Novel mutations in the antifolate drug resistance marker genes among Plasmodium vivax isolates exhibiting severe manifestations.

Plasmodium vivax is the predominant species of the human malaria parasite present in the Indian subcontinent. There have been recent reports on Chloroquine (CQ) resistance and severe manifestations shown by P. vivax from different regions of the world including India. This study focuses on Bikaner, India where during the last few years there have been continuous reports of severe manifestations by both Plasmodium falciparum and P. vivax. This region has a widespread use of Chloroquine and Sulfadoxine-Pyrimethamine for the treatment of malaria, but the resistance profiles of these drugs are not available. We report here the profile of mutations in marker genes associated with Chloroquine and antifolate drug resistance among the P. vivax parasites obtained from patients with severe (n=30) and non-severe (n=48) manifestations from this region. Most isolates showed the wild type alleles for both the Chloroquine and antifolate resistance markers (P<0.0005). Except for one isolate showing Y976F mutation in the Pvmdr-1 gene, no reported mutation was observed in the Pvmdr-1 or Pvcrt gene. This is in accordance with the fact that till date no Chloroquine resistance has been reported from this region. However, the single isolate with a mutation in Pvmdr-1 may suggest the beginning of the trend towards decreased susceptibility to Chloroquine. The frequency of PvDHFR-PvDHPS two locus mutations was higher among the patients showing severe manifestations than the patient group with non-severe (uncomplicated) malaria (P<0.003). None of the parasites from patients with uncomplicated P. vivax malaria showed the mutant PvDHPS genotype. Novel mutations in PvDHFR (S117H) and PvDHPS (F365L, D459A and M601I) were observed only in the parasite population obtained from patients exhibiting severe complications. Preliminary homology modeling and molecular docking studies predicted that these mutations apparently do not have any effect on the binding of the drug molecule to the enzyme. However, the presence of novel mutations in the PvDHPS gene indicate a degree of polymorphism of this molecule which is in contrast to available published information.

Genetic variation in the Plasmodium falciparum circumsporozoite protein in India and its relevance to RTS,S malaria vaccine.

RTS,S is the most advanced malaria vaccine candidate, currently under phase-III clinical trials in Africa. This Plasmodium falciparum vaccine contains part of the central repeat region and the complete C-terminal T cell epitope region (Th2R and Th3R) of the circumsporozoite protein (CSP). Since naturally occurring polymorphisms at the vaccine candidate loci are critical determinants of the protective efficacy of the vaccines, it is imperative to investigate these polymorphisms in field isolates. In this study we have investigated the genetic diversity at the central repeat, C-terminal T cell epitope (Th2R and Th3R) and N-terminal T cell epitope regions of the CSP, in P. falciparum isolates from Madhya Pradesh state of India. These isolates were collected through a 5-year prospective study aimed to develop a well-characterized field-site for the future evaluation of malaria vaccine in India. Our results revealed that the central repeat (63 haplotypes, n = 161) and C-terminal Th2R/Th3R epitope (24 haplotypes, n = 179) regions were highly polymorphic, whereas N-terminal non-repeat region was less polymorphic (5 haplotypes, n = 161) in this population. We did not find any evidence of the role of positive natural selection in maintaining the genetic diversity at the Th2R/Th3R regions of CSP. Comparative analysis of the Th2R/Th3R sequences from this study to the global isolates (n = 1160) retrieved from the GenBank database revealed two important points. First, the majority of the sequences (~61%, n = 179) from this study were identical to the Dd2/Indochina type, which is also the predominant Th2R/Th3R haplotype in Asia (~59%, n = 974). Second, the Th2R/Th3R sequences in Asia, South America and Africa are geographically distinct with little allele sharing between continents. In conclusion, this study provides an insight on the existing polymorphisms in the CSP in a parasite population from India that could potentially influence the efficacy of RTS,S vaccine in this region.

Differential genetic hitchhiking around mutant pfcrt alleles in the Indian Plasmodium falciparum population.

OBJECTIVES: To study the origin and spread of the chloroquine-resistant Plasmodium falciparum population in the Indian subcontinent. METHODS: Fourteen microsatellites spanning a ∼120 kb region, flanking the P. falciparum chloroquine resistance transporter (pfcrt) gene, were analysed in 185 parasite isolates. RESULTS: The Indian P. falciparum population exhibited a selective valley of reduced genetic variation in the flanking microsatellites of the mutant pfcrt alleles (up to ±29 kb) as compared with the wild-type allele. This valley is much narrower than the ±200 kb valley reported from African and South-East Asian countries. The majority of the isolates showed asymmetry in the selective valley, where upstream microsatellites showed less genetic variation than the downstream microsatellites. Regional variation in the width and symmetry of the selective valley was noticed, which seems to be related to the number of pfcrt alleles present in the parasite population of a region. Forty-six different microsatellite haplotypes were observed among the P. falciparum isolates containing mutant pfcrt alleles. Parasite populations from different regions of mainland India shared microsatellite haplotypes between them, but they shared none with the isolates from the Andaman and Nicobar Islands, and vice versa. Indian isolates shared microsatellite haplotypes with the isolates from Papua New Guinea and Thailand. CONCLUSIONS: With regard to chloroquine there is regional variation in the selection pressure on the P. falciparum population in India. These findings will help the regional implementation of drug policy in India's malaria control programme.

Multiple origins of Plasmodium falciparum dihydropteroate synthetase mutant alleles associated with sulfadoxine resistance in India.

With the spread of chloroquine (CQ)-resistant malaria in India, sulfadoxine-pyrimethamine (SP) alone or in combination with artesunate is used as an alternative antimalarial drug. Due to continuous drug pressure, the Plasmodium falciparum parasite is exhibiting resistance to antifolates because of mutations in candidate genes dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps). Our earlier study on flanking microsatellite markers of dhfr mutant alleles from India had shown a single origin of the pyrimethamine resistance and some minor haplotypes which shared haplotypes with Southeast Asian (Thailand) strains. In the present study, we have analyzed 193 of these Indian P. falciparum isolates for 15 microsatellite loci around dhps to investigate the genetic lineages of the mutant dhps alleles in different parts of the country. Eighty-one of these samples had mutant dhps alleles, of which 62 were from Andaman and Nicobar Islands and the remaining 19 were from mainland India. Of 112 isolates with a wild-type dhps allele, 109 were from mainland India and only 3 were from Andaman and Nicobar Islands. Consistent with the model of selection, the mean expected heterozygosity (H(e)) around mutant dhps alleles (H(e) = 0.55; n = 81) associated with sulfadoxine resistance was lower (P ≤ 0.05) than the mean H(e) around the wild-type dhps allele (H(e) = 0.80; n = 112). There was more genetic diversity in flanking microsatellites of dhps than dhfr among these isolates, which confirms the assertion that dhps mutations are at a very early stage of fixation in the parasite population. Microsatellite haplotypes around various mutant dhps alleles suggest that the resistant dhps alleles have multiple independent origins in India, especially in Andaman and Nicobar Islands. Determining the genetic lineages of the resistant dhps alleles on Andaman and Nicobar Islands and mainland India is significant, given the role of Asia in the intercontinental spread of chloroquine- and pyrimethamine-resistant parasites in the past.

Novel K540N mutation in Plasmodium falciparum dihydropteroate synthetase confers a lower level of sulfa drug resistance than does a K540E mutation.

Sulfadoxine (SDX) and sulfamethoxazole (SMX) each inhibit the Plasmodium falciparum dihydropteroate synthetase (PfDHPS), and certain point mutations in this enzyme yield the drug-resistant parasite. Using a simple Escherichia coli model system, we describe here the effect of the recently reported novel K540N mutation in PfDHPS on the level of SDX/SMX resistance. The survival rate of the transformed E. coli (DHPS-deficient strain) under different SDX or SMX concentrations revealed that the K540N mutation confers a lower level of drug resistance than its contemporary K540E mutation. Further, SMX was more effective than SDX in the E. coli system.

High chloroquine treatment failure rates and predominance of mutant genotypes associated with chloroquine and antifolate resistance among falciparum malaria patients from the island of Car Nicobar, India.

OBJECTIVES: An in vivo chloroquine efficacy study was undertaken on the island of Car Nicobar because a temporal rise in the Plasmodium falciparum parasite population containing mutations in the chloroquine resistance transporter (PfCRT) protein has been reported there. METHODS: A WHO protocol with a 28 day follow-up schedule was used for chloroquine efficacy studies. Finger-prick blood from P. falciparum malaria patients was used for sequencing the genes encoding PfCRT (exon 2), dihydrofolate reductase (PfDHFR) and dihydropteroate synthetase (PfDHPS). RESULTS: The majority of patients showed chloroquine treatment failure (60.42%, n=48). A higher early treatment failure (ETF) rate was recorded among non-responders (23 of 29, 79.31%). Each patient, irrespective of their chloroquine response, was infected with P. falciparum that contained mutated PfCRT (predominantly genotype C72V73I74E75T76) associated with high chloroquine resistance and none with the wild-type pfcrt gene. Therefore, mutated PfCRT was also present in the P. falciparum isolates of all the chloroquine responders. The majority of individuals from both groups also contained parasites with a high number of two-locus PfDHFR-PfDHPS mutations, associated with a high level of antifolate resistance. CONCLUSIONS: There is a predominance of chloroquine- and antifolate-resistant P. falciparum malaria in Car Nicobar, requiring an alternative antimalarial drug treatment policy, such as implementation of artesunate combination therapy (ACT), for this island.

Prevalence of clinical remission in patients with sporadic idiopathic hypoparathyroidism.

BACKGROUND: Remission of disease activity is a characteristic feature of autoimmune endocrine disorders such as Graves' disease, Addison's disease and occasionally in patients with premature ovarian failure. Autoimmunity is also implicated in sporadic idiopathic hypoparathyroidism (SIH) with clinical remission of disease reported in three cases. OBJECTIVE: To assess the rate of remission in patients with sporadic idiopathic hypoparathyroidism and review the cases reported so far. SUBJECTS AND METHODS: Subjects included 53 patients (M:F, 24:29) with SIH who had been symptomatic for at least 1 year (range 1-31 years). They were treated with calcium and 1-alpha-(OH)D(3)/cholecalciferol therapy and had a mean duration of follow up of 5.0 +/- 3.2 years. Treatment was withdrawn in two stages in the patients who maintained normal levels of serum total calcium during the preceding year of treatment. In stage-1, the dose of therapy was reduced to half and subsequently all treatment was stopped (stage 2) in those patients who maintained normal serum total calcium levels on the reduced dose. Remission of SIH was defined as maintenance of normal serum total (>or=2.12 mmol/l) and ionized calcium, inorganic phosphorus and serum intact parathyroid hormone (iPTH) for at least 3 months after withdrawal of calcium and 1-alpha-(OH)D(3)/cholecalciferol therapy. Calcium sensing receptor autoantibodies (CaSRAb) were determined by Western blot. RESULTS: Two of the 53 patients (3.8%) with SIH stayed in remission for 1 year after complete withdrawal of therapy. CaSRAb was absent in both the cases. The clinical features, age at onset and duration of hypocalcaemic symptoms in cases with remission were comparable to those who did no show remission. CONCLUSION: Sporadic idiopathic hypoparathyroidism is not irreversible as is widely believed and spontaneous remission of disease may occur in 3.8% of patients.

Characteristics of genetic hitchhiking around dihydrofolate reductase gene associated with pyrimethamine resistance in Plasmodium falciparum isolates from India.

Sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum has been widespread across continents, causing the major hurdle of controlling malaria. Resistance is encoded mainly by point mutations in P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) target genes. To study the origin and evolution of pyrimethamine resistance on the Indian subcontinent, microsatellite markers flanking the pfdhfr gene were mapped. Here we describe the characteristics of genetic hitchhiking around the pfdhfr gene among 190 P. falciparum isolates. These isolates were collected from five different geographical regions of India (Uttar Pradesh, Madhya Pradesh, Assam, Orissa, and Andaman and Nicobar Islands) where malarial transmission rates and levels of drug resistance vary across regions. Among the isolates, we observed a significant reduction in genetic variation in the +/-20-kb vicinity of the mutant pfdhfr alleles due to hitchhiking. This reduction in genetic diversity was more prominent around quadruple pfdhfr alleles (heterozygosity [H(e)] = 0.23) than around double (H(e) = 0.365) and single (H(e) = 0.465) mutant alleles. Asymmetry in the selective sweep flanking the pfdhfr alleles was observed with regional isolates, emphasizing the drug usage with the parasite population. All the pfdhfr alleles share a single microsatellite haplotype and seem to have originated from a single progenitor similar to that of Southeast Asian (Thailand) pfdhfr mutants. Results of the present study also indicate that the emergence of drug-resistant alleles is a recent phenomenon in India compared to Southeast Asian countries.

Emergence of an unusual sulfadoxine-pyrimethamine resistance pattern and a novel K540N mutation in dihydropteroate synthetase in Plasmodium falciparum isolates obtained from Car Nicobar Island, India, after the 2004 Tsunami.

BACKGROUND: Enormous amounts of drugs were used to contain the outbreak of infectious diseases in areas of India affected by the tsunami in December 2004. The impact of this drug use on the Plasmodium falciparum population needs to be investigated. METHODS: The nucleotide sequence of the pfcrt, pfdhps, and pfdhfr genes was determined for 229 clinical P. falciparum isolates collected from patients on Car Nicobar Island at 6 different time points between May 2004 and May 2008. RESULTS: Over time, there was an increase in the proportion of the P. falciparum population that had mutations in the pfcrt, pfdhps, and pfdhfr genes associated with higher levels of chloroquine, sulfadoxine, and pyrimethamine resistance, respectively. However, the parasites collected during October 2005 had mutations associated with a lower level of pyrimethamine resistance and a higher level of sulfadoxine resistance (a rare combination), as well as a novel K540N mutation in P. falciparum dihydropteroate synthetase (PfDHPS). The emergence of this parasite population coincided with the widespread use of an additional antifolate drug, trimethoprim-sulfamethoxazole, to treat other infections during January- March 2005. Molecular modeling revealed that the sulfadoxine binding affinity of the new PfDHPS triple mutant A436G437N540A581A613 was similar to that of A436G437E540A581A613 (bold type indicates mutated amino acids). CONCLUSIONS: The use of 2 antifolate drugs in combination should be avoided to prevent the selection of parasites with newer mutations and altered drug susceptibilities

Prevalence of mutations associated with higher levels of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum isolates from Car Nicobar Island and Assam, India.

To assess sulfadoxine and pyrimethamine resistance (SPR), we describe here the dihydropteroate synthetase (DHPS) mutations among the Plasmodium falciparum isolates in which dihydrofolate reductase (DHFR) mutations had recently been described by us (A. Ahmed, M. K. Das, V. Dev, M. A. Saifi, Wajihullah, and Y. D. Sharma, Antimicrob. Agents Chemother. 50:1546-1549, 2006). A majority of isolates from Car Nicobar island showed double DHPS mutations, whereas a majority of isolates from Uttar Pradesh (U.P.) and Assam contained the wild-type DHPS. Based on DHFR-DHPS mutations, the expected level of SPR was lowest in U.P., higher in Assam, and highest in Car Nicobar, suggesting that a region-wise drug policy is needed in India.