Routine and molecular diagnostic tools for mutations raising concerns monitoring in plasmodium falciparum endemic disease proteome: multiclonal infection multiplicity, allelic marker msp genetic polymorphism, pf-haplotypes circulating recombinant form, HRP-2/3 gene deletions and anti-malarial drug resistance mutations

Background: Malaria is one of the top three major diseases, caused most importantly P. falciparum (Pf). The fight against malaria continues to have obstacles in the Malaria elimination as difficulties to identify Plasmodium with gene deletion or drug resistance mutation. Here, we used different diagnostic tools. Information on circulating Malaria genotypes and mutations can help to take the appropriate decisions. Methods: Febrile children aged 10 days to 15 years suspected of malaria were recruited from March 8 to 13, 2021 at the medical emergencies of the Bangui paediatric hospital in EDTA tube/haematology on site and Dried Blood Spot (DBS)/molecular analyses in Basel, Switzerland. Nucleic acids (DNA and RNA) Plasmodium spp. and Merozoite Surface Protein (MSP) 1 and 2 based Parasite were extracted. Their respective allelic markers again to detect the extent of genetic diversity of clinical isolates of P. falciparum. Demographic, clinical-haematological parameters, and parasitological complete analyses results obtained from the various techniques (automate, RDT, Microscopy, molecular: PCR) were carried out. Plasmodium species identification and analysis of antimalarial resistance markers by multiplex assay Copy Number Variations (CNV) determination in the pf-plasmepsin-2 and pfmdr1 gene and Single Nucleotide Polymorphisms (SNPs) detection in pfmdr1, pfcrt, pfdhps, pfdhfr and pfK13. Results: 41/100 children were positive in a P. falciparum RDT/microscopy on admission to hospital while 85/100 were positive in PCR. The Plasmodium spp. DNA and RNA, successfully extracted from DBS allowed the determination of 70 Plasmodium species/100 samples (69 P. falciparum/1 P. ovale), while in 15/100 samples Plasmodium spp. nucleic acid was detected but no species could be assigned. In another 15/100 samples, no Plasmodium spp. was found. No pfhrp-2/3 gene deletions were found. We found a high level of allelic markers between 6 to 22 by P. falciparum isolate, in total, MSP 1 (118) and MSP 2 (125). Furthermore, we observed that the multiplicity of multiclonal infection seems to occur frequently. High parasite density was significantly associated with age under 1 year-old and anaemia and leucocytosis (pairwise Wilcox p < 0.05). In the malaria genotyping, the prevalence of resistance markers was high in pfdhfr and pfdhps (I431V) indicating elevated Sulfadoxine/Pyrimethamine (SP) resistance. In the current study, the west African agK and SgK haplotypes were found with a frequency of 21.3% and 10.6%, an increasing since 2004, from 6.8% and 8.1%, respectively. The East African Sge haplotype occurred in 12.8% of patients (increased from 1.4% in 2004). These three haplotypes formed 8,5% of Pf-haplotype recombinant form in Pf positive cases. Fortunately, no resistance markers were found in pfK13, indicating high efficacy of artemisinin derivatives. Additionally, no CNV were found in pfmdr1 and pf-plasmepsin-2, indicating a lack of resistance against mefloquine (MQ) and piperaquine (PPQ). Interestingly, Chloroquine resistance markers were only found in one out of 59 samples, seems that P. falciparum are susceptible to chloroquine again, only 17 years after it was replaced by Artemether/Lumefantrine (AL) due to the high resistance prevalence. Conclusion: 9/10 febrile children suffer from malaria with anaemia multiclonal infections multiplicity, RDT PfHRP still provide sufficient sensitivity to detect P. falciparum infections with high and median parasite density. The CAR is the transitional Plasmodium haplotypes country from the East to the West Africa with the Pf-haplotype circulating recombinant form suggesting importance for best prevention of SP, the intermittent preventive treatment during pregnancy (IPTp. Artemisinin derivatives, MQ and PPQ remain the sustainable therapeutic solutions. Chloroquine resistance markers were only found in one. The results obtained demonstrate the importance of the complementarity of the tests in malaria control or other diseases control for their elimination in developing countries.