الملخص الإنجليزي
Introduction: Malaria is a major disease, caused by Plasmodia parasites, and is widely distributed in tropical and subtropical areas. The Arabian Peninsula lies at fringes of malaria endemicity, where successful control efforts have brought local transmission to halt in many parts of this region. However, limited foci in Yemen and southern Saudi Arabia remain malarious, with a high prevalence of drug-resistant P. falciparum parasites. The aims of this thesis are to assess the prevalence of drug resistance P. falciparum genotypes in Yemen, and characterize the genetic structure of the parasites. This includes analysis of genes associated with resistance to commonly used anti-ma drugs, including chloroquine, Fansidar, mefloquine and artemisinin. In addition, the study examined microsatellites flanking these genes to assess the source of resistance in Yemen, and its relationship to drug resistance genotypes seen in Jazan area, south west Saudi Arabia.
Material and methods: Hundred and fifteen, parasitologically confirmed, malaria patients from three areas (Dhamar, Hodeidah, and Taiz) in Yemen were recruited for this study. In addition, 90 P. falciparum isolates were collected from patients in Jazan area, south west Saudi Arabia. Single nucleotide polymorphisms (SNPs) in the P. falciparum chloroquine resistance transporter (Pfort) (C725, M741, N75E, K76T), multidrug resistance (Pfmdrl) (N86Y, Y184F, $1034C, N1042D, D1246Y), dihydrofolate reductase (dhfr) (N511, C59R, $108N, 1164L) and dihydropteroate synthase (dhps) (S436F, A437G, K540E, A6139) genes were examined using PCR and sequencing. In addition, 13 single copy microsatellites flanking the above genes were examined by capillary electrophoresis. Moreover, alleles of two polymorphic genes (MSP-2 and Pfg377) not involved in drug resistance were tested, to examine the genetic background of parasites in Yemen and Saudi Arabia.
Results: High prevalence of SNPs and mutant genotypes of Pfort and Pfmdr/ associated with chloroquine resistance (COR) were seen in Yemen. Two mutant Pfcrt genotypes CVIET and SVMNT existed in Yemen at a prevalence of 89% and 4%, respectively. Analysis of microsatellites flanking Pfort, revealed presence of 33 haplotypes, 8 carried the wild-type, 22 (66.7%) harboured genotype CVIET and 3 (9%) contained genotype SVMNT. Regarding Pfmdr1, four SNPs were detected, 86Y (2%), 184F (99%), 10340 (70%) and 1042D (70%). The majority of P. falciparum isolates carried the Pfmdr/ mutant genotype NFCDD (57%). Analysis of microsatellites flanking Pfmdrl, revealed presence of 72 haplotypes, 1 (1.4%), 19 (26.4%), 7 (9.7%), 36 (50%) and 9 (12.5%) haplotypes harboured the wild-type, NFSND, YFSND, NFCDD and YFCDD genotype, respectively. With regard to dhfr and dhps, 58 (54%) of isolates carried the double mutant dhfr genotype ICNI; however no dhps mutations were detected. Analysis of microsatellites flanking dhfr, revealed presence of 47 haplotypes, 34 (72.3%) harboured the wild-type and 13 (27.7%) contained genotype ICNI.
High diversity was seen among the other genes, Pfg377 (expected heterozygosity, He= 0.66) and MSP-2 (He =0.80). Five alleles of Pfg377 were detected, their sizes varied between 269 bp and 353 bp. Similarly, 15 and 30 alleles of the MSP-2 allelic types, FC27 and 3D7 were seen among parasites, respectively. The rate of mixed genotype infection was high (57%) with an average of 1.8 genotypes/ patient. This agrees with the absence of LD between the drug resistance genes (Pfort, Pfmdr), dhfr and dhps) and the non drug resistance genes (Pfg377 and MSP-2) (p<0.05). Comparison between P. falciparum parasites in Yemen and Saudi Arabia showed lower Fs (0.003) for the neutral gene (Pfg377). This suggest gene flow between parasites in the two regions. Nonetheless, a relatively higher F (0.091 to 0.111) was seen for the microsatellites, on chromosome 8 and MSP-2 (F= 0.062). In contrast to the above genes, there was a significant difference in distribution of drug resistance genes as well as the MSP-2 genes between the two regions. The Pfcrt CVIET was the predominant mutant genotype in both countries, however, genotype SVMNT was only seen in Yemen. Similar to Pfort, the double mutant dhfr ICNI existed at significant difference prevalence in both Yemen and Saudi Arabia. Moreover, the genetic background (flanking microsatellite) of the most dominant mutant genotypes of Pfort (CVIET) and dhfr (ICNI) are different in the two sites.
Conclusion: Pfort and Pfmdr/ mutations implicated in resistance to CQ, mefloquine and lumefantrine existed at high prevalence in Yemen. However, mutations in dhfr and dhps associated with sulfadoxine-pyrimethamine (SP) resistance were limited or absent, suggesting high efficacy of SP. The presence of two distinct haplotypes of Pfort suggests that CQR in Yemen has evolved from two independent origins. The source of these CQR lineages can be linked to Africa and Asia, however, the high degree of parasite diversity and recombination between different genotypes have generated an array of haplotypes of the two Pfcrt genotypes (CVIET and SVMNT). In contrast to parasites in Yemen, limited genetic diversity was seen among parasites in Saudi Arabia. Difference in distribution of MSP-2 alleles and drug resistance genes between the two regions can be explained by variation in selection pressure on these genes. However, the shared drug resistance haplotypes in the two regions support the findings of low Fs for the neutral gene (Pfg377), suggest gene flow between of parasites in the two regions, as a result of human movement.
