Simultaneous detection of chikungunya virus, dengue virus and human pathogenic Leptospira genomes using a multiplex TaqMan® assay

Abstract Background In 2005–2006 a major epidemics of Chikungunya disease occurred in South-West Indian Ocean islands. In Reunion Island, the magnitude of Chikungunya infection related symptoms was high and with over 38% of serological prevalence in the population. This epidemics illustrated the potential threat of emerging arboviral diseases for inhabitants of Reunion Island and elsewhere since vectors are worldwide distributed. A sentinel surveillance network was set-up to detect emerging pathogens associated with fever over 38 °C and in the absence of known etiologic causes. Leptospirosis is caused by a pathogenic spirochete of the Leptospira genus and is an endemic and recurrent seasonal disease of great concern in Reunion Island. To accurately diagnose potentially infected patients and to advise Health authorities on the presence of emerging pathogens, a rapid diagnostic test was needed that could differentiate between these 3 pathogens. Methods A one-step multiplex real-time PCR assay was developed that can simultaneously detect RNA of Chikungunya and Dengue viruses and leptospiral DNA with good performance for a routine diagnostic use. Results Simplex protocols already published were used with key modifications to implement a triplex assay which was set-up with a small reaction volume to improve cost efficiency. Conclusions This approach has enabled greater diagnostic capacity in our laboratory. We established a multiplex approach validated and valuable for cost savings, and with the concurrent detection of 3 pathogens of public health concern

Improved detection of genus-specific Alphavirus using a generic TaqMan® assay

International audienceBackground: Alphaviruses are arthropod borne RNA viruses of medical importance. Geographical expansion of mosquitoes of the Aedes genus in the past decades has been associated with major Alphavirus-associated outbreaks. Climate changes and intensification of air travels have favored vector expansion and virus dissemination in new territories leading to virus emergence not only in tropical areas but also in temperate regions. The detection of emergence is based upon surveillance networks with epidemiological and laboratory investigation.Method: A specific, sensitive and rapid screening test for genus-specific Alphavirus is critically required. To address this issue, we developed a new molecular assay targeting nsP4 gene and using a TaqMan® real time RT-PCR method for the specific detection of all major Alphavirus genus members.Results: This assay was tested for specificity using several Alphavirus species. We also tested successfully clinical sensitivity using patient’s samples collected during the Chikungunya outbreak of 2005–2006 in the Indian Ocean.Conclusions: This new pan-Alphavirus molecular diagnostic tool offers great potential for exclusion diagnosis and emergence detection given its broad specificity restricted to Alphavirus genu

The SARS-CoV-2 B.1.351 lineage (VOC β) is outgrowing the B.1.1.7 lineage (VOC α) in some French regions in April 2021

International audienceTo assess SARS-CoV-2 variants spread, we analysed 36,590 variant-specific reverse-transcription-PCR tests performed on samples from 12 April–7 May 2021 in France. In this period, contrarily to January–March 2021, variants of concern (VOC) β (B.1.351 lineage) and/or γ (P.1 lineage) had a significant transmission advantage over VOC α (B.1.1.7 lineage) in Île-de-France (15.8%; 95% confidence interval (CI): 15.5–16.2) and Hauts-de-France (17.3%; 95% CI: 15.9–18.7) regions. This is consistent with VOC β’s immune evasion abilities and high proportions of prior-SARS-CoV-2-infected persons in these regions

Rapid spread of the SARS-CoV-2 Delta variant in some French regions, June 2021

International audienceWe analysed 9,030 variant-specific RT-PCR tests performed on SARS-CoV-2-positive samples collected in France between 31 May and 21 June 2021. This analysis revealed rapid growth of the Delta variant in three of the 13 metropolitan French regions and estimated a +79% (95% confidence interval: 52–110%) transmission advantage compared with the Alpha variant. The next weeks will prove decisive and the magnitude of the estimated transmission advantages of the Delta variant could represent a major challenge for public health authorities

Diagnostic Relevance of Immunoglobulin G Avidity for Hepatitis A Virus

Diagnosis of acute hepatitis A virus (HAV) infection is based on the detection of HAV immunoglobulin M (IgM). However, IgM could be detected due to nonspecific polyclonal activation of the immune system. An avidity test for anti-HAV IgG was developed to distinguish acute infection, where low-avidity antibodies are detected, from immune reactivation. The assay was tested on 104 samples, including 11 sera from patients with past infection, 15 sera from patients with acute infection and 4 collected after recovery, 10 sera from vaccinated subjects, 4 sera from patients with suspected immune reactivation, and 60 unselected HAV-IgM positive sera, collected over 1 year in a routine laboratory. The avidity index (AI) was expressed as percentage. The results were provided as the mean ± one standard deviation. Patients with a history of prior infection had AIs of >70% (mean, 86% ± 10), whereas the mean AI was 36% ± 16 during acute HAV infection (P < 0.001). Within the first month after the onset of hepatitis, avidity was either noncalculable due to a very low IgG titer or <50%. In patients with immune reactivation, avidity was >70% (88% ± 10%), a finding consistent with a prior infection. Among the 60 unselected sera, 35 (58%) had a noncalculable or <50% avidity, and most of them had a detectable HAV RNA, confirming HAV infection. In contrast, 16 (27%) had an avidity of >70%, and none was reverse transcription-PCR positive, suggesting immune reactivation. These 16 patients were significantly older than the others (50 ± 16 years versus 26 ± 14 years). The new anti-HAV IgG avidity assay we developed could improve HAV infection diagnosis, particularly in elderly patients

Cinétique intra-hôte des variants du SARS-CoV2

International audienceSince early 2021, SARS-CoV-2 variants of concern (VOCs) have been causing epidemic rebounds in many countries. Their properties are well characterised at the epidemiological level but the potential underlying within-host determinants remain poorly understood. We analyse a longitudinal cohort of 6,944 individuals with 14,304 cycle threshold (Ct) values of RT-qPCR VOC screening tests performed in the general population and hospitals in France between February 6 and August 21, 2021. To convert Ct values into numbers of virus copies, we performed an additional analysis using droplet digital PCR (ddPCR). We find that the number of viral genome copies reaches a higher peak value and has a slower decay rate in infections caused by Alpha variant compared to that caused by historical lineages. Following the evidence that viral genome copies in upper respiratory tract swabs are informative on contagiousness, we show that the kinetics of the Alpha variant translate into significantly higher transmission potentials, especially in older populations. Finally, comparing infections caused by the Alpha and Delta variants, we find no significant difference in the peak viral copy number. These results highlight that some of the differences between variants may be detected in virus load variations.Depuis le début de l'année 2021, les variants préoccupants du SRAS-CoV-2 (VOC) provoquent des rebonds épidémiques dans de nombreux pays. Leurs propriétés sont bien caractérisées au niveau épidémiologique, mais les déterminants potentiels au niveau intra-hôte restent mal compris. Nous analysons une cohorte longitudinale de 6 944 individus avec 14 304 valeurs de cycle threshold (Ct) de tests de dépistage des COV par RT-qPCR réalisés dans la population générale et les hôpitaux en France entre le 6 février et le 21 août 2021. Pour convertir les valeurs Ct en nombre de copies virales, nous avons effectué une analyse supplémentaire en utilisant la PCR digitale. Nous constatons que le nombre de copies du génome viral atteint un pic plus élevé et présente un taux de décroissance plus lent dans les infections causées par le variant Alpha par rapport à celles causées par les lignées historiques. En se basant sur certaines observations montrant que le nombre copies virales dans les écouvillons des voies respiratoires supérieures sont informatives sur la contagiosité, nous montrons que la cinétique du variant Alpha se traduit par des potentiels de transmission significativement plus élevés, en particulier dans les populations plus âgées. Enfin, en comparant les infections causées par les variants Alpha et Delta, nous ne trouvons pas de différence significative dans le nombre maximal de copies virales. Ces résultats soulignent que certaines des différences entre les variants peuvent être détectées dans les variations de la charge virale

Detecting Rapid Spread of SARS-CoV-2 Variants, France, January 26–February 16, 2021

International audienceVariants of severe acute respiratory syndrome coronavirus 2 raise concerns regarding the control of coronavirus disease epidemics. We analyzed 40,000 specific reverse transcription PCR tests performed on positive samples during January 26–February 16, 2021, in France. We found high transmission advantage of variants and more advanced spread than anticipated

Severe bilateral pleuropneumonia caused by Legionella sainthelensi: a case report

International audienceBackground: Legionella spp. are ubiquitous freshwater bacteria responsible for rare but potentially severe cases of Legionnaires' disease (LD). Legionella sainthelensi is a non-pneumophila Legionella species that was first isolated in 1980 from water near Mt. St-Helens (USA). Although rare cases of LD caused by L. sainthelensi have been reported, very little data is available on this pathogen. Case presentation: We describe the first documented case of severe bilateral pleuropneumonia caused by L. sainthelensi. The patient was a 35-year-old woman with Sharp's syndrome treated with long-term hydroxychloroquine and corticosteroids who was hospitalized for an infectious illness in a university hospital in Reunion Island (France). The patient's clinical presentation was complicated at first (bilateral pneumonia, multiloculated pleural effusion, then bronchopleural fistula) but her clinical condition eventually improved with the reintroduction of macrolides (spiramycin) in intensive care unit. Etiological diagnosis was confirmed by PCR syndromic assay and culture on bronchoalveolar lavage. Conclusions: To date, only 14 documented cases of L. sainthelensi infection have been described worldwide. This pathogen is difficult to identify because it is not or poorly detected by urinary antigen and molecular methods (like PCR syndromic assays that primarily target L. pneumophila and that have only recently been deployed in microbiology laboratories). Pneumonia caused by L. sainthelensi is likely underdiagnosed as a result. Clinicians should consider the possibility of non-pneumophila Legionella infection in patients with a compatible clinical presentation when microbiological diagnostic tools targeted L. pneumophila tested negative

Risk Factors for Selection of the L74I Reverse Transcriptase Mutation in Human Immunodeficiency Virus Type 1-Infected Patients

We analyzed 3,475 human immunodeficiency virus sequences and 241 therapeutic histories. The L74I mutation was carried by 7% of viruses. L74I was strongly associated with T215F, K70R, and V75M/S/T/A mutations and increased with the number of thymidine analog mutations. It seemed to be linked to the use of abacavir or efavirenz