Influenza viruses cause a remarkable disease burden and significant morbidity and mortality worldwide, and these impacts vary between seasons. To understand the mechanisms associated with these differences, a comprehensive approach is needed to characterize the impact of influenza genomic traits on the burden of disease. During 2016–2017, a year with severe A(H3N2), we sequenced 176 A(H3N2) influenza genomes using next generation sequencing (NGS) for routine surveillance of circulating influenza viruses collected via the French national influenza community-based surveillance network or from patients hospitalized in the intensive care units of the University Hospitals of Lyon, France. Taking into account confounding factors, sequencing and clinical data were used to identify genomic variants and quasispecies associated with influenza severity or vaccine failure. Several amino acid substitutions significantly associated with clinical traits were found, including NA V263I and NS1 K196E which were associated with severity and co-occurred only in viruses from the 3c.2a1 clade. Additionally, we observed that intra-host diversity as a whole and on a specific set of gene segments increased with severity. These results support the use of whole genome sequencing as a tool for the identification of genetic traits associated with severe influenza in the context of influenza surveillance.

Background: Sensitive and easy-to-perform methods for the diagnosis of malaria are not yet available. Improving the limit of detection and following the requirements for certification are issues to be addressed in both endemic and non-endemic settings. The aim of this study was to test whether loop-mediated isothermal amplification of DNA (LAMP) may be an alternative to microscopy or real-time PCR for the screening of imported malaria cases in non-endemic area. Results: 310 blood samples associated with 829 suspected cases of imported malaria were tested during a one year period. Microscopy (thin and thick stained blood slides, reference standard) was used for the diagnosis. Real-time PCR was used as a standard of truth, and LAMP (Meridian Malaria Plus) was used as an index test in a prospective study conducted following the Standards for Reporting Diagnosis Accuracy Studies. In the 83 positive samples, species identification was P. falciparum (n = 66), P. ovale (n = 9), P. vivax (n = 3) P. malariae (n = 3) and 2 co-infections with P. falciparum + P.malariae. Using LAMP methods, 93 samples gave positive results, including 4 false-positives. Sensitivity, specificity, positive predictive value and negative predictive value for LAMP tests were 100%, 98.13%, 95.51%, and 100% compared to PCR. Conclusion: High negative predictive value, and limit of detection suggest that LAMP can be used for screening of imported malaria cases in non-endemic countries when expert microscopists are not immediately available. However, the rare occurrence of non-valid results and the need for species identification and quantification of positive samples preclude the use of LAMP as a single reference method. Contexte : Des méthodes sensibles et faciles pour le diagnostic du paludisme sont encore attendues. Améliorer la limite de détection et répondre aux besoins de la certification sont des questions auxquelles il faut répondre tant en zone endémique que non-endémique. L'objectif de cette étude était de tester si la méthode d'amplification isothermique d'ADN en boucle (LAMP) peut être une alternative à la microscopie ou à la PCR temps réel pour le dépistage des cas de paludisme d'importation en zone non-endémique. Résultats : 310 échantillons de sang provenant de 829 suspicions de paludisme importé ont été testés pendant l'année de l'étude. La microscopie (frottis sanguins et gouttes épaisses colorées, standard de référence) a été utilisée pour le diagnostic. La PCR temps réel a été utilisée comme standard de vérité, et la LAMP (Meridian Malaria Plus) a été utilisée en méthode index dans une étude prospective conduite selon les standards pour rapporter les études d'efficacité diagnostique. Parmi les 83 échantillons positifs, l'identification des espèces était P. falciparum (n = 66), P. ovale (n = 9), P. vivax (n = 3) P. malariae (n = 3) et 2 coïnfections P. falciparum + P. malariae. La méthode LAMP a donné 93 échantillons positifs, dont 4 faux positifs. La sensitivité, la spécificité, la valeur prédictive positive et la valeur prédictive négative pour le LAMP étaient 100 %, 98.13 %, 95.51 %, 100 % comparées à la PCR. Conclusion : Une valeur prédictive négative élevée et la limite de détection suggèrent que le LAMP peut être utilisé pour dépister les cas de paludisme d'importation en zone non-endémique lorsque des microscopistes experts ne sont pas immédiatement disponibles. Cependant, la rare possibilité de résultats non valides et le besoin d'une identification d'espèce et d'une quantification empêchent que la méthode LAMP soit utilisée comme seule méthode de référence.

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Plasmodium vivax is the second most important human malaria parasite, widely spread across the world. This parasite is associated with important issues in the process toward malaria elimination, including potential for relapse and increased resistance to chloroquine. Plasmodium vivax multi-drug resistant (pvmdr1) is suspected to be a marker of resistance although definitive evidence is lacking. Progress has been made in knowledge of biological factors affecting parasite growth, including mechanisms of regulated cell death and the suspected role of metacaspase. Plasmodium vivax metacaspase1 (PvMCA1-cd) has been described with a catalytic domain composed of histidine (H372) and cysteine (C428) residues. The aim of this study was to test for a link between the conserved histidine and cysteine residues in PvMCA1-cd, and the polymorphism of the P. vivax multi-drug resistant gene (pvmdr1). Thirty P. vivax isolates were collected from Mauritania, Sudan, and Oman. Among the 28 P. vivax isolates successfully sequenced, only 4 samples showed the conserved His (372)–Cys (428) residues in PvMCA1-cd. Single nucleotide polymorphisms observed were H372T (46.4%), H372D (39.3%), and C428R (85.7%). A new polymorphic catalytic domain was observed at His (282)–Cys (305) residues. Sequences alignment analysis of pvmdr1 showed SNP in the three codons 958, 976 and 1076. A single SNP was identified at the codon M958Y (60%), 2 SNPs were found at the position 976: Y976F (13%) and Y976V (57%), and 3 SNPs were identified at the position 1076: F1076L (40%), F1076T (53%) and F1076I (3%). Only one isolate was wildtype in all three codons (MYF), 27% were single MYL mutants, and 10% were double MFL mutants. Three new haplotypes were also identified: the triple mutant YVT was most prevalent (53.3%) distributed in the three countries, while triple YFL and YVI mutants (3%), were only found in samples from Sudan and Mauritania. Triple or quadruple mutants for metacaspase genes and double or triple mutants for Pvmdr1 were observed in 24/28 and 19/28 samples. There was no difference in the frequency of mutations between PvMCA1-cd and Pvmdr1 (P > 0.2). Histidine and cysteine residues in PvMCA1-cd are highly polymorphic and linkage disequilibrium with SNPs of Pvmdr1 gene may be expected from these three areas with different patterns of P. vivax transmission.

Introduction: Minor frequency viruses play many important roles during viral infection that cannot be explained by the consensus sequence alone. In influenza, immunosuppressed individuals appear to generate numerous viral variants, leading to subpopulations with important role in infection. The objective of the present study was to describe viral diversification over time in immunocompetent patients during influenza virus infection. Methods: All clinical records of patients admitted to the Lyon university hospital (Lyon, France) during the influenza infection epidemics of the 2010-2015 period and sampled at least twice during their clinical management were retrospectively analyzed. To estimate performance of the sequencing procedures, well-characterized plasmids containing each of the 8 segments of influenza viruses were used as quality controls. Diversity, i.e. the number of validated single nucleotide variants, was analyzed to compare characteristics over time and according to clinical severity (mild, severe with neurological complications, severe with respiratory complications). Results: After validation on quality controls (n=51), and verification of possible confusion bias, a 5%-threshold of detection was applied to clinical viral sequences (n=29). At this threshold, amino-acid coordinates (n=183/4,246, 4.31%) were identified as having at least one mutation during clinical course, independently of the clinical severity. Considering a threshold of 4 days of symptoms, as a limit for early and late sampling, diversity was significantly higher in late samples for the mild group, compared to both early mild and severe groups (p<0.05). At a single-segment scale, for PB2-coding segment, diversity was significantly higher in early samples of the neurological group than in both early and late samples in the respiratory group and for late samples in the mild group (p<0.05). For the NS1-coding segment, significant differences were observed between initial diversity of mild and severe patients, as for early and late samples in mild patients (p<0.01). Discussion. This study is the first describing diversity through time, associating biological and clinical information during viral diversification, during the infection of an immunocompetent human host. This latter opens a large field of investigation in infectious disease management using next-generation sequencing and suggest development of new therapies, focusing on non-antigenic viral properties, in non-vaccine fields of research