Characterization of drug resistance genes in Indian Plasmodium falciparum and Plasmodium vivax field isolates.

One of the major challenges for malaria control and elimination is the spread and emergence of antimalarial drug resistance. Mutations in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) field isolates for five drug resistance genes viz. crt, mdr1, dhps, dhfr and kelch known to confer resistance to choloroquine (CQ), sulfadoxine-pyrimethamine (SP) and artemisinin (ART) and its derivatives were analyzed. A total of 342 symptomatic isolates of P. falciparum (Pf) and P. vivax (Pv) from 1993 to 2014 were retrieved from malaria parasite repository at National Institute of Malaria Research (NIMR). Sample DNA was extracted from dried blood spots and various targeted single nucleotide polymorphisms (SNPs) associated with antimalarial drug resistance were analysed for these isolates. 72S (67.7%) and 76T (83.8%) mutations along with SVMNT haplotype (67.7%) predominated the study population for Pfcrt. The most prevalent SNPs were 108N (73.2%) and 437G (24.8%) and the most prevalent haplotypes were ACNRNI (51.9%) and SAKAA (74.5%) in Pfdhfr and Pfdhps respectively. Only two mutations in Pfmdr1, 86Y (26.31%) and 184F (56.26%), were seen frequently in our study population. No mutations associated with Pfk13 were observed. For Pv, all the studied isolates showed two Pvdhps mutations, 383G and 553G, and two Pfdhfr mutations, 58R and 117N. Similarly, three mutations, viz. 958M, 908L and 1076L were found in Pvmdr1. No variations were observed in Pvcrt-o and Pvk12 genes. Overall, our study demonstrates an increase in mutations associated with SP resistance in both Pf and Pv, however, no single nucleotide polymorphisms (SNPs) associated with ART resistance have been observed for either species. Various SNPs associated with CQ resistance were seen in Pf; whereas only Pvmdr1 associated resistant SNPs were observed in Pv. Therefore, molecular characterization of drug resistance genes is essential for timely monitoring and prevention of malaria by identifying the circulating drug resistant parasites in the country.

Deletions of Histidine-Rich Protein 2/3 Genes in Natural Plasmodium falciparum Populations from Cameroon and India: Role of Asymptomatic and Submicroscopic Infections.

The bulk of malaria rapid diagnostic tests (RDTs) target histidine-rich protein 2 of Plasmodium falciparum, the deadliest malaria species. The WHO considers pfhrp2/3 deletions as one of the main threats to successful malaria control and/or elimination; as such, parasites that lack part or all of the pfhrp2 gene are missed by pfHRP2-targeting RDTs. Such deletions have been reported in several African and Asian countries, but little is known in Cameroon and India. Blood samples were collected from individuals living in four areas of Cameroon (Douala, Maroua, Mayo-Oulo, Pette) and India (Mewat, Raipur, Ranchi, Rourkela). Deletions in pfhrp2/3 genes were confirmed if samples 1) had ≥100 parasites/µL by quantitative polymerase chain reaction (PCR), 2) PCR negative for pfhrp2/3, and 3) PCR positive for at least two single-copy genes. The overall proportion of pfhrp2 and pfhrp3 deletions in Cameroon was 13.5% and 3.1%. In India, the overall proportion was 8% for pfhrp2 and 4% for pfhrp3. The overall proportions of samples with both gene deletions (pfhrp2-/3-) were 3.1% in Cameroon and 1.3% in India. In Cameroon, pfhrp2-/3+ and pfhrp2-/3- deletions were common in Maroua (P = 0.02), in asymptomatic parasitemia (P = 0.006) and submicroscopic parasitemia (P <0.0001). In both countries, pfhrp2/3 deletions, including pfhrp2-/3- deletions, were mainly seen in monoclonal infections. This study outlines that double deletions that result in false negative RDTs are uncommon in our settings, and highlights the importance of active molecular surveillance for pfhrp2/3 deletions in Cameroon and India.

First Insight into Drug Resistance Genetic Markers, Glucose-6-Phosphate Dehydrogenase and Phylogenetic Patterns of Misdiagnosed Plasmodium vivax Malaria in Far North Region, Cameroon.

Plasmodium falciparum (Pf) is the predominant malaria species in Africa, but growing rates of non-falciparum species such as P. vivax (Pv) have been reported recently. This study aimed at characterizing drug resistance genes, glucose-6-phosphate dehydrogenase gene (G6PD), and phylogenetic patterns of a Pv + Pf co-infection misdiagnosed as a Pf mono-infection in the Far North region of Cameroon. Only one non-synonymous mutation in the pvdhps gene A383G was found. Pv drug resistance gene sequences were phylogenetically closer to the reference SAL-I strain and isolates from Southeast Asia and Western Pacific countries. Analyzing co-infecting Pf revealed no resistance mutations in Pfmdr1 and Pfk13 genes, but mutations in Pfcrt (C72V73I74E75T76) and Pfdhfr-Pfdhps genes (A16C50I51R59N108L164 - A436A437K540G581S613) were observed. No G6PD deficiency-related mutations were found. This is first study from Cameroon reporting presence of putative drug resistance mutations in Pv infections, especially in the pvdhps gene, and also outlined the absence of a G6PD-deficiency trait in patients.

Global polymorphism of Plasmodium falciparum histidine rich proteins 2/3 and impact on malaria rapid diagnostic test detection: a systematic review and meta-analysis.

BACKGROUND: This review presents an overview of field findings on sequence variation of Plasmodium falciparum histidine-rich proteins 2/3 (PfHRP2/3) for which reference types (1-24) have been identified, and its critical impact on PfHRP2-based rapid diagnostic test (RDT) detection. RESEARCH DESIGN AND METHODS: This systematic review and meta-analysis was registered with PROSPERO, CRD42022316027, and conducted as per the PRISMA guidelines, and the methodological quality of studies was assessed. RESULTS: Of the 2184 records identified, 34 studies were included mostly from Africa (47.1%) and Asia (35.3%). The reference PfHRP2 types 1, 2, 3, 6, and 7 are invariably found at proportions ≥ 80-100% in all areas with the exception of The Americas where their proportion is very low. The proteins exhibited high diversity of variants/unknown types, especially for types 1, 2, 4, and 7. Eleven major PfHRP2 epitopes were found at pooled proportion > 90%. The existing models to predict RDT detection are greatly limited by the impact of factors such as low (very low) parasitemia, RDT brand, and PfHRP3 cross-reactivity. PfHRP2 length and presence/number of a given reference repeat type/variant did not seem to impact RDT detection. CONCLUSIONS: PfHRP2/3 are highly polymorphic and current findings are insufficient, conflicting and not convincing enough to conclude on the role of PfHRP2/3 sequence polymorphism in PfHRP2-based RDT detection.

Nationwide spatiotemporal drug resistance genetic profiling from over three decades in Indian Plasmodium falciparum and Plasmodium vivax isolates.

BACKGROUND: Drug resistance is a serious impediment to efficient control and elimination of malaria in endemic areas. METHODS: This study aimed at analysing the genetic profile of molecular drug resistance in Plasmodium falciparum and Plasmodium vivax parasites from India over a ~ 30-year period (1993-2019). Blood samples of P. falciparum and/or P. vivax-infected patients were collected from 14 regions across India. Plasmodial genome was extracted and used for PCR amplification and sequencing of drug resistance genes in P. falciparum (crt, dhps, dhfr, mdr1, k13) and P. vivax (crt-o, dhps, dhfr, mdr1, k12) field isolates. RESULTS: The double mutant pfcrt SVMNT was highly predominant across the country over three decades, with restricted presence of triple mutant CVIET from Maharashtra in 2012. High rates of pfdhfr-pfdhps quadruple mutants were observed with marginal presence of "fully resistant" quintuple mutant ACIRNI-ISGEAA. Also, resistant pfdhfr and pfdhps haplotype has significantly increased in Delhi between 1994 and 2010. For pfmdr1, only 86Y and 184F mutations were present while no pfk13 mutations associated with artemisinin resistance were observed. Regarding P. vivax isolates, the pvcrt-o K10 "AAG" insertion was absent in all samples collected from Delhi in 2017. Pvdhps double mutant SGNAV was found only in Goa samples of year 2008 for the first time. The pvmdr1 908L, 958M and 1076L mutations were highly prevalent in Delhi and Haryana between 2015 and 2019 at complete fixation. One nonsynonymous novel pvk12 polymorphism was identified (K264R) in Goa. CONCLUSIONS: These findings support continuous surveillance and characterization of P. falciparum and P. vivax populations as proxy for effectiveness of anti-malarial drugs in India, especially for independent emergence of artemisinin drug resistance as recently seen in Africa.

The spectrum of clinical biomarkers in severe malaria and new avenues for exploration.

Globally, malaria is a public health concern, with severe malaria (SM) contributing a major share of the disease burden in malaria endemic countries. In this context, identification and validation of SM biomarkers are essential in clinical practice. Some biomarkers (C-reactive protein, angiopoietin 2, angiopoietin-2/1 ratio, platelet count, histidine-rich protein 2) have yielded interesting results in the prognosis of Plasmodium falciparum severe malaria, but for severe P. vivax and P. knowlesi malaria, similar evidence is missing. The validation of these biomarkers is hindered by several factors such as low sample size, paucity of evidence-evaluating studies, suboptimal values of sensitivity/specificity, poor clinical practicality of measurement methods, mixed Plasmodium infections, and good clinical value of the biomarkers for concurrent infections (pneumonia and current COVID-19 pandemic). Most of these biomarkers are non-specific to pathogens as they are related to host response and hence should be regarded as prognostic/predictive biomarkers that complement but do not replace pathogen biomarkers for clinical evaluation of SM patients. This review highlights the importance of research on diagnostic/predictive/therapeutic biomarkers, neglected malaria species, and clinical practicality of measurement methods in future studies. Finally, the importance of omics technologies for faster identification/validation of SM biomarkers is also included.