Alternatively spliced transcripts and novel pseudogenes of the Plasmodium falciparum resistance-associated locus pfcrt detected in East African malaria patients.

OBJECTIVES: Polymorphisms in the lysosomal transporter encoded by the pfcrt gene directly impact on Plasmodium falciparum susceptibility to aminoquinolines. The Lys76Thr mutation is the critical change conferring chloroquine resistance in vitro and in vivo, but always occurs with additional non-synonymous changes in the pfcrt coding sequence. We sought to better describe pfcrt polymorphisms distal to codon 76. METHODS: Small-volume samples (≤ 500 µL) of parasite-infected blood collected directly from malaria patients presenting for treatment in Sudan and Tanzania were immediately preserved for RNA extraction. The pfcrt locus was amplified from cDNA preparations by nested PCR and sequenced directly to derive full-length mRNA sequences. RESULTS: In one of two sites in Sudan, two patients were found with an unorthodox spliced form of pfcrt mRNA in which two exons were skipped, but it was not possible to test for the presence of the putative protein products of these aberrant transcripts. Genomic DNA sequencing from dried blood spots collected in parallel confirmed the presence of spliced pfcrt pseudogenes in a minority of parasite isolates. Full-length cDNA from conventionally spliced mRNA molecules in all study sites demonstrated the existence of a variety of pfcrt haplotypes in East Africa, and thus provides evidence of intragenic recombination. CONCLUSIONS: The presence of pseudogenes, although unlikely to have any direct public health impact, may confound results obtained from simple genotyping methods that consider only codon 76 and the adjacent residues of pfcrt.

A multi-country phase 2 study to evaluate the suitcase lab for rapid detection of SARS-CoV-2 in seven Sub-Saharan African countries: Lessons from the field.

BACKGROUND: The COVID-19 pandemic led to severe health systems collapse, as well as logistics and supply delivery shortages across sectors. Delivery of PCR related healthcare supplies continue to be hindered. There is the need for a rapid and accessible SARS-CoV-2 molecular detection method in low resource settings. OBJECTIVES: To validate a novel isothermal amplification method for rapid detection of SARS-CoV-2 across seven sub-Sharan African countries. STUDY DESIGN: In this multi-country phase 2 diagnostic study, 3,231 clinical samples in seven African sites were tested with two reverse transcription Recombinase-Aided Amplification (RT-RAA) assays (based on SARS-CoV-2 Nucleocapsid (N) gene and RNA-dependent RNA polymerase (RdRP) gene). The test was performed in a mobile suitcase laboratory within 15 min. All results were compared to a real-time RT-PCR assay. Extraction kits based on silica gel or magnetic beads were applied. RESULTS: Four sites demonstrated good to excellent agreement, while three sites showed fair to moderate results. The RdRP gene assay exhibited an overall PPV of 0.92 and a NPV of 0.88. The N gene assay exhibited an overall PPV of 0.93 and a NPV 0.88. The sensitivity of both RT-RAA assays varied depending on the sample Ct values. When comparing sensitivity between sites, values differed considerably. For high viral load samples, the RT-RAA assay sensitivity ranges were between 60.5 and 100% (RdRP assay) and 25 and 98.6 (N assay). CONCLUSION: Overall, the RdRP based RT-RAA test showed the best assay accuracy. This study highlights the challenges of implementing rapid molecular assays in field conditions. Factors that are important for successful deployment across countries include the implementation of standardized operation procedures, in-person continuous training for staff, and enhanced quality control measures.

Increased pfmdr1 copy number and sequence polymorphisms in Plasmodium falciparum isolates from Sudanese malaria patients treated with artemether-lumefantrine.

Molecular markers for surveillance of Plasmodium falciparum resistance to current antimalarials are sorely needed. A 28-day efficacy study of artemether-lumefantrine in eastern Sudan identified 5 treatment failures among 100 evaluable patients; 9 further individuals were parasite positive by PCR during follow-up. Polymorphisms in pfatpase6 and pfmdr1 were evaluated by DNA sequencing. One individual carried parasites with a novel pfmdr1 polymorphism (F1044L). pfmdr1 gene amplification in parasites prior to treatment occurred in three individuals who had recurrent infection during follow-up.

A comparative study on the efficacy of artesunate plus sulphadoxine/pyrimethamine versus artemether-lumefantrine in eastern Sudan.

BACKGROUND: A combination of artesunate (AS) plus sulphadoxine/pyrimethamine (SP) as first-line and artemether-lumefantrine (AL) as second-line treatment are currently recommended against uncomplicated P. falciparum infection in Sudan. However, there is limited information on the efficacy of ACTs in the country and only one report of PCR-corrected results for AS/SP only. METHODS: The WHO protocol for the assessment of antimalarial drug efficacy for the treatment of uncomplicated falciparum malaria was employed. Artesunate plus sulphadoxine/pyrimethamine (AS/SP) was compared to artemether-lumefantrine (AL) in a 28-day follow up. Samples that were classified as early treatment failure (ETF), late treatment failure (LCF) or late parasitological failure (LPF) were genotyped for msp-1 and msp-2 genes to differentiate recrudescence from reinfection. RESULTS: A total of 178 patients were screened and 160 met the enrollment criteria and were recruited to the study of which 157 (98.1%) completed the follow up and had an analysed treatment outcome. On the AS/SP arm, three (0.038%) patients were lost during the follow-up, two on day 1 and one on day 7, and 77 (96.3) completed the study, while all 80 (100%) patients completed the follow up in the AL arm. In the per protocol analysis for AS/SP the treatment outcome for patients who completed the follow-up were as follows: adequate clinical and parasitological response (ACPR); 84.4% ETF; 1.3%, LCF; 3.9%, (LPF); 10.4%. For the AL arm the out come was as follows, ACPR; 90%, ETF; 0%, LCF; 6.3% and LPF; 3.8%. However, when PCR-corrected, 6.5% (5/77) of patients treated with AS/SP maintained parasites from their primary infection, while (7/80) in the AL group maintained their initial parasite genotype. Therefore, PCR-corrected efficacy was 93.5% in the AS/SP treated group and for AL it was 91.3%. CONCLUSION: Both AS/SP and AL are highly effective for the treatment of uncomplicated falciparum malaria in eastern Sudan. However, AS/SP appears to have a slightly higher efficacy than AL, this may be due to patient compliance with the repeated dose rather than drug efficacy.