Marked differences in the prevalence of chloroquine resistance between urban and rural communities in Burkina Faso

BACKGROUND: Chloroquine (CQ) resistance has reached high levels in Africa in recent years. Little is known about variations of resistance between urban and rural areas. OBJECTIVES: To compare the rates of in vivo resistance to CQ and the prevalences of the main molecular marker for CQ resistance among young children from urban and rural areas in Burkina Faso. METHODS: The current analysis used the frame of a randomized controlled trial (ISRCTN27290841) on the combination CQ-methylene blue (MB) (n=177) compared to CQ alone (n=45) in young children with uncomplicated malaria. We examined clinical and parasitological failure rates as well as the prevalence of the Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) T76 mutation. RESULTS: Clinical and parasitological failure rates of CQ-MB differed significantly between urban (70%) and rural areas (29%, p<0.0001). Likewise, CQ failure rates were higher in the urban setting. Matching this pattern, pfcrt T76 was more frequently seen among parasite strains from urban areas (81%) when compared to rural ones (64%, p=0.01). In the presence of parasites exhibiting pfcrt T76, the odds of overall clinical failure were increased to 2.6-fold ([1.33, 5.16], p(LR)=0.005). CQ was detected at baseline in 21% and 2% of children from the urban and the rural study area, respectively (p(Chi)=0.002). CONCLUSION: Even within circumscribed geographical areas, CQ efficacy can vary dramatically. The differences in the prevalence of pfcrt T76 and in CQ failure rates are probably explained by a higher drug pressure in the urban area compared to the rural study area. This finding has important implications for national malaria policies.     

In vivo chloroquine resistance and prevalence of the pfcrt codon 76 mutation in Plasmodium falciparum isolates from the Republic of Congo

Chloroquine (CQ) resistance in Plasmodium falciparum has been particularly associated with mutations in the pfcrt gene. The present study was carried out in the malaria hyperendemic town of Brazzaville (Republic of Congo, Central Africa) where CQ is still recommended and used as a first-line drug for P. falciparum malaria. We assessed the efficacy of CQ in vivo, and the association between pfcrt mutation at codon 76 and treatment outcome in 50 children with uncomplicated malaria. The failure rate on day 28 was 95.7% and the pfcrt K76T mutation was present in 100% of isolates. No variation in the multiplicity of infection was observed in pre- and post-treatment isolates. In further 87 isolates from uncomplicated patients not treated with CQ, the mutation was detected in 98.5% of isolates. This study confirms the high level of in vivo resistance to CQ and shows the high prevalence of pfcrt K76T mutation in the Republic of Congo.     

Usefulness of the Plasmodium falciparum chloroquine resistance transporter T76 genotype failure index for the estimation of in vivo chloroquine resistance in Burkina Faso

The prevalence of chloroquine (CQ) treatment failure and the genotype failure index was determined in four sentinel sites in Burkina Faso. In three sites, the genotype failure index varied between 1.7 and 3, a result confirming the relationship between the Plasmodium falciparum CQ resistance transporter (Pfcrt) T76 mutation and CQ resistance. In the remaining site, the genotype failure index was unusually low, 1.1, which was significantly different than that in the other sites (P < 0.00001). These findings are discussed. Often but not always, the prevalence of CQ resistance can be correctly estimated by the Pfcrt T76 genotype failure index.     

Association of Plasmodium falciparum chloroquine resistance transporter variant T76 with age-related plasma chloroquine levels

The mutation leading to the substitution of a threonine (T76) for a lysine at position 76 of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was genotyped in 100 Nigerian children with asymptomatic parasitemia. Isolates containing both pfcrt variants were found to harbor higher numbers of parasite clones (P < 0.002). The prevalence of the pfcrt T76 variant decreased with age (P < 0.0001) and increased with blood levels of chloroquine (CQ) (P < 0.0001). Whereas the K76 allele was more frequent in individuals without detectable plasma CQ levels (79.7%), only the pfcrt T76 variant was observed in children with CQ levels > 150 nmol/L. In individuals without detectable plasma CQ, the proportion of those with pfcrt T76 decreased from 60% in children < 2 years old to 23.5% in children > or = 6 years old (P < 0.01). The association of actual blood levels of CQ and the occurrence of pfcrt T76 underlines that the pfcrt T76 variant is in fact the mediator of CQ resistance.     

Falciparum malaria in the north of Laos: the occurrence and implications of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene haplotype SVMNT

OBJECTIVE: The Pfcrt-gene encodes a transmembrane protein located in the Plasmodium falciparum digestive vacuole. Chloroquine resistant (CQR) strains of African and Southeast Asian origin carry the Pfcrt-haplotype (c72-76) CVIET, whereas most South American and Papua New Guinean CQR stains carry the SVMNT haplotype. METHOD: Eighty-eight samples from an area with reported in vivo Chloroquine and in vitro Amodiaquine-resistance were screened for the K76T mutation and their Pfcrt-haplotype (c72-76) using a new SSOP-ELISA. RESULTS: Hundred percent of the analysed samples showed the K76T mutation which is highly associated with in vivo drug failure. This very high rate of a CQR-marker is alarming in an area were CQ is still used as first line drug. The distribution of the three main Pfcrt-haplotypes was as follows: 68% CVIET, 31% SVMNT, 0% CVMNT. CONCLUSIONS: These data show, for the first time, the South American/PNG -haplotype (SVMNT) on mainland Southeast Asia. SVMNT-haplotype and others might be associated with a decreased efficacy of Amodiaquine and could therefore be potential markers for of amodiaquine resistance (AQR). If there is a correlation between AQR and the SVMNT-haplotype as suggested, 31% prevalence of a potential resistance marker is cause for concern.     

Association of pfcrt but not pfmdr1 alleles with chloroquine resistance in Iranian isolates of Plasmodium falciparum

This study was designed to analyze the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) mutations as markers of chloroquine (CQ) resistance in 200 blood samples collected from malaria patients in south-eastern Iran during 2002-2005. Among these, 25 (post-treatment) fulfilled the 28-day follow-up study. A high number of Iranian P. falciparum (97%) strains harbored quadruple mutations at codons 76T, 220S, 326D, and 356L. All post-treatment isolates harbored the mutant allele 76T, but low rates of the mutant allele 86Y (44%) of the pfmdr1 gene were detected. No wild haplotype of pfcrt (72-CVMNKAQNIR-371) was found in post-treatment samples; however, 56% of clinical "failure" samples carried the wild type of pfmdr1 (NYSND). The present results suggest a strong association between pfcrt 76T, but not pfmdr1 86Y mutation and in vivo CQ resistance. Furthermore, we found the CQ resistance-associated SVMNT haplotype, which previously had been seen in South American isolates. Although Iran is located more proximally to Southeast Asia than to South America, no CQ resistance-associated CVIET haplotye has been observed in this region. Therefore, these results were not consistent with the earlier presumed spread of CQR parasites from Southeast Asia to Africa via the Indian subcontinent. In conclusion, P. falciparum mutations associated with resistance to CQ are abundant in south-eastern Iran and this finding strongly supports that CQ as the first line drug is inadequate for treatment of uncomplicated falciparum malaria in Iran.     

The T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia.

The T76 mutation in the pfcrt gene has been linked to chloroquine (CQ) resistance in Plasmodium falciparum. PCR-based analysis of pfcrt alleles was performed on pre-treatment samples from 107 individuals who had P. falciparum infections and lived in Papua, Indonesia. The results of a 28-day, in-vivo test revealed clinical resistance to CQ in 79 (74%) of the samples. The crude sensitivity of the pfcrt T76 assay for detecting the CQ-resistant infections in the samples was 96% and the crude specificity 52%. Discordance between pfcrt genotype and in-vivo phenotype was analysed either by genotyping of the merozoite surface protein-2 (to distinguish re-infection from recrudescence) or by amplification of the P. falciparum-specific small-subunit ribosomal RNA (ssrRNA) gene, using nested PCR (to detect any sub-patent but resistant parasites in infections misclassified as sensitive by the in-vivo test). When adjusting for the results of these analyses, the sensitivity and specificity of the pfcrt T76 assay for detecting the CQ-resistant infections became 93% and 82%, respectively. Overall, the present results indicate that the pfcrt T76 assay may be used to forecast therapeutic failure caused by CQ resistance. Validation requires exploration of the phenotype classifications based on the results of in-vivo tests, using genetic analyses that distinguish re-infection from recrudescence and detect microscopically subpatent parasitaemias.     

Detection of in-vivo chloroquine resistance in Plasmodium falciparum from Rameswaram Island, a pilgrim centre in southern India

Resistance to chloroquine (CQ) in Plasmodium falciparum is one of the main causes of the wide-spread resurgence of malaria in India and a challenge to the effective control of the disease. In the pilgrim centre of Rameswaram Island, malaria has persisted despite the various control measures undertaken over the years. When CQ resistance in Rameswaram was investigated in vivo, recrudescent parasitaemias were observed in 25 (58%) of the 43 study subjects who were given CQ and completed follow-up, all occurring between days 10 and 28 (late treatment failures). The results of the msp(1), msp(2) and glurp genotyping of paired samples of P. falciparum, collected on day 0 and the day of recrudescence from 23 of the apparent treatment failures, indicated that 21 (91%) of the 23 were probably true treatment failures. All of the paired samples harboured parasites with the K76T mutation in their pfcrt genes, and subsequent sequencing of nine day-0 samples revealed the SVMNT haplotype in all nine. This is the first report of in-vivo drug resistance in P. falciparum from Rameswaram Island. Such resistance, which is probably the result of the indiscriminate use of CQ and/or the import of malaria from mainland India, warrants a change in the drug regimen used locally for the first-line treatment of uncomplicated, P. falciparum malaria, to make treatment more effective and slow the development and spread of more foci of CQ resistance.     

Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi

In 1993, Malawi stopped treating patients with chloroquine for Plasmodium falciparum malaria because of a high treatment failure rate (58%). In 1998, the in vitro resistance rate to chloroquine was 3% in the Salima District of Malawi; in 2000, the in vivo resistance rate was 9%. We assayed two genetic mutations implicated in chloroquine resistance (N86Y in the P. falciparum multiple drug resistance gene 1 and K76T in the P. falciparum chloroquine resistance transporter gene) in 82 P. falciparum isolates collected during studies in 1998 and 2000. The prevalence of N86Y remained similar to that in neighboring African countries that continued to use chloroquine. In contrast, the prevalence of K76T was substantially lower than in neighboring countries, decreasing significantly from 17% in 1998 to 2% in 2000 (P < 0.02). However, neither mutation was significantly associated with in vivo or in vitro resistance (P > 0.29). Withdrawal of the use of chloroquine appears to have resulted in the recovery of chloroquine efficacy and a reduction in the prevalence of K76T. However, other polymorphisms are also expected to contribute to resistance.     

In vitro susceptibility and genetic variations for chloroquine and mefloquine in Plasmodium falciparum isolates from Thai-Myanmar border

In vitro drug susceptibility to chloroquine (CQ) and mefloquine (MF) were assessed in 39 P. falciparum isolates from the Thai-Myanmar border area. To further characterize CQ- and MF-resistance profiles in this area, we also analyzed pfcrt K76T mutation that is critical for CQ resistance, and pfmdr1 polymorphism that has an association with MF resistance. Eighteen isolates were successfully examined by in vitro tests for CQ, and 17 of them had resistance to the drug. Geometric mean concentration of CQ that inhibited the growth of parasites at 50% (IC50) was 371 +/- 227 nM (105-971 nM). Sixteen isolates were successfully examined by in vitro tests for MF, and 8 of them were resistant to the drug. Geometric mean of IC50 for MF was 41 +/- 31 nM (4-125 nM). Genotypes of drug resistance, such as pfcrt and pfmdr1 mutations, were also analyzed. All the 39 isolates had the same haplotype (CVIET) for PfCRT at its 72-76th amino acids. A pfmdr1 Y86 mutation was found in 95% of isolates. A pfmdr1 D1042 mutation was also present in 7 isolates, while no pfmdr1 Y1246 mutation was observed. These results indicated a correlation between CQ resistance and the pfcrt T76 and pfmdr1 Y86 mutations.     

Polymorphism of the Plasmodium falciparum multidrug resistance and chloroquine resistance transporter genes and in vitro susceptibility to aminoquinolines in isolates from the Peruvian Amazon

In vitro drug sensitivity to chloroquine (CQ), mefloquine (MQ) and quinine was investigated in 60 culture-adapted Plasmodium falciparum isolates from malaria patients in Padrecocha, a village in the Amazonian Department of Loreto, Peru. All isolates showed resistance to CQ, decreased susceptibility to quinine, and sensitivity to MQ. These isolates were examined for mutations in the P. falciparum multidrug resistance 1 (pfmdr1) and chloroquine resistance transporter (pfcrt) genes previously linked to CQ resistance. The mutations N86Y and D1246Y, two of the five mutations commonly observed in the pfmdr1 gene of CQ-resistant clones, were not found. The pfcrt mutation K76T, associated with CQ resistance, was identified in all the isolates tested. Sequence analysis of codons 72-76 in the pfcrt gene showed the haplotypes SVMNT and CVMNT.     

Molecular monitoring of Plasmodium falciparum resistance to antimalarial drugs after adoption of sulfadoxine-pyrimethamine plus artesunate as the first line treatment in Iran

The main objective of this investigation was whether the combination therapy of sulfadoxine pyrimethamine (SP) plus artesunate (AS) protects against the spread of resistance to SP in malaria-endemic south-eastern Iran. Infected blood samples of Plasmodium falciparum (n=170) were collected during 2008-2010 after the adoption of SP-AS as the first line treatment in Iran. Four different genes of P. falciparum [dihydropteroate synthetase (pfdhps), dihydrofolate reductase (pfdhfr), chloroquine (CQ) resistance transporter (pfcrt K76T) and multidrug resistance1 (pfmdr1 N86Y)], associated with SP and CQ resistance were analyzed using PCR-RFLP methods. The result showed 4.1, 95.9 and 100% prevalence of pfdhfr 51I, 59R and 108N, respectively and the majority of patients (95.9%) were found to carry both 59R and 108N. The prevalence of single mutation at pfdhps 437G gene was 26.9% before the adoption of SP-AS, but as SP was used as the first line treatment; this mutation started to increase and reached a high level of 55.5% in 2008 (χ(2) test, P<0.05). However, three years after the introduction of SP-AS, this prevalence was reduced from 55.5% (in 2008) to 39.1% (in 2009) and then 40.5% (in 2010). The frequency of parasites carrying pfdhfr/pfdhps mutations (N(51)R(59)N(108)/G(437)) decreased from 53.3% in 2008 to 39.1% in 2009 and 38% in 2010. In addition, no significant reduction was seen in the frequency of mutant alleles of pfcrt 76T and pfmdr1 86Y after CQ was discontinued from study areas as a treatment for P. falciparum. This is explained by the fixation of pfcrt 76T in the falciparum populations that need more time to recover from CQ sensitivity in the absence of drug pressure in this region. In conclusion, the present findings suggest that in Iran, SP is still effective for the treatment of uncomplicated falciparum malaria as a partner drug of Artemisinin Combination Therapy (ACT) in this region.     

Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China

Resistance of Plasmodium falciparum to chloroquine (CQ) is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene and modified by other mutations in this gene and in the P. falciparum multidrug resistance 1 (pfmdr1) gene. To determine the extent of polymorphisms in these genes in field P. falciparum isolates from Yunnan province of China, we genotyped the pfcrt codon 76, pfmdr1 codons 86 and 1246. Our results showed that although CQ has been withdrawn from treating falciparum malaria for over two decades, 90.3% of the parasites still carried the pfcrt K76T mutation. In contrast, mutations at pfmdr1 codons 86 and 1246 were rare. Sequencing analysis of the pfcrt gene in 34 parasite field isolates revealed CVIET at positions 72-76 as the major type, consistent with the theory of Southeast Asian origin of CQ resistance in the parasite. In addition, two novel pfcrt haplotypes (75D/144Y/220A and 75E/144Y/220A) were identified. Real-time polymerase chain reaction was used to determine pfmdr1 gene amplification, which is associated with mefloquine resistance. Our result indicated that in agreement with that mefloquine has not been used in this area, most (>90%) of the parasites had one pfmdr1 copy. Genotyping at two hypervariable loci showed relatively low levels of genetic diversity of the parasite population. Meanwhile, 28.4% of cases were found to contain mixed clones, which favour genetic recombination. Furthermore, despite a unique history of antimalarial drugs in Yunnan, its geographical connections with three malarious countries facilitate gene flow among parasite populations and evolution of novel drug-resistant genotypes. Therefore, continuous surveillance of drug resistance in this area is necessary for timely adjustment of local drug policies and more effective malaria control.     

Molecular assessment of drug resistance in Plasmodium falciparum from Bahr El Gazal province, Sudan

AIMS: To assess resistance to chloroquine (CQ) and sulphadoxine/pyrimethamine (SP) in a Sudanese parasite population. METHODS: Recurrent security problems in Akuem, Sudan, prevented us from conducting a classical in vivo treatment efficacy study. Instead we genotyped key mutations in the chloroquine resistance transporter (pfcrt), the multidrug resistance gene (pfmdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps). We genotyped the K76T mutation in pfcrt and the N86Y mutation in (pfmdr) by restriction digestion of fluorescent end-labelled polymerase chain reaction (PCR) products, while we genotyped codons 16, 51, 59, 108 and 164 in dhfr and codons 436, 437, 540, 581 and 613 in dhps by primer extension in 100 blood samples. RESULTS: Sixty-three percent of parasites carried the 76T mutation at pfcrt critical for CQ resistance, while 31% carried the 86Y mutation at pfmdr that is associated with, although not essential, for CQ resistance. We found five dhfr alleles: 60% of infections contained wild-type dhfr alleles, 3% had one mutation, 34% had two mutations, while 3% had three mutations. We found three dhps alleles: 47% were wild type, 44% had one mutation, while 9% had two mutations. CONCLUSIONS: We expect high levels of treatment failure (RI-RIII) with CQ (20-40%) and predict efficient treatment with SP. However, dhfr alleles with three mutations (51I, 59R, 108N) are present as are dhps alleles with two mutations (437G, 540E). Successful treatment with SP is therefore likely to be short-lived.     

Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene haplotype SVMNT in P. falciparum malaria in India

The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) and the P. falciparum multidrug resistance 1 (Pfmdr1) mutation (N86Y) were analyzed as markers of chloroquine resistance in the DNAs of 73 blood samples from patients with P. falciparum malaria in India. Seventy of the 73 DNAs had the Pfcrt K76T mutation. Of these, 66 had the SVMNT haplotype and four had CVIET, the African/Southeast Asian haplotype. Only 20 of 69 DNAs had the Pfmdr1 N86Y mutation. It is surprising that the Pfcrt haplotype in India is predominantly SVMNT, rather than that seen in Southeast Asia. The widespread prevalence of the Pfcrt K76T mutation is a cause for concern.     

Low level genotypic chloroquine resistance near Malawi's northern border with Tanzania

We conducted a prevalence study of mutations in Plasmodium falciparum that are associated with antimalarial drug resistance at a rural site in Karonga near Malawi's northern border with Tanzania. We found a higher prevalence of the key chloroquine resistance-conferring mutation in the pfcrt gene (K76T) at this site in comparison with the prevalence in Blantyre, a city in the south of Malawi, far from an international border (9% vs. 0%; P  < 0.0005). In contrast we found a lower prevalence of the quintuple dhfr/dhps mutation, which is highly predictive of SP treatment failure, at the Karonga site compared to Blantyre (76% vs. 88%; P  <   0.005). The prevalence of the K76T pfcrt mutation at two Tanzanian sites close to the border with Malawi was recently reported to be over 50%. Our findings suggest a considerable 'leakage' of parasite antimalarial drug resistance across the border between two countries with different national malaria control policies and with different levels of resistance. Neighbouring countries should consider implementing common regional rather than national malaria treatment policies to prevent the spread of antimalarial drug resistance alleles across their borders.     

Short report: polymorphisms in the chloroquine resistance transporter gene in Plasmodium falciparum isolates from Lombok, Indonesia

The polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) and P. falciparum chloroquine resistance transporter (pfcrt) genes, which are associated with chloroquine resistance, were examined in 48 P. falciparum isolates from uncomplicated malaria patients from the West Lombok District in Indonesia. The point mutation N86Y in pfmdr1 was present in 35.4% of the isolates and mutation K76T in pfcrt was found in all but one of the samples studied. Identified pfcrt haplotypes were mainly identical to the Papua New Guinea type S(agt)VMNT (42 of 48, 87.5%), and a few isolates had the Southeast Asia type CVIET (5 of 48, 10.4%). Moreover, one P. falciparum isolate harbored the K76N mutation, giving rise to the haplotype CVMNN, which was not previously reported in field isolates. Our findings suggest that chloroquine resistance in this area might have the same origin as in Papua New Guinea.     

Monitoring chloroquine resistance using Plasmodium falciparum parasites isolated from wild mosquitoes in Tanzania

Monitoring antimalarial drug resistance is a useful epidemiologic tool and provides early detection of resistance foci. Using DNA extracted from the head/thorax of wild mosquitoes collected from Bagamoyo Coastal Tanzania, samples infected by Plasmodium falciparum (N = 89, in 2002 and N = 249 in 2004) were screened by nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for mutations at Pfcrt76 and Pfmdr1-86 associated with chloroquine (CQ) resistance. The majority of isolates were of single infection (71%), and the prevalence of mutant alleles of Pfcrt76 decreased from 64.5% in 2002 to 16% in 2004; likewise, mutant Pfmdr1-86 alleles decreased from 46.6% to 2.7%. Overall, there was a decline of mutant isolates by a factor of 17 and 4 for Pfmdr1 and Pfcrt, respectively. In contrast, isolates with wild-type alleles increased significantly from < 20% in 2002 to 67.6% for Pfcrt76 and 83.5% for Pfmdr1-86 in 2004. This observation suggest a biologic trend of decrease of CQ mutants and a subsequent increase of CQ susceptible parasites in circulation after the discontinued use of CQ in 2001 as a first-line drug in Tanzania. High prevalence of susceptible P. falciparum found in circulation not only supports other reports of a decline of mutant parasites after a reduction of drug selection pressure but suggests that the fitness cost is high in mutant parasites. Typing parasite isolates from infected mosquitoes, an alternative means of data collection, has the potential to increase the spatial and temporal coverage, and this approach is practical in highly endemic regions of Africa.     

High prevalence of pfcrt K76t mutants among Plasmodium falciparum isolates from Sabah, Malaysia

Chloroquine (CQ) remains the first line drug for the prevention and treatment of malaria in Malaysia in spite of the fact that resistance to CQ has been observed in Malaysia since the 1960s. CQ-resistance is associated with various mutations in pfcrt, which encodes a putative transporter located in the digestive vacuolar membrane of P. falciparum. Substitution of lysine (K) to threonine (T) at amino acid 76 (K76T) in pfcrt is the primary genetic marker conferring resistance to CQ. To determine the presence of T76 mutation in pfcrt from selected areas of Kalabakan, Malaysia 619 blood samples were screened for P. falciparum, out of which 31 were positive. Blood samples were collected on 3 MM Whatman filter papers and DNA was extracted using QIAmp DNA mini kit. RFLP-PCR for the detection of the CQ-resistant T76 and sensitive K76 genotype was carried out. Twenty-five samples were shown to have the point mutation in pfcrt whereas the remaining samples were classified as CQ-sensitive (wild-type). In view of the fact that CQ is the first line anti-malarial drug in Malaysia, this finding could be an important indication that treatment with CQ may no longer be effective in the future.     

Prevalence of severe chloroquine resistance associates the point mutation in pfcrt and pfmdrI gene in eastern India

ObjectiveTo evaluate the molecular mechanism of chloroquine resistant Plasmodium falciparum malaria in West Bengal, eastern part, India.MethodsParasitic bloods were collected from patients in Kolkata and Purulia, in vitro drug susceptibility test were performed in those 179 isolates. Now parasitic DNA was isolated by phenol chloroform extraction method and then polymerase chain reaction and restriction fragment length polymorphism analysis of different codons of pfcrt gene (76) and the pfmdrI gene (86, 1246) were assessed.ResultsThe response of 179 patients to chloroquine was determined. The prevalence of both pfcrt K76T (68.72%) and pfmdrI N86Y (43.1%) mutation was found here. But most importantly severe in vitro chloroquine resistance has been found (53.07%) here, with either double 76T + 86Y mutation or triple 76T + 86Y + 1246Y mutation.ConclusionsOur present findings implicate that due to enormous drug (chloroquine) pressure, double mutation with pfcrt 76T + pfmdrI 86Y and triple mutation with pfcrt 76T + pfmdrI 86Y + pfmdrI 1246Y was highly correlated (P = 0.001) with in vitro chloroquine resistance for the first time in eastern India.