Are pvcrt-o and pvmdr1 Gene Mutations Associated with Plasmodium vivax Chloroquine-Resistant Parasites?

(1) Background: Malaria remains a significant global public health issue. Since parasites quickly became resistant to most of the available antimalarial drugs, treatment effectiveness must be constantly monitored. In Brazil, up to 10% of cases of vivax malaria resistant to chloroquine (CQ) have been registered. Unlike P. falciparum, there are no definitive molecular markers for the chemoresistance of P. vivax to CQ. This work aimed to investigate whether polymorphisms in the pvcrt-o and pvmdr1 genes could be used as markers for assessing its resistance to CQ. (2) Methods: A total of 130 samples from P. vivax malaria cases with no clinical and/or parasitological evidence of CQ resistance were studied through polymerase chain reaction for gene amplification followed by target DNA sequencing. (3) Results: In the pvcrt-o exons, the K10 insert was present in 14% of the isolates. Regarding pvmdr1, T958M and F1076L haplotypes showed frequencies of 95% and 3%, respectively, while the SNP Y976F was not detected. (4) Conclusions: Since K10-pvcrt-o and F1076L/T958M-pvmdr1 polymorphisms were detected in samples from patients who responded well to CQ treatment, it can be concluded that mutations in these genes do not seem to have a potential for association with the phenotype of CQ resistance.

Surveillance of drug resistance molecular markers in Plasmodium vivax before and after introduction of dihydroartemisinin and piperaquine in Thailand: 2009-2019.

Resistance to antimalarial drugs is a serious issue around the world. Widespread Plasmodium vivax and P. falciparum coinfections are commonly found in Thailand. Dihydroartemisinin and piperaquine (DHA-PPQ) have been used as first-line treatments for P. falciparum since 2015, and chloroquine (CQ) and primaquine (PQ) have remained first-line drugs for P. vivax for more than 60 years. Coinfections may lead parasites to evolve with regard to genetics under selective drug pressure. This study is aimed at investigating genes linked to antimalarial resistance in P. vivax before and after introduction of DHA-PPQ as a new drug regimen in Thailand. A total of 400 P. vivax isolates were collected from samples along the Thai-Myanmar and Thai-Malaysian borders before (2009-2015) and after (2016-2019) introduction of DHA-PPQ. Genomic DNA of P. vivax was obtained and subjected to analysis of five drug resistance-associated genes (Pvdhfr, Pvdhps, Pvmdr1, Pvcrt-o, and PvK12) by nested polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and nucleotide sequencing. A high prevalence of Pvdhfr was found in both endemic areas over the period. The quadruple (57I/58R/61M/117T) Pvdhfr haplotype was predominant in both periods in both endemic areas. Although the wild-type haplotype of Pvdhps was predominant in Thai-Malaysian isolates in both periods, a single mutant haplotype (383G) was dominant in Thai-Myanmar isolates during both periods. A low prevalence of the Pvmdr1 976F mutation was found in both periods among Thai-Myanmar isolates. A significant decrease in Pvmdr1 976F was identified in Thai-Malaysian isolates from the second period (p < 0.01). Only one nonsynonymous mutation of Pvcrt-o (193E) and one synonymous mutation of PvK12 (R584) were detected in four isolates (4.7%) and one isolate (0.5%) in the first period among Thai-Myanmar isolates, respectively. Thus, with limited clinical efficacy data, the low prevalence of drug-resistance markers may suggest that there is a low prevalence of P. vivax-resistant strains and that the current drug regimen for P. vivax is still effective for treating this P. vivax parasite population. Continued surveillance of antimalarial drug resistance markers and monitoring of clinical drug efficacy should be conducted for epidemiological and policy implications.

Polymorphisms of pvcrt-o and pvmdr1 genes of 48 Plasmodium vivax isolates from the China-Myanmar border / 中国热带医学

@#Abstract: Objective To detect the polymorphisms of drug resistance-related genes pvcrt-o and pvmdr1 of Plasmodium vivax in lazan city in the China-Myanmar border, in order to guide the treatment plan of Plasmodium vivax. Methods A total of 48 Plasmodium vivax samples were collected from Lazan in the China-Myanmar border in 2007, and fragments of pvcrt-o and pvmdr1 genes were amplified by PCR and sequenced. The sequences were aligned with the Salvador I (Sal-I) strain reference genome sequences to determine the presence of SNPs. Results The target fragments of pvcrt-o gene were amplified from 39 Plasmodium vivax samples, while pvmdr1 genes were amplified from 40 samples. Amongst them, 25 samples had AAG insertion before the 10th amino acid (K10 insertion) of pvcrt-o gene, accounting for 64.1%. Non-synonymous mutations were detected at three loci of pvmdr1 gene (T958M, Y976F, and F1076L), the mutation rates were 100%, 22.5%, and 55.0%, respectively. There were three haplotypes of pvmdr1 gene, of which the triple mutant 958M/976F/1076L accounted for 22.5% (9/40), the double mutant 958M/Y976/1076L accounted for 32.5% (13/40), and the single mutant 958M/Y976/F1076 accounted for 45.0% (18/40). The proportion of strains with pvcrt-o and pvmdr1 gene mutation is 63.16%, which is significantly different from those only with pvmdr1 mutation. Conclusions The proportion of pvcrt-o and pvmdr1 gene mutation of 48 Plasmodium vivax isolates is high in the China-Myanmar border, and there is a certain degree of correlation between the two gene mutations. To assess changes in Plasmodium vivax drug resistance in this region, it is required to improve the surveillance of these two molecular markers.

Subpatent Plasmodium with mutant pfmdr1, pfcrt, and pvmdr1 alleles from endemic provinces in Mindanao, the Philippines: implications for local malaria elimination.

OBJECTIVES: This study was performed to identify and characterize circulating Plasmodium species in three provinces of Mindanao approaching malaria elimination. METHODS: Rapid diagnostic tests (RDT), microscopic examination, and PCR were used to detect malaria parasites. PCR-positive isolates were genotyped for polymorphisms in loci of interest. RESULTS: A total of 2639 participants were surveyed in Mindanao between 2010 and 2013. Malaria prevalence by PCR was 3.8% (95% confidence interval (CI): 2.7-5.2%) in Sarangani, 10% (95% CI: 7.7-12.7%) in South Cotabato, and 4.2% (95% CI: 3.2-5.6%) in Tawi-Tawi. P. falciparum and P. vivax were identified in all three provinces, and there was one case of P. malariae in South Cotabato. RDT was inferior to PCR for detecting asymptomatic P. falciparum and P. vivax. In Tawi-Tawi, microscopy failed to identify 46 PCR-positive malaria infections. The presence of pfcrt haplotypes CVMNK, CVIET, and SMNT (codons 72-76), pfmdr1 haplotype NFSND (codons 86, 184, 1034, 1042, 1246), and pvmdr1 haplotype NFL (codons 91, 976, 1076) was confirmed in Mindanao. CONCLUSIONS: Asymptomatic Plasmodium infections persisted in local communities between 2010 and 2013. PCR successfully identified subpatent malaria infections, and can better characterize malaria epidemiology in communities seeking malaria control and elimination at the local level.

An unlabelled probe-based real time PCR and modified semi-nested PCR as molecular tools for analysis of chloroquine resistant Plasmodium vivax isolates from Afghanistan.

BACKGROUND: Plasmodium vivax resistance to chloroquine (CQ) has been reported from many endemic regions in the world. Plasmodium vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are possible markers for CQ-resistance in P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. The present work aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax. METHODS: Plasmodium vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. A new asymmetric qPCR and melting analysis assay based on unlabelled probe developed for scanning of K10 insertion in pvcrt-o gene. RESULTS: The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces. Of the 36 samples evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province, respectively, possessed K10 insertion, confirmed by either sequencing and unlabelled probes. CONCLUSION: Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance in P. vivax populations in Afghanistan. Furthermore, unlabelled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

Plasmodium vivax drug resistance markers: Genetic polymorphisms and mutation patterns in isolates from Malaysia.

Transmission of Plasmodium vivax still persist in Malaysia despite the government's aim to eliminate malaria in 2020. High treatment failure rate of chloroquine monotherapy was reported recently. Hence, parasite drug susceptibility should be kept under close monitoring. Mutation analysis of the drug resistance markers is useful for reconnaissance of anti-malarial drug resistance. Hitherto, information on P. vivax drug resistance marker in Malaysia are limited. This study aims to evaluate the mutations in four P. vivax drug resistance markers pvcrt-o (putative), pvmdr1 (putative), pvdhfr and pvdhps in 44 isolates from Malaysia. Finding indicates that 27.3%, 100%, 47.7%, and 27.3% of the isolates were carrying mutant allele in pvcrt-o, pvmdr1, pvdhfr and pvdhps genes, respectively. Most of the mutant isolates had multiple point mutations rather than single point mutation in pvmdr1 (41/44) and pvdhfr (19/21). One novel point mutation V111I was detected in pvdhfr. Allelic combination analysis shows significant strong association between mutations in pvcrt-o and pvmdr1 (X2 = 9.521, P < 0.05). In the present study, 65.9% of the patients are non-Malaysians, with few of them arrived in Malaysia 1-2 weeks before the onset of clinical manifestations, or had previous history of malaria infection. Besides, few Malaysian patients had travel history to vivax-endemic countries, suggesting that these patients might have acquired the infections during their travel. All these possible imported cases could have placed Malaysia in a risk to have local transmission or outbreak of malaria. Six isolates were found to have mutations in all four drug resistance markers, suggesting that the multiple-drugs resistant P. vivax strains are circulating in Malaysia.

Emergence of Plasmodium vivax Resistance to Chloroquine in French Guiana.

In South America, Plasmodium vivax resistance to chloroquine was recently reported in Brazil and Bolivia. The objective of this study was to collect data on chloroquine resistance in French Guiana by associating a retrospective evaluation of therapeutic efficacy with an analysis of recurrent parasitemia from any patients. Patients with P. vivax infection, confirmed by microscopy and a body temperature of ≥37.5°C, were retrospectively identified at Cayenne Hospital between 2009 and 2015. Follow-up and treatment responses were performed according to the World Health Organization protocol. Parasite resistance was confirmed after dosage of a plasma concentration of chloroquine and microsatellite characterization. The pvmdr1 and pvcrt-o genes were analyzed for sequence and gene copy number variation. Among the 172 patients followed for 28 days, 164 presented adequate clinical and parasitological responses. Eight cases of treatment failures were identified (4.7%; n = 8/172), all after 14 days. The therapeutic efficacy of chloroquine was estimated at 95.3% (95% confidence interval [CI], 92.5 to 98.1%; n = 164/172). Among the eight failures, five were characterized: two cases were true P. vivax chloroquine resistance (1.2%; 95% CI, 0 to 2.6%; n = 2/172), and three cases were found with subtherapeutic concentrations of chloroquine. No particular polymorphism in the Plasmodium vivaxpvmdr1 and pvcrt-o genes was identified in the resistant parasites. This identified level of resistance of P. vivax to chloroquine in French Guiana does not require a change in therapeutic recommendations. However, primaquine should be administered more frequently to limit the spread of resistance, and there is still a need for a reliable molecular marker to facilitate the monitoring of P. vivax resistance to chloroquine.

Polymorphisms in genes associated with drug resistance of Plasmodium vivax in India.

Malaria is a sterning public health concern in India and contribute to a major part of malaria burden in Southeast Asia. Being more populated and diverse geographic conditions makes more suitable place for sustaining malaria parasite in India. Anti-malarial resistance is a major concern in the battle against malaria, and the identified molecular markers will aid us to monitor the drug resistance in endemic areas. The aim of the current study is to determine the genotype of drug resistance associated genes pvmdr-1 and pvcrt-o from four different regions of India. Especially from Puducherry and Jodhpur, there were no prior studies focused on screening of drug resistance genes in P. vivax parasite. A total of 240 positive P. vivax infected patient samples were collected from four tertiary care hospitals from four different regions of India, namely, Puducherry (PDY), Mangaluru (MAQ), Cuttack (CTC), Jodhpur (JDH). All samples were screened by microscopy, RDT, QBC, and further DNA was extracted and vivax mono-infection was confirmed by nested PCR. Randomly selected amplicons were further subjected to nucleotide sequencing. The prevalence of K10 insertion in pvcrt-o gene was detected with 18.8% in PDY, 12.5% in MAQ and 6.3% in CTC P. vivax isolates, whereas no change in nucleotide was identified in P. vivax isolates collected from JDH region. Based on the F1076L mutation in pvmdr-1 gene, resistant P. vivax isolates was highly predominant in both the regions, JDH and CTC, with 100%, followed by MAQ with 93.3% and PDY with 73.3%. This study showed less frequency of pvcrt-o and high frequency of pvmdr-1 gene variants associated with CQ resistance, which act as an indicator and the onset of P. vivax drug resistance trend in four different regions of India. Due to the poor phenotypic studies available for P. vivax parasite, the present study data for CQ resistance based on pvcrt-o and pvmdr-1 markers should assist by providing base-line data for future monitoring of drug resistance.

Simultaneous detection of Plasmodium vivax dhfr, dhps, mdr1 and crt-o resistance-associated mutations in the Colombian Amazonian region.

BACKGROUND: Malaria continues being a public health problem worldwide. Plasmodium vivax is the species causing the largest number of cases of malaria in Asia and South America. Due to the lack of a completely effective anti-malarial vaccine, controlling this disease has been based on transmission vector management, rapid diagnosis and suitable treatment. However, parasite resistance to anti-malarial drugs has become a major yet-to-be-overcome challenge. This study was thus aimed at determining pvmdr1, pvdhfr, pvdhps and pvcrt-o gene mutations and haplotypes from field samples obtained from an endemic area in the Colombian Amazonian region. METHODS: Fifty samples of parasite DNA infected by a single P. vivax strain from symptomatic patients from the Amazonas department in Colombia were analysed by PCR and the pvdhfr, pvdhps, pvmdr1 and pvcrt-o genes were sequenced. Diversity estimators were calculated from the sequences and the haplotypes circulating in the Colombian Amazonian region were obtained. CONCLUSION: pvdhfr, pvdhps, pvmdr1 and pvcrt-o genes in the Colombian Amazonian region are characterized by low genetic diversity. Some resistance-associated mutations were found circulating in this population. New variants are also being reported. A selective sweep signal was located in pvdhfr and pvmdr1 genes, suggesting that these mutations (or some of them) could be providing an adaptive advantage.

Measuring ex vivo drug susceptibility in Plasmodium vivax isolates from Cambodia.

BACKGROUND: While intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013-2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates. RESULTS: Ex vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013-4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification. CONCLUSION: The findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method.

Chloroquine efficacy for Plasmodium vivax in Myanmar in populations with high genetic diversity and moderate parasite gene flow.

BACKGROUND: Plasmodium vivax malaria remains a major public health burden in Myanmar. Resistance to chloroquine (CQ), the first-line treatment for P. vivax, has been reported in the country and has potential to undermine local control efforts. METHODS: Patients over 6 years of age with uncomplicated P. vivax mono-infection were enrolled into clinical efficacy studies in Myawaddy in 2014 and Kawthoung in 2012. Study participants received a standard dose of CQ (25 mg/kg over 3 days) followed by weekly review until day 28. Pvmdr1 copy number (CN) and microsatellite diversity were assessed on samples from the patients enrolled in the clinical study and additional cross-sectional surveys undertaken in Myawaddy and Shwegyin in 2012. RESULTS: A total of 85 patients were enrolled in the CQ clinical studies, 25 in Myawaddy and 60 in Kawthoung. One patient in Myawaddy (1.2%) had an early treatment failure and two patients (2.3%) in Kawthoung presented with late treatment failures on day 28. The day 28 efficacy was 92.0% (95% CI 71.6-97.9) in Myawaddy and 98.3% (95% CI 88.7-99.8) in Kawthoung. By day 2, 92.2% (23/25) in Myawaddy and 85.0% (51/60) in Kawthoung were aparasitaemic. Genotyping and pvmdr1 CN assessment was undertaken on 43, 52 and 46 clinical isolates from Myawaddy, Kawthoung and Shwegyin respectively. Pvmdr1 amplification was observed in 3.2% (1/31) of isolates in Myawaddy, 0% (0/49) in Kawthoung and 2.5% (1/40) in Shwegyin. Diversity was high in all sites (H E 0.855-0.876), with low inter-population differentiation (F ST 0.016-0.026, P < 0.05). CONCLUSIONS: Treatment failures after chloroquine were observed following chloroquine monotherapy, with pvmdr1 amplification present in both Myawaddy and Shwegyin. The results emphasize the importance of ongoing P. vivax drug resistance surveillance in Myanmar, particularly given the potential connectivity between parasite population at different sites.

Genetic diversity and natural selection of Plasmodium vivax multi-drug resistant gene (pvmdr1) in Mesoamerica.

BACKGROUND: The Plasmodium vivax multidrug resistant 1 gene (pvmdr1) codes for a transmembrane protein of the parasite's digestive vacuole. It is likely that the pvmdr1 gene mutations occur at different sites by convergent evolution. In here, the genetic variation of pvmdr1 at three sites of the Mesoamerican region was studied. Since 1950s, malarious patients of those areas have been treated only with chloroquine and primaquine. METHODS: Blood samples from patients infected with P. vivax were obtained in southern Mexico (SMX), in the Northwest (NIC-NW) and in the northeast (NIC-NE) of Nicaragua. Genomic DNA was obtained and fragments of pvmdr1 were amplified and sequenced. The nucleotide and amino acid changes as well as the haplotype frequency in pvmdr1 were determined per strain and per geographic site. The sequences of pvmdr1 obtained from the studied regions were compared with homologous sequences from the GenBank database to explore the P. vivax genetic structure. RESULTS: In 141 parasites, eight nucleotide changes (two changes were synonymous and other six were nonsynonymous) were detected in 1536 bp. The PvMDR1 amino acid changes Y976F, F1076FL were predominant in endemic parasites from NIC-NE and outbreak parasites in NIC-NW but absent in SMX. Thirteen haplotypes were resolved, and found to be closely related, but their frequency at each geographic site was different (P = 0.0001). The pvmdr1 codons 925-1083 gene fragment showed higher genetic and haplotype diversity in parasites from NIC-NE than the other areas outside Latin America. The haplotype networks suggested local diversification of pvmdr1 and no significant departure from neutrality. The F ST values were low to moderate regionally, but high between NIC-NE or NIC-NW and other regions inside and outside Latin America. CONCLUSIONS: The pvmdr1 gene might have diversified recently at regional level. In the absence of significant natural, genetic drift might have caused differential pvmdr1 haplotype frequencies at different geographic sites in Mesoamerica. A very recent expansion of divergent pvmdr1 haplotypes in NIC-NE/NIC-NW produced high differentiation between these and parasites from other sites including SMX. These data are useful to set a baseline for epidemiological surveillance.

Genetic diversity of Plasmodium vivax metacaspase 1 and Plasmodium vivax multi-drug resistance 1 genes of field isolates from Mauritania, Sudan and Oman.

BACKGROUND: Plasmodium vivax is the second most important human malaria parasite, widely spread across the world. This parasite is associated with important issues in the process toward malaria elimination, including potential for relapse and increased resistance to chloroquine. Plasmodium vivax multi-drug resistant (pvmdr1) is suspected to be a marker of resistance although definitive evidence is lacking. Progress has been made in knowledge of biological factors affecting parasite growth, including mechanisms of regulated cell death and the suspected role of metacaspase. Plasmodium vivax metacaspase1 (PvMCA1-cd) has been described with a catalytic domain composed of histidine (H372) and cysteine (C428) residues. The aim of this study was to test for a link between the conserved histidine and cysteine residues in PvMCA1-cd, and the polymorphism of the P. vivax multi-drug resistant gene (pvmdr1). RESULTS: Thirty P. vivax isolates were collected from Mauritania, Sudan, and Oman. Among the 28 P. vivax isolates successfully sequenced, only 4 samples showed the conserved His (372)-Cys (428) residues in PvMCA1-cd. Single nucleotide polymorphisms observed were H372T (46.4%), H372D (39.3%), and C428R (85.7%). A new polymorphic catalytic domain was observed at His (282)-Cys (305) residues. Sequences alignment analysis of pvmdr1 showed SNP in the three codons 958, 976 and 1076. A single SNP was identified at the codon M958Y (60%), 2 SNPs were found at the position 976: Y976F (13%) and Y976V (57%), and 3 SNPs were identified at the position 1076: F1076L (40%), F1076T (53%) and F1076I (3%). Only one isolate was wildtype in all three codons (MYF), 27% were single MYL mutants, and 10% were double MFL mutants. Three new haplotypes were also identified: the triple mutant YVT was most prevalent (53.3%) distributed in the three countries, while triple YFL and YVI mutants (3%), were only found in samples from Sudan and Mauritania. CONCLUSIONS: Triple or quadruple mutants for metacaspase genes and double or triple mutants for Pvmdr1 were observed in 24/28 and 19/28 samples. There was no difference in the frequency of mutations between PvMCA1-cd and Pvmdr1 (P > 0.2). Histidine and cysteine residues in PvMCA1-cd are highly polymorphic and linkage disequilibrium with SNPs of Pvmdr1 gene may be expected from these three areas with different patterns of P. vivax transmission.

Plasmodium vivax drug resistance genes; Pvmdr1 and Pvcrt-o polymorphisms in relation to chloroquine sensitivity from a malaria endemic area of Thailand.

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 µl) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.

Plasmodium vivax Drug Resistance Genes; Pvmdr1 and Pvcrt-o Polymorphisms in Relation to Chloroquine Sensitivity from a Malaria Endemic Area of Thailand

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 mul) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.

Plasmodium vivax Drug Resistance Genes; Pvmdr1 and Pvcrt-o Polymorphisms in Relation to Chloroquine Sensitivity from a Malaria Endemic Area of Thailand

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 mul) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.

Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates

Plasmodium vivax control is now being hampered by drug resistance. Orthologous Plasmodium falciparum genes linked to chloroquine or sulfadoxine-pyrimethamine chemoresistance have been identified in P. vivax parasites, but few studies have been performed. The goal of the present work is to characterise pvmdr1 and pvdhfr genes in parasite isolates from a Brazilian endemic area where no molecular investigation had been previously conducted. The pvmdr1 analysis revealed the existence of single (85.7 percent) and double (14.3 percent) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2 percent) and triple (42.8 percent) mutant haplotypes. The implications of these findings are discussed.