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.

Evaluation of two commercial kits and two laboratory-developed qPCR assays compared to LAMP for molecular diagnosis of malaria.

BACKGROUND: Malaria is an infectious disease considered as one of the biggest causes of mortality in endemic areas. This life-threatening disease needs to be quickly diagnosed and treated. The standard diagnostic tools recommended by the World Health Organization are thick blood smears microscopy and immuno-chromatographic rapid diagnostic tests. However, these methods lack sensitivity especially in cases of low parasitaemia and non-falciparum infections. Therefore, the need for more accurate and reliable diagnostic tools, such as real-time polymerase chain reaction based methods which have proven greater sensitivity particularly in the screening of malaria, is prominent. This study was conducted at the French National Malaria Reference Centre to assess sensitivity and specificity of two commercial malaria qPCR kits and two in-house developed qPCRs compared to LAMP. METHODS: 183 blood samples received for expertise at the FNMRC were included in this study and were subjected to four different qPCR methods: the Biosynex Ampliquick® Malaria test, the BioEvolution Plasmodium Typage test, the in-house HRM and the in-house TaqMan qPCRs. The specificity and sensitivity of each method and their confidence intervals were determined with the LAMP-based assay Alethia® Malaria as the reference for malaria diagnosis. The accuracy of species diagnosis of the Ampliquick® Malaria test and the two in-house qPCRs was also evaluated using the BioEvolution Plasmodium Typage test as the reference method for species identification. RESULTS: The main results showed that when compared to LAMP, a test with excellent diagnostic performances, the two in-house developed qPCRs were the most sensitive (sensitivity at 100% for the in-house TaqMan qPCR and 98.1% for the in-house HRM qPCR), followed by the two commercial kits: the Biosynex Ampliquick® Malaria test (sensitivity at 97.2%) and the BioEvolution Plasmodium Typage (sensitivity at 95.4%). Additionally, with the in-house qPCRs we were able to confirm a Plasmodium falciparum infection in microscopically negative samples that were not detected by commercial qPCR kits. This demonstrates that the var genes of P. falciparum used in these in-house qPCRs are more reliable targets than the 18S sRNA commonly used in most of the developed qPCR methods for malaria diagnosis. CONCLUSION: Overall, these results accentuate the role molecular methods could play in the screening of malaria. This may represent a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis, which could be essential for the detection and treatment of malaria carriers and even for the eradication of this disease.

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%.

Potent Antiplasmodial Derivatives of Dextromethorphan Reveal the Ent-Morphinan Pharmacophore of Tazopsine-Type Alkaloids.

The alkaloid tazopsine 1 was introduced in the late 2000s as a novel antiplasmodial hit compound active against Plasmodium falciparum hepatic stages, with the potential to develop prophylactic drugs based on this novel chemical scaffold. However, the structural determinants of tazopsine 1 bioactivity, together with the exact definition of the pharmacophore, remained elusive, impeding further development. We found that the antitussive drug dextromethorphan (DXM) 3, although lacking the complex pattern of stereospecific functionalization of the natural hit, was harboring significant antiplasmodial activity in vitro despite suboptimal prophylactic activity in a murine model of malaria, precluding its direct repurposing against the disease. The targeted N-alkylation of nor-DXM 15 produced a small library of analogues with greatly improved activity over DXM 3 against P. falciparum asexual stages. Amongst these, N-2'-pyrrolylmethyl-nor-DXM 16i showed a 2- to 36-fold superior inhibitory potency compared to tazopsine 1 and DXM 3 against P. falciparum liver and blood stages, with respectively 760 ± 130 nM and 2.1 ± 0.4 µM IC50 values, as well as liver/blood phase selectivity of 2.8. Furthermore, cpd. 16i showed a 5- to 8-fold increase in activity relative to DXM 3 against P. falciparum stages I-II and V gametocytes, with 18.5 µM and 13.2 µM IC50 values, respectively. Cpd. 16i can thus be considered a promising novel hit compound against malaria in the ent-morphinan series with putative pan cycle activity, paving the way for further therapeutic development (e.g., investigation of its prophylactic activity in vivo).