MEASURING ALLELIC HETEROGENEITY IN PLASMODIUM FALCIPARUM BY A HETERODUPLEX TRACKING ASSAY

We developed a novel Plasmodium falciparum genotyping strategy based on the heteroduplex tracking assay (HTA) method commonly used to genotype viruses. Because it can detect both sequence and size polymorphisms, we hypothesized that HTA is more sensitive than current methods. To test this hypothesis, we compared the ability of HTA and a nested polymerase chain reaction (PCR) to detect genetic diversity in 17 Thai samples. The HTA detected more MSP1 sequence variants in eight isolates (47%), less sequence variants in three isolates (18%), and an equal number of sequence variants in six isolates (35%), suggesting that HTA is equal to or more sensitive than the nested PCR. This study is a proof of concept that HTA is a sensitive allelic discrimination method able to determine genetic diversity in P. falciparum and warrants its use in studies of antimalarial drug efficacy

Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar

<p>Abstract</p> <p>Background</p> <p>Strains of <it>Plasmodium falciparum </it>genetically resistant to chloroquine (CQ) due to the presence of <it>pfcrt </it>76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant <it>P. falciparum </it>isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect <it>pfcrt </it>76T-bearing <it>P. falciparum </it>minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings.</p> <p>Methods</p> <p>This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect <it>pfcrt </it>76T-bearing minority variant <it>P. falciparum</it>. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples.</p> <p>Results</p> <p>Thirty one clinical <it>P. falciparum </it>isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the <it>pfcrt </it>K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant <it>P. falciparum </it>strains after therapy as detected by HTA. RFLP analysis failed to detect any <it>pfcrt </it>K76T-bearing isolates.</p> <p>Conclusion</p> <p>These findings indicate that genetically CQ-resistant <it>P. falciparum </it>are more common than previously thought in Madagascar even though the fitness of the minority variant <it>pfcrt </it>76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.</p

A Randomized Controlled Pilot Trial of Azithromycin or Artesunate Added to Sulfadoxine-Pyrimethamine as Treatment for Malaria in Pregnant Women

New anti-malarial regimens are urgently needed in sub-Saharan Africa because of the increase in drug resistance. We investigated the safety and efficacy of azithromycin or artesunate combined with sulfadoxine-pyrimethamine used for treatment of malaria in pregnant women in Blantyre, Malawi.This was a randomized open-label clinical trial, conducted at two rural health centers in Blantyre district, Malawi. A total of 141 pregnant women with uncomplicated Plasmodium falciparum malaria were recruited and randomly allocated to 3 treatment groups: sulfadoxine-pyrimethamine (SP; 3 tablets, 500 mg sulfadoxine and 25 mg pyrimethamine per tablet); SP plus azithromycin (1 g/dayx2 days); or SP plus artesunate (200 mg/dayx3 days). Women received two doses administered at least 4 weeks apart. Heteroduplex tracking assays were performed to distinguish recrudescence from new infections. Main outcome measures were incidence of adverse outcomes, parasite and fever clearance times and recrudescence rates. All treatment regimens were well tolerated. Two women vomited soon after ingesting azithromycin. The parasite clearance time was significantly faster in the SP-artesunate group. Recrudescent episodes of malaria were less frequent with SP-azithromycin [Hazard Ratio 0.19 (95% confidence interval 0.06 to 0.63)] and SP-artesunate [Hazard Ratio 0.25 (95% confidence interval 0.10 to 0.65)] compared with SP monotherapy. With one exception (an abortion in the SP-azithromycin group), all adverse pregnancy outcomes could be attributed to known infectious or obstetrical causes. Because of the small sample size, the effect on birth outcomes, maternal malaria or maternal anemia could not be evaluated.Both SP-artesunate and SP-azithromycin appeared to be safe, well tolerated and efficacious for the treatment of malaria during pregnancy. A larger study is needed to determine their safety and efficacy in preventing poor birth outcomes.ClinialTrials.gov NCT00287300

Differing patterns of selection and geospatial genetic diversity within two leading Plasmodium vivax candidate vaccine antigens

Although Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens--Plasmodium vivax merozoite surface protein 1 (pvmsp-1) and Plasmodium vivax circumsporozoite protein (pvcsp). Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n = 44) and the complete gene of pvcsp (n = 47) from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess for selection and population structure, we found strong evidence of balancing selection in the 42 kDa region of pvmsp-1, which varied significantly over the length of the gene, consistent with immune-mediated selection. In pvcsp, the highly variable central repeat region also showed patterns consistent with immune selection, which were lacking outside the repeat. The patterns of selection seen in both genes differed from their P. falciparum orthologs. In addition, we found that, similar to merozoite antigens from P. falciparum malaria, genetic diversity of pvmsp-1 sequences showed no geographic clustering, while the non-merozoite antigen, pvcsp, showed strong geographic clustering. These findings suggest that while immune selection may act on both vivax vaccine candidate antigens, the geographic distribution of genetic variability differs greatly between these two genes. The selective forces driving this diversification could lead to antigen escape and vaccine failure. Better understanding the geographic distribution of genetic variability in vaccine candidate antigens will be key to designing and implementing efficacious vaccines

Genetic diversity of Plasmodium vivax malaria in China and Myanmar

Genetic diversity and population structure of Plasmodium vivax parasites are valuable to the prediction of the origin and spread of novel variants within and between populations, and to the program evaluation of malaria control measures. Using two polymorphic genetic markers, the merozoite surface protein genes PvMSP-3α and PvMSP-3β, we investigated the genetic diversity of four Southeast Asian P. vivax populations, representing both subtropical and temperate strains with dramatically divergent relapse patterns. PCR amplification of PvMSP-3α and PvMSP-3β genes detected three and four major size polymorphisms among the 235 infections examined, respectively, while restriction analysis detected 15 and 19 alleles, respectively. Samples from different geographical areas differed dramatically in their PvMSP-3α and PvMSP-3β allele composition and frequency. Samples tended to cluster on the basis of their PCR-RFLP polymorphism. These results indicated that different parasite genotypes were circulating in each endemic area, and that geographic isolation may exist. Multiple infections were detected in all four parasite populations, ranging from 20.5% to 31.8%, strongly indicating that P. vivax populations were highly diverse and multiple clonal infections are common in these malaria-hypoendemic regions of Southeast Asia

Molecular screening of Plasmodium infections among migrant workers in Thailand

Background & objective: A cross-sectional study was conducted to determine the prevalence of Plasmodiuminfections among migrant workers in Thailand.Methods: A total of 241 migrants at Kanchanaburi, Pathumthani and Nakornpathom provinces of Thailand wererecruited in our surveillance. Blood samples were examined for human malaria parasites by using microscopyand semi-nested multiplex PCR (SnM-PCR).Results: Laboratory diagnosis revealed 6.2% total positive rate. As compared to microscopy (26.7%), SnM-PCRwas more sensitive (93.3%) for malaria. Plasmodium falciparum was predominant than P. vivax (53% : 40%,respectively). The majority of positive cases were from Myanmar workers who had low parasitaemia and withoutsymptoms. The highest prevalence (13.7%) was found among migrant workers from Kanchanaburi province inwestern Thailand.Conclusion: These findings indicate risk of malaria transmission from migrant workers. Malaria surveillanceshould be included in the health-screening program for migrants in Thailand to manage this health risk

Asymptomatic malaria infections among foreign migrant workers in Thailand

AbstractObjectiveTo determine the prevalence of malaria infections among foreign migrant workers in Thailand.MethodsGiemsa-stained thin and thick blood films were prepared from blood samples of 294 foreign migrant workers recruited in the study. Microscopic examination of these blood films was performed for malaria detection.ResultsBlood film examination revealed 1.36% malaria infections in these 294 subjects. All positive cases were male Myanmar workers in which their blood films only ring stage of Plasmodium spp. was found at low parasite density (mean= 144 parasites/μ L of blood). The prevalence of malaria infections was not significantly different among foreign migrant workers classified by age, gender, and resident province (P>0.05). Thin blood films of these workers also showed 78.91% hypochromic erythrocytes and 61.9% relative Eosinophilia.ConclusionsThese findings indicate a high risk of malaria transmission. Therefore active malaria surveillance by using molecular methods with more sensitive and specific than microscopy should be considered for malaria control in foreign migrant workers

Development of Nested PCR-Heteroduplex Mobility Assay for Determination of Genetic Diversity in the Block 2 Region of the Plasmodium falciparum Merozoite Surface Protein 1 Gene

Genetic diversity of Plasmodium parasite has significantly related to malaria control and vaccine development. The P. falciparum merozoite surface protein 1 (Pfmsp1) gene is a commonly used molecular marker to differentiate genetic diversity. This study is aimed at developing a nested PCR-Heteroduplex Mobility Assay (nPCR-HMA) for determination of the block 2 of the Pfmsp1 gene. The MAD20 family allele of P. falciparum was used as a control for optimization of the annealing and polyacrylamide gel electrophoresis conditions. In order to evaluate the developed nPCR-HMA, 8 clinical P. falciparum isolates were examined for allelic variants. The results revealed 9 allelic variants. Our study indicated that the successful nPCR-HMA with good precision and accuracy offers a more rapid, efficient, and cheap method for large-scale molecular epidemiological studies as compared to nucleotide sequencing