PET-PCR method for the molecular detection of malaria parasites in a national malaria surveillance study in Haiti, 2011.

BACKGROUND: Recently, a real-time PCR assay known as photo-induced electron transfer (PET)-PCR which relies on self-quenching primers for the detection of Plasmodium spp. and Plasmodium falciparum was described. PET-PCR assay was found to be robust, and easier to use when compared to currently available real-time PCR methods. The potential of PET-PCR for molecular detection of malaria parasites in a nationwide malaria community survey in Haiti was investigated. METHODS: DNA from the dried blood spots was extracted using QIAGEN methodology. All 2,989 samples were screened using the PET-PCR assay in duplicate. Samples with a cycle threshold (CT) of 40 or less were scored as positive. A subset of the total samples (534) was retested using a nested PCR assay for confirmation. In addition, these same samples were also tested using a TaqMan-based real-time PCR assay. RESULTS: A total of 12 out of the 2,989 samples screened (0.4%) were found to be positive by PET-PCR (mean CT value of 35.7). These same samples were also found to be positive by the nested and TaqMan-based methods. The nested PCR detected an additional positive sample in a subset of 534 samples that was not detected by either PET-PCR or TaqMan-based PCR method. CONCLUSION: While the nested PCR was found to be slightly more sensitive than the PET-PCR, it is not ideal for high throughput screening of samples. Given the ease of use and lower cost than the nested PCR, the PET-PCR provides an alternative assay for the rapid screening of a large number of samples in laboratory settings.

Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti.

BACKGROUND: Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. METHODS: DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. RESULTS: Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch's T-test p-value = 0.53 and permutations p-value = 0.59). CONCLUSION: This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti.

Plasmodium falciparum Drug-Resistant Haplotypes and Population Structure in Postearthquake Haiti, 2010.

Chloroquine (CQ) remains the first-line treatment of malaria in Haiti. Given the challenges of conducting in vivo drug efficacy trials in low-endemic settings like Haiti, molecular surveillance for drug resistance markers is a reasonable approach for detecting resistant parasites. In this study, 349 blood spots were collected from suspected malaria cases in areas in and around Port-au-Prince from March to July 2010. Among them, 121 samples that were Plasmodium falciparum positive by polymerase chain reaction were genotyped for drug-resistant pfcrt, pfdhfr, pfdhps, and pfmdr1 alleles. Among the 108 samples that were successfully sequenced for CQ resistant markers in pfcrt, 107 were wild type (CVMNK), whereas one sample carried a CQ-resistant allele (CVIET). Neutral microsatellite genotyping revealed that the CQ-resistant isolate was distinct from all other samples in this study. Furthermore, the remaining parasite specimens appeared to be genetically distinct from other reported Central and South American populations.

New baseline environmental assessment of mosquito ecology in northern Haiti during increased urbanization.

The catastrophic 2010 earthquake in Port-au-Prince, Haiti, led to the large-scale displacement of over 2.3 million people, resulting in rapid and unplanned urbanization in northern Haiti. This study evaluated the impact of this unplanned urbanization on mosquito ecology and vector-borne diseases by assessing land use and change patterns. Land-use classification and change detection were carried out on remotely sensed images of the area for 2010 and 2013. Change detection identified areas that went from agricultural, forest, or bare-land pre-earthquake to newly developed and urbanized areas post-earthquake. Areas to be sampled for mosquito larvae were subsequently identified. Mosquito collections comprised five genera and ten species, with the most abundant species being Culex quinquefasciatus 35% (304/876), Aedes albopictus 27% (238/876), and Aedes aegypti 20% (174/876). All three species were more prevalent in urbanized and newly urbanized areas. Anopheles albimanus, the predominate malaria vector, accounted for less than 1% (8/876) of the collection. A set of spectral indices derived from the recently launched Landsat 8 satellite was used as covariates in a species distribution model. The indices were used to produce probability surfaces maps depicting the likelihood of presence of the three most abundant species within 30 m pixels. Our findings suggest that the rapid urbanization following the 2010 earthquake has increased the amount of area with suitable habitats for urban mosquitoes, likely influencing mosquito ecology and posing a major risk of introducing and establishing emerging vector-borne diseases.