Characteristics of respiratory viruses' circulation through a six-year period (2016-2022) in a pediatric population in Normandy, France, and the impact of COVID-19 pandemic.

IMPORTANCE: The report highlights an epidemiological change in the circulation of respiratory viruses in pediatric populations due to strategies adopted against COVID-19 pandemic. COVID-19 has resulted in a significant increase in requests for multiplex respiratory research to identify the virus responsible for the symptoms. The diagnostic needs have increased, and the number of samples analyzed in 2021-2022 is equal to the samples analyzed over the four epidemic periods preceding the pandemic. The report suggests the importance of active surveillance of respiratory viruses' circulation and new recommendations for respiratory virus detection in pediatric patients.

[Virological diagnosis of lower respiratory tract infections]. / Diagnostic virologique des infections respiratoires basses.

INTRODUCTION: The etiological diagnosis of bronchopulmonary infections cannot be assessed with clinical, radiological and epidemiological data alone. Viruses have been demonstrated to cause a large proportion of these infections, both in children and adults. BACKGROUND: The diagnosis of viral bronchopulmonary infections is based on the analysis of secretions, collected from the lower respiratory tract when possible, by techniques that detect either influenza and respiratory syncytial viruses, or a large panel of viruses that can be responsible for respiratory disease. The latter, called multiplex PCR assays, allow a syndromic approach to respiratory infection. Their high cost for the laboratory raises the question of their place in the management of patients in terms of antibiotic economy and isolation. In the absence of clear recommendations, the strategy and equipment are very unevenly distributed in France. OUTLOOK: Medico-economic analyses need to be performed in France to evaluate the place of these tests in the management of patients. The evaluation of the role of the different viruses often detected in co-infection, especially in children, also deserves the attention of virologists and clinicians. CONCLUSIONS: The availability of new diagnostic technologies, the recent emergence of SARS-CoV-2, together with the availability of new antiviral drugs are likely to impact future recommendations for the management of viral bronchopulmonary infections.

Severe underquantification of HIV-1 group O isolates by major commercial PCR-based assays.

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

Comparative performance of the Biocentric Generic Viral Load, Roche CAP/CTM v1.5, Roche CAP/CTM v2.0 and m2000 Abbott assays for quantifying HIV-1 B and non-B strains: Underestimation of some CRF02 strains.

BACKGROUND: HIV-1 viral load testing is now recommended by the World Health Organization for every patient receiving antiretroviral therapy (ART). OBJECTIVES: The objective of this study is to evaluate the performance of commercial assays for their ability to quantify HIV-1 strains currently circulating in France. STUDY DESIGN: The performances of the Generic HIV-RNA assay from Biocentric were compared to those of the Roche CAP/CTM v1.5, Roche CAP/CTM v2.0 and Abbott m2000 RealTime HIV-1 assays. A total of 1885 HIV-1 plasma samples were tested, including 684 samples from patients included in the ANRS-Primo Cohort. RESULTS: We found a good concordance of quantification between the Roche v2.0 and the Biocentric assays, both of which were superior to the Roche v1.5 assay. We show moderate agreement between techniques; however, CRF02_AG strains and undetermined viruses were underestimated when quantified with the Roche CAP/CTM v2.0. In contrast, a comparison of the Biocentric and Abbott assay results showed strong agreement between assays, indicating that both are well suited for quantification of CRF02_AG strains. Moreover, a 2% underestimation of the B subtypes was observed with the Biocentric assay. CONCLUSIONS: These results have implications for viral load monitoring in Western Africa, where CRF02_AG strains are highly prevalent. Closer epidemiological surveillance and evaluation of commercial assays are still necessary to better evaluate the impact of the genetic evolution of circulating viruses on HIV-RNA quantification in the regions most affected by the HIV-1 epidemic.

HIV-1 non-group M phenotypic susceptibility to integrase strand transfer inhibitors.

Objectives: To determine natural phenotypic susceptibility of non-group M HIV-1 to integrase strand transfer inhibitors (INSTIs) in a large panel of 39 clinical strains from groups O, N and P and to identify genotypic polymorphisms according to susceptibility levels. Methods: Susceptibility to raltegravir, elvitegravir and dolutegravir was evaluated in 36 HIV-1/O, 2 HIV-1/N and 1 HIV-1/P strains plus an HIV-1/M reference strain. IC50 values were determined after 3 days, and fold changes (FCs) were calculated relative to the HIV-1/M strain. Genotypic polymorphism was determined by amplification of codons 19-263 of the integrase; the natural occurrence of resistance-associated mutations was analysed using the main resistance algorithms and the IAS-USA list. VESPA analysis of the strain sequences was used to determine a signature pattern associated with higher FC. Results: Similar IC50 results were observed for the three drugs. Based on the value for the HIV-1/M reference strain, the data showed FC values <2.5 for raltegravir and dolutegravir, whereas the distribution for elvitegravir was heterogeneous, with FC > 10 for six strains (15%). Analysis of the non-M integrase sequences showed a high level of polymorphism without a major genotypic impact; it also revealed mutations that may be associated with the highest FC values obtained for elvitegravir. Conclusions: Our phenotypic data showed that non-M strains are globally susceptible to the three currently used INSTIs, but the impact of the high FC values observed for some strains with elvitegravir needs to be explored. Clinical data are now needed to confirm these phenotypic results.

Prevalence of HIV-1 drug resistance in treated patients with viral load >50 copies/mL: a 2014 French nationwide study.

Background: Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. Methods: The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. Results: The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. Conclusion: In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.

Multi-site clinical evaluation of the Xpert(®) HIV-1 viral load assay.

BACKGROUND: The most important reason for measuring HIV-1 viral load (VL) is to monitor the effectiveness of antiretroviral therapy (ART), both for the initial therapeutic response and sustained responses. Maintaining low or undetectable HIV-1 VL levels can reduce both the risks of progression to AIDS and transmission of infection to others. OBJECTIVES: To evaluate the diagnostic accuracy of Xpert(®) HIV-1 Viral Load (VL) assay compared to the Abbott RealTime HIV-1 assay, including assessing specificity by testing plasma specimens from confirmed HIV-1 negative blood donors. STUDY DESIGN: Subjects were enrolled from 4 participating sites, 2 in Europe and 2 in the USA. Fresh plasma samples were tested prospectively, while frozen plasma samples were collected prospectively, and tested retrospectively after selection of specimens to cover the assay's quantification range (40cp/mL-10,000,000 cp/mL). Eligibility criteria included a clinician ordered HIV-1 VL test from a confirmed HIV-1 positive adult (≥18 years) with a known antiviral treatment status. Exclusion criteria included previous enrollment in this study or improper specimen collection. Human blood donor specimens determined to be HIV-1 negative by standard blood bank antibody and nucleic acid amplification methods were used to assess specificity. RESULTS: Of the 764 specimens collected, 752 were eligible for inclusion but 5 were not tested by the Xpert, leaving 747 specimens tested (28.2% from females and 71.8% from males). Valid results were obtained for 724/747 (96.9%) specimens tested using the Xpert HIV-1 VL assay. The Xpert HIV-1 VL assay detected or quantified 568/724 (78.5%) specimens, while the RealTime HIV-1 assay detected or quantified 559/724 (77.2%). Of the 724 specimens tested by both assays, 390 were quantified by both assays and showed strong correlation: r=0.9847, with an R(2)=0.9696. Bland-Altman analysis showed good agreement between the two assays (381/390; 97.7%) with a distribution within 0.5 log10 cp/mL centered around zero. Xpert yielded VLs for 393 (80%) of the 494 quantifiable samples by Abbott. VLs of those specimens quantified by one of the assays, and either detected but not quantified or not detected by the other assay were all <170cp/mL. Specificity of the Xpert assay was found to be 100% (109/109), 95% CI: 96.7-100.0. CONCLUSION: Very good correlation was seen between the Xpert HIV-1 VL and Abbott RealTime HIV-1 assays, with added benefits for Xpert HIV-1 VL of: (1) lot-to-lot consistency traceable to WHO International Standard, (2) requiring both high and low level internal controls to be in range to have a valid result, (3) use of a single HIV-1 target for PCR and (4) faster turn-around-time for results, no need to wait to do batch testing of specimens. In summary, Xpert HIV-1 VL generated accurate VL results that if implemented could allow for actionable and timely treatment decisions during the same clinic visit. This scenario could reduce the loss to follow up often seen when these test results take days to weeks to become available to the clinician and patient.

[Respiratory syncytial virus (RSV) infections in the pediatric teaching hospital Charles de Gaulle of Ouagadougou, Burkina Faso]. / Infections à virus respiratoire syncytial (VRS) au CHU pédiatrique Charles de Gaulle de Ouagadougou, Burkina Faso.

Human respiratory syncytial virus (RSV) infections are little known in Burkina Faso. The objective of our work is to study the epidemiological and clinical aspects of RSV infections in infants in the Pediatric Teaching Hospital Charles de Gaulle of Ouagadougou. Between July 1(st) 2010 and June 30(th) 2011, we analyzed by direct immunofluorescence and PCR nasopharyngeal swabs from children from 0 to 36 months old. All in all, 210 patients among whom 74 from the external consultation (35.2%) and 136 hospitalized (64.7%) benefited from a nasopharyngeal aspiration. The motives for consultation were cough (91.7%), rhinitis (79.2%), fever (79.2%) and respiratory distress syndrome (66.7%). The evoked diagnoses were predominantly the acute bronchiolitis in 14 cases (58.3%) followed by the acute pulmonary disease in 7 patients (26.2%) then flue in 1 patient (16.7%). We detected by direct immunofluorescence the antigens of the respiratory viruses in 21 nasopharyngeal aspirations with 10 cases of respiratory syncytial virus (RSV) infections (47.6%). The PCR realized on 208 samples allowed to identify 153 positive samples (73.2%) with 24 RSV, i.e. a global prevalence of 16.1% with a peak of 18 cases (75%). In October, all the patients benefited from an often multiple antibiotic treatment of at least 10 days which was not still necessary. The evolution was favorable for all patients. This study confirms the important place of the viruses which are detected in 70% of the cases. The PCR multiplex, certainly expensive but effective and successful, deserves to be used in our developing countries to avoid the irrational prescription of antibiotic.

[Specific diagnosis and follow-up of HIV-1 group O infection: RES-O data]. / Diagnostic spécifique et prise en charge des infections par un VIH-1 groupe O: données de RES-O.

INTRODUCTION: HIV-1 group O (HIV-O), mainly found in Cameroon, has a very high genetic diversity with consequences on the diagnosis and treatment of patients, requiring the development of specific tools. OBJECTIVE: We present the currently available tools for the specific detection of HIV-O and its therapeutic monitoring, and the first RES-O data, a French network for the identification and monitoring of patients infected by HIV-O. METHOD: The diagnosis of infection was confirmed by group-specific envelope serotyping. The viral load was assessed by a specific technique, LTR-O, developed in the laboratory and compared to the nonspecific kit RealTime HIV-1 (Abbott). The sequencing of antiretroviral target regions (Protease, Reverse Transcriptase (RT), Integrase and Gp41), was performed by specific primers. The analysis of resistance mutations was performed with the ANRS algorithm used for HIV-M. RESULTS: HIV-O infection was confirmed for 117 patients. Measuring viral load showed the two techniques were equivalent, but with a tendency to under-quantification for the Abbott technique greater than 1 log for 5% of samples. 70 to 100% of the studied strains had at least 10 mutations in the Protease, four 4 in the RT, and one in Gp41, resulting in a natural genotypic resistance to some anti-retroviral molecules. DISCUSSION: The diagnosis and monitoring of HIV-O infection is now possible. However, the impact of this variant's natural polymorphism on response to treatment remains undocumented.

Evaluation of an upgraded version of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test for HIV-1 load quantification.

We evaluated the performance of the prototype Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, using prospective and archived clinical samples initially underquantitated by the Cobas AmpliPrep/Cobas TaqMan HIV-1 test. The performance of the new test was significantly improved, and the majority of the underquantitation observed with the first-version test was eliminated.

Census and analysis of persistent false-negative results in serological diagnosis of human immunodeficiency virus type 1 group O infections.

Human immunodeficiency viruses (HIV) have a high level of genetic diversity. The outlier variants of HIV type 1 (HIV-1) group O are distantly related to HIV-1 group M. Their divergence has an impact on serological diagnosis, with a risk of false-negative results. In this study, we report 20 failure cases, involving patients with primary or chronic infection, in France and Cameroon between 2001 and 2008. Our results indicate that some assays detected group O infection much less efficiently than others. Two major reasons for these false-negative results were identified: the presence or absence of a group O-specific antigen (and the designed sequence) for the detection of antibodies and the greater envelope variability of group O than of group M strains. This study highlights the complexity of screening for these divergent variants and the need to evaluate test performance with a large panel of strains, due to the extensive diversity of group O variants.

RNA amplification of the HIV-1 Pol and env regions on dried serum and plasma spots.

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

[Human immunodeficiency virus type 1 subtypes in cohort of infected patients, Saint-Etienne, France, from 1984 to 2003]. / Sous-types du virus de l'immunodéficience humaine de type 1 dans une cohorte de sujets dépistés à Saint-Etienne, France, de 1984 à 2003.

OBJECTIVE: The authors had for aim to study the distribution of HIV-1 subtypes in a cohort of HIV-1 positive patients in the University hospital of Saint-Etienne, France, and to describe the epidemiological characteristics of patients infected with a non-B subtype strain. DESIGN: An epidemiological study was made on 271 HIV-1 positive patients followed up in the Infectious Diseases Department over 20 years. All patients sample were subtyped by serotyping and some samples were also tested by genotyping. RESULTS: Two hundred and sixty-four patients (191 men and 73 women) were found infected by an HIV-1 strain belonging to the M group. After combining serotyping and genotyping results, 195 patients were found infected by a B subtype and 69 by a non-B subtype. Most of the latter strains belonged to an A subtype or related ones. The following factors were shown to be linked to an infection by a non-B strain: being born abroad, having contracted the infection though heterosexual practice, and being a woman. The incidence of non-B strains increased regularly over time (to reach more than 40% in 2003). This progression was especially noted for men born in France with risky sexual behaviour. CONCLUSION: These results indicate that more than 40% of HIV-1 new cases detected in the Saint-Etienne area are related to non-B strains and that strains of A and related subtypes are common in the local population with risky sexual behaviour.

V3 serological subtyping of human immunodeficiency virus type 2 infection is not relevant.

V3 enzyme immunoassays have been shown to discriminate effectively between human immunodeficiency virus type 1 (HIV-1) subtypes. The aim of this study was to investigate the feasibility of V3 serotyping for HIV-2 infection. We serotyped 29 sera with three peptides, corresponding to the V3 loop of subtypes A, B, and D of HIV-2. Sera were collected from HIV-2-infected patients, whose infecting strains were sequenced and subjected to phylogenetic analysis. Our results indicate that HIV-2 serotyping using V3 peptides is not relevant. V3 serotyping data were not consistent with genotyping results. The V3-A and V3-D peptides displayed poor discrimination, and the V3-B peptide was not representative of circulating viruses. Comparison of amino acid sequences and serotype reactivities demonstrated the importance of positions 309 and 314, located on either side of the tip of the V3 loop, in antibody binding.

Synthetic peptide strategy for the detection of and discrimination among highly divergent primate lentiviruses.

We developed a simple, rapid, inexpensive, and highly sensitive and specific strategy for the detection and lineage differentiation of primate lentiviruses (PIV-ELISA). It is based on the use of two indirect ELISA methods using synthetic peptides mapping the gp41/36 region (detection component) and the V3 region (differentiation component) of four lentivirus lineages, namely SIVcpz/HIV-1 (groups M, O, N, and SIVcpz-gab), SIVmnd, SIVagm, and SIVsm/SIVmac/HIV-2. This strategy was evaluated with panels of sera originating from both humans and nonhuman primates. The human reference panel consisted of 144 HIV Western blot (WB)-positive sera in which the corresponding virus had been genotyped (HIV-1: 72 group M, 28 group O, and 6 group N; HIV-2: 21 subtype A and 10 subtype B; and 7 HIV-1+2) and 105 HIV WB-negative samples. The nonhuman primate reference panel consisted of 24 sera from monkeys infected by viruses belonging to the four lineages included in the PIV-ELISA strategy (5 chimpanzees, 5 macaques, 8 mandrills, and 6 vervets) and 42 samples from seronegative animals. Additional field evaluation panels consisted of 815 human sera from Gabon, Cameroon, and France and 537 samples from 25 nonhuman primate species. All the samples from the two reference panels were correctly detected and discriminated by PIV-ELISA. In the human field evaluation panel, the gp41/36 component correctly identified all the test samples, with 98% specificity. The V3 component discriminated 206 HIV-1 group M, 98 group O, 12 group M+O, and 128 HIV-2 sera. In the primate field evaluation panel, both gp41/36 and V3 detected and discriminated all the WB-positive samples originating from monkeys infected with SIVcpz, SIVagm-ver, SIVmnd-1, SIVmnd-2, SIVdrl, or SIVsun. These results were confirmed by genotyping in every case. Four SIV-infected red-capped mangabeys (confirmed by PCR) were correctly identified by gp41/36, but only two reacted with the V3 peptides in the absence of a specific SIVrcm V3 peptide. Addition of a V3 SIVrcm peptide discriminated all the SIVrcm-positive samples. Fourteen Papio papio samples were positive for SIVsm gp 36 and by WB, but negative by PCR, whereas three Papio cynocephalus samples were positive by gp41/36 but indeterminate by WB and negative by PCR. This combined ELISA system is thus highly sensitive and specific for antibodies directed against HIV and SIV. In addition, the V3-based serotyping results always agreed with genotyping results. This method should prove useful for studies of lentivirus prevalence and diversity in human and nonhuman primates, and may also have the potential to detect previously undescribed SIVs.