Immunogenicity and efficacy of          heterologous ChAdOx1-BNT162b2 vaccination.

Following severe adverse reactions to the AstraZeneca ChAdOx1-S-nCoV-19 vaccine1,2, European health authorities recommended that patients under the age of 55 years who received one dose of ChAdOx1-S-nCoV-19 receive a second dose of the Pfizer BNT162b2 vaccine as a booster. However, the effectiveness and the immunogenicity of this vaccination regimen have not been formally tested. Here we show that the heterologous ChAdOx1-S-nCoV-19 and BNT162b2 combination confers better protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection than the homologous BNT162b2 and BNT162b2 combination in a real-world observational study of healthcare workers (n = 13,121). To understand the underlying mechanism, we conducted a longitudinal survey of the anti-spike immunity conferred by each vaccine combination. Both combinations induced strong anti-spike antibody responses, but sera from heterologous vaccinated individuals displayed a stronger neutralizing activity regardless of the SARS-CoV-2 variant. This enhanced neutralizing potential correlated with increased frequencies of switched and activated memory B cells that recognize the SARS-CoV-2 receptor binding domain. The ChAdOx1-S-nCoV-19 vaccine induced a weaker IgG response but a stronger T cell response than the BNT162b2 vaccine after the priming dose, which could explain the complementarity of both vaccines when used in combination. The heterologous vaccination regimen could therefore be particularly suitable for immunocompromised individuals.

Use of genotypic HIV DNA testing: a DELPHI-type consensus.

OBJECTIVES: As many disparities in the clinical use of HIV DNA sequencing are observed, a DELPHI-type consensus was initiated in France to homogenize use, techniques and interpretation of results. METHODS: Based on a literature review and clinical experience, a steering committee (SC) of eight virologists and one infectious disease specialist formulated statements. Statements were submitted to an independent and anonymous electronic vote of virologists and HIV clinicians in France, between October 2022 and December 2022. RESULTS: The SC developed 20 statements grouped into six categories: clinical situations for the use of HIV DNA genotyping; techniques for performing HIV DNA genotyping; consideration of apolipoprotein B mRNA editing enzyme (APOBEC) mutations; genotyping results reporting; recycling of antiretrovirals; and availability of HIV DNA genotyping tests and delays. Twenty-one virologists and 47 clinicians participated in two voting rounds and 18/20 (90%) assertions reached a 'strong' consensus. For example, that prior genotyping on HIV DNA is useful for clinical decision-making when considering switching to some long-acting regimens or to reduce the number of antiretroviral agents in virologically suppressed patients for whom RNA data are unavailable/not exploitable/not sufficiently informative. Two statements achieved no consensus: reporting any detected viral minority population for discussion in multidisciplinary meetings (virologists), and possible risk of virological failure when using a second-generation InSTI plus lamivudine or emtricitabine regimen in patients with undetectable viral load within ≥1 year and in the presence of a documented M184V mutation within the last 5 years (clinicians). CONCLUSIONS: This DELPHI-type consensus will facilitate the strengthening and harmonization of good practice when performing HIV DNA sequencing.

Monitoring of Both Humoral and Cellular Immunities Could Early Predict COVID-19 Vaccine Efficacy Against the Different SARS-CoV2 Variants.

Reliable immunoassays are essential to early predict and monitor vaccine efficacy against SARS-CoV-2. The performance of an Interferon Gamma Release Assay (IGRA, QuantiFERON® SARS-CoV-2), and a current anti-spike serological test, compared to a plaque reduction neutralization test (PRNT) taken as gold standard were compared. Eighty vaccinated individuals, whose 16% had a previous history of COVID-19, were included in a longitudinal prospective study and sampled before and two to four weeks after each dose of vaccine. In non-infected patients, 2 doses were required for obtaining both positive IGRA and PRNT assays, while serology was positive after one dose. Each dose of vaccine significantly increased the humoral and cellular response. By contrast, convalescent subjects needed a single dose of vaccine to be positive on all 3 tests. Both IGRA and current serology assay were found predictive of a positive titer of neutralizing antibodies that is correlated with vaccine protection. Patients over 65 or 80 years old had a significantly reduced response. The response tended to be better with the heterologous scheme (vs. homologous) and with the mRNA-1273 vaccine (vs. BNT162b2) in the homologous group, in patients under 55 and under 65 years old, respectively. Finally, decrease intensity or absence of IGRA response and to a less extent of anti-spike serology were also correlated to reinfection which has occurred during the follow up. In conclusion, both IGRA and current anti-spike serology assays could be used at defined thresholds to monitor the vaccine response against SARS-CoV-2 and to simply identify non-responding individuals after a complete vaccination scheme. Two available specific tests (IGRA and anti-spike antibodies) could early assess the vaccine-induced immunity against SARS-CoV-2 at the individual scale, to potentially adapt the vaccination scheme in non-responder patients.

Urine-based detection of HPV for cervical cancer screening: Time for standardized tests.

Cervical cancer is preventable because it has an established etiology, mainly attributed to a detectable pathogen, human papillomavirus (HPV). In 2018, the world health organization issued an unprecedented call for global action to eliminate cervical cancer by 2030. The adaptation of regular screening programs is fundamental to achieve the goal of cervical cancer elimination. However, it is still difficult to achieve satisfactory coverage rates of screening in developing countries as well as in developed countries because many women are reluctant to participate in gynecologic examination. HPV detection in urine is a convenient, widely acceptable by women and relatively affordable without the necessity for clinical visits to improve the coverage rates of cervical cancer screening. Unfortunately, the clinical implementation of urine-based tests for HPV detection has been hindered by the lack of standardized tests. Further optimization of protocols and standardization of urinary HPV detection are expected to be realized. With the advantages of urine sampling to overcome cost, personal, and cultural barriers, time has come for the standardized tests to facilitate a wide clinical implementation of urinary HPV detection that will significantly contribute to the WHO's goal, that is, to eliminate the cervical cancer globally.

[Viruses present in the environment: virological considerations and examples of their impact on human health]. / Les virus dans l'environnement, considérations virologiques et exemples de leur impact en santé humaine.

Animal viruses are present in most human environments. Their viability in these media is very variable and the most important element that conditions this viability is the existence or not of a phospholipid envelope surrounding the nucleocapsid. After some general considerations on the structure of viruses, their multiplication cycle and their resistance to different physico-chemical agents, some examples of the impact of animal viruses present in the environment on human health will be presented. The situations that are related concern recent epidemiological events: circulation of type 2 polioviruses derived from the Sabin vaccine strain in the wastewater of New York, London and Jerusalem; risk of transmission of Sars-CoV-2 during the spreading of sludge from wastewater treatment plants on agricultural land in the era of the Covid-19 pandemic; « new ¼ forms of food-borne poisoning of viral origin (hepatitis E, tick-borne encephalitis, Nipah virus infection); contamination by epidemic viruses of mobile phones used by pediatricians; role of fomites in the spread of orthopoxvirus infections (smallpox, cowpox, monkeypox). The risk attached to animal viruses present in the environment must be assessed in a measured way without overestimating or underestimating their potential consequences for human health.

Exploration of the ocular surface infection by SARS-CoV-2 and implications for corneal donation: An ex vivo study.

BACKGROUND: The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors. METHODS AND FINDINGS: To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors. CONCLUSIONS: In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms. TRIAL REGISTRATION: Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/.

Performance of the COVID19SEROSpeed IgM/IgG Rapid Test, an Immunochromatographic Assay for the Diagnosis of SARS-CoV-2 Infection: a Multicenter European Study.

This study assessed the diagnostic performance of the new COVID19SEROSpeed IgM/IgG rapid test (BioSpeedia, a spinoff of the Pasteur Institute of Paris) for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in comparison to other commercial antibody assays through a large cross-European investigation. The clinical specificity was assessed on 215 prepandemic sera (including some from patients with viral infections or autoimmune disorders). The clinical sensitivity was evaluated on 710 sera from 564 patients whose SARS-CoV-2 infection was confirmed by quantitative reverse transcription-PCR (qRT-PCR) and whose antibody response was compared to that measured by five other commercial tests. The kinetics of the antibody response were also analyzed in seven symptomatic patients. The specificity of the test (BioS) on prepandemic specimens was 98.1% (95% confidence interval [CI], 96.2% to 99.4%). When tested on the 710 pandemic specimens, BioS showed an overall clinical sensitivity of 86.0% (95% CI, 0.83 to 0.89), with good concordance with the Euroimmun assay (overall concordance of 0.91; Cohen's kappa coefficient of 0.62). Due in part to simultaneous detection of IgM and IgG for both S1 and N proteins, BioS exhibited the highest positive percent agreement at ≥11 days post-symptom onset (PSO). In conclusion, the BioS IgM/IgG rapid test was highly specific and demonstrated a higher positive percentage of agreement than all the enzyme-linked immunosorbent assay/chemiluminescence immunoassay (ELISA/CLIA) commercial tests considered in this study. Moreover, by detecting the presence of antibodies prior to 11 days PSO in 78.2% of the patients, the BioS test increased the efficiency of the diagnosis of SARS-CoV-2 infection in the early stages of the disease.

Platelet depletion limits the severity but does not prevent the occurrence of experimental transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a severe pulmonary reaction due to blood transfusions. The pathophysiology of this complication is still not widely elucidated by the scientific community, especially regarding the direct role of blood platelets within the cellular mechanism responsible for the development of TRALI. STUDY DESIGN AND METHODS: In this study, a mouse model was used to induce the development of antibody-mediated acute lung injury through injections of lipopolysaccharide and an anti-major histocompatibility complex Class I antibody. BALB/c mice were pretreated with an anti-GPIbα antibody, which induces platelet depletion, or ML354, a protease receptor 4 pathway inhibitor, 30 minutes before TRALI induction. RESULTS: Depletion of platelets before TRALI induction appeared to reduce the severity of TRALI without completely inhibiting its development. Also, inhibition of platelet activation by ML354 did not prevent the onset of TRALI. Finally, the stimuli used for TRALI induction also triggered specific platelet activation upon ex vivo stimulation. CONCLUSIONS: This study suggests that blood platelets are not critically required for TRALI induction, although they are to some extent involved in its pathophysiology.

A serum protein factor mediates maturation and apoB-association of HCV particles in the extracellular milieu.

BACKGROUND & AIMS: In the sera of infected patients, hepatitis C virus (HCV) particles display heterogeneous forms with low-buoyant densities (<1.08), underscoring their lipidation via association with apoB-containing lipoproteins, which was proposed to occur during assembly or secretion from infected hepatocytes. However, the mechanisms inducing this association remain poorly-defined and most cell culture grown HCV (HCVcc) particles exhibit higher density (>1.08) and poor/no association with apoB. We aimed to elucidate the mechanisms of lipidation and to produce HCVcc particles resembling those in infected sera. METHODS: We produced HCVcc particles of Jc1 or H77 strains from Huh-7.5 hepatoma cells cultured in standard conditions (10%-fetal calf serum) vs. in serum-free or human serum conditions before comparing their density profiles to patient-derived virus. We also characterized wild-type and Jc1/H77 hypervariable region 1 (HVR1)-swapped mutant HCVcc particles produced in serum-free media and incubated with different serum types or with purified lipoproteins. RESULTS: Compared to serum-free or fetal calf serum conditions, production with human serum redistributed most HCVcc infectious particles to low density (<1.08) or very-low density (<1.04) ranges. In addition, short-time incubation with human serum was sufficient to shift HCVcc physical particles to low-density fractions, in time- and dose-dependent manners, which increased their specific infectivity, promoted apoB-association and induced neutralization-resistance. Moreover, compared to Jc1, we detected higher levels of H77 HCVcc infectious particles in very-low-density fractions, which could unambiguously be attributed to strain-specific features of the HVR1 sequence. Finally, all 3 lipoprotein classes, i.e., very-low-density, low-density and high-density lipoproteins, could synergistically induce low-density shift of HCV particles; yet, this required additional non-lipid serum factor(s) that include albumin. CONCLUSIONS: The association of HCV particles with lipids may occur in the extracellular milieu. The lipidation level depends on serum composition as well as on HVR1-specific properties. These simple culture conditions allow production of infectious HCV particles resembling those of chronically-infected patients. LAY SUMMARY: Hepatitis C virus (HCV) particles may associate with apoB and acquire neutral lipids after exiting cells, giving them low-buoyant density. The hypervariable region 1 (HVR1) is a majorviral determinant of E2 that controls this process. Besides lipoproteins, specific serum factors including albumin promote extracellular maturation of HCV virions. HCV particle production in vitro, with media of defined serum conditions, enables production of infectious particles resembling those of chronically infected patients.

Impact of HIV-1 primary drug resistance on the efficacy of a first-line antiretroviral regimen in the blood of newly diagnosed individuals in Bamako, Mali.

Background: To achieve the 90-90-90 targets assigned by UNAIDS, it is crucial to monitor ART in HIV-1-infected patients, especially in resource-limited countries. Objectives: To evaluate the immunovirological response after 12 months of ART in newly HIV-1-diagnosed people in Bamako, Mali; to determine primary and acquired resistance rates to antiretroviral drugs; and to evaluate the impact of primary resistance on the efficacy of ART. Patients and methods: One hundred and nineteen HIV-1-infected people (88.2% women; median age 34 years) were enrolled between January and June 2014. HIV-1 RNA loads (Abbott RealTime HIV-1 assay) were tested in the blood before and at months 3, 6 and 12 after initiation of ART. Primary and acquired resistances to ART were evaluated by the Viroseq™ HIV-1 genotyping assay. Results: During the study, 8.4% of people died and 37% were lost to follow-up. After 1 year of ART, an undetectable HIV-1 RNA viral load was found in 87.7% of cases. The overall rate of primary drug resistance mutations was 17.5% (3.2%, 15.9% and 0% for NRTIs, NNRTIs and PIs, respectively). These mutations were not associated with either higher mortality rates or larger numbers of virological failures. The acquired resistance rate was estimated at 3.1%. Conclusions: Our study showed a high primary resistance level and a huge proportion of people non-adherent to the treatment programme. Reassuringly, almost 90% virological success and a low level of acquired mutations were observed in adherent people at month 12. Reinforced education, regular virological monitoring and early HIV-1 diagnosis may help to improve retention in the care system.

The amino-terminus of the hepatitis C virus (HCV) p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types.

Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.

Inhibition of the CD40/CD40L complex protects mice against ALI-induced pancreas degradation.

BACKGROUND: Acute lung injury (ALI) is a severe complication of transfusion. In a previous study, we saw that inhibition of the CD40/CD40L complex allowed restoration of ALI lesions in an experimental mouse model. OBJECTIVES: This study focused on pancreas-associated injury development during experimental ALI pathogenesis and its limitation through CD40/CD40L complex inhibition. MATERIALS AND METHODS: An ALI mouse model was established through intraperitoneal lipopolysaccharide and intravenous anti-major histocompatibility complex class I monoclonal antibody injection. Preemption of lesions was achieved with intravenous injection of neutralizing anti-CD40L monoclonal antibody 30 minutes before the trigger, that is, anti-major histocompatibility complex class I monoclonal antibody administration. Histology and immunoassay analyses were used to evaluate pancreatic lesions. RESULTS: ALI development induced significant degradation of the lungs and pancreas and was associated with pancreatic lesions. Different scores were established showing more severe injury to the pancreas in ALI conditions; however, injury was significantly reduced through CD40/CD40L complex inhibition. CONCLUSION: This study supports the idea that several organs are exposed during ALI development, and particularly when such experimental ALI aims at mimicking transfusion-associated ALI; nevertheless, preventive treatment inhibiting CD40/CD40L (sCD40L) complex formation provides protection from lung disease as well as disease of other organs.

Platelet toll-like receptors are crucial sensors of infectious danger moieties.

In addition to their haemostatic role and function in the repair of damaged vascular epithelium, platelets play a defensive role in innate immunity, having the capacity to produce and secrete various anti-infectious factors, as well as cytokines, chemokines and related products, to interact with other immune cells to modulate immune responses to pathogens. Thus, it is now widely acknowledged that platelets participate in inflammatory processes and infection resolution, most notably by expressing and using receptors to bind infectious pathogen moieties and contributing to pathogen clearance. The ability of platelets to sense external danger signals relates to the expression of certain innate immunity receptors, such as toll-like receptors (TLRs), and the activation of efficient cell signalling machinery. TLR engagement triggers platelet response, which results in adapted degranulation according to: the type of TLR engaged, the nature of the ligand and the milieu; together, the TLR-mediated event and other signalling events may be followed by aggregation. Platelets thus use complex tools to mediate a whole range of functions upon sensing danger. By linking the inflammatory and haemostatic platelet response to infection, TLRs play a central role. The extent of the inflammatory response to pathogen clearance is still a debatable issue and is discussed in this short review.

Major influence of CD4 count at the initiation of cART on viral and immunological reservoir constitution in HIV-1 infected patients.

BACKGROUND: A persistent immune activation is observed in gut during HIV-1 infection, which is not completely reversed by a combined antiretroviral therapy (cART). The impact of the time of cART initiation may highly influence the size of the viral reservoir and the ratio of CD4(+)/CD8(+) T cells in the gut. In this study, we analyzed the characteristics of HIV rectal reservoir of long-term treated patients, regarding their blood CD4(+) T cells count at the time of cART initiation. RESULTS: Twenty-four consenting men were enrolled: 9 exhibiting a CD4(+) T cells count >350/mm(3) ("high-level CD4 group") and 15 < 350/mm(3) ("low-level CD4 group") in blood, at the start of cART. An immunophenotypical analysis of T and B cells subpopulations was performed in blood and rectal biopsies. HIV cell-associated DNA loads and qualitative intra-cellular RNA were determined in both compartments. The ratio of CD4(+)/CD8(+) T cells was significantly decreased in the blood but not in the rectum of the "low-level CD4 group" of patients. The alteration in ß7(+) CD4(+) T cells homing was higher in this group and was correlated to a low ratio of CD4(+)/CD8(+) T cells in blood. An initiation of cART in men exhibiting a low-level CD4 count was also associated with an alteration of B cells maturation. HIV blood and gut DNA reservoirs were significantly lower in the "high-level CD4 group" of men. A high HIV DNA level was associated to a detectable intracellular HIV RNA in rectum. CONCLUSIONS: An early initiation of cART could significantly preserve gut immunity and limit the viral reservoir constitution.

The Genotyping Resistance Profile of the Pol Gene Detected in Blood of Newly Diagnosed HIV-Positive Men Is Durably Archived in the Gut Whatever the Time of Initiation of cART.

OBJECTIVE: To evaluate the mutational patterns on the pol gene of the main HIV-1 strain archived in cell genome of 10 chronically infected men according to their clinical and therapeutic history. The genotyping resistance profiles were compared between the first blood plasma available at the time of HIV diagnosis and rectal biopsies and PBMC sampled 1-5 years after the initiation of combined antiretroviral therapy (cART). METHODS: HIV-1 RNA and cell-associated HIV-1 DNA were quantified by Abbott Real-Time HIV-1 and Generic HIV® DNA cell (Biocentric) assays. The mutations in protease and reverse transcriptase genes were assessed by the Trugene® assay (Siemens). The C2V3 region was amplified to determine the viral tropism. RESULTS: In 9 patients, slight or no differences were observed between the 3 resistance profiles. Those mostly detected were related to the resistance to nucleos(t)ide (D67N, L210W, T215A, T69D) and nonnucleoside (K103N, V106I, V179I) inhibitors. In 1 rilpivirine-treated patient, the M230I mutation was detected in PBMC. No change of viral tropism was observed between samples. CONCLUSION: These data suggest that resistance mutations harbored by the main HIV strain in plasma at the time of diagnosis are durably archived in DNA cells whatever the delay between infection and initiation of therapy in patients well controlled by cART.

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens.

A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis.

Comparative performances of HIV-1 RNA load assays at low viral load levels: results of an international collaboration.

Low-level viremia during antiretroviral therapy and its accurate measurement are increasingly relevant. Here, we present an international collaboration of 4,221 paired blood plasma viral load (pVL) results from four commercial assays, emphasizing the data with low pVL. The assays compared were the Abbott RealTime assay, the Roche Amplicor assay, and the Roche TaqMan version 1 and version 2 assays. The correlation between the assays was 0.90 to 0.97. However, at a low pVL, the correlation fell to 0.45 to 0.85. The observed interassay concordance was higher when detectability was defined as 200 copies/ml than when it was defined as 50 copies/ml. A pVL of ∼100 to 125 copies/ml by the TaqMan version 1 and version 2 assays corresponded best to a 50-copies/ml threshold with the Amplicor assay. Correlation and concordance between the viral load assays were lower at a low pVL. Clear guidelines are needed on the clinical significance of low-level viremia.

Potential contribution of saliva to the sexual transmission of HIV through the secretion of CCL20 by genital epithelial cells.

Saliva can be considered as an important actor during sexual intercourse. However, there is no data concerning its influence on HIV sexual transmission. The aim of this study was to evaluate the role of whole saliva on the in vitro secretion of CCL20 by monolayered HEC-1A endocervical epithelium cells. HEC-1A cells were cultivated in 96-well microplates and incubated with specimens of whole saliva collected from 57 subjects tested seropositive (n = 34) or seronegative (n = 23) for HIV and presenting different oral conditions (healthy periodontally, n = 22, and gingivitis/periodontitis, n = 35). The production of CCL20 in the supernatants of HEC-1A cells after overnight incubation at 37°C was quantified using ELISA. The salivary concentration of lactoferrin (Lf) and IL-1ß was tested by ELISA. Saliva samples were found able to stimulate dramatically the production of CCL20 by epithelial cells, increasing this synthesis by a mean factor of 38.1 with reference to untreated cells. This stimulation was equivalent to that observed with IL-1ß used as positive control. Although no difference was observed according to oral condition, HIV status or salivary concentration of Lf and IL-1ß, the high salivary concentration of the latter protein could acknowledge in large part for the overproduction of CCL20 by HEC-1A cells when stimulated by saliva. Saliva was shown to significantly increase CCL20 secretion and may be responsible for an enhanced recruitment of dendritic/Langerhans cells at the genital level. These results suggest that saliva could facilitate HIV entry and possibly other pathogens through the genital mucosa during heterosexual intercourse.

Human immunodeficiency virus and oral microbiota: mutual influence on the establishment of a viral gingival reservoir in individuals under antiretroviral therapy.

The role of the oral microbiota in the overall health and in systemic diseases has gained more importance in the recent years, mainly due to the systemic effects that are mediated by the chronic inflammation caused by oral diseases, such as periodontitis, through the microbial communities of the mouth. The chronic infection by the human immunodeficiency virus (HIV) interacts at the tissue level (e.g. gut, genital tract, brain) to create reservoirs; the modulation of the gut microbiota by HIV infection is a good example of these interactions. The purpose of the present review is to assess the state of knowledge on the oral microbiota (microbiome, mycobiome and virome) of HIV-infected patients in comparison to that of HIV-negative individuals and to discuss the reciprocal influence of HIV infection and oral microbiota in patients with periodontitis on the potential establishment of a viral gingival reservoir. The influence of different clinical and biological parameters are reviewed including age, immune and viral status, potent antiretroviral therapies, smoking, infection of the airway and viral coinfections, all factors that can modulate the oral microbiota during HIV infection. The analysis of the literature proposed in this review indicates that the comparisons of the available studies are difficult due to their great heterogeneity. However, some important findings emerge: (i) the oral microbiota is less influenced than that of the gut during HIV infection, although some recurrent changes in the microbiome are identified in many studies; (ii) severe immunosuppression is correlated with altered microbiota and potent antiretroviral therapies correct partially these modifications; (iii) periodontitis constitutes a major factor of dysbiosis, which is exacerbated in HIV-infected patients; its pathogenesis can be described as a reciprocal reinforcement of the two conditions, where the local dysbiosis present in the periodontal pocket leads to inflammation, bacterial translocation and destruction of the supporting tissues, which in turn enhances an inflammatory environment that perpetuates the periodontitis cycle. With the objective of curing viral reservoirs of HIV-infected patients in the future years, it appears important to develop further researches aimed at defining whether the inflamed gingiva can serve of viral reservoir in HIV-infected patients with periodontitis.

Comparison of four concentration methods of adenovirus, norovirus and rotavirus in tap water.

Human enteric viruses, as adenovirus (HAdV), norovirus (HuNoV) and rotavirus (RVA) are significant causes of gastroenteritis associated with consumption of contaminated water worldwide. Various methods have been described for their detection and monitoring in water. The aim of this study was to compare the performance of four conditions for concentrating HAdV, HuNoV and RVA from water matrices, in order to develop a single protocol that could simultaneously concentrate all target viruses from tap water. The tested conditions were based on the adsorption-elution using electronegative filters, in which we evaluated cation-coated filtration by MgCl2 with or without acid rinse by H2SO4 and two elution buffers, namely NaOH and tris-glycine-beef extract. Genomic material was extracted and amplified by real-time PCR and real-time RT-PCR using commercial kits. Based on the statistical analysis of amplification results (cycles of quantification), the condition involving cation-coated filtration by MgCl2 using electronegative filters with acid rinse by H2SO4 combined with NaOH elution allowed efficient recovery of both HAdV, HuNoV and RVA from tap water compared to the other conditions. These findings confirm the effectiveness of the approach used to monitor three major enteric viruses in tap water.