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.

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.

Indigenous emergence and spread of kelch13 C469Y artemisinin-resistant Plasmodium falciparum in Uganda.

Artemisinin-based combination therapies (ACTs) were introduced as the standard of care for uncomplicated malaria in Africa almost two decades ago. Recent studies in East Africa have reported a gradual increase in kelch13 (k13) mutant parasites associated with reduced artesunate efficacy. As part of the Community Access to Rectal Artesunate for Malaria project, we collected blood samples from 697 children with signs of severe malaria in northern Uganda between 2018 and 2020, before and after the introduction of rectal artesunate (RAS) in 2019. K13 polymorphisms were assessed, and parasite editing and phenotyping were performed to assess the impact of mutations on parasite resistance. Whole-genome sequencing was performed, and haplotype networks were constructed to determine the geographic origin of k13 mutations. Of the 697 children, 540 were positive for Plasmodium falciparum malaria by PCR and were treated with either RAS or injectable artesunate monotherapy followed in most cases by ACT. The most common k13 mutation was C469Y (6.7%), which was detected more frequently in samples collected after RAS introduction. Genome editing confirmed reduced in vitro susceptibility to artemisinin in C469Y-harboring parasites compared to wild-type controls (P < 0.001). The haplotypic network showed that flanking regions of the C469Y mutation shared the same African genetic background, suggesting a single and indigenous origin of the mutation. Our data provide evidence of selection for the artemisinin-resistant C469Y mutation. The realistic threat of multiresistant parasites emerging in Africa should encourage careful monitoring of the efficacy of artemisinin derivatives and strict adherence to ACT treatment regimens.

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.

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.

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.

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.

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).

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.

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.

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.

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%.

Effect of Drug Pressure on Promoting the Emergence of Antimalarial-Resistant Parasites among Pregnant Women in Ghana.

The continuous spread of antimalarial drug resistance is a threat to current chemotherapy efficacy. Therefore, characterizing the genetic diversity of drug resistance markers is needed to follow treatment effectiveness and further update control strategies. Here, we genotyped Plasmodium falciparum resistance gene markers associated with sulfadoxine-pyrimethamine (SP) and artemisinin-based combination therapy (ACT) in isolates from pregnant women in Ghana. The prevalence of the septuple IRN I- A/FG K GS/Tpfdhfr/pfdhps haplotypes, including the pfdhps A581G and A613S/T mutations, was high at delivery among post-SP treatment isolates (18.2%) compared to those of first antenatal care (before initiation of intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine [IPTp-SP]; 6.1%; P = 0.03). Regarding the pfk13 marker gene, two nonsynonymous mutations (N458D and A481C) were detected at positions previously related to artemisinin resistance in isolates from Southeast Asia. These mutations were predicted in silico to alter the stability of the pfk13 propeller-encoding domain. Overall, these findings highlight the need for intensified monitoring and surveillance of additional mutations associated with increased SP resistance as well as emergence of resistance against artemisinin derivatives.

Pneumocystis Cytochrome b Mutants Associated With Atovaquone Prophylaxis Failure as the Cause of Pneumocystis Infection Outbreak Among Heart Transplant Recipients.

Background: Although trimethoprim-sulfamethoxazole is the more efficient drug for prophylactic and curative treatment of pneumocystosis, atovaquone is considered a second-line prophylactic treatment in immunocompromised patients. Variations in atovaquone absorption and mutant fungi selection after atovaquone exposure have been associated with atovaquone prophylactic failure. We report here a Pneumocystis jirovecii cytochrome b (cyt b) mutation (A144V) associated with such prophylactic failure during a pneumocystosis outbreak among heart transplant recipients. Methods: Analyses of clinical data, serum drug dosage, and molecular modeling of the P. jirovecii Rieske-cyt b complex were performed to investigate these prophylactic failures. Results: The cyt b A144V mutation was detected in all infected, heart transplant recipient patients exposed to atovaquone prophylaxis but in none of 11 other immunocompromised, infected control patients not treated with atovaquone. Serum atovaquone concentrations associated with these prophylactic failures were similar than those found in noninfected exposed control patients under a similar prophylactic regimen. Computational modeling of the P. jirovecii Rieske-cyt b complex and in silico mutagenesis indicated that the cyt b A144V mutation might alter the volume of the atovaquone-binding pocket, which could decrease atovaquone binding. Conclusions: These data suggest that the cyt b A144V mutation confers diminished sensitivity to atovaquone, resulting in spread of Pneumocystis pneumonia among heart transplant recipients submitted to atovaquone prophylaxis. Potential selection and interhuman transmission of resistant P. jirovecii strain during atovaquone prophylactic treatment has to be considered and could limit its extended large-scale use in immucompromised patients.

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.

Ex vivo susceptibility to antimalarial drugs and polymorphisms in drug resistance genes of African Plasmodium falciparum, 2016-2023: a genotype-phenotype association study.

Background: Given the altered responses to both artemisinins and lumefantrine in Eastern Africa, monitoring antimalarial drug resistance in all African countries is paramount. Methods: We measured the susceptibility to six antimalarials using ex vivo growth inhibition assays (IC50) for a total of 805 Plasmodium falciparum isolates obtained from travelers returning to France (2016-2023), mainly from West and Central Africa. Isolates were sequenced using molecular inversion probes (MIPs) targeting fourteen drug resistance genes across the parasite genome. Findings: Ex vivo susceptibility to several drugs has significantly decreased in 2019-2023 versus 2016-2018 parasite samples: lumefantrine (median IC50: 23·0 nM [IQR: 14·4-35·1] in 2019-2023 versus 13·9 nM [8·42-21·7] in 2016-2018, p<0·0001), monodesethylamodiaquine (35·4 [21·2-51·1] versus 20·3 nM [15·4-33·1], p<0·0001), and marginally piperaquine (20·5 [16·5-26·2] versus 18.0 [14·2-22·4] nM, p<0·0001). Only four isolates carried a validated pfkelch13 mutation. Multiple mutations in pfcrt and one in pfmdr1 (N86Y) were significantly associated with altered susceptibility to multiple drugs. The susceptibility to lumefantrine was altered by pfcrt and pfmdr1 mutations in an additive manner, with the wild-type haplotype (pfcrt K76-pfmdr1 N86) exhibiting the least susceptibility. Interpretation: Our study on P. falciparum isolates from West and Central Africa indicates a low prevalence of molecular markers of artemisinin resistance but a significant decrease in susceptibility to the partner drugs that have been the most widely used since a decade -lumefantrine and amodiaquine. These phenotypic changes likely mark parasite adaptation to sustained drug pressure and call for intensifying the monitoring of antimalarial drug resistance in Africa. Funding: This work was supported by the French Ministry of Health (grant to the French National Malaria Reference Center) and by the Agence Nationale de la Recherche (ANR-17-CE15-0013-03 to JC). JAB was supported by NIH R01AI139520. JR postdoctoral fellowship was funded by Institut de Recherche pour le Développement.

Ex vivo susceptibility to antimalarial drugs and polymorphisms in drug resistance genes of African Plasmodium falciparum, 2016-2023: a genotype-phenotype association study.

Background: Given the altered responses to both artemisinins and lumefantrine in Eastern Africa, monitoring antimalarial drug resistance in all African countries is paramount. Methods: We measured the susceptibility to six antimalarials using ex vivo growth inhibition assays (IC 50 ) for a total of 805 Plasmodium falciparum isolates obtained from travelers returning to France (2016-2023), mainly from West and Central Africa. Isolates were sequenced using molecular inversion probes (MIPs) targeting fourteen drug resistance genes across the parasite genome. Findings: Ex vivo susceptibility to several drugs has significantly decreased in 2019-2023 versus 2016-2018 parasite samples: lumefantrine (median IC 50 : 23·0 nM [IQR: 14·4-35·1] in 2019-2023 versus 13·9 nM [8·42-21·7] in 2016-2018, p<0·0001), monodesethylamodiaquine (35·4 [21·2-51·1] versus 20·3 nM [15·4-33·1], p<0·0001), and marginally piperaquine (20·5 [16·5-26·2] versus 18.0 [14·2-22·4] nM, p<0·0001). Only four isolates carried a validated pfkelch13 mutation. Multiple mutations in pfcrt and one in pfmdr1 (N86Y) were significantly associated with altered susceptibility to multiple drugs. The susceptibility to lumefantrine was altered by pfcrt and pfmdr1 mutations in an additive manner, with the wild-type haplotype ( pfcrt K76- pfmdr1 N86) exhibiting the least susceptibility. Interpretation: Our study on P. falciparum isolates from West and Central Africa indicates a low prevalence of molecular markers of artemisinin resistance but a significant decrease in susceptibility to the partner drugs that have been the most widely used since a decade -lumefantrine and amodiaquine. These phenotypic changes likely mark parasite adaptation to sustained drug pressure and call for intensifying the monitoring of antimalarial drug resistance in Africa.

Plasmodium ovale spp dhfr mutations associated with reduced susceptibility to pyrimethamine in sub-Saharan Africa: a retrospective genetic epidemiology and functional study.

BACKGROUND: Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised. METHODS: We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug 31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility. FINDINGS: We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts. INTERPRETATION: The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp. FUNDING: French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division.

Genomic epidemiology of SARS-CoV-2 in Senegal in 2020-2021.

INTRODUCTION: In Senegal, molecular diagnosis was widely used for the detection and management of COVID-19 patients. However, genomic surveillance was very limited in the public sector. This study aimed to share the experience of a Senegalese public sector laboratory in response to the COVID-19 pandemic, and to describe the distribution of variants circulating in 2020 and 2021. METHODOLOGY: From July 2020 to December 2021, SARS-CoV-2 qRT-PCR was performed on nasopharyngeal samples from travelers and symptomatic patients at the Bacteriology and Virology Laboratory (LBV) of the Aristide le Dantec University Teaching Hospital. Samples with a cycle threshold (Ct) ≤ 30 were selected for whole-genome sequencing (WGS) using the Nanopore technology. In-house scripts were developed to study the spatial and temporal distribution of SARS-CoV-2 variants in Senegal, using our sequences and those retrieved from the GISAID database. RESULTS: Of 8,207 patients or travelers screened for SARS-CoV-2, 970 (11.8%) were positive and 386 had a Ct ≤ 30. WGS was performed on 133 samples. Concomitantly with high-quality sequences deposited in the GISAID database covering nine cities in Senegal in 2020 and 2021 (n = 1,539), we observed a high circulation of the 20A (B.1, B.1.416 and B.1.620) and 20B (B.1.1.420) lineages in 2020, while most of the samples belonged to Delta variants (AY34 and AY.34.1, 22%) in 2021. CONCLUSIONS: Despite its late involvement, COVID-19 diagnosis was routinely performed in LBV, but genomic characterization remained challenging. The genomic diversity of SARS-CoV-2 strains in Senegal reflected that observed worldwide during the first waves of the pandemic.

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.