients and 234 health care workers positive for SARS-CoV-2, diagnosed at 2 hospitals in Paris, France, were analyzed using whole-genome sequencing using Nanopore technology. Custom scripts were developed to assess the SARS-CoV-2 genetic diversity between the 2 groups and within the host. RESULTS: Most infections were SARS-CoV-2 Delta or Omicron lineages. Viral genetic diversity was significantly higher in infections of immunocompromised patients than those of controls. Minor mutations were identified in viruses sequenced from immunocompromised individuals, which became signature mutations for newer SARS-CoV-2 variants as the epidemic progressed. Two patients were coinfected with Delta and Omicron variants. The follow-up of immunocompromised patients revealed that the SARS-CoV-2 genome evolution differed in the upper and lower respiratory tracts. CONCLUSIONS: This study found that SARS-CoV-2 infection in immunocompromised patients is associated with higher genetic diversity, which could lead to the emergence of new SARS-CoV-2 variants with possible immune evasion or different virulence characteristics.

Poor Antibody Response After Two Doses of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccine in Transplant Recipients.

A low anti-spike antibody response of 28.6% was observed 28 days after BNT162b2 vaccine second dose among 133 solid organ transplant recipients without previous coronavirus disease 2019 (COVID-19). No serious adverse events were recorded. Four severe COVID-19 cases were reported between or after the 2 doses. Our data suggest to change the vaccine strategy.

HIV-1 protease, Gag and gp41 baseline substitutions associated with virological response to a PI-based regimen.

OBJECTIVES: To assess, at ART initiation, the impact of baseline substitutions in protease, Gag and gp41 regions on the virological response to a first-line PI-based regimen. PATIENTS AND METHODS: One hundred and fifty-four HIV-infected ART-naive patients initiating a PI-based regimen including darunavir (n = 129) or atazanavir (n = 25) were assessed, including 36 experiencing virological failure (VF). Whole pol, gag and gp41 genes were sequenced at ART baseline by ultra-deep sequencing (UDS) using Illumina® technology. Supervised data-mining analyses were performed to identify mutations associated with virological response. Structural analyses were performed to assess the impact of mutations on protease conformation. RESULTS: UDS was successful in 127, 138 and 134 samples for protease, Gag and gp41, respectively (31% subtype B and 38% CRF02_AG). Overall, T4A and S37T mutations in protease were identified as being associated with VF (P = 0.02 and P = 0.005, respectively). Among CRF02_AG sequences, I72M and E21D mutations were associated with VF (P = 0.03 for both). They all induced some conformational changes of some protease side-chain residues located near mutated residues. In Gag, mutations associated with VF were G62D, N315H and Y441S (P = 0.005, P = 0.007 and P = 0.0003, respectively). All were localized outside Gag cleavage sites (G62D, matrix; N315H, capsid; and Y441S, p1). In gp41, the I270T mutation, localized in the cytoplasmic tail, was associated with VF (P = 0.003), and the I4L mutation, in the fusion peptide, was associated with virological success (P = 0.004). CONCLUSIONS: In this study, new baseline substitutions in Gag, protease and g41, potentially impacting PI-based regimen outcome, were evidenced. Phenotypic analyses are required to confirm their role in the PI-resistance mechanism.

New mechanisms of resistance in virological failure to protease inhibitors: selection of non-described protease, Gag and Gp41 mutations.

OBJECTIVES: To further characterize HIV-1 viruses of patients experiencing unexplained virological failure (VF) on PI-containing regimens, ultradeep sequencing was performed on protease, gag and gp41 genes in patients failing a first-line treatment. METHODS: All naive patients initiating an antiretroviral treatment based on boosted darunavir, atazanavir or lopinavir and experiencing VF without any transmitted drug resistance mutation detected by Sanger sequencing on protease and reverse transcriptase genes were selected. Ultradeep sequencing (IlluminaTM Nextera®) was performed on protease, gag and gp41 genes in plasma before initiation of treatment and at VF to identify emergent mutations. RESULTS: Among the 32 patients included in the study, emergent and previously undescribed mutations in the viral protease gene were identified in five patients at VF: 64M (1 CRF02_AG), 64M/70R with mutation 15V (2 CRF02_AG), 79A (1 CRF06_cpx) and 79A with mutation 15V (1 CRF02_AG). Two patients showed the emergence of R286K in the gag region, outside of cleavage sites (2 CRF02_AG). In the gp41 region, the V321I mutation emerged inside the cytoplasmic tail (1 subtype A and 1 subtype B). All these patients were treated with a darunavir/ritonavir-based regimen. CONCLUSIONS: In some cases of VF to PIs, we observed the emergence of protease, Gag or Gp41 mutations that had not previously been associated with VF or PI resistance. These mutations should be further studied, in particular the 15V/64M/70R pattern in the protease gene identified among CRF02_AG viruses.

No impact of HIV-1 protease minority resistant variants on the virological response to a first-line PI-based regimen containing darunavir or atazanavir.

OBJECTIVES: To evaluate, in a clinical cohort of HIV-1-infected patients, the prevalence of PI minority resistant variants (MRV) at ART baseline and their impact on the virological response to a first-line PI-based regimen. PATIENTS AND METHODS: In an observational single-centre cohort, we assessed all ART-naive patients initiating a first-line regimen including two NRTI and one boosted PI, darunavir/ritonavir or atazanavir/ritonavir, between January 2012 and March 2015. Ultra-deep sequencing of the pol gene was performed using Illumina® technology. Protease mutations were identified using the WHO transmitted drug resistance list and major PI resistance mutations (IAS-USA drug resistance mutations list). RESULTS: Ninety-four and 16 patients initiating a darunavir/ritonavir-based regimen and an atazanavir/ritonavir-based regimen, respectively, were assessed. Twenty-eight percent of the patients were HIV-1 subtype B, 39% CRF02_AG and 33% other non-B subtypes. Thirteen patients (13.8%) in the darunavir group and three patients (18.8%) in the atazanavir group experienced a virological failure (VF). Overall, 13 (11.8%) subjects had PI MRV at baseline in the median proportion of 1.3% (IQR = 1.1-1.7). The most prevalent PI MRV were G73C (n = 5) and M46I (n = 3). The proportion of patients harbouring baseline PI MRV was similar between those with virological success (10.6%) and those experiencing VF (18.8%) (P = 0.40). No difference was observed in the rate of PI MRV by viral subtype (P = 0.51) or by PI drug (P = 0.40). CONCLUSIONS: This study showed a prevalence of 11.8% of PI MRV among 110 ART-naive subjects, without significant impact on the virological response to a first-line PI-based regimen containing darunavir or atazanavir.

Week 96 efficacy of lopinavir/ritonavir monotherapy in virologically suppressed patients with HIV: a randomized non-inferiority trial (ANRS 140 DREAM).

Background: Sparing of antiretroviral drug classes could reduce the toxicity and cost of maintenance treatment for HIV infection. Objectives: To evaluate the non-inferiority of efficacy and the safety of lopinavir/ritonavir (r) monotherapy versus a single-tablet regimen of efavirenz, emtricitabine and tenofovir (EFV/FTC/TDF) over 2 years. Methods: Adults on stable ART with plasma HIV-1 RNA viral load <50 copies/mL for the past 12 months and no documented treatment failure were randomized to receive either lopinavir/r or EFV/FTC/TDF for 2 years. The primary endpoint was the proportion of patients without treatment failure at week 96 (viral load <50 copies/mL at week 96, confirmed at week 98), without study treatment discontinuation, a new AIDS-defining illness, or death. Results: In the ITT analysis, the primary endpoint was reached by, respectively, 64% and 71% of patients in the lopinavir/r (n = 98) and EFV/FTC/TDF arms (n = 97), yielding a difference of -6.8% (lower limit of the 95% two-sided CI: -19.9%). Sanger and UltraDeep sequencing showed the occurrence of PI mutations in the lopinavir/r arm (n = 4) and of NNRTI and/or NRTI mutations in the EFV/FTC/TDF arm (n = 2). No unexpected serious clinical events occurred. Conclusions: Lopinavir/r monotherapy cannot be considered non-inferior to EFV/FTC/TDF. PI resistance rarely emerged in the lopinavir/r arm and did not undermine future PI options. Two years of lopinavir/r monotherapy had no deleterious clinical impact when compared with EFV/FTC/TDF.

Qualitative and quantitative HIV antibodies and viral reservoir size characterization in vertically infected children with virological suppression.

Background: Absence of detectable viraemia after treatment cessation in some vertically HIV-infected (VHIV) children suggests that early initiation of HAART could lead to functional cure. Objectives: We described the factors associated with HIV antibody levels and the viral reservoir size in HAART-treated VHIV children. Methods: Study included 97 VHIV children with virological suppression, in Bamako, Mali. The anti-gp41 antibody activities and HIV serostatus were assessed. The viral reservoir size was measured by quantifying total cell-associated HIV DNA. Results: Among the children studied, the median total HIV DNA level was 445 copies/10 6 cells (IQR = 187-914) and the median anti-gp41 antibody activity was 0.29 OD (IQR = 0.18-0.75). Low activity of anti-gp41 antibodies was associated with a younger age of HAART initiation ( P = 0.01). Overall, eight HIV-1 seroreversions were identified. Conclusions: Study identified potential candidates with low viral reservoir and low antibody levels or activities for future trials aiming to reduce HIV-1 reservoir to limit HAART duration.

Low-level HIV-1 viraemia in patients on HAART: risk factors and management in clinical practice.

OBJECTIVES: Characterization of the conditions favouring HIV-1 low-level viraemia (LLV) during treatment is required to guide strategies for prevention and cure. METHODS: The characteristics and treatments of 171 patients experiencing a confirmed LLV of 50-1000 copies/mL (PLLVs) were compared with those of 146 patients with persistently controlled viraemia. We analysed the risk factors for LLV, the parameters affecting the level of viraemia and the presence of resistance-associated mutations (RAMs). We compared outcomes for PLLVs on fully effective HAART as a function of treatment modifications. RESULTS: LLV was <500 copies/mL in at least 90% of cases. A higher zenith viral load (VL) (5.27 versus 4.91 log10 copies/mL, OR 2.23; P = 0.0003), a shorter time on continuous HAART (4.3 versus 6.8 years, OR 0.88; P = 0.0003) and previously detected RAMs (43% versus 23%, OR 2.42; P = 0.0033) were independent predictors of LLV. NNRTIs were less frequently used in PLLVs and were associated with more stable treatment. The presence of any RAM during LLV was associated with a lower zenith VL and a higher LLV. In the absence of resistance, virological success was achieved in similar proportions of patients with and without treatment modification. CONCLUSIONS: Viraemia >500 copies/mL should no longer be considered to be LLV. In patients with a high zenith VL, several years on continuous HAART may be required to decrease the HIV reservoir and prevent LLV. Resistance testing is useful to detect RAMs, leading if necessary to treatment modifications. In the absence of resistance, treatment changes seemed dispensable.

Improved detection of resistance at failure to a tenofovir, emtricitabine and efavirenz regimen by ultradeep sequencing.

OBJECTIVES: Resistant minority variants present before ART can be a source of virological failure. This has been shown for NRTIs, NNRTIs and CCR5 inhibitors. However, very few data are available for the detection of such minority resistant variants that could be selected at virological failure and not detected using classical Sanger sequencing. METHODS: We studied 26 patients treated with tenofovir, emtricitabine and efavirenz with their first virological failure (defined as two consecutive viral loads >50 copies/mL). We performed standard Sanger sequencing and ultradeep sequencing (UDS; Roche 454(®) Life Sciences) in plasma at failure. For UDS, mutations >1% were considered. We compared the presence of reverse transcriptase mutations between the two techniques, using the latest ANRS algorithm. RESULTS: UDS detected more resistance mutations in 38.5% of cases (10/26 patients) and the genotypic sensitivity score (GSS) was reduced for 6 of them (23.1%). The GSS was impacted more often for NRTIs than for NNRTIs, for which most mutations were already detected by Sanger sequencing. Resistant minority variants were detected even in patients with low viral load at failure. CONCLUSIONS: These results strongly argue for the use of next-generation sequencing in patients failing on an NRTI+NNRTI regimen, as UDS has the potential to modify the choice of the subsequent regimen.

Highly multidrug-resistant HIV: clonal analysis and therapeutic strategies.

OBJECTIVES: There are today HIV-infected patients in therapeutic impasses because of highly multidrug-resistant (HMDR) viruses. We studied the distribution of resistance mutations at clonal level, and we analysed the therapeutic strategies used in such cases to achieve undetectable viraemia. METHODS: The HMDR profile was defined as a genotypic sensitivity score (GSS) ≤ 1.5 for etravirine and raltegravir with full resistance to darunavir. About 30 clones per gene and per patient were sequenced. Virtual phenotypes were determined. Efficacy of therapeutic strategies was evaluated by follow-up of viral loads, CD4 cell counts and trough concentrations of drugs. RESULTS: Among 1310 patients on treatment and with genotypic resistance testing, 25 (2%) were resistant to darunavir and 11 (0.8%) had an HMDR profile. Five-hundred clones could be analysed for four of them. HMDR profiles were harboured by the great majority of clones and all resistance mutations were located on the same strains for all genes. Despite this and a regimen with a GSS <2.0 in three patients, they achieved a viraemia <20 copies/mL. These results were obtained using different strategies: high doses of drugs; combination of antiretrovirals with full or intermediate susceptibility, such as tipranavir, etravirine or maraviroc; and use of alternative compounds, such as foscarnet or interferon. CONCLUSION: Patients with HMDR HIV were uncommon, but, in such cases, all resistance mutations were borne on the same majority strains. In this study, tipranavir was the only protease inhibitor with full or intermediate susceptibility. Despite very limited therapeutic options, an undetectable viraemia can be achieved by combining different strategies.

Prolonged replication of BA.1 and BA.2 Omicron lineages compared to Delta variant in nasopharyngeal samples from COVID-19 patients.

OBJECTIVES: We aimed to characterize and compare the viral loads (VL) of the Omicron BA.1 and BA.2 lineages and the Delta variant in nasopharyngeal samples from newly diagnosed COVID-19 patients and their kinetics over time. PATIENTS AND METHODS: The kinetics of the VL were measured on the CT data from 215 SARS-CoV-2 positive patients who presented at least two positive PCRs a day apart and were screened for SARS-CoV-2 viral lineages. RESULTS: We observed no significant difference in median CT value during the first diagnostic test between the Delta variant and the two Omicron lineages. However, the kinetics of CT decreases for the BA.1 and BA.2 lineage were significantly lengthier in time than the kinetics for the Delta variant. The BA.2 lineage presented lower median CT value (-2 CT) (inversely proportional to the VL) than the BA.1 lineage. CONCLUSIONS: BA.2 Omicron lineage presented higher VL than BA.1 Omicron lineage at diagnostic. Omicron BA.1 and BA.2 lineages have more prolonged replication than the Delta variant.

Evaluating interventions to reduce behaviour associated with HCV reinfection in men who have sex with men: study protocol for a non-blinded, phase 2, randomised trial.

BACKGROUND: As highly effective therapy against hepatitis C virus (HCV) infection is available with rapid uptake, there is newfound optimism for HCV elimination. Nevertheless, certain key populations have a high risk of HCV reinfection, in particular men who have sex with men (MSM) in Western European countries. Modelling data indicate that HCV elimination will not be feasible without reduction in risk behaviour, thus supporting the need for effective interventions aimed at reducing risk behaviour and preventing reinfections in MSM. METHODS: The ICECREAM study is an international, multi-centred, phase 2, 3-arm randomised trial comparing run-in and intervention periods enrolling MSM with a history of a cured or spontaneously cleared HCV infection. Individuals are followed in routine care for 6 months (i.e. run-in period) and then randomly allocated (1:1:1) to one of the following: a tailored, interactive online risk-reduction behavioural intervention, a validated home-based HCV-RNA self-sampling test service using dried blood spots, or a combination of both. After randomisation, individuals are followed every 6 months until 18 months (i.e. intervention period). Interventions are delivered in addition to standard of care. Online questionnaire measuring risk behaviour over the past 6 months is administered at every visit. The primary outcome is the proportion at risk of HCV infection during run-in versus intervention periods assessed by using the HCV-MOSAIC risk score. The risk score consists of six self-reported HCV-related risk behaviours. Secondary outcomes include incidence of HCV reinfection, changes in the individual risk behaviour items and changes in sexual well-being since changes in sexual behaviour may have an impact on sexual experience. Two hundred forty-six MSM aged 18 years or older will be invited to participate. DISCUSSION: The ICECREAM study is a trial aimed at establishing interventions that could effectively decrease the incidence of HCV re-infection in MSM with a previous HCV infection. By offering an online behavioural risk-reduction intervention and HCV-RNA self-sampling, both of which are aimed to influence risk behaviour, we are able to provide products to at-risk MSM that could further reduce population-level HCV incidence and ultimately help reach HCV micro-elimination. TRIAL REGISTRATION: ClinicalTrials.gov NCT04156945. Registered on November 8, 2019.

Viral loads in clinical samples of men with monkeypox virus infection: a French case series.

BACKGROUND: Monkeypox virus (MPXV) is currently spreading among men who have sex with men, outside of sub-Saharan Africa, and close contact during sex seems to be one of the key pathways of viral transmission in the current outbreak. Our aim was to describe the distribution of MPXV in the human body, as it might play a role in its dissemination through sexual contact. METHODS: The study population in this case series consisted of patients with confirmed MPXV infection attending the Pitié-Salpêtrière Hospital (Paris, France), who had been sampled from multiple anatomical sites, including skin, anus, throat, blood, urine, and semen, at diagnosis and 2 weeks later. We compared the proportion of positive samples and MPXV viral loads (given as PCR cycle thresholds [Ct]) between anatomical sites, and between day 0 (D0) and D14. FINDINGS: Overall, 356 samples were collected between May 20 and June 13, 2022, from 50 men with a median age of 34 years (IQR 29-40). 22 (44%) of the 50 men were classified as HIV-negative on day (D)0, and 22 (44%) were living with HIV. At D0, MPXV detection was more frequent from skin (44 [88%] of 50), anus (30 [71%] of 42), and throat (36 [77%] of 47) than from blood (13 [29%] of 45), urine (nine [22%] of 41), or semen (13 [54%] of 24). Viral loads were significantly higher from skin lesions (Ct 19·8) and anal samples (Ct 20·9) than from throat (Ct 27·2), blood (Ct 32·8), urine (31·1), or semen samples (Ct 27·8). When analysing the 107 samples taken from 24 patients at D14, the proportion of positive samples strongly decreased between D0 and D14 at all sites: skin (four [22%] of 18), anus (two [9%] of 22), throat (none of 21), blood (one [5%] of 21), urine (none of 14), and semen (two [9%] of 11). INTERPRETATION: These data contribute to a better understanding of how the virus might spread between sexual partners over a relatively short period of time. High MPXV viral loads from skin and mucosa, including genital and anal sites, suggest that transmission most likely occurs through direct body contact rather than through the respiratory route or contact with body fluids, which should help to refine the prevention messages delivered to individuals most exposed to the virus. FUNDING: None.

Epidemiology and Characteristics of SARS-CoV-2 Variants of Concern: The Impacts of the Spike Mutations.

SARS-CoV-2 expresses on its surface the Spike protein responsible for binding with the ACE2 receptor and which carries the majority of immunodominant epitopes. Mutations mainly affect this protein and can modify characteristics of the virus, giving each variant a unique profile concerning its transmissibility, virulence, and immune escape. The first lineage selected is the B.1 lineage characterized by the D614G substitution and from which all SARS-CoV-2 variants of concern have emerged. The first three variants of concern Alpha, Beta, and Gamma spread in early 2021: all shared the N501Y substitution. These variants were replaced by the Delta variant in summer 2021, carrying unique mutations like the L452R substitution and associated with higher virulence. It was in turn quickly replaced by the Omicron variant at the end of 2021, which has predominated since then, characterized by its large number of mutations. The successive appearance of variants of concern showed a dynamic evolution of SARS-CoV-2 through the selection and accumulation of mutations. This has not only allowed progressive improvement of the transmissibility of SARS-CoV-2, but has also participated in a better immune escape of the virus. This review brings together acquired knowledge about SARS-CoV-2 variants of concern and the impacts of the Spike mutations.

Low level of baseline resistance in recently HCV-infected men who have sex with men with high-risk behaviours.

OBJECTIVES: Presence of baseline hepatitis C virus (HCV) resistance-associated substitutions (RASs) can impair treatment outcome of direct-acting antivirals. We investigated the prevalence of pre-treatment HCV resistance among recently HCV-infected men who have sex with men (MSM) with high risk behaviours, either human immunodeficiency virus (HIV) co-infected or at high risk of HIV acquisition and under pre-exposure prophylaxis (PrEP). METHODS: NS5A and NS3 fragments were deep sequenced on pre-treatment samples of 72 subjects using Illumina MiSeq paired-end sequencing technology. Ultra-deep sequencing data were analysed by SmartGene® platform. RASs mentioned in the literature were analysed and interpreted depending on genotype (GT) at 10% cut-off. RESULTS: HCV genotyping showed 36 (50.0%) GT1a, 31 (43.1%) GT4d and 5 (6.9%) GT3a infections. Fifty-five patients (76.4%) were co-infected with HIV and 15 (20.8%) received PrEP. In GT1a viruses, NS3 RASs were found in 4/30 viruses (13.3%; S122 G/N, R155 K and I170 V) and Q80 K polymorphism was present in 14/30 viruses (46.7%). No NS3 RASs were detected in GT4d and GT3a viruses. NS5A RASs were detected in 3/36 GT1a viruses (8.3%; Q30E/R, L31 M and H58 L). NS5A subtype-specific polymorphisms L30R and T58 P were found at high frequencies in 31/31 (100%) and 16/31 (51.6%) GT4d viruses, respectively. One RAS M31 L was also observed along with the polymorphisms L30R and T58 P. No NS5A RASs were detected in GT3a viruses. CONCLUSION: A low level of RASs to NS3 and NS5A inhibitors in pre-treatment samples was detected in the study population. Our findings reassure the clinical management of HCV infection in this high-risk population.