Pharmaco-virological outcomes and genotypic resistance profiles among children and adolescents receiving a DTG-based regimen in Togo.

BACKGROUND: Few data are available on the real-world efficacy of receiving tenofovir-lamivudine-dolutegravir (DTG) as HIV treatment, particularly among young people in West Africa. Here, we evaluated pharmaco-virological outcomes and resistance profiles among Togolese children and adolescents. METHODS: A cross-sectional study was conducted in Lomé, Togo, enrolling antiretroviral-treated people with HIV aged from 18 months to 24 years. Plasma HIV-1 viral load and antiretroviral concentrations were measured. Next-Generation Sequencing (NGS) of protease, Reverse Transcriptase (RT) and integrase was performed on all samples with viral load >200 c/mL. Drug resistance mutations (DRMs) were identified and interpreted using the ANRS-MIE algorithm. RESULTS: 264 participants were enrolled (median age=17 years), 226 received a DTG-based regimen for a median of 20.5 months. Among them, virological suppression at the 200 c/mL threshold in 80.0% of the participants. Plasma DTG concentrations were adequate (i.e., >640 ng/mL), suboptimal and below the limit of quantification in 74.1%, 6.7% and 19.2% of participants receiving DTG, respectively. Overall, viruses resistant to any of Nucleoside RT Inhibitors, Non-NRTIs, and protease inhibitors were found in 52%, 66% and 1.6% of participants, respectively. A major integrase inhibitor DRM was observed in 9.4% (n=3/32, R263K, E138A-G140A-Q148R, and N155H) of participants with a viral load >200 c/mL. CONCLUSIONS: These first findings in such a large series of adolescents in a low-income country, showed a good virological response of 80% and the presence of an integrase DRM in 9.4% of the virological failures, supporting the need to monitor DTG drug resistance to reduce the risk of resistance acquisition.

RSV-GenoScan: An automated pipeline for whole-genome human respiratory syncytial virus (RSV) sequence analysis.

BACKGROUND: Advances in high-throughput sequencing (HTS) technologies and reductions in sequencing costs have revolutionised the study of genomics and molecular biology by making whole-genome sequencing (WGS) accessible to many laboratories. However, the analysis of WGS data requires significant computational effort, which is the major drawback in implementing WGS as a routine laboratory technique. OBJECTIVE: Automated pipelines have been developed to overcome this issue, but they do not exist for all organisms. This is the case for human respiratory syncytial virus (RSV), which is a leading cause of lower respiratory tract infections in infants, the elderly, and immunocompromised adults. RESULTS: We present RSV-GenoScan, a fast and easy-to-use pipeline for WGS analysis of RSV generated by HTS on Illumina or Nanopore platforms. RSV-GenoScan automates the WGS analysis steps directly from the raw sequence data. The pipeline filters the sequence data, maps the reads to the RSV reference genomes, generates a consensus sequence, identifies the RSV subgroup, and lists amino acid mutations, insertions and deletions in the F and G viral genes. This enables the rapid identification of mutations in these coding genes that are known to confer resistance to monoclonal antibodies. AVAILABILITY: RSV-GenoScan is freely available at https://github.com/AlexandreD-bio/RSV-GenoScan.

Higher Levels of SARS-CoV-2 Genetic Variation in Immunocompromised Patients: A Retrospective Case-Control Study.

BACKGROUND: A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection lasts longer in immunocompromised hosts than in immunocompetent patients. Prolonged infection is associated with a higher probability of selection for novel SARS-CoV-2 mutations, particularly in the spike protein, a critical target for vaccines and therapeutics. METHODS: From December 2020 to September 2022, respiratory samples from 444 immunocompromised patients and 234 health care workers positive for SARS-CoV-2, diagnosed at 2 hospitals in Paris, France, were analyzed using whole-genome sequencing using Nanopore technology. Custom scripts were developed to assess the SARS-CoV-2 genetic diversity between the 2 groups and within the host. RESULTS: Most infections were SARS-CoV-2 Delta or Omicron lineages. Viral genetic diversity was significantly higher in infections of immunocompromised patients than those of controls. Minor mutations were identified in viruses sequenced from immunocompromised individuals, which became signature mutations for newer SARS-CoV-2 variants as the epidemic progressed. Two patients were coinfected with Delta and Omicron variants. The follow-up of immunocompromised patients revealed that the SARS-CoV-2 genome evolution differed in the upper and lower respiratory tracts. CONCLUSIONS: This study found that SARS-CoV-2 infection in immunocompromised patients is associated with higher genetic diversity, which could lead to the emergence of new SARS-CoV-2 variants with possible immune evasion or different virulence characteristics.

Proportion of APOBEC3-induced defective HIV DNA after 1†year of dolutegravir + lamivudine simplification in the ANRS 167 LAMIDOL trial.

BACKGROUND: Hypermutated viruses induced by APOBEC3 (apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3) proteins comprise some of the defective viruses in the HIV reservoir. Here, we assessed the proportion of APOBEC3-induced defective proviruses in HIV-positive patients before and after receiving dolutegravir + lamivudine dual therapy. METHODS: PBMCs of virologically suppressed patients enrolled in the ANRS 167 LAMIDOL trial, evaluating a switch from triple therapy to dolutegravir + lamivudine, were collected 8 weeks before (W-8) and 48 weeks after (W48) dual-therapy initiation. The Vif and RT regions were subject to next-generation sequencing. Bioinformatic algorithms were developed to identify APOBEC3-defective sequences and APOBEC3-related drug resistance mutations (APOMuts). All hypermutated sequences and those containing at least one stop codon were considered as defective. RESULTS: One hundred and four patients were enrolled (median virological suppression duration: 4.2 years; IQR: 2.0-9.1). Proviral defective reads at W-8 and W48 were detected in Vif in 22% and 29% of patients, respectively, and in RT in 38% and 42% of patients, respectively. At least one APOMut was present in proviruses of 27% and 38% of patients at W-8 and W48, respectively. The ratio of APOMuts/number of potential APOMut sites was significantly higher at W48 (16.5%) than at W-8 (9.8%, P = 0.007). The presence of APOBEC3-defective viruses at W-8 was not associated with HIV total DNA level, nor with the third drug class received prior to switching to dolutegravir + lamivudine, nor with the duration of virological suppression. CONCLUSIONS: Whereas no significant change in the proportion of patients with APOBEC3-defective proviruses was evidenced after 1 year of dolutegravir + lamivudine maintenance, enrichment in APOMuts was observed. Further longer-term studies are needed to assess the other forms of defective viruses with dual-therapy.

Evolution and spread of Plasmodium falciparum mutations associated with resistance to sulfadoxine-pyrimethamine in central Africa: a cross-sectional study.

BACKGROUND: Efficacy of sulfadoxine-pyrimethamine, the malaria chemoprophylaxis used in pregnant women, and in children when combined with amodiaquine, is threatened by the accumulation of mutations in the Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes. Data on the prevalence of resistant alleles in central Africa and the new pfdhps I431V mutation, particularly associated with other mutations to form the pfdhps vagKgs allele, are scarce. We explored the frequency and geographical distribution of pfdhps and pfdhfr mutations in central Africa in 2014-18, and assessed the evolutionary origin of the vagKgs allele. METHODS: Samples were collected at 18 health-care centres in seven countries (Angola, Cameroon, Central African Republic, Democratic Republic of the Congo, Gabon, Nigeria, and Republic of the Congo) from patients who showed possible symptoms of malaria between March 1, 2014, and Oct 31, 2018. Samples that were positive for P falciparum were transported to a laboratory in Toulouse, France, and genotyped. The frequency of pfdhfr and pfdhps mutations was studied in 1749 samples. Microsatellites in pfdhps flanking regions and whole-genome analysis compared with parasite genomes from the data-sharing network MalariaGEN were performed on samples carrying the vagKgs allele. FINDINGS: Mapping of the prevalence of single nucleotide polymorphisms and corresponding alleles of pfdhfr and pfdhps showed a substantial spread of alleles associated with sulfadoxine-pyrimethamine resistance in central Africa during the 2014-18 period, especially an increase going west to east in pfdhps alleles carrying the K540E and A581G mutations. A high prevalence of the pfdhps I431V mutation was observed in Cameroon (exceeding 50% in the northern region) and Nigeria. Genomic analysis showed a recent African emergence and a clonal expansion of the most frequent pfdhps vagKgs allele. INTERPRETATION: Reduced sulfadoxine-pyrimethamine efficacy due to increased resistance is a worrying situation, especially because the malaria transmission level is high in central Africa. Although the resistance phenotype remains to be confirmed, the emergence and spread of the vagKgs allele in west and central Africa could challenge the use of sulfadoxine-pyrimethamine. FUNDING: Toulouse Institute for Infectious and Inflammatory Diseases.

Genomic diversity of mpox virus in Paris area (France) during the 2022 outbreak.

In May 2022, several countries reported mpox cases from patients without history of traveling to endemic areas. France was one of the most affected European countries by this outbreak. In this study, the clinical characteristics of mpox cases in France were described, and the genetic diversity of the virus was studied. Patients diagnosed with mpox infection (quantitative polymerase chain reaction ct < 28) between May 21, and July 4, 2022 and between 16th August and 10th September 2022 were included to this study. Twelve amplicons corresponding to the most polymorphic regions of the mpox genome and covering ~30 000 nucleotides were generated and sequenced using the S5 XL Ion Torrent technology to evaluate the genetic diversity of mpox sequences. One hundred and forty-eight patients were diagnosed with mpox-infection. 95% were men, 5% transgender (M-to-F), 50% were taking human immunodeficiency virus (HIV) pre-exposure prophylaxis, and 25% were HIV seropositive. One hundred and sixty-two samples (some patients had two samples) were sequenced and compared to GenBank sequences. Overall, low genetic diversity of mpox sequences was found compared with pre-epidemic Western-African sequences, with 32 distinct mutational patterns. This study provides a first glance at the mutational landscape of early mpox 2022 circulating strains in Paris (France).

Genetic Profiling of Plasmodium ovale wallikeri Relapses With Microsatellite Markers and Whole-Genome Sequencing.

Like Plasmodium vivax, both Plasmodium ovale curtisi and Plasmodium ovale wallikeri have the ability to cause relapse in humans, defined as recurring asexual parasitemia originating from liver-dormant forms subsequent to a primary infection. Here, we investigated relapse patterns in P ovale wallikeri infections from a cohort of travelers who were exposed to the parasite in sub-Saharan Africa and then experienced relapses after their return to France. Using a novel set of 8 highly polymorphic microsatellite markers, we genotyped 15 P ovale wallikeri relapses. For most relapses, the paired primary and relapse infections were highly genetically related (with 12 being homologous), an observation that was confirmed by whole-genome sequencing for the 4 relapses we further studied. This is, to our knowledge, the first genetic evidence of relapses in P ovale spp.

Phylodynamics of SARS-CoV-2 in France, Europe, and the world in 2020.

Although France was one of the most affected European countries by the COVID-19 pandemic in 2020, the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) movement within France, but also involving France in Europe and in the world, remain only partially characterized in this timeframe. Here, we analyzed GISAID deposited sequences from January 1 to December 31, 2020 (n = 638,706 sequences at the time of writing). To tackle the challenging number of sequences without the bias of analyzing a single subsample of sequences, we produced 100 subsamples of sequences and related phylogenetic trees from the whole dataset for different geographic scales (worldwide, European countries, and French administrative regions) and time periods (from January 1 to July 25, 2020, and from July 26 to December 31, 2020). We applied a maximum likelihood discrete trait phylogeographic method to date exchange events (i.e., a transition from one location to another one), to estimate the geographic spread of SARS-CoV-2 transmissions and lineages into, from and within France, Europe, and the world. The results unraveled two different patterns of exchange events between the first and second half of 2020. Throughout the year, Europe was systematically associated with most of the intercontinental exchanges. SARS-CoV-2 was mainly introduced into France from North America and Europe (mostly by Italy, Spain, the United Kingdom, Belgium, and Germany) during the first European epidemic wave. During the second wave, exchange events were limited to neighboring countries without strong intercontinental movement, but Russia widely exported the virus into Europe during the summer of 2020. France mostly exported B.1 and B.1.160 lineages, respectively, during the first and second European epidemic waves. At the level of French administrative regions, the Paris area was the main exporter during the first wave. But, for the second epidemic wave, it equally contributed to virus spread with Lyon area, the second most populated urban area after Paris in France. The main circulating lineages were similarly distributed among the French regions. To conclude, by enabling the inclusion of tens of thousands of viral sequences, this original phylodynamic method enabled us to robustly describe SARS-CoV-2 geographic spread through France, Europe, and worldwide in 2020.

Ex vivo RSA and pfkelch13 targeted-amplicon deep sequencing reveal parasites susceptibility to artemisinin in Senegal, 2017.

BACKGROUND: Malaria control is highly dependent on the effectiveness of artemisinin-based combination therapy (ACT), the current frontline malaria curative treatment. Unfortunately, the emergence and spread of parasites resistant to artemisinin (ART) derivatives in Southeast Asia and South America, and more recently in Rwanda and Uganda (East Africa), compromise their long-term use in sub-Saharan Africa, where most malaria deaths occur. METHODS: Here, ex vivo susceptibility to dihydroartemisinin (DHA) was evaluated from 38 Plasmodium falciparum isolates collected in 2017 in Thiès (Senegal) expressed in the Ring-stage Survival Assay (RSA). Both major and minor variants were explored in the three conserved-encoding domains of the pfkelch13 gene, the main determinant of ART resistance using a targeted-amplicon deep sequencing (TADS) approach. RESULTS: All samples tested in the ex vivo RSA were found to be susceptible to DHA (parasite survival rate < 1%). The non-synonymous mutations K189T and K248R in pfkelch13 were observed each in one isolate, as major (99%) or minor (5%) variants, respectively. CONCLUSION: The results suggest that ART is still fully effective in the Thiès region of Senegal in 2017. Investigations combining ex vivo RSA and TADS are a useful approach for monitoring ART resistance in Africa.

Plasmodium sporozoites require the protein B9 to invade hepatocytes.

Plasmodium sporozoites are transmitted to a mammalian host during blood feeding by an infected mosquito and invade hepatocytes for initial replication of the parasite into thousands of erythrocyte-invasive merozoites. Here we report that the B9 protein, a member of the 6-cysteine domain protein family, is secreted from sporozoite micronemes and is required for productive invasion of hepatocytes. The N-terminus of B9 forms a beta-propeller domain structurally related to CyRPA, a cysteine-rich protein forming an essential invasion complex in Plasmodium falciparum merozoites. The beta-propeller domain of B9 is essential for sporozoite infectivity and interacts with the 6-cysteine proteins P36 and P52 in a heterologous expression system. Our results suggest that, despite using distinct sets of parasite and host entry factors, Plasmodium sporozoites and merozoites may share common structural modules to assemble protein complexes for invasion of host cells.

Ex vivo RSA and Pfkelch13 targeted-amplicon deep sequencing reveal parasites susceptibility to artemisinin in Senegal, 2017.

INTRODUCTION: Malaria control is highly dependent on the effectiveness of artemisinin-based combination therapies (ACTs), the current frontline malaria curative treatments. Unfortunately, the emergence and spread of parasites resistant to artemisinin (ART) derivatives in Southeast Asia and South America, and more recently in Rwanda and Uganda (East Africa), compromise their long-term use in Sub-Saharan Africa where most malaria deaths occur. METHODS: Here, we evaluated ex vivo susceptibility to dihydroartemisinin (DHA) from 38 P. falciparum isolates collected in 2017 in Thiès (Senegal) expressed with the Ring-stage Survival Assay (RSA). We explored major and minor variants in the full Pfkelch13 gene, the main determinant of ART resistance using a targeted-amplicon deep sequencing (TADS) approach. RESULTS: All samples tested in the ex vivo RSA were found to be susceptible to DHA. Both non-synonymous mutations K189T and K248R were observed each in one isolate, as major (99%) or minor (5%) variants, respectively. CONCLUSION: Altogether, investigations combining ex vivo RSA and TADS are a useful approach for monitoring ART resistance in Africa.

Pharmaco-virological algorithm to target risk of drug resistance among a population of HIV-infected key populations in Togo.

No data about antiretroviral (ARV) treatment coverage and virological response are available among key populations (female sex workers [FSW] and Men having Sex with Men [MSM]) in Togo. This study aimed to both describe Human Immunodeficiency Virus (HIV) immunovirological status and evaluate the pertinence of an original algorithm combining pharmacology (PK) and viral load (VL) to identify subjects at risk of ARV drug resistance. A cross-sectional multicentric study was conducted in 2017 in Togo. Our PK-virological algorithm (PK-VA) defines subjects at risk of resistance when exhibiting both detectable plasma drug concentrations and VL > 200 c/mL. Among the 123 FSW and 136 MSM included, 50% and 66% were receiving ARV, with 69% and 80% of them successfully-treated, respectively. Genotypes showed drug-resistance mutation in 58% and 63% of nonvirologically controlled (VL > 200 c/mL) ARV-treated FSW and MSM, respectively. PK-VA would have enabled to save 75% and 72% of genotypic tests, for FSW and MSM, respectively. We reported first data about HIV care cascade among key populations in Togo, highlighting they are tested for HIV but linkage to care remains a concern. Furthermore, 70%-80% of ARV-treated participants experienced virological success. In limited resources settings, where genotyping tests are beyond reach, PK-VA might be an easiest solution to sort out patients needing ARV adaptation due to resistance.

Temporal dynamics of RSV shedding and genetic diversity in adults during the COVID-19 pandemic in a French hospital, early 2021.

Human respiratory syncytial virus (RSV) is responsible of lower respiratory tract infections which may be severe in infants, elderly and immunocompromised adults. Europe and North-American countries have observed a massive reduction of RSV incidence during the 2020-2021 winter season. Using a systematic RSV detection coupled to SARS-CoV-2 for all adult patients admitted at the Foch hospital (Suresnes, France) between January and March 2021 (n = 11,324), only eight RSV infections in patients with prolonged RNA shedding were diagnosed. RSV whole-genome sequencing revealed that six and two patients were infected by RSV groups A and B, respectively. RSV carriage lasted from 7 to at least 30 days disregarding of RSV lineage. The most prolonged RSV shedding was observed in an asymptomatic patient. We detected novel patient-specific non-synonymous mutations in the G glycoprotein gene, including a double identical mutation in the repeated region for one patient. No additional mutation occurred in the RSV genome over the course of infection in the four patients tested for. In conclusion, our results suggest that the temporal shift in the RSV epidemic is not likely to be explained by the emergence of a high frequency, unreported variant. Moreover, prolonged RSV carriages in asymptomatic patients could play a role in virus spread.

Nosocomial Malaria Transmissions Resolved by Genomic Analyses-A Retrospective Case Report Study in France: 2007-2021.

BACKGROUND: Exposure of blood to malaria parasites can lead to infection even in the absence of the mosquito vector. During a stay in a healthcare facility, accidental inoculation of the skin with blood from a malaria patient might occur, referred to as nosocomial malaria. METHODS: Between 2007 and 2021, we identified 6 autochthonous malaria cases that occurred in different French hospitals, originating from nosocomial transmission and imported malaria cases being the infection source. Four cases were observed during the coronavirus disease 2019 pandemic. The genetic relatedness between source and nosocomial infections was evaluated by genome-wide short tandem repeats (STRs) and single-nucleotide polymorphisms (SNPs). RESULTS: None of the patients with autochthonous malaria had travel history to an endemic area nor had been transfused. For each case, both the source and recipient patients stayed a few hours in the same ward. After diagnosis, autochthonous cases were treated with antimalarials and all recovered except 1. Genetically, each pair of matched source/nosocomial parasite infections showed <1% of different STRs and <6.9% (<1.5% for monoclonal infections) of different SNPs. Similar levels of genetic differences were obtained for parasite DNA samples that were independently sequenced twice as references of identical infections. Parasite phylogenomics were consistent with travel information reported by the source patients. CONCLUSIONS: Our study demonstrates that genomics analyses may resolve nosocomial malaria transmissions, despite the uncertainty regarding the modes of contamination. Nosocomial transmission of potentially life-threatening parasites should be taken into consideration in settings or occasions where compliance with universal precautions is not rigorous.

Dual Monoclonal Antibodies on Sars-Cov-2 Alpha and Delta Variants: Clinical and Virological Efficacy.

Monoclonal antibodies (MAbs) targeting the Spike glycoprotein of SARS-CoV-2 is a key strategy to prevent severe COVID-19. Here, the efficacy of two monoclonal antibody bitherapies against SARS-CoV-2 was assessed on 92 patients at high risk of severe COVID-19 between March and October 2021 (Bichat-Claude Bernard Hospital, Paris, France). Nine patients died despite appropriate management. From 14 days following treatment initiation, we observed a slower viral load decay for patients treated with the bitherapy Bamlanivimab/Etsevimab compared to the Casirivimab/Imdevimab association therapy (P = 0.045). The emergence of several mutations on the Spike protein known to diminish antiviral efficacy was observed from 1 to 3 weeks after infusion. The Q493R mutation was frequently selected, located in a region of joint structural overlap by Bamlanivimab/Etsevimab antibodies. Despite that this study was done on former SARS-CoV-2 variants (Alpha and Delta), the results provide new insights into resistance mechanisms in SARS-CoV-2 antibodies neutralization escape and should be considered for current and novel variants. IMPORTANCE Monoclonal antibody bitherapies (MAbs) are commonly prescribed to treat severe SARS-CoV-2-positive patients, and the rapid growth of resistance mutation emergence is alarming globally. To explore this issue, we conducted both clinical and genomic analyses of SARS-CoV-2 in a series of patients treated in 2021. We first noticed that the two dual therapies prescribed during the study had different kinetics of viral load decay. Rapidly after initiation of the treatments, resistance mutations emerged in the interface between the MAbs and the target Spike glycoprotein, demonstrating the importance to continuously screen the viral genome during treatment course. Taken together, the results highlight that viral mutations may emerge under selective pressure, conferring a putative competitive advantage, and could rapidly spread, as observed for the Omicron variant.

Circulation of an Artemisinin-Resistant Malaria Lineage in a Traveler Returning from East Africa to France.

A returned traveler to Uganda presented with a Plasmodium falciparum kelch13 A675V mutant infection that exhibited delayed clearance under artesunate therapy. Parasites were genetically related to recently reported Ugandan artemisinin-resistant A675V parasites. Adequate malaria prevention measures and clinical and genotypic surveillance are important tools to avoid and track artemisinin resistance.

5WBF: a low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates.

BACKGROUND: Whole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programmes. A major drawback of this approach is the large amount of human DNA compared to parasite DNA extracted from unprocessed whole blood. As red blood cells (RBCs) have a diameter of about 7-8 µm and exhibit some deformability, it was hypothesized that cheap and commercially available 5 µm filters might retain leukocytes but much less of Plasmodium falciparum-infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for 5 µm Whole Blood Filtration), may provide highly enriched parasite material suitable for P. falciparum WGS. METHODS: Whole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitaemia range: 0.04-5.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured P. falciparum 3D7 parasites with uninfected human whole blood (final parasitaemia range: 0.02-1.1%). These whole blood samples (50 to 400 µL) were diluted in RPMI 1640 medium or PBS 1× buffer and filtered with a syringe connected to a 5 µm commercial filter. DNA was extracted from 5WBF-treated and unfiltered counterpart blood samples using a commercial kit. The 5WBF method was evaluated on the ratios of parasite:human DNA assessed by qPCR and by sequencing depth and percentages of coverage from WGS data (Illumina NextSeq 500). As a comparison, the popular selective whole-genome amplification (sWGA) method, which does not rely on blood filtration, was applied to the unfiltered counterpart blood samples. RESULTS: After applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN18S gene) and from 4096- to 65,536-fold loss of human template DNA (human ß actin gene). WGS analyses revealed that > 95% of the  parasite nuclear and organellar genomes were all covered at ≥ 10× depth for all samples tested. In sWGA counterparts, the organellar genomes were poorly covered and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10× depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5460 genes; mean coverage: 91×; median coverage: 93×; 5th percentile: 70×; 95th percentile: 103×), allowing the identification of gene copy number variations such as for gch1. This later analysis was not possible for sWGA-treated samples, as a much more heterogeneous distribution of reads across gene sequences was observed (mean coverage: 80×; median coverage: 51×; 5th percentile: 7×; 95th percentile: 245×). CONCLUSIONS: The novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5 µm filters which cost from 1.0 to 1.7€ per unit depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum ring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%.

Mutation in the Plasmodium falciparum BTB/POZ Domain of K13 Protein Confers Artemisinin Resistance.

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 (pfk13) gene. Here, we carried out in vitro selection over a 1-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

Earlier In Vitro Viral Production With SARS-CoV-2 Alpha Than With Beta, Gamma, B, or A.27 Variants.

Since its emergence in China at the end of 2019, SARS-CoV-2 has rapidly spread across the world to become a global public health emergency. Since then, the pandemic has evolved with the large worldwide emergence of new variants, such as the Alpha (B.1.1.7 variant), Beta (B.1.351 variant), and Gamma (P.1 variant), and some other under investigation such as the A.27 in France. Many studies are focusing on antibody neutralisation changes according to the spike mutations, but to date, little is known regarding their respective replication capacities. In this work, we demonstrate that the Alpha variant provides an earlier replication in vitro, on Vero E6 and A549 cells, than Beta, Gamma, A.27, and historical lineages. This earlier replication was associated with higher infectious titres in cell-culture supernatants, in line with the higher viral loads observed among Alpha-infected patients. Interestingly, Beta and Gamma variants presented similar kinetic and viral load than the other non-Alpha-tested variants.