Evaluation of Analytical and Clinical Performance and Usefulness in a Real-Life Hospital Setting of Two in-House Real-Time RT-PCR Assays to Track SARS-CoV-2 Variants of Concern.

Since the end of 2020, multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have emerged and spread worldwide. Tracking their evolution has been a challenge due to the huge number of positive samples and limited capacities of whole-genome sequencing. Two in-house variant-screening RT-PCR assays were successively designed in our laboratory in order to detect specific known mutations in the spike region and to rapidly detect successively emerging VOCs. The first one (RT-PCR#1) targeted the 69-70 deletion and the N501Y substitution simultaneously, whereas the second one (RT-PCR#2) targeted the E484K, E484Q, and L452R substitutions simultaneously. To evaluate the analytical performance of these two RT-PCRs, 90 negative and 30 positive thawed nasopharyngeal swabs were retrospectively analyzed, and no discordant results were observed. Concerning the sensitivity, for RT-PCR#1, serial dilutions of the WHO international standard SARS-CoV-2 RNA, corresponding to the genome of an Alpha variant, were all detected up to 500 IU/mL. For RT-PCR#2, dilutions of a sample harboring the E484K substitution and of a sample harboring the L452R and E484Q substitutions were all detected up to 1000 IU/mL and 2000 IU/mL, respectively. To evaluate the performance in a real-life hospital setting, 1308 and 915 profiles of mutations, obtained with RT-PCR#1 and RT-PCR#2, respectively, were prospectively compared to next-generation sequencing (NGS) data. The two RT-PCR assays showed an excellent concordance with the NGS data, with 99.8% for RT-PCR#1 and 99.2% for RT-PCR#2. Finally, for each mutation targeted, the clinical sensitivity, the clinical specificity and the positive and negative predictive values showed excellent clinical performance. Since the beginning of the SARS-CoV-2 pandemic, the emergence of variants-impacting the disease's severity and the efficacy of vaccines and therapies-has forced medical analysis laboratories to constantly adapt to the strong demand for screening them. Our data showed that in-house RT-PCRs are useful and adaptable tools for monitoring such rapid evolution and spread of SARS-CoV-2 VOCs.

Evidence of Transmission and Circulation of Deltacron XD Recombinant Severe Acute Respiratory Syndrome Coronavirus 2 in Northwest France.

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

Investigation of outbreak cases infected with the SARS-CoV-2 B.1.640 variant in a fully vaccinated elderly population, Normandy, France, November to December 2021.

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

New HIV-1 circulating recombinant form 94: from phylogenetic detection of a large transmission cluster to prevention in the age of geosocial-networking apps in France, 2013 to 2017.

BackgroundEnding the HIV pandemic must involve new tools to rapidly identify and control local outbreaks and prevent the emergence of recombinant strains with epidemiological advantages.AimThis observational study aimed to investigate in France a cluster of HIV-1 cases related to a new circulating recombinant form (CRF). The confirmation this CRF's novelty as well as measures to control its spread are presented.MethodsPhylogenetic analyses of HIV sequences routinely generated for drug resistance genotyping before 2018 in French laboratories were employed to detect the transmission chain. The CRF involved was characterised by almost full-length viral sequencing for six cases. Cases' clinical data were reviewed. Where possible, epidemiological information was collected with a questionnaire.ResultsThe transmission cluster comprised 49 cases, mostly diagnosed in 2016-2017 (n = 37). All were infected with a new CRF, CRF94_cpx. The molecular proximity of this CRF to X4 strains and the high median viraemia, exceeding 5.0 log10 copies/mL, at diagnosis, even in chronic infection, raise concerns of enhanced virulence. Overall, 41 cases were diagnosed in the Ile-de-France region and 45 were men who have sex with men. Among 24 cases with available information, 20 reported finding partners through a geosocial networking app. Prevention activities in the area and population affected were undertaken.ConclusionWe advocate the systematic use of routinely generated HIV molecular data by a dedicated reactive network, to improve and accelerate targeted prevention interventions. Geosocial networking apps can play a role in the spread of outbreaks, but could also deliver local targeted preventive alerts.

Detection of numerous HIV-1/MO recombinants in France.

BACKGROUND: The broad genetic divergence of HIV-1/O relative to HIV-1/M has important implications for diagnosis, monitoring and treatment. Despite this divergence, some HIV-1/M+O dual infections and HIV-1/MO recombinant forms have been reported, mostly in Cameroon, where both groups are prevalent. Here, we describe the characteristics of such infections detected in France in 10 new patients, and discuss their implications for biological and clinical practice, owing to the presence of group O species. METHODS: The French National Reference Centre for HIV received samples within the framework of mandatory notification of HIV infections, and for expert analysis. A strategy combining serotyping, viral quantification, group-specific molecular amplification and whole-genome sequencing was used for strain characterization and complementary investigations. RESULTS: We identified one patient with M+O infection, three patients with M+O infection associated with an MO recombinant, and six patients with only an MO recombinant. These atypical infections were detected upon strain characterization (n = 4) or because of anomalies during patient monitoring (n = 6). We identified eight new URF_MO, all but one originating from Cameroon. Interestingly, two distinct recombinant strains were found in two unrelated patients, representing possible precursors of a CRF_MO. CONCLUSION: Our work highlights the fact that the continuous evolution of HIV can hinder diagnosis and complicate clinical practice. We stress that unexpected results during diagnosis or monitoring necessitate further serological and molecular exploration, these atypical infections influence biological and therapeutic management and necessitate appropriate tools, and specific surveillance is necessary, especially as the frequency of such infections may be underestimated.

HIV-1 group P infection: towards a dead-end infection?

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

Short Communication: Characterization of a New HIV-1 Group N Isolate Originating from a Cameroonian Patient.

HIV-1 group N (HIV-1/N) remains rare and mainly restricted to Cameroon. In this study, we report a new HIV-1/N infected case identified during routine HIV screening activities in Yaounde. The genetic characterization of the near full-length genome of this virus strain revealed that it is genetically distinct to all HIV-1/N described to date. However, the Vpu protein responsible for tetherin antagonism displayed the same amino acid substitutions (E15A, V19A, I25L, and V26L) as other HIV-1/N from Cameroon.

Misidentification of recombinant hepatitis C virus leading to treatment failure with direct acting antivirals.

The use of new Direct Acting Antivirals, specific of HCV, has greatly improved the HCV treatment. Most of the DAA are specific of HCV genotypes. Genotyping methods may target different regions of the HCV genome, though only the whole genome sequencing could confirm the correct genotype. The present study describes the virological investigation of a treatment failure due to the false identification of an unusual 2k/1b recombinant HCV form. It describes the sequencing methods, and the similarity analysis of the sequences to different genotype query sequences, to identify the recombination breakpoint.

HIV-1 sequences in the epidemic suggest an alternative pathway for the generation of the Long Terminal Repeats.

To generate the long-terminal repeats (LTR) that border the integrated viral genome, two-strand transfer steps must occur during reverse transcription. Analysis of the genetic polymorphisms that are present in the LTR of HIV-1 heterozygous virions in single infection cycle studies has revealed which of the two copies of genomic RNAs is used for each transfer event. Thus, the first event of strand transfer has been described to be either intra- or intermolecular, while the second event is generally intramolecular. Here, we repeated these analyses using sequences from HIV databases and extended the study to the regions surrounding the LTR. We observed a striking correlation between the pattern of recombination in the LTR and the phylogenetic origin of the surrounding sequences. This correlation suggests that the second-strand transfer can be either intra- or intermolecular and, interestingly, could reflect an effect of proximity between nucleic acids that would guide this transfer. This factor could be particularly relevant for heterozygous viruses containing highly divergent genomic RNAs, such as those considered in the present study.

Multiple HIV-1/M + HIV-1/O dual infections and new HIV-1/MO inter-group recombinant forms detected in Cameroon.

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

Simian Immunodeficiency Virus seroreactivity in inhabitants from rural Cameroon frequently in contact with non-human primates.

Central African tropical forests are home to several species of non-human primates (NHPs), infected by Simian Immunodeficiency Virus (SIV). It is well-known that HIV-1 epidemic is due to cross-transmission and adaptation of SIV to humans. The main goal of this work was to investigate if a NHP bite is a risk factor for SIV acquisition. A cross-sectional study was performed in rural Cameroon on 246 bitten individuals (mostly by adult NHPs), matched, according to sex, age, and ethnicity (Bantus and Pygmies), with an equal number of not-bitten subjects. Following a serological assay for a wide range of SIVs, we observed a high level of indeterminate seroreactivity (25.8%) in the total population, whereas 68.9% were sero-negative and 5.3% HIV-1 positive. Bites do not appear to be a risk factor for SIV seroreactivity, in contrast to Simian Foamy Virus and Simian T-Lymphotropic Virus type 1 in the same studied population.

Origin, diffusion and evolution of HIV-1 group O viruses.

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

Origine, diffusion et évolution des VIH-1 de groupe O.

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

A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants.

The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns.

First evidence of transmission of an HIV-1 M/O intergroup recombinant virus.

OBJECTIVE: Despite the genetic divergence between HIV-1 groups M and O, HIV-1  M/O intergroup recombinants were reported. Actually, there is no data on the transmissibility of such recombinant forms. During a surveillance of HIV genetic diversity in Cameroon, we investigated the possible direct transmission of an HIV-1  M/O recombinant virus in an HIV-infected couple. METHODS: Consecutive samples obtained from the couple were analysed for detection of dual HIV-1 groups M and O infections, and HIV-1  M/O recombinant forms. Analyses were performed using a serological and molecular algorithm based on HIV serotyping and group-specific PCRs targeting the polymerase and envelope genes. Pattern characterization of the strains found in both patients was based on complete genome sequencing. Phylogenetic and similarity profile analyses were performed to investigate the genetic relationship between viruses from both spouses and the previously described recombinant forms. RESULTS: The sero-molecular algorithm data showed a group O serotype confirmed by molecular analysis in the envelope regions, whereas molecular tests identified HIV-1 group M in the polymerase. Phylogenetic analyses and similarity profiles of the full-length genome sequences showed that both spouses were infected with a unique recombinant virus having two recombination breakpoints in the vpr gene and LTR region. No phylogenetic link was found with the previous M/O recombinants. CONCLUSION: We provide, for the first time, molecular evidence of direct transmission of an HIV-1  M/O recombinant, highlighting the potential spread of these divergent viruses. The importance of HIV-1 recombination on genetic evolution and public health when implying divergent strains as group O has to be carefully considered.

The Two-Phase Emergence of Non Pandemic HIV-1 Group O in Cameroon.

Unlike the pandemic form of HIV-1 (group M), group O viruses are endemic in west central Africa, especially in Cameroon. However, little is known about group O's genetic evolution, and why this highly divergent lineage has not become pandemic. Using a unique and large set of group O sequences from samples collected from 1987 to 2012, we find that this lineage has evolved in successive slow and fast phases of diversification, with a most recent common ancestor estimated to have existed around 1930 (1914-1944). The most rapid periods of diversification occurred in the 1950s and in the 1980s, and could be linked to favourable epidemiological contexts in Cameroon. Group O genetic diversity reflects this two-phase evolution, with two distinct populations potentially having different viral properties. The currently predominant viral population emerged in the 1980s, from an ancient population which had first developed in the 1950s, and is characterized by higher growth and evolutionary rates, and the natural presence of the Y181C resistance mutation, thought to confer a phenotypic advantage. Our findings show that although this evolutionary pattern is specific to HIV-1 group O, it paralleled the early spread of HIV-1 group M in the Democratic Republic of Congo. Both viral lineages are likely to have benefited from similar epidemiological contexts. The relative role of virological and social factors in the distinct epidemic histories of HIV-1 group O and M needs to be reassessed.

Performance of the Liaison XL Murex HIV Ab/Ag test on clinical samples representing current epidemic HIV variants.

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

Field evaluation of the Abbott ARCHITECT HIV Ag/Ab Combo immunoassay.

BACKGROUND: Fourth generation assays for HIV diagnosis are progressively being introduced into routine services, due to their improvement of diagnosis. In spite of this, HIV diagnosis remains a challenge in sub-Saharan Africa, due to false positive reactivity. There is a continuous need for field evaluations and routine validations of fourth generation HIV tests in African populations. OBJECTIVES: Evaluate the performances of the ARCHITECT HIV Ag/Ab kit (Abbott) in a population living in an African setting-Cameroon compared to a population living in a European setting-France. STUDY DESIGN: 645 HIV samples from both France and Cameroon were evaluated. The positive panel (378 samples) included a diverse series of HIV-1 variants (groups M, N, O, and P) as well as HIV-2 samples. Results were compared to original diagnosis done with other 4th generation assays (AxSYM HIV Ag/Ab (Abbott) and Vidas HIV DUO QUICK) (bioMérieux). RESULTS: Sensitivity of the ARCHITECT was 100% in both sites. It diagnosed all variants of the panel with different reactivity profiles following strain diversity. A wider range of reactivity was observed for group O. Specificity was slightly lower (97.6%) in Cameroon than in France (98.6%), probably due to a higher rate of false positive reactivity. ARCHITECT HIV Ag/Ab assay had high performances in clinical sensitivity and specificity and is adapted to the wide genetic diversity of viruses circulating in West Central Africa. CONCLUSION: Our results further highlight the need to evaluate HIV diagnostic tests before introduction into routine diagnostic services both in the North and in the South.

A new real-time quantitative PCR for diagnosis and monitoring of HIV-1 group O infection.

The correct diagnosis and monitoring of HIV-1 group O (HIV-O) infection are essential for appropriate patient management, for the prevention of mother-to-child transmission, and for the detection of dual HIV-M/HIV-O infections. HIV-O RNA quantification is currently possible with two commercial kits (from Abbott and Roche), which quantify HIV-M and HIV-O strains indifferently; therefore, they cannot be used for the specific identification of HIV-O infection. We designed a new real-time quantitative reverse transcription PCR (RT-qPCR assay) (INT-O), which we compared with our previous version, LTR-O, and with the Abbott RealTime HIV-1 kit. Specificity was assessed with 27 HIV-1 group M strains and the prototype strain of group P. Clinical performances were analyzed by using 198 stored plasma samples, representative of HIV-O genetic diversity. Analytical sensitivity, repeatability, and reproducibility were also determined. The detection limit of the INT-O assay was 40 copies/ml, and its specificity was 100%. The repeatability and reproducibility were excellent. Analysis of clinical samples showed a good correlation between the INT-O and LTR-O assays (r = 0.8240), with an improvement of analytical sensitivity. A good correlation was also obtained between the INT-O and Abbott assays (r = 0.8599) but with significantly higher values (0.19 logs) for the INT-O method, due to marked underquantifications for some patients. These results showed that HIV-O genetic diversity still has an impact on RNA quantification. The new assay, INT-O, allows both the specific diagnosis of HIV-O infection and the quantification of diverse HIV-O strains. Its detection limit is equivalent to that of commercial kits. This assay is cheap and suitable for use in areas in which strains of HIV-1 groups M and O cocirculate.