Temporal changes in genetic diversity of msp-1, msp-2, and msp-3 in Plasmodium falciparum isolates from Grande Comore Island after introduction of ACT.

BACKGROUND: Malaria is still one of the serious public health problems in Grande Comore Island, although the number of annual cases has been greatly reduced in recent years. A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate temporal changes in genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) in Grande Comore 10 years after introduction of ACT. METHODS: A total of 232 P. falciparum clinical isolates were collected from the Grande Comore Island during two sampling periods (118 for 2006‒2007 group, and 114 for 2013‒2016 group). Parasite isolates were characterized for genetic diversity and complexity of infection by genotyping polymorphic regions in merozoite surface protein gene 1 (msp-1), msp-2, and msp-3 using nested PCR and DNA sequencing. RESULTS: Three msp-1 alleles (K1, MAD20, and RO33), two msp-2 alleles (FC27 and 3D7), and two msp-3 alleles (K1 and 3D7) were detected in parasites of both sampling periods. The RO33 allele of msp-1 (84.8%), 3D7 allele of msp-2 (90.8%), and K1 allele of msp-3 (66.7%) were the predominant allelic types in isolates from 2006-2007 group. In contrast, the RO33 allele of msp-1 (63.4%), FC27 allele of msp-2 (91.1%), and 3D7 allele of msp-3 (53.5%) were the most prevalent among isolates from the 2013-2016 group. Compared with the 2006‒2007 group, polyclonal infection rates of msp-1 (from 76.7 to 29.1%, P < 0.01) and msp-2 (from 62.4 to 28.3%, P < 0.01) allelic types were significantly decreased in those from 2013‒2016 group. Similarly, the MOIs for both msp-1 and msp-2 were higher in P. falciparum isolates in the 2006-2007 group than those in 2013-2016 group (MOI = 3.11 vs 1.63 for msp-1; MOI = 2.75 vs 1.35 for msp-2). DNA sequencing analyses also revealed reduced numbers of distinct sequence variants in the three genes from 2006‒2007 to 2013‒2016: msp-1, from 32 to 23 (about 28% decline); msp-2 from 29 to 21 (about 28% decline), and msp-3 from 11 to 3 (about 72% decline). CONCLUSIONS: The present data showed dramatic reduction in genetic diversity and MOI among Grande Comore P. falciparum populations over the course of the study, suggesting a trend of decreasing malaria transmission intensity and genetic diversity in Grande Comore Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.

Prevalence of crt and mdr-1 mutations in Plasmodium falciparum isolates from Grande Comore island after withdrawal of chloroquine.

BACKGROUND: In Comoros, the widespread of chloroquine (CQ)-resistant Plasmodium falciparum populations was a major obstacle to malaria control, which led to the official withdrawal of CQ in 2004. Continuous monitoring of CQ-resistant markers of the P. falciparum CQ resistant transporter (pfcrt) and the P. falciparum multiple drug resistance 1 (pfmdr-1) is necessary inder to obtain first-hand information on CQ susceptibility of parasite populations in the field. The objective of this study is to assess the prevalence and evolution of CQ-resistance in the P. falciparum populations on the Comoros' Grande Comore island after withdrawal of CQ. METHODS: A total of 207 P. falciparum clinical isolates were collected from the island, including 118 samples from 2006 to 2007 and 89 samples from 2013 to 2014. Nucleotide substitutions in the pfcrt and pfmdr-1 genes linked to CQ response in parasite isolates were assessed using nested PCR and DNA sequencing. RESULTS: From the pfcrt gene segment sequenced, we detected C72S, M74I, N75E, and K76T substitutions in the parasite isolates collected from both 2006-2007 to 2013-2014 periods. Significant decline of pfcrt resistant alleles at C72S (42.6 to 6.9 %), M74I (39.1 to 14.9 %), N75E (63.5 to 18.3 %), and K76T (72.2 to 19.5 %) from 2006-2007 to 2013-2014 were observed, and the frequency of pfcrt wild type allele was significantly increased from 19.1 % in 2006-2007 to 75.8 % in 2013-2014. Sequence analysis of pfmdr-1 also detected point mutations at codons N86Y, Y184F, and D1246Y, but not S1034C and N1042D, in the isolates collected from both examined periods. An increasing trend in the prevalence of the pfmdr-1 wild type allele (NYD, 4.3 % in 2006-2007; and 28.7 % in 2013-2014), and a decreasing trend for pfmdr-1 N86Y mutation (87.0 % in 2006-2007; and 40.2 % in 2013-2014) were observed in our samples. CONCLUSIONS: The present data indicate that the prevalence and patterns of mutant pfcrt and pfmdr-1 dramatically decreased in the Grande Comore isolates from 2006 to 2014, suggesting that the CQ-sensitive P. falciparum strains have returned after the withdrawal of CQ. The data also suggests that the parasites with wild type pfcrt/pfdmr-1 genes may have growth and/or transmission advantages over the mutant parasites. The information obtained from this study will be useful for developing and updating anti-malarial treatment policy in Grande Comore island.

Polymorphisms of the artemisinin resistant marker (K13) in Plasmodium falciparum parasite populations of Grande Comore Island 10 years after artemisinin combination therapy.

BACKGROUND: Plasmodium falciparum malaria is a significant public health problem in Comoros, and artemisinin combination therapy (ACT) remains the first choice for treating acute uncomplicated P. falciparum. The emergence and spread of artemisinin-resistant P. falciparum in Southeast Asia, associated with mutations in K13-propeller gene, poses a potential threat to ACT efficacy. Detection of mutations in the P. falciparum K13-propeller gene may provide the first-hand information on changes in parasite susceptibility to artemisinin. The objective of this study is to determinate the prevalence of mutant K13-propeller gene among the P. falciparum isolates collected from Grande Comore Island, Union of Comoros, where ACT has been in use since 2004. METHODS: A total of 207 P. falciparum clinical isolates were collected from the island during March 2006 and October 2007 (n = 118) and March 2013 and December 2014 (n = 89). All isolates were analysed for single nucleotide polymorphisms (SNPs) and haplotypes in the K13-propeller gene using nested PCR and DNA sequencing. RESULTS: Only three 2006-2007 samples carried SNPs in the K13-propeller gene, one having a synonymous (G538G) and the other having two non-synonymous (S477Y and D584E) substitutions leading to two mutated haplotypes (2.2%, 2/95). Three synonymous mutations (R471R, Y500Y, and G538G) (5.9%, 5/85) and 7 non-synonymous substitutions (21.2%, 18/85) with nine mutated haplotypes (18.8%, 16/85) were found in isolates from 2013 to 2014. However, none of the polymorphisms associated with artemisinin-resistance in Southeast Asia was detected from any of the parasites examined. CONCLUSION: This study showed increased K13-propeller gene diversity among P. falciparum populations on the Island over the course of 8 years (2006-2014). Nevertheless, none of the polymorphisms known to be associated with artemisinin resistance in Asia was detected in the parasite populations examined. Our data suggest that P. falciparum populations in Grande Comore are still effectively susceptible to artemisinin. Our results provide insights into P. falciparum populations regarding mutations in the gene associated with artemisinin resistance and will be useful for developing and updating anti-malarial guidance in Comoros.