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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.

ABSTRACT: 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.

SUBMITTER: Mosawi SH

PROVIDER: S-EPMC7362495 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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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.

Mosawi Sayed Hussain SH Dalimi Abdolhossein A Safi Najibullah N Fotouhi-Ardakani Reza R Ghaffarifar Fatemeh F Sadraei Javid J

Malaria journal 20200714 1

<h4>Background</h4>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 w ...[more]

PMID: 32664942

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Project description:BackgroundAnalysis of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations in Plasmodium vivax wild isolates has been considered to be a valuable molecular approach for mapping resistance to sulphadoxine-pyrimethamine (SP). The present study investigates the frequency of SNPs-haplotypes in the dhfr and dhps genes in P. vivax clinical isolates circulating in two malaria endemic areas in Afghanistan.MethodsP. vivax clinical isolates (n = 171) were collected in two different malaria endemic regions in north-west (Herat) and east (Nangarhar) Afghanistan in 2008. All collected isolates were analysed for SNP-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the pvdhfr and 383 and 553 of the pvdhps genes using PCR-RFLP methods.ResultsAll 171 examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 4.1% and 12.3% of Afghan isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B was the most prevalent variant among Herat (86%) and Nangarhar (88.4%) isolates. Mixed genotype infections (type A/B and A/B/C) were detected in only 2.3% (2/86) of Herat and 1.2% (1/86) of Nangarhar isolates, respectively. The combination of pvdhfr and pvdhps haplotypes among all 171 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were wild-type (86%) and single mutant haplotype I13P33F57S58T61N 117I173/A383A553 (6.4%).Double (I13P33S57R58T61N117I173/A383A553) and triple mutant haplotypes (I13P33S57R 58T61N117I173/G383A553) were found in 1.7% and 1.2% of Afghan isolates, respectively. This triple mutant haplotype was only detected in isolates from Herat, but in none of the Nangarhar isolates.ConclusionThe present study shows a limited polymorphism in pvdhfr from Afghan isolates and provides important basic information to establish an epidemiological map of drug-resistant vivax malaria, and updating guidelines for anti-malarial policy in Afghanistan. The continuous usage of SP as first-line anti-malarial drug in Afghanistan might increase the risk of mutations in the dhfr and dhps genes in both P. vivax and Plasmodium falciparum isolates, which may lead to a complete SP resistance in the near future in this region. Therefore, continuous surveillance of P. vivax and P. falciparum molecular markers are needed to monitor the development of resistance to SP in the region.

Project description:BackgroundChloroquine (CQ) and primaquine (PQ) are still the drugs of choice to treat Plasmodium vivax malaria in many endemic areas, Brazil included. There is in vivo evidence for the P. vivax resistance to CQ in the Brazilian Amazon, where the increase in the proportion of P. vivax malaria parallels the increase of unusual clinical complications related to this species. In this study, in vitro CQ and mefloquine (MQ)-susceptibility of P. vivax isolates from the Western Brazilian Amazon was tested using the double-site enzyme-linked lactate dehydrogenase immunodetection (DELI) assay.MethodsA total of 112 P. vivax isolates were tested in vitro for CQ-susceptibility and out of these 47 were also tested for MQ-susceptibility. The DELI assay was used to detect P. vivax growth at 48-hour short-term culture in isolates with ring stages ranging from 50 to %. Each isolate was tested in triplicate and geometric means of IC50's was obtained. Nineteen isolates were genetically characterized for pvdhfr, pvmrp1, pvmdr1 and pvdhps candidate genes likely related to CQ resistance (10 with IC50<40 nM and 9 with IC50 >100 nM).ResultsTwelve out of 112 isolates were considered resistant to CQ, resulting in 10.7% (IC95% 5.0-16.4), while 3 out of 47 (6.4%; IC95% 0.0-12.8) were resistant to MQ. A discrete correlation was observed between IC50's of CQ and MQ (Spearman=0.294; p=0.045). For pvdhps gene, a non-synonymous mutation was found at codon 382 (S→C) in 5/8 CQ-sensitive samples and 1/9 CQ-resistant samples (p=0.027). The other molecular markers were not associated to CQ-susceptibility.ConclusionsIn vitro CQ-resistance estimated in this study, estimated by the DELI test, was very similar to that observed in clinical trials, suggesting that in vitro procedures developed by capable local laboratories are useful in the surveillance of CQ-resistance in the Amazon; concurrent Amazon P. vivax strains with both CQ and MQ resistance may be common; and a non-synonymous mutation at pvdhps codon 382 (S→C) was associated to in vitro susceptibility to CQ, needing further studies to be confirmed.

Project description:BACKGROUND: With 75% of the Ethiopian population at risk of malaria, accurate diagnosis is crucial for malaria treatment in endemic areas where Plasmodium falciparum and Plasmodium vivax co-exist. The present study evaluated the performance of regular microscopy in accurate identification of Plasmodium spp. in febrile patients visiting health facilities in southern Ethiopia. METHODS: A cross-sectional study design was employed to recruit study subjects who were microscopically positive for malaria parasites and attending health facilities in southern Ethiopia between August and December 2011. Of the 1,416 febrile patients attending primary health facilities, 314 febrile patients, whose slides were positive for P. falciparum, P. vivax or mixed infections using microscopy, were re-evaluated for their infection status by PCR. Finger-prick blood samples were used for parasite genomic DNA extraction. Phylogenetic analyses were performed to reconstruct the distribution of different Plasmodium spp. across the three geographical areas. RESULTS: Of the 314 patients with a positive thick blood smear, seven patients (2%) were negative for any of the Plasmodium spp. by nested PCR. Among 180 microscopically diagnosed P. falciparum cases, 111 (61.7%) were confirmed by PCR, 44 (24.4%) were confirmed as P. vivax, 18 (10%) had mixed infections with P. falciparum and P. vivax and two (1.1%) were mixed infections with P. falciparum and P. malariae and five (2.8%) were negative for any of the Plasmodium spp. Of 131 microscopically diagnosed P. vivax cases, 110 (84%) were confirmed as P. vivax, 14 (10.7%) were confirmed as P. falciparum, two (1.5%) were P. malariae, three (2.3%) with mixed infections with P. falciparum and P. vivax and two (1.5%) were negative for any of the Plasmodium spp. Plasmodium falciparum and P. vivax mixed infections were observed. Plasmodium malariae was detected as mono and mixed infections in four individuals. CONCLUSION: False positivity, under-reporting of mixed infections and a significant number of species mismatch needs attention and should be improved for appropriate diagnosis. The detection of substantial number of false positive results by molecular methodologies may provide the accurate incidence of circulating Plasmodium species in the geographical region and has important repercussions in understanding malaria epidemiology and subsequent control.

Dataset Information

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.

Publications

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.

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