Yagya D. Sharma

Plasmodium falciparum Isolates in India Exhibit a Progressive Increase in Mutations Associated with Sulfadoxine-Pyrimethamine Resistance

ABSTRACT The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant Plasmodium falciparum malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of P. falciparum collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double dhfr mutations and single dhps mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing.

Discordant Patterns of Genetic Variation at Two Chloroquine Resistance Loci in Worldwide Populations of the Malaria Parasite Plasmodium falciparum

ABSTRACT Mutations in the chloroquine resistance (CQR) transporter gene of Plasmodium falciparum (Pfcrt; chromosome 7) play a key role in CQR, while mutations in the multidrug resistance gene (Pfmdr1; chromosome 5) play a significant role in the parasites resistance to a variety of antimalarials and also modulate CQR. To compare patterns of genetic variation at Pfcrt and Pfmdr1 loci, we investigated 460 blood samples from P. falciparum-infected patients from four Asian, three African, and three South American countries, analyzing microsatellite (MS) loci flanking Pfcrt (five loci [∼40 kb]) and Pfmdr1 (either two loci [∼5 kb] or four loci [∼10 kb]). CQR Pfmdr1 allele-associated MS haplotypes showed considerably higher genetic diversity and higher levels of subdivision than CQR Pfcrt allele-associated MS haplotypes in both Asian and African parasite populations. However, both Pfcrt and Pfmdr1 MS haplotypes showed similar levels of low diversity in South American parasite populations. Median-joining network analyses showed that the Pfcrt MS haplotypes correlated well with geography and CQR Pfcrt alleles, whereas there was no distinct Pfmdr1 MS haplotype that correlated with geography and/or CQR Pfmdr1 alleles. Furthermore, multiple independent origins of CQR Pfmdr1 alleles in Asia and Africa were inferred. These results suggest that variation at Pfcrt and Pfmdr1 loci in both Asian and African parasite populations is generated and/or maintained via substantially different mechanisms. Since Pfmdr1 mutations may be associated with resistance to artemisinin combination therapies that are replacing CQ, particularly in Africa, it is important to determine if, and how, the genetic characteristics of this locus change over time.

Progressive Increase in Point Mutations Associated with Chloroquine Resistance in Plasmodium falciparum Isolates from India

BACKGROUND Effective malaria control programs require continuous monitoring of drug pressure in the field, using molecular markers. METHODS We used sequence analysis to investigate the pfcrt and pfmdr1 mutations in Indian Plasmodium falciparum isolates. To evaluate the chloroquine drug pressure in the field, isolates were collected from 5 different areas at 2 time points, with an interval of 2 years. RESULTS In 265 P. falciparum isolates, pfcrt mutations were observed at codons 72, 74, 75, 76, and 220, resulting in 8 different genotypes: SMNTS (61.89%), CIETS (12.08%), CMNKS (0.38%), CMNTA (2.64%), CMNTS (4.91%), SMNTA (0.38%), CIDTS (2.26%), and wild-type CMNKA (15.47%). During the 2-year period, there was a significant decrease in the number of isolates with the SMNTS genotype and an increase in the number of isolates with the highly chloroquine-resistant pfcrt genotype CIETS (P < .05). The N86Y mutation was less prevalent (30.13%) than the Y184F mutation (99.16%) in the pfmdr1 gene in 239 isolates, but the number of isolates with the N86Y mutation increased significantly during the 2-year period (P < .05). The number of isolates with higher total numbers of pfcrt and pfmdr1 2-loci mutations, therefore, increased significantly during this period. There was a regional bias in the mutation rate of these genes, because isolates from areas where chloroquine resistance was high had higher numbers of 2-loci mutations, and areas where chloroquine resistance was low had isolates with lower numbers of 2-loci mutations. CONCLUSION There was a temporal increase in the number of pfcrt and pfmdr1 2-loci mutations, and this led to the higher level of chloroquine resistance. This is a cause for concern for the antimalarial drug policy in India.

Similar Trends of Pyrimethamine Resistance-Associated Mutations in Plasmodium vivax and P. falciparum

ABSTRACT The antifolate drugs sulfadoxine and pyrimethamine are commonly used to treat Plasmodium falciparum malaria. However, they can also affect the Plasmodium vivax parasite if it coexists with P. falciparum, as both species have common drug targets. Resistance to the antifolate drugs arises due to point mutations in the target enzymes of the respective parasite. To assess the cross-species impact of antifolate drug treatment, we describe here the dihydrofolate reductase (DHFR) mutations among field isolates of P. vivax and P. falciparum. The overall DHFR mutation rate for P. vivax was lower than that for P. falciparum. However, both species of Plasmodium followed similar trends of DHFR mutations. Similar to P. falciparum, the DHFR mutation rate of P. vivax also varied from region to region. It was lower in P. vivax-dominant regions but higher in the P. falciparum-dominated areas and highest where antifolates are used as the first line of antimalarial treatment. In conclusion, the antifolate treatment of falciparum malaria is proportionately affecting the DHFR mutations of P. vivax, suggesting that the drug should be used with caution to minimize the development of cross-species resistance in the field.

Therapeutic efficacy of artemether-lumefantrine in uncomplicated falciparum malaria in India

BackgroundArtemisinin-based combination therapy (ACT) is the treatment of choice for uncomplicated falciparum malaria. Artemether-lumefantrine (AL), a fixed dose co-formulation, has recently been approved for marketing in India, although it is not included in the National Drug Policy for treatment of malaria. Efficacy of short course regimen (4 × 4 tablets of 20 mg artemether plus 120 mg lumefantrine over 48 h) was demonstrated in India in the year 2000. However, low cure rates in Thailand and better plasma lumefantrine concentration profile with a six-dose regimen over three days, led to the recommendation of higher dose globally. This is the first report on the therapeutic efficacy of the six-dose regimen of AL in Indian uncomplicated falciparum malaria patients. The data generated will help in keeping the alternative ACT ready for use in the National Programme as and when required.MethodsOne hundred and twenty four subjects between two and fifty-five years of age living in two highly endemic areas of the country (Assam and Orissa) were enrolled for single arm, open label prospective study. The standard six-dose regimen of AL was administered over three days and was followed-up with clinical and parasitological evaluations over 28 days. Molecular markers msp-1 and msp-2 were used to differentiate the recrudescence and reinfection among the study subjects. In addition, polymorphism in pfmdr 1 was also carried out in the samples obtained from patients before and after the treatment.ResultsThe PCR corrected cure rates were high at both the sites viz. 100% (n = 53) in Assam and 98.6% (n = 71) in Orissa. The only treatment failure case on D7 was a malnourished child. The drug was well tolerated with no adverse events. Patients had pre-treatment carriage of wild type codons at positions 86 (41.7%, n = 91) and 184 (91.3%, n = 91) of pfmdr1 gene.ConclusionAL is safe and effective drug for the treatment of acute uncomplicated falciparum malaria in India. The polymorphism in pfmdr 1 gene is not co-related with clinical outcome. However, treatment failure can also occur due to incomplete absorption of the drug as is suspected in one case of failure at D7 in the study. AL can be a viable alternative of artesunate plus sulphadoxine/pyrimethamine (AS + SP), however, the drug should be used rationally and efficacy needs to be monitored periodically.

Prevalence of the K76T mutation in the pfcrt gene of Plasmodium falciparum among chloroquine responders in India

Chloroquine-resistant Plasmodium falciparum needs to be monitored in the field for effective malaria control strategies. A point mutation K76T in the P. falciparum chloroquine resistance transporter (Pfcrt) protein has recently been proposed as a molecular marker for the faster detection of chloroquine-resistant falciparum malaria in field. We describe here the evaluation of this marker in Indian P. falciparum isolates. A total of 274 Indian P. falciparum isolates were analyzed for the K76T mutation. This mutation was detected in all the clinical isolates obtained from the in vivo chloroquine non-responders. But majority of the clinical isolates from chloroquine responders (71 of 74 patients, i.e. 96%) also harbored this mutation. The K76T mutation was indeed highly prevalent (91%) among 213 clinical isolates. There was a significant association between K76T mutation and the in vitro chloroquine response (P<0.05) but six isolates showed discordant results. In conclusion, the K76T mutation fails to differentiate majority of the chloroquine responders from that of the non-responders and thus will be of limited use in the field in India.

Discordance in drug resistance-associated mutation patterns in marker genes of Plasmodium falciparum and Plasmodium knowlesi during coinfections

OBJECTIVES Human Plasmodium knowlesi infections have been reported from several South-East Asian countries, excluding India, but its drug susceptibility profile in mixed-infection cases remains unknown. METHODS The chloroquine resistance transporter (CRT) and dihydrofolate reductase (DHFR) genes of P. knowlesi and other Plasmodium species were sequenced from clinical isolates obtained from malaria patients living in the Andaman and Nicobar Islands, India. The merozoite surface protein-1 and 18S rRNA genes of P. knowlesi were also sequenced from these isolates. RESULTS Among 445 samples analysed, only 53 of them had P. knowlesi-specific gene sequences. While 3 of the 53 cases (5.66%) had P. knowlesi monoinfection, the rest were coinfected with Plasmodium falciparum (86.79%, n = 46) or Plasmodium vivax (7.55%, n = 4), but none with Plasmodium malariae or Plasmodium ovale. There was discordance in the drug resistance-associated mutations among the coinfecting Plasmodium species. This is because the P. knowlesi isolates contained wild-type sequences, while P. falciparum isolates had mutations in the CRT and DHFR marker genes associated with a higher level of chloroquine and antifolate drug resistance, respectively. The mutation pattern indicates that the same patient, having a mixed infection, may be harbouring the drug-susceptible P. knowlesi parasite and a highly drug-resistant P. falciparum parasite. CONCLUSIONS A larger human population in South-East Asia may be at risk of P. knowlesi infection than reported so far. The different drug susceptibility genotypes of P. knowlesi from its coinfecting Plasmodium species in mixed infections adds a new dimension to the malaria control programme, requiring formulation of an appropriate drug policy.

Sequence diversity and natural selection at domain I of the apical membrane antigen 1 among Indian Plasmodium falciparum populations

BackgroundThe Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate antigen. The complete AMA1 protein is comprised of three domains where domain I exhibits high sequence polymorphism and is thus named as the hyper-variable region (HVR). The present study describes the extent of genetic polymorphism and natural selection at domain I of the ama1 gene among Indian P. falciparum isolates.MethodsThe part of the ama 1 gene covering domain I was PCR amplified and sequenced from 157 P. falciparum isolates collected from five different geographical regions of India. Statistical and phylogenetic analyses of the sequences were done using DnaSP ver. 4. 10. 9 and MEGA version 3.0 packages.ResultsA total of 57 AMA1 haplotypes were observed among 157 isolates sequenced. Forty-six of these 57 haplotypes are being reported here for the first time. The parasites collected from the high malaria transmission areas (Assam, Orissa, and Andaman and Nicobar Islands) showed more haplotypes (H) and nucleotide diversity π as compared to low malaria transmission areas (Uttar Pradesh and Goa). The comparison of all five Indian P. falciparum subpopulations indicated moderate level of genetic differentiation and limited gene flow (Fixation index ranging from 0.048 to 0.13) between populations. The difference between rates of non-synonymous and synonymous mutations, Tajimas D and McDonald-Kreitman test statistics suggested that the diversity at domain I of the AMA1 antigen is due to positive natural selection. The minimum recombination events were also high indicating the possible role of recombination in generating AMA1 allelic diversity.ConclusionThe level of genetic diversity and diversifying selection were higher in Assam, Orissa, and Andaman and Nicobar Islands populations as compared to Uttar Pradesh and Goa. The amounts of gene flow among these populations were moderate. The data reported here will be valuable for the development of AMA1-based malaria vaccine.

Quadruple Mutations in Dihydrofolate Reductase of Plasmodium falciparum Isolates from Car Nicobar Island, India

ABSTRACT Quadruple mutations in the Plasmodium falciparum dihydrofolate reductase (PFDHFR) enzyme give rise to the highest level of pyrimethamine resistance leading to treatment failures. We describe here the presence of these quadruple mutations in a majority of P. falciparum isolates from Car Nicobar (Andaman and Nicobar) Island, India. Isolates from the mainland, however, continue to show a prevalence of double PFDHFR mutations and some with triple but none with quadruple mutations. In conclusion, the antifolate drug pressure is very high in the island, which should be a cause of concern for the malaria control program in the country.

Plasmodium vivax: Sequence polymorphism and effect of natural selection at apical membrane antigen 1 (PvAMA1) among Indian population

Present study describes the characterization of apical membrane antigen 1 (PvAMA1) polymorphisms among Indian Plasmodium vivax isolates. The partial PvAMA1 gene (covering domain I and domain II regions) sequenced from sixty-one (n=61) isolates in this study resulted into 49 haplotypes. Comparison with the previously available PvAMA1 sequences in the GenBank database revealed that 45 of these were new haplotypes that have never been reported till date. For further analyses, we also included 11 previously reported PvAMA1 sequences from India available in the database. Thus genetic diversity and effect of natural selection were analyzed both at domain I and domain II of this promising malaria vaccine candidate among 72 Indian P. vivax isolates. Non-synonymous mutations were found at 25 codons (16 at domain I and 9 at domain II) where 17 codons were dimorphic while rest of them (8 codons) were trimorphic. Thus codon polymorphisms were observed to be more at domain I as compared to domain II. Although the difference between the rate of non-synonymous (dN) and synonymous (dS) mutations was positive (dN-dS, 0.002+/-0.004SE) at domain II, it was not significantly different from each other (P=0.272), indicating tendency of stronger diversifying selection at this domain. The dN-dS difference for domain I (-0.006+/-0.009SE, P=0.268) and for entire 900 bp region (-0.002+/-0.005E, P=0.320) being negative and statistically insignificant suggests the role of both positive as well as purifying selection. Three-dimensional distributions of all polymorphic residues were mapped on a modeled PvAMA1 structure. Results suggested that almost all of the observed polymorphisms were located at one surface of the antigen. In conclusion, PvAMA1 antigen displays high diversity among Indian isolates with more diversifying selection at domain II. The result has significant value in malaria vaccine development using this antigen.