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

In vivo and molecular evaluation of artesunate+sulfadoxine-pyrimethamine efficacy for uncomplicated falciparum malaria in Tehama region, Yemen / Wahib Mohammed Mohsein Atroosh

Yemen has updated its national malaria policies in 2009 to introduce histidine rich protein 2 – based rapid diagnostic test (HRP2-RDT) as a method of diagnosis in peripheral malaria-endemic areas, and to replace chloroquine (CQ) by artemisinin combination therapy (ACT) for treating uncomplicated falciparum malaria infection. The present study aimed to evaluate the HRP2-RDT and to assess the therapeutic efficacy of artesunate + sulfadoxine/pyrimethamine (AS+SP) as a first line treatment. A total of 622 febrile individuals from two malaria-endemic areas in Tehama region, Yemen (Hodeidah and Al-Mahwit governorates) were screened by CareStartTM malaria HRP2-RDT and confirmed later by microscopy, followed by gene sequencing analysis of Pfhrp2. Evaluation of AS+SP therapeutic efficacy was performed through in-vivo evaluation of the clinical and parasitological response over 28 days of follow-up according to standard protocols. Moreover, frequency of mutations associated with drugs resistance was obtained for the pfdhfr, pfdhps, and pfK13 genes for AS + SP, as well as pfcrt and pfmdr1 genes for other antimalarials. A total of 188 (30.2%) participants were found positive for P. falciparum by the RDT compared to 189 (30.4%) by microscopy. The sensitivity and specificity of the RDT were 90.5% and 96.1%, respectively. Eighty-six patients completed the AS+SP in-vivo study, with a cure rate of 96.5% (94.2% PCR-uncorrected). However, the efficacy of gametocyte clearance was poor, with gametocytes persisting throughout the study in some patients. All the isolates sequenced had the pfk13 propeller domain wild-type allele, and mutations associated with SP failure were observed only for pfdhfr, with the double mutation (S108N+N51I) was reported in 65.4% of the isolates. Pfcrt gene showed wide prevalence of mutations, with the predominance of pfcrt 76T CQ resistance (97.7%). Mutated pfcrt haplotypes were highly prevalent (98.8%) with CVIET classic, old-world African/Southeast Asian haplotype being the most predominant, and was mostly found in the isolates from Khamis Bani Saad and AdDahi districts (93.1% and 88.9% respectively). Interestingly, the SVMNT new-world South American haplotype was exclusively detected in isolates from Bajil districts of Hodeidah (9.3%). Mutations at Y184F of pfmdr1 were found at a fixation level (100%) in all districts, while mutations of codons 1034C and 86Y were only found in the isolates from AdDahi and Khamis Bani Saad districts. In conclusion, CareStartTM Malaria HRP2-based RDT showed high level of sensitivity and specificity in malaria-endemic areas in Yemen. Moreover, AS+SP therapy remains effective for the treatment of uncomplicated falciparum malaria iv with poor gametocidal activity. No polymorphism in pfk13 was detected in all isolates studied. Adding a single dose primaquine, which minimizes transmission potential, to the current ACT drug policy is strongly recommended. The high prevalence of mutations in pfcrt, 5 years after official cessation of CQ suggests a sustained CQ pressure on P. falciparum isolates in the study area. Moreover, the low prevalence of mutations in the pfmdr1 gene could be a good indicator of the high susceptibility of P. falciparum isolates to antimalarials other than CQ. Therefore, a new strategy to ensure the complete nationwide withdrawal of CQ from the private drug market is recommended

Awareness of cervical cancer and its associated socio-demographic factors among Yemeni immigrant women in Malaysia

Abstract Background Studies have revealed that a higher proportion of women affected by cervical cancer are from some minority groups of immigrant women. Hence, this study was conducted to assess Yemeni immigrant women’s awareness of cervical cancer and its associated socio-demographic factors. Methods A cross-sectional study was conducted among 370 Yemeni women in Selangor and Kuala Lumpur, Malaysia. Data on the awareness of symptoms/signs, risk factors, and screening programme were collected using Cervical Cancer Awareness Measurement (Cervical CAM) questionnaire. Results More than 74% of the study participants were unable to recall any warning symptoms/signs, and 73% were unable to recall any risk factors. The factors associated with the awareness of symptoms and risk factors were age (95% CI 4.22–5.22, p = 0.039), marital status (95% CI 4.05–7.87, p = 0.021), employment (95% CI 3.89–5.77, p = 0.046) and the number of children (95% CI 5.33–6.54, p = 0.041). Conclusion The findings underline the need for public awareness campaigns to improve public awareness of cancer symptoms and risk factors among underserved communities

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

Systematic Review and Meta-Analysis: Epidemiology of Human <i>Blastocystis</i> spp. Infection in Malaysia

Blastocystis spp. is a unicellular enteric protozoan parasite in humans with a controversial role in disease etiology. It is common in developing countries among immunocompromised patients and people who have close contact with animals. In this study, we have systematically reviewed previous studies on the distribution and genotypes of human Blastocystis infection in Peninsular Malaysia. Studies examining the prevalence of Blastocystis in diverse demographics, including rural, urban, comorbid conditions, and high-risk populations, were taken into consideration. The infection has been reported in nine states; the total percentage of infection was 17.8% (1671/9397), with the most cases in Pahang (27.3%) and the least in Johor (3.4%). Molecular studies revealed the presence of six subtypes: ST1, ST2, ST3, ST4, ST5, and ST6. ST3 was reported as the predominant subtype in all the states, with a prevalence of 54.7% (338/618). The findings provide greater clarity on the epidemiology of Blastocystis in Malaysia, which will help in policy making towards planning and strategizing control measures against the parasite

Occurrence of Acanthamoeba genotypes in Central West Malaysian environments

Acanthamoeba species are ubiquitous free-living protozoa that can be found worldwide. Occasionally, it can become parasitic and the causative agent of acanthamoebic keratitis (AK) and Granulomatous Amoebic Encephalitis (GAE) in man. A total of 160 environmental samples and 225 naturally-infected animal corneal swabs were collected for Acanthamoeba cultivation. Acanthamoeba was found to be high in samples collected from environments (85%, 136/160) compared to infected animal corneas (24.89%, 56/225) by microscopic examination. Analysis of nucleotide sequence of 18S rRNA gene of all the 192 cultivable Acanthamoeba isolates revealed 4 genotypes (T3, T4. T5 and T15) with T4 as the most prevalent (69.27%, 133/192) followed by T5 (20.31%), T15 (9.90%) and T3 (0.52%). Genotype T4 was from the strain of A. castellanii U07401 (44.27%), A. castellanii U07409 (20.83%) and A. polyphagaAY026243 (4.17%), but interestingly, only A. castellanii U07401 was detected in naturally infected corneal samples. In environmental samples, T4 was commonly detected in all samples including dry soil, dust, wet debris, wet soil and water. Among the T4, A. castellanii (U07409) strains were detected high occurrence in dry (45%) followed by aquatic (32.50%) and moist (22.50%) samples but however A. castellanii (U07401) strains were dominant in dry samples of soil and dust (93.10%). Subsequently, genotype T5 of A. lenticulata (U94741) strains were dominant in samples collected from aquatic environments (58.97%). In summary, A. castellanii (U07401) strains were found dominant in both environmental and corneal swab samples. Therefore, these strains are possibly the most virulent and dry soil or dusts are the most possible source of Acanthamoeba infection in cats and dogs corneas

Epidemiology of intestinal polyparasitism among Orang Asli school children in rural Malaysia.

This cross-sectional study aimed to investigate the current prevalence and risk factors associated with intestinal polyparasitism (the concurrent infection with multiple intestinal parasite species) among Orang Asli school children in the Lipis district of Pahang state, Malaysia.Fecal samples were collected from 498 school children (50.6% boys and 49.4% girls), and examined by using direct smear, formalin-ether sedimentation, trichrome stain, modified Ziehl Neelsen stain, Kato-Katz, and Harada Mori techniques. Demographic, socioeconomic, environmental, and personal hygiene information were collected by using a pre-tested questionnaire. Overall, 98.4% of the children were found to be infected by at least one parasite species. Of these, 71.4% had polyparasitism. The overall prevalence of Trichuris trichiura, Ascaris lumbricoides, hookworm, Giardia duodenalis, Entamoeba spp., and Cryptosporidium spp. infections were 95.6%, 47.8%, 28.3%, 28.3%, 14.1% and 5.2%, respectively. Univariate and multivariate analyses showed that using an unsafe water supply as a source for drinking water, presence of other family members infected with intestinal parasitic infections (IPI), not washing vegetables before consumption, absence of a toilet in the house, not wearing shoes when outside, not cutting nails periodically, and not washing hands before eating were significant risk factors associated with intestinal polyparasitism among these children.Intestinal polyparasitism is highly prevalent among children in the peninsular Malaysian Aboriginal communities. Hence, effective and sustainable control measures, including school-based periodic chemotherapy, providing adequate health education focused on good personal hygiene practices and proper sanitation, as well as safe drinking water supply should be implemented to reduce the prevalence and consequences of these infections in this population