A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR

Background

The erythrocyte binding antigen-175 (EBA-175) on Plasmodium falciparum merozoites mediates sialic acid dependent binding to glycophorin A on host erythrocytes and, therefore, plays a crucial role in cell invasion. Dimorphic allele segments have been found in its encoding gene with a 342 bp segment present in FCR-3 strains (F-segment) and a 423 bp segment in CAMP strains (C-segment). Possible associations of the dimorphism with severe malaria have been analysed in a case-control study in northern Ghana.

Methods

Blood samples of 289 children with severe malaria and 289 matched parasitaemic but asymptomatic controls were screened for eba- 175 F- and C-segments by nested polymerase chain reaction.

Results

In children with severe malaria, prevalences of F-, C- and mixed F-/C-segments were 70%, 19%, and 11%, respectively. The C-segment was found more frequently in severe malaria cases whereas mixed infections were more common in controls. Infection with strains harbouring the C-segment significantly increased the risk of fatal outcome.

Conclusion

The results show that the C-segment is associated with fatal outcome in children with severe malaria in northern Ghana, suggesting that it may contribute to the virulence of the parasite.     

SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination of amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1)

The pfmdr1 gene, which encodes P-glycoprotein homolog 1, has been shown to be a reliable marker of resistance for Plasmodium falciparum related to artesunate and mefloquine combination therapy. The aims of this study are to investigate the copy number of pfmdr1 in P. falciparum isolates collected from the 4 malaria-endemic areas of Thailand (Kanchanaburi, Mae Hongson, Ranong, and Tak) along the Thailand-Myanmar (Burma) border (Thai-Myanmar border) by using SYBR Green I and the standard method TaqMan real-time polymerase chain reaction (RT-PCR) and to compare the efficiency (sensitivity and specificity) of SYBR Green I with TaqMan RT-quantitative (q)PCR methods in determining pfmdr1 gene copy number. Ninety-six blood samples were collected onto filter paper from patients with uncomplicated falciparum malaria who attended malaria clinics in the Kanchanaburi (n = 45), Mae Hongson (n = 18), Ranong (n = 11), and Tak (n = 22) provinces in Thailand. Parasite genomic DNA was extracted from dried blood spots by using QIAcube? automated sample preparation. Pfmdr1 gene copy number was determined by TaqMan (63 samples) and SYBR Green I (96 samples) real-time PCR. Seventy-one (74.0%), 14 (14.6%), 10 (10.4%), and 1 (1%) isolates carried 1, 2, 3, and 4 pfmdr1 gene copies, respectively. Forty-three of 48 (89.6%), 6 of 11 (54.5%), and 3 of 4 (75.0%) samples, respectively, showed agreement with results of 1, 2, and 3 pfmdr1 gene copies as determined by both methods. The efficiency of SYBR Green I in identifying pfmdr1 gene copy number was found to be significantly correlated with that of TaqMan. Considering its simplicity and relatively low cost, SYBR Green I RT-qPCR is therefore a promising alternative technique for the determination of pfmdr1 copy number.     

Mass blood survey for malaria: pooling and realtime PCR combined with expert microscopy in north-west Thailand

ABSTRACT: BACKGROUND: Asymptomatic carriage of Plasmodium falciparum and Plasmodium vivax is common in both low-and high-transmission settings and represents an important reservoir of infection that needs to be targeted if malaria elimination is to succeed. METHODS: Mass blood examinations (475 individuals) were conducted in two villages in Mae Hong Son, an area of endemic but low-transmission malaria in the north-west of Thailand. The microscopist at the local malaria clinic did not detect any infections. Pools of four samples were screened by real-time PCR; individual members of all of the positive pools were then re-examined by expert microscopy and by a second species-specific PCR reaction. RESULTS: Eight subjects were found to be positive by both PCR and expert microscopy and one was found to be positive by PCR alone. The slides contained asexual stage parasites of P. vivax, P. falciparum and Plasmodium malariae, but no gametocytes. The local clinic was notified within two to eight days of the survey. CONCLUSION: A combination of pooling, real-time PCR and expert microscopy provides a feasible approach to identifying and treating asymptomatic malaria infections in a timely manner.     

Selection and Spread of Artemisinin-Resistant Alleles in Thailand Prior to the Global Artemisinin Resistance Containment Campaign

The recent emergence of artemisinin resistance in the Greater Mekong Subregion poses a major threat to the global effort to control malaria. Tracking the spread and evolution of artemisinin-resistant parasites is critical in aiding efforts to contain the spread of resistance. A total of 417 patient samples from the year 2007, collected during malaria surveillance studies across ten provinces in Thailand, were genotyped for the candidate Plasmodium falciparum molecular marker of artemisinin resistance K13. Parasite genotypes were examined for K13 propeller mutations associated with artemisinin resistance, signatures of positive selection, and for evidence of whether artemisinin-resistant alleles arose independently across Thailand. A total of seven K13 mutant alleles were found (N458Y, R539T, E556D, P574L, R575K, C580Y, S621F). Notably, the R575K and S621F mutations have previously not been reported in Thailand. The most prevalent artemisinin resistance-associated K13 mutation, C580Y, carried two distinct haplotype profiles that were separated based on geography, along the Thai-Cambodia and Thai-Myanmar borders. It appears these two haplotypes may have independent evolutionary origins. In summary, parasites with K13 propeller mutations associated with artemisinin resistance were widely present along the Thai-Cambodia and Thai-Myanmar borders prior to the implementation of the artemisinin resistance containment project in the region.     

Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1_270-290, 3D7_610-630, G_650-690, while 2 variants, K1_150-170, and 3D7_670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.     

Determination of artemether and its major metabolite, dihydroartemisinin, in plasma using high-performance liquid chromatography with electrochemical detection

A rapid, selective, sensitive and reproducible HPLC with recutive electrochemical detection for quantitatvie determination of artemether (ART) and its plasma metabolite, dihydroartemisinin (DHA: and β isomers) in plasma is described. The procedure involved the extraction of ART, DHA and the internal standard, artemisinin (ARN) with dichloromethane-tert.-methylbutyl ether (1:1, v/v) or n-butyl chloride-ethyl acetate (9:1, v/v). Chromatographic separation was performed with a mobile phase of acetonitrile-water (20:80, v/v) containing 0.1 M acetic acid pH 5.0, running through a μBondapak CN column. The method was capable of separating the two isomeric forms of DHA (, β). The retention times of -DHA, β-DHA, ARN and ART were 4.6, 5.9, 7.9 and 9.6 min, respectively. Validation of the assay method was performed using both extraction systems. The two extraction systems produced comparable recoveries of the various analytes. The average recoveries of ART, DHA and ARN over the concentration range 80–640 ng/ml were 86–93%. The coefficients of variation were below 10% for all three drugs (ART, -DHA, ARN). The minimum detectable concentrations for ART and -DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for use in clinical pharmacokinetic study.