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Objective: To elucidate the genetic diversity of Plasmodium falciparum in residual transmission foci of northern India., Methods: Clinically suspected patients with malaria were screened for malaria infection by microscopy. 48 P. falciparum-infected patients were enrolled from tertiary care hospital in Chandigarh, India. Blood samples were collected from enrolled patients, genomic DNA extraction and nested PCR was performed for further species confirmation. Sanger sequencing was carried out using block 2 region of msp1, R2 region of glurp and pfs25-specific primers., Results: Extensive diversity was found in msp1 alleles with predominantly RO33 alleles. Overall allelic prevalence was 55.8% for RO33, 39.5% for MAD20 and 4.7% for K1. Six variants were observed in MAD20, whereas no variant was found in RO33 and K1 alleles. A phylogenetic analysis of RO33 alleles indicated more similarity to South African isolates, whereas MAD20 alleles showed similarity with South-East Asian isolates. In glurp, extensive variation was observed with eleven different alleles based on the AAU repeats. However, pfs25 showed less diversity and was the most stable among the targeted genes., Conclusion: Our findings document the genetic diversity among circulating strains of P. falciparum in an area of India with low malaria transmission and could have implications for control strategies to reach the national goal of malaria elimination., (© 2017 John Wiley & Sons Ltd.)

Objective: We report for the first-time detection of Plasmodium ovale in central India., Methods: From 2010, all hospitalised suspected malaria cases at the malaria clinic of the Regional Medical Research Centre for Tribals in Bastar district, central India, were screened. Plasmodium species were identified by microscopy and species-specific nested PCR of 18s rRNA., Results: Of 256 enrolled cases of confirmed P. falciparum malaria by microscopy, P. ovale infection was detected in three cases (1.2%) by PCR and sequencing. Of these three cases, one had cerebral malaria and another had severe malaria anaemia. In both of these cases, P. ovale infection was mixed with P. falciparum, while in third case the infection was mixed with both P. falciparum and P. vivax. Phylogenetic analysis revealed that these isolates showed closed homology with West African genotypes., Conclusion: All three hospitalised patients were originally residents of remote inaccessible forest villages and never moved out of their residence. This finding also has implications in malaria control and elimination as P. ovale causes relapses. This study highlights the need of molecular diagnosis of malaria species for appropriate treatment and control., (© 2013 John Wiley & Sons Ltd.)

Objective: During an epidemiological study (January-July 2012) on malaria in forest villages of Central India, Plasmodium malariae-like malaria parasites were observed in blood smears of fever cases. We aimed to confirm the presence of P. malariae using molecular tools i.e. species-specific nested polymerase chain reaction (PCR) and DNA sequencing., Methods: All fever cases or cases with history of fever in 25 villages of Balaghat district were screened for malaria parasite using bivalent rapid diagnostic test and microscopy after obtaining written informed consent. Nested PCR was employed on microscopically suspected P. malariae cases. DNA sequences in the target region for PCR diagnosis were analysed for all the suspected cases of P. malariae., Results: Among the 22 microscopy suspected P. malariae cases, nested PCR confirmed the identity of P. malariae in 19 cases. Among these 14 were mono P. malariae infections, three were mixed infection of P. malariae with Plasmodium falciparum and two were mixed infection of P. malariae with Plasmodium vivax. Clinically P. malariae subjects generally presented with fever and headache. However, the typical 3-day pattern of quantum malaria was not observed. The parasite density of P. malariae was significantly lower than that of P. vivax and P. falciparum., Discussions: Plasmodium malariae may have been in existence in forest villages of central India but escaped identification due to its close resemblance to P. vivax. The results re-affirm the importance of molecular methods of testing on routine basis for efficacious control strategies against malaria., (© 2012 Blackwell Publishing Ltd.)

Objectives: To assess the therapeutic efficacy of chloroquine (CQ) treatment against uncomplicated Plasmodium falciparum infections in a tribal population of central India (Madhya Pradesh) and to investigate the prevalence of mutant P. falciparum chloroquine-resistant transporter (pfcrt) gene in the parasite population., Methods: Clinical and parasitological response was determined by in-vivo testing. For molecular testing, the parasite DNA was extracted from blood samples and used to amplify and sequence parts of the pfcrt (44-177 codons), MSP1 (block 2) and MSP2 (central repeat region) genes., Results: Of 463 patients presenting fever, 137 tested positive for P. falciparum. They were treated with CQ. Of these, 58% participated in the study. Overall, treatment failure occurred in 53% of participants. Children under 5 years of age showed significantly more CQ resistance than adults. Mutant genotype S(72)V(73)M(74)N(75)T(76) was prevalent among both CQ responders (61.29%) and non-responders (66.7%). Interestingly, several patients from the CQ non-responder group (33.3%, n = 39) were harbouring parasite with wild type C(72)V(73)M(74)N(75)K(76) genotype of the pfcrt gene. Microsatellite sequences downstream of exon 2 varied widely among both wild type and mutant pfcrt haplotypes., Conclusion: The high rate of treatment failure in the present study clearly indicates the need to reassess the use of CQ as first-line antimalarial therapy in central India. This is supported by the presence of mutant pfcrt genotype among majority of the parasite population of the CQ non-responder group of patients. However, the presence of wild type amino acid at codon 76 of the pfcrt gene among several patients with CQ non-responders requires further investigations.