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Characterization of drug resistance and genetic diversity of Plasmodium falciparum parasites from Tripura, Northeast India.

ABSTRACT: Monitoring of anti-malarial drug resistance is vital in Northeast India as this region shares its international border with Southeast Asia. Genetic diversity of Plasmodium parasites regulates transmission dynamics, disease severity and vaccine efficacy. P. falciparum chloroquine resistance transporter (Pfcrt), multidrug resistance-1 (Pfmdr-1) and kelch 13 propeller (PfK-13) genes which govern antimalarial drug resistance and three genetic diversity markers, merozoite surface protein 1 and 2 (Pfmsp-1, Pfmsp-2) and glutamate rich protein (Pfglurp) were evaluated from Tripura, Northeast India using molecular tools. In the Pfcrt gene, 87% isolates showed triple mutations at codons M74I, N75E and K76T. 12.5% isolates in Pfmdr-1 gene showed mutation at N86Y. No polymorphism in PfK-13 propeller was found. Polyclonal infections were observed in 53.85% isolates and more commonly in adults (p?=?0.0494). In the Pfmsp-1 locus, the K1 allelic family was predominant (71.2%) followed by the 3D7/IC family (69.2%) in the Pfmsp-2 locus. RII region of Pfglurp exhibited nine alleles with expected heterozygosity of 0.85. The multiplicity of infection for Pfmsp-1, Pfmsp-2 and Pfglurp were 1.56, 1.31 and 1.06 respectively. Overall, the study demonstrated a high level of chloroquine resistance and extensive parasite diversity in the region, necessitating regular surveillance in this population group.

SUBMITTER: Patgiri SJ

PROVIDER: S-EPMC6757058 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Characterization of drug resistance and genetic diversity of Plasmodium falciparum parasites from Tripura, Northeast India.

Patgiri S J SJ Sarma K K Sarmah N N Bhattacharyya N N Sarma D K DK Nirmolia T T Bhattacharyya D R DR Mohapatra P K PK Bansal D D Bharti P K PK Sehgal R R Mahanta J J Sultan A A AA

Scientific reports 20190923 1

Monitoring of anti-malarial drug resistance is vital in Northeast India as this region shares its international border with Southeast Asia. Genetic diversity of Plasmodium parasites regulates transmission dynamics, disease severity and vaccine efficacy. P. falciparum chloroquine resistance transporter (Pfcrt), multidrug resistance-1 (Pfmdr-1) and kelch 13 propeller (PfK-13) genes which govern antimalarial drug resistance and three genetic diversity markers, merozoite surface protein 1 and 2 (Pfm ...[more]

PMID: 31548652

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Project description:BACKGROUND: Since 2011, artesunate?+?sulphadoxine-pyrimethamine (ASP), instead of chloroquine, has been recommended for treatment of uncomplicated malaria in India. In Ujjain, central India, with an annual parasite index <0.1, the prevalence of drug-resistant Plasmodium falciparum is unknown. In other parts of India chloroquine and sulphadoxine-pyrimethamine-resistant P. falciparum is prevalent. The aim of this study was to determine the prevalence of anti-malarial drug resistance-associated genetic polymorphisms in P. falciparum collected in Ujjain in 2009 and 2010, prior to the introduction of ASP. METHODS: Blood samples from 87 patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons Pfcrt 72-76, pfmdr1 1034-1246, pfdhfr 16-185, pfdhps 436-632 and pfnhe1 ms4760 haplotypes were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism, and pfmdr1 gene copy number by real-time PCR. RESULTS: Sulphadoxine-pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 75/78 (96%) and 70/78 (90%) samples, respectively, and pfdhps 437G was found in 7/77 (9%) samples. Double mutant pfdhfr 59R?+?108 N were found in 62/76 (82%) samples. Triple mutant pfdhfr 59R?+?108 N and pfdhps 437G were found in 6/76 (8%) samples. Chloroquine-resistance-associated pfcrt 76 T was found in 82/87 (94%). The pfcrt 72-76 haplotypes found were: 80/84 (95%) SVMNT, 3/84 (4%) CVMNK and 1/84 (1%) CVMNT. Pfmdr1 N86 and 86Y were identified in 70/83 (84%) and 13/83 (16%) samples, respectively. Pfmdr1 S1034?+?N1042?+?D1246 were identified together in 70/72 (97%) of successfully sequenced samples. One pfmdr1 gene copy was found in 74/75 (99%) successfully amplified samples. CONCLUSION: This is the first characterization of key anti-malarial drug resistance-associated genetic markers among P. falciparum collected in Ujjain, Madhya Pradesh, India. The results indicate that the efficacy of standard dose chloroquine at the time of the study was likely to be poor, whereas ASP was likely to be efficacious, supporting the changed drug treatment policy. However, P. falciparum with reduced susceptibility to sulphadoxine-pyrimethamine is highly prevalent, highlighting the need for continuous surveillance of ASP efficacy in the study area.

Project description:BackgroundCape Verde is an archipelago located off the West African coast and is in a pre-elimination phase of malaria control. Since 2010, fewer than 20 Plasmodium falciparum malaria cases have been reported annually, except in 2017, when an outbreak in Praia before the rainy season led to 423 autochthonous cases. It is important to understand the genetic diversity of circulating P. falciparum to inform on drug resistance, potential transmission networks and sources of infection, including parasite importation.MethodsEnrolled subjects involved malaria patients admitted to Dr Agostinho Neto Hospital at Praia city, Santiago island, Cape Verde, between July and October 2017. Neighbours and family members of enrolled cases were assessed for the presence of anti-P. falciparum antibodies. Sanger sequencing and real-time PCR was used to identify SNPs in genes associated with drug resistance (e.g., pfdhfr, pfdhps, pfmdr1, pfk13, pfcrt), and whole genome sequencing data were generated to investigate the population structure of P. falciparum parasites.ResultsThe study analysed 190 parasite samples, 187 indigenous and 3 from imported infections. Malaria cases were distributed throughout Praia city. There were no cases of severe malaria and all patients had an adequate clinical and parasitological response after treatment. Anti-P. falciparum antibodies were not detected in the 137 neighbours and family members tested. No mutations were detected in pfdhps. The triple mutation S108N/N51I/C59R in pfdhfr and the chloroquine-resistant CVIET haplotype in the pfcrt gene were detected in almost all samples. Variations in pfk13 were identified in only one sample (R645T, E668K). The haplotype NFD for pfmdr1 was detected in the majority of samples (89.7%).ConclusionsPolymorphisms in pfk13 associated with artemisinin-based combination therapy (ACT) tolerance in Southeast Asia were not detected, but the majority of the tested samples carried the pfmdr1 haplotype NFD and anti-malarial-associated mutations in the the pfcrt and pfdhfr genes. The first whole genome sequencing (WGS) was performed for Cape Verdean parasites that showed that the samples cluster together, have a very high level of similarity and are close to other parasites populations from West Africa.

Project description:BackgroundDespite evident success of malaria control in many sites in the Arabian Peninsula, malaria remains endemic in a few spots, in Yemen and south-west of Saudi Arabia. In addition to local transmission, imported malaria sustains an extra source of parasites that can challenge the strengths of local control strategies. This study examined the genetic diversity of Plasmodium falciparum in Yemen and mutations of drug resistant genes, to elucidate parasite structure and distribution of drug resistance genotypes in the region.MethodsFive polymorphic loci (MSP-2, Pfg377 and three microsatellites on chromosome 8) not involved in anti-malarial drug resistance, and four drug resistant genes (pfcrt, pfmdr1, dhfr and dhps) were genotyped in 108 P. falciparum isolates collected in three sites in Yemen: Dhamar, Hodeidah and Taiz.ResultsHigh diversity was seen in non-drug genes, pfg377 (He?=?0.66), msp-2 (He?=?0.80) and three microsatellites on chr 8, 7.7 kb (He?=?0.88), 4.3 kb (He?=?0.77) and 0.8 kb (He?=?0.71). There was a high level of mixed-genotype infections (57%), with an average 1.8 genotypes per patient. No linkage disequilibrium was seen between drug resistant genes and the non-drug markers (p?<?0.05). Genetic differentiation between populations was low (most pair-wise FST values <0.03), indicating extensive gene flow between the parasites in the three sites.ConclusionThe high diversity of P. falciparum in Yemen is indicative of a large parasite reservoir, which represents a challenge to control efforts. The presence of two distinct pfcrt genotype, CVIET and SVMNT, suggests that chloroquine resistance can possibly be related to a migratory path from Africa and Asia. The absence of the triple mutant dhfr genotype (IRN) and dhps mutations supports the use of artesunate?+?sulphadoxine-pyrimethamine as first-line therapy. However, the prevalent pfmdr1 genotype NFSND [21%] has previously been associated with tolerance/resistance response to artemisinin combination therapy (ACT). Regular surveys are, therefore, important to monitor spread of pfmdr1 and dhfr mutations and response to ACT.

Project description:BackgroundDespite significant progress in eliminating malaria from the state of Odisha, India, the disease is still considered endemic. Artesunate plus sulfadoxine-pyrimethamine (AS + SP) has been introduced since 2010 as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study aimed to investigate the prevalence of mutations associated with resistance to chloroquine (CQ), sulfadoxine-pyrimethamine (SP), and artesunate (ART) in P. falciparum parasites circulating in the state.MethodsA total of 239 isolates of P. falciparum mono infection were collected during July 2018-November 2020 from the four different geographical regions of the state. Genomic DNA was extracted from 200 µL of venous blood and amplified using nested polymerase chain reaction. Mutations on gene associated with CQ (Pfcrt and Pfmdr1) were assessed by PCR amplification and restriction fragment length polymorphism, artemisinin (Pfk13) gene by DNA sequencing and SP (Pfdhfr and Pfdhps) genes by allele-specific polymerase chain reaction (AsPCR).ResultsThe point mutation in Pfcrt (K76T) was detected 2.1%, in Pfmdr1 (N86Y) 3.4%, and no mutations were found in Pfkelch13 propeller domain. Prevalence of Pfdhfr, Pfdhps and Pfhdfr-Pfdhps (two locus) gene mutations were 50.43%, 47.05% and 49.79% respectively. The single, double, triple and quadruple point mutations in Pfdhfr gene was 11.2%, 8.2%, 17.2% and 3.4% while, in Pfdhps gene was 10.9%,19.5%, 9.5% and 2.7% respectively. Of the total 13 haplotypes found in Pfdhfr, 8 were detected for the first time in the state and of the total 26 haplotypes found in Pfdhps, 7 were detected for the fisrt time in the state. The linked quintuple mutation Pfdhfr (N51I-C59R-S108N)-Pfdhps (A437G-K540E) responsible for clinical failure (RIII level of resistance) of SP resistance and A16V-S108T mutation in Pfdhfr responsible for cycloguanil was absent.ConclusionThe study has demonstrated a low prevalence of CQ resistance alleles in the study area. Despite the absence of the Pfkelch13 mutations, high prevalence of Pfdhfr and Pfdhps point mutations undermine the efficacy of SP partner drug, thereby threatening the P. falciparum malaria treatment policy. Therefore, continuous molecular and in vivo monitoring of ACT efficacy is warranted in Odisha.

Dataset Information

Characterization of drug resistance and genetic diversity of Plasmodium falciparum parasites from Tripura, Northeast India.

Publications

Characterization of drug resistance and genetic diversity of Plasmodium falciparum parasites from Tripura, Northeast India.

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