Molecular epidemiology of Plasmodium species prevalent in Yemen based on 18 s rRNA

<p>Abstract</p> <p>Background</p> <p>Malaria is an endemic disease in Yemen and is responsible for 4.9 deaths per 100,000 population per year and 43,000 disability adjusted life years lost. Although malaria in Yemen is caused mainly by <it>Plasmodium falciparum </it>and <it>Plasmodium vivax</it>, there are no sequence data available on the two species. This study was conducted to investigate the distribution of the <it>Plasmodium </it>species based on the molecular detection and to study the molecular phylogeny of these parasites.</p> <p>Methods</p> <p>Blood samples from 511 febrile patients were collected and a partial region of the 18 s ribosomal RNA (18 s rRNA) gene was amplified using nested PCR. From the 86 positive blood samples, 13 <it>Plasmodium falciparum </it>and 4 <it>Plasmodium vivax </it>were selected and underwent cloning and, subsequently, sequencing and the sequences were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods.</p> <p>Results</p> <p>Malaria was detected by PCR in 86 samples (16.8%). The majority of the single infections were caused by <it>P. falciparum </it>(80.3%), followed by <it>P. vivax </it>(5.8%). Mixed infection rates of <it>P. falciparum </it>+ <it>P. vivax </it>and <it>P. falciparum </it>+ <it>P. malariae </it>were 11.6% and 2.3%, respectively. All <it>P. falciparum </it>isolates were grouped with the strain 3D7, while <it>P. vivax </it>isolates were grouped with the strain Salvador1. Phylogenetic trees based on 18 s rRNA placed the <it>P. falciparum </it>isolates into three sub-clusters and <it>P. vivax </it>into one cluster. Sequence alignment analysis showed 5-14.8% SNP in the partial sequences of the 18 s rRNA of <it>P. falciparum</it>.</p> <p>Conclusions</p> <p>Although <it>P. falciparum </it>is predominant, <it>P. vivax</it>, <it>P. malariae </it>and mixed infections are more prevalent than has been revealed by microscopy. This overlooked distribution should be considered by malaria control strategy makers. The genetic polymorphisms warrant further investigation.</p

Genetic diversity of Plasmodium falciparum isolates from Pahang, Malaysia based on MSP-1 and MSP-2 genes

<p>Abstract</p> <p>Background</p> <p>Malaria is still a public health problem in Malaysia especially in the interior parts of Peninsular Malaysia and the states of Sabah and Sarawak (East Malaysia). This is the first study on the genetic diversity and genotype multiplicity of <it>Plasmodium falciparum </it>in Malaysia.</p> <p>Methods</p> <p>Seventy-five <it>P. falciparum </it>isolates were genotyped by using nested-PCR of <it>MSP-1 </it>(block 2) and <it>MSP-2 </it>(block 3).</p> <p>Results</p> <p><it>MSP-1 </it>and <it>MSP-2 </it>allelic families were identified in 65 blood samples. RO33 was the predominant <it>MSP-1 </it>allelic family identified in 80.0% (52/65) of the samples while K1 family had the least frequency. Of the <it>MSP-2 </it>allelic families, 3D7 showed higher frequency (76.0%) compared to FC27 (20.0%). The multiplicity of <it>P. falciparum </it>infection (MOI) was 1.37 and 1.20 for <it>MSP-1 </it>and <it>MSP-2</it>, respectively. A total of seven alleles were detected; of which three <it>MSP-1 </it>allelic families (RO33, MAD20 and K1) were monomorphic in terms of size while <it>MSP-2 </it>alleles were polymorphic (two 3D7 and two FC27). Heterozygosity (H_E) was 0.57 and 0.55 for <it>MSP-1 </it>and <it>MSP-2</it>, respectively.</p> <p>Conclusions</p> <p>The study showed that the MOI of <it>P. falciparum </it>is low, reflected the low intensity of malaria transmission in Pahang, Malaysia; RO33 and 3D7 were the most predominant circulating allelic families. The findings showed that <it>P. falciparum </it>has low allelic diversity with a high frequency of alleles. As a result, antimalarial drug efficacy trials based on MSP genotyping should be carefully interpreted.</p

High frequency of Plasmodium falciparum chloroquine resistance marker (pfcrt T76 mutation) in Yemen: An urgent need to re-examine malaria drug policy

<p>Abstract</p> <p>Background</p> <p>Malaria remains a significant health problem in Yemen with <it>Plasmodium falciparum </it>being the predominant species which is responsible for 90% of the malaria cases. Despite serious concerns regarding increasing drug resistance, chloroquine is still used for the prevention and treatment of malaria in Yemen. This study was carried out to determine the prevalence of choloroquine resistance (CQR) of <it>P. falciparum </it>isolated from Yemen based on the <it>pfcrt </it>T76 mutation.</p> <p>Methods</p> <p>A cross-sectional study was carried out among 511 participants from four governorates in Yemen. Blood samples were screened using microscopic and species-specific nested PCR based on the 18S rRNA gene to detect and identify <it>Plasmodium </it>species. Blood samples positive for <it>P. falciparum </it>were used for detecting the <it>pfcrt </it>T76 mutation using nested-PCR.</p> <p>Results</p> <p>The prevalence of <it>pfcrt </it>T76 mutation was 81.5% (66 of 81 isolates). Coastal areas/foothills had higher prevalence of <it>pfcrt </it>T76 mutation compared to highland areas (90.5% <it>vs </it>71.8%) (p = 0.031). The <it>pfcrt </it>T76 mutation had a significant association with parasitaemia (p = 0.045). Univariate analysis shows a significant association of <it>pfcrt </it>T76 mutation with people aged > 10 years (OR = 9, 95% CI = 2.3 - 36.2, p = 0.001), low household income (OR = 5, 95% CI = 1.3 - 19.5, p = 0.027), no insecticide spray (OR = 3.7, 95% CI = 1.16 - 11.86, p = 0.025) and not sleeping under insecticide treated nets (ITNs) (OR = 4.8, 95% CI = 1.38 - 16.78, p = 0.01). Logistic regression model confirmed age > 10 years and low household income as predictors of <it>pfcrt </it>T76 mutation in Yemen <it>P. falciparum </it>isolates.</p> <p>Conclusions</p> <p>The high prevalence of <it>pfcrt </it>T76 mutation in Yemen could be a predictive marker for the prevalence of <it>P. falciparum </it>CQR. This finding shows the necessity for an in-vivo therapeutic efficacy test for CQ.<it> P. falciparum </it>CQR should be addressed in the national strategy to control malaria.</p

The detection of <it>pfcrt</it> and <it>pfmdr1</it> point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia

Abstract Background Malaria is still a public health problem in Malaysia with chloroquine (CQ) being the first-line drug in the treatment policy of uncomplicated malaria. There is a scarcity in information about the magnitude of Plasmodium falciparum CQ resistance. This study aims to investigate the presence of single point mutations in the P. falciparum chloroquine-resistance transporter gene (pfcrt) at codons 76, 271, 326, 356 and 371 and in P. falciparum multi-drug resistance-1 gene (pfmdr1) at codons 86 and 1246, as molecular markers of CQ resistance. Methods A total of 75 P. falciparum blood samples were collected from different districts of Pahang state, Malaysia. Single nucleotide polymorphisms in pfcrt gene (codons 76, 271, 326, 356 and 371) and pfmdr1 gene (codons 86 and 1246) were analysed by using mutation-specific nested PCR and restriction fragment length polymorphism (PCR-RFLP) methods. Results Mutations of pfcrt K76T and pfcrt R371I were the most prevalent among pfcrt gene mutations reported by this study; 52% and 77%, respectively. Other codons of the pfcrt gene and the positions 86 and 1246 of the pfmdr1 gene were found mostly of wild type. Significant associations of pfcrt K76T, pfcrt N326S and pfcrt I356T mutations with parasitaemia were also reported. Conclusion The high existence of mutant pfcrt T76 may indicate the low susceptibility of P. falciparum isolates to CQ in Peninsular Malaysia. The findings of this study establish baseline data on the molecular markers of P. falciparum CQ resistance, which may help in the surveillance of drug resistance in Peninsular Malaysia.</p