Frequencies distribution of dihydrofolate reductase and dihydropteroate synthetase mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum population from Hadhramout Governorate, Yemen

Background: Malaria in Yemen is mainly caused by Plasmodium falciparum and 25 % of the population is at high risk. Sulfadoxine-pyrimethamine (SP) had been used as monotherapy against P. falciparum. Emergence of chloroquine resistance led to the shift in anti-malarial treatment policy in Yemen to artemisinin-based combination therapy, that is artesunate (AS) plus SP as first-line therapy for uncomplicated malaria and artemether-lumefantrine as second-line treatment. This study aimed to screen mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes associated with SP resistance among P. falciparum population in Hadhramout governorate, Yemen. Methods: Genomic DNA was extracted from dried blood spots of 137 P. falciparum isolates collected from a community-based study. DNA was amplified using nested polymerase chain reaction (PCR) and subsequently sequenced for Pfdhfr and Pfdhps genes. Sequences were analysed for mutations in Pfdhfr gene codons 51, 59, 108, and 164 and in Pfdhps gene codons 436, 437, and 540. Results: A total of 128 and 114 P. falciparum isolates were successfully sequenced for Pfdhfr and Pfdhps genes, respectively. Each Pfdhfr mutant allele (I-51 and N-108) in P. falciparum population had a frequency of 84 %. Pfdhfr R-59 mutant allele was detected in one isolate. Mutation at codon 437 (G(437)) in the Pfdhps gene was detected in 44.7 % of falciparum malaria isolates. Frequencies of Pfdhfr double mutant genotype ((I) under bar C-51(59)(N) under bar I-108(164)) and Pfdhfr/Pfdhps triple mutant genotype ((I) under bar C-51(59)(N) under bar I-108(164)-S-436(G) under bar K-437(540)) were 82.8 and 39.3 %, respectively. One isolate harboured Pfdhfr triple mutant genotype ((I) under bar (51),(R) under bar (59),(N) under bar (108),I-164) and Pfdhfr/Pfdhps quadruple mutant genotype ((I) under bar (51)(R) under bar (59)(N) under bar I-108(164)-S-436(G) under bar K-437(540)). Conclusion: High frequencies of Pfdhfr and Pfdhps mutant alleles and genotypes in P. falciparum population in Hadhramout, Yemen, highlight the risk of developing resistance for SP, the partner drug of AS, which subsequently will expose the parasite to AS monotherapy increasing then the potential of the emergence of AS resistance. Study findings necessitate the continuous monitoring of the efficacy of the national anti-malarial drugs policy in Yemen. In addition, monitoring SP efficacy using molecular markers that has shown to be a practical and informative method for monitoring the partner drug of AS

Detection and molecular characterization of Giardia isolated from recreational lake water in Malaysia

Nine 50-l surface water samples from a Malaysian recreational lake were examined microscopically using an immunomagnetisable separation-immunofluorescent method. No Cryptosporidium oocysts were detected, but 77.8 of samples contained low numbers of Giardia cysts (range, 0.17-1.1 cysts/l), which were genetically characterised by SSU rRNA gene sequencing. Genotype analyses indicated the presence of Giardia duodenalis assemblage A suggesting potential risk to public health. The present study represents the first contribution to our knowledge of G. duodenalis assemblages in Malaysian recreational water

Molecular characterization of Giardia duodenalis in Yemen

Giardia duodenalis is an important intestinal protozoan in Yemen with infection rates ranging from 18 to 27. To date, there has been no genotyping study to provide a better understanding of the transmission dynamic. This study was conducted to genotype and subtype G. duodenalis in Yemen. Stool samples were collected from 503 Yemeni outpatients between I and 80 years old, including 219 males and 284 females. Giardia cysts were detected via microscopy after the formal-ether concentration. Genotyping of Giardia was carried out using PCR and sequence analysis of the 16s rRNA and b-giardin genes. Of the 89 microscopy-positive Giardia samples, 65 were successfully sequenced, of which 66 (43 of 65) were identified as G. duodenalis assemblage A and 34 (22 of 65) as assemblage B. Further subtyping analysis based on b-giardin gene identified the presence of subtypes A2 and A3, which belong to the anthroponotic sub-assemblage All. Data of the study suggest that anthroponotic transmission played a potential role in the transmission of giardiasis in the community. However, further genotyping and subtyping studies of specimens from humans and animals living in the same households are needed for a more definitive understanding of giardiasis transmission in Yemen. (C) 2013 Elsevier Inc. All rights reserved

Childhood Cryptosporidium infection among aboriginal communities in Peninsular Malaysia

Cryptosporidium is a coccidian parasite that is prevalent worldwide, some species of which cause morbidity in both immunocompromised and immunocompetent individuals. The prevalence and predictors of Cryptosporidium infection, and its effect on nutritional status, have recently been explored among 276 children (141 boys and 135 girls, aged 2-15 years) in aboriginal (Orang Asli) villages in the Malaysian state of Selangor. Faecal smears were examined by the modified Ziehl-Neelsen staining technique while socio-economic data were collected using a standardized questionnaire. Nutritional status was assessed by anthropometric measurements. Cryptosporidium infection, which was detected in 7.2 of the aboriginal children, was found to be significantly associated with low birthweight (2 years). The output of a binary logistic regression confirmed that large household size was a significant predictor of Cryptosporidium infection (giving an odds ratio of 2.15, with a 95 confidence interval of 1.25-5.02). Cryptosporidium infection is clearly a public-health problem among the aboriginal children of Selangor, with person-to-person the most likely mode of transmission

Human Malaria in the highlands of Yemen

Between June 2008 and March 2009, a cross-sectional study of human malaria was carried out in four governorates of Yemen, two (Taiz and Hodiedah) representing the country's highlands and the others (Dhamar and Raymah) the country's coastal plains/foothills. The main aims were to determine the prevalences of Plasmodium infection among 455 febrile patients presenting for care at participating health facilities and to investigate the potential risk factors for such infection. Malarial infection was detected in 78 (17.1) of the investigated patients and was more likely to be detected among the febrile patients from the highlands than among those presenting in the coastal plains/foothills (22.6 v. 13.9; chi(2)=10.102; P=0.018). Binary logistic-regression models identified low household income odds ratio (OR)=13.52; 95% confidence interval (CI)=2.62-69.67; P=0.002, living in a household with access to a water pump (OR=4.18; CI=1.60-10.96; P=0.004) and living in a household near a stream (OR=4.43; CI=1.35-14.56; P=0.014) as significant risk factors for malarial infection in the highlands. Low household income was the only significant risk factor identified for such infection in the coastal plains and foothills (OR=8.20; CI=1.80-37.45; P=0.007). It is unclear why febrile patients in the highlands of Yemen are much more likely to be found to have malarial infection than their counterparts from the coastal plains and foothills. Although it is possible that malarial transmission is relatively intense in the highlands, it seems more likely that, compared with those who live at lower altitudes, those who live in the highlands are less immune to malaria, and therefore more likely to develop febrile illness following malarial infection. Whatever the cause of the symptomatic malarial infection commonly found in the highlands of Yemen, it is a matter of serious concern that should be addressed in the national strategy to control malaria

Multilocus genotyping of giardia duodenalis in Malaysia. Infection, genetics and evolution

Giardia duodenalis is considered the most common intestinal parasite in humans worldwide. In Malaysia, 26 many studies have been conducted on the epidemiology of giardiasis. However, there is a scarcity of 27 information on the genetic diversity and the dynamics of transmission of G. duodenalis. The present study 28 was conducted to identify G. duodenalis assemblages and sub-assemblages based on multilocus analysis 29 of the glutamate dehydrogenase (gdh), beta-giardin (bg) and triose phosphate isomerase (tpi) genes. Fae- 30 cal specimens were collected from 484 Orang Asli children with a mean age of 7 years and examined 31 using light microscopy. Specimens positive for Giardia were subjected to PCR analysis of the three genes 32 and subsequent sequencing in both directions. Sequences were edited and analysed by phylogenetic 33 analysis. G. duodenalis was detected in 17% (84 of 484) of the examined specimens. Among them, 71 were 34 successfully sequenced using at least one locus. Genotyping results showed that 30 (42%) of the isolates 35 Q2 belonged to assemblage A, 32 (45%) belonged to assemblage B, while discordant genotype results were 36 observed in 9 specimens. Mixed infections were detected in 43 specimens using a tpi-based assemblage 37 specific protocol. At the sub-assemblages level, isolates belonged to assemblage A were AII. High nucle- 38 otide variation found in isolates of assemblage B made subtyping difficult to achieve. The finding of 39 assemblage B and the anthroponotic genotype AII implicates human-to-human transmission as the most 40 possible mode of transmission among Malaysian aborigines. The high polymorphism found in isolates of 41 assemblage B warrants a more defining tool to discriminate assemblage B at the sub-assemblage level

First molecular characterization of Giardia duodenalis from goats in Malaysia

In the present study, 310 faecal samples from goats from eight different farms in Malaysia were tested for the presence of Giardia using a PCR-coupled approach. The nested PCR for SSU amplified products of the expected size (�200 bp) from 21 of 310 (6.8) samples. Sixteen of these 21 products could be sequenced successfully and represented six distinct sequence types. Phylogenetic analysis of the SSU sequence data using Bayesian Inference (BI) identified Giardia assemblages A, B and E. The identification of the 'zoonotic' assemblages A and B suggests that Giardia-infected goats represent a possible reservoir for human giardiasis in Malaysia

Multi locus genotyping of Giardia duodenalis in Malaysia

Giardia duodenalis is considered the most common intestinal parasite in humans worldwide. In Malaysia, many studies have been conducted on the epidemiology of giardiasis. However, there is a scarcity of information on the genetic diversity and the dynamics of transmission of G. duodenalis. The present study was conducted to identify G. duodenalis assemblages and sub-assemblages based on multilocus analysis of the glutamate dehydrogenase (gdh), beta-giardin (bg) and triose phosphate isomerase (tpi) genes. Faecal specimens were collected from 484 Orang Asli children with a mean age of 7 years and examined using light microscopy. Specimens positive for Giardia were subjected to PCR analysis of the three genes and subsequent sequencing in both directions. Sequences were edited and analysed by phylogenetic analysis. G. duodenalis was detected in 17 (84 of 484) of the examined specimens. Among them, 71 were successfully sequenced using at least one locus. Genotyping results showed that 30 (42) of the isolates belonged to assemblage A, 32 (45) belonged to assemblage B, while discordant genotype results were observed in 9 specimens. Mixed infections were detected in 43 specimens using a tpi-based assemblage specific protocol. At the sub-assemblages level, isolates belonged to assemblage A were AII. High nucleotide variation found in isolates of assemblage B made subtyping difficult to achieve. The finding of assemblage B and the anthroponotic genotype All implicates human-to-human transmission as the most possible mode of transmission among Malaysian aborigines. The high polymorphism found in isolates of assemblage B warrants a more defining tool to discriminate assemblage B at the sub-assemblage level. (C) 2013 Elsevier B.V. All rights reserved

Field evaluation of a PfHRP-2/pLDH rapid diagnostic test and light microscopy for diagnosis and screening of falciparum malaria during the peak seasonal transmission in an endemic area in Yemen

Background: Malaria is a public health threat in Yemen, with 149,451 cases being reported in 2013. Of these, Plasmodium falciparum represents 99 %. Prompt diagnosis by light microscopy (LM) and rapid diagnostic tests (RTDs) is a key element in the national strategy of malaria control. The heterogeneous epidemiology of malaria in the country necessitates the field evaluation of the current diagnostic strategies, especially RDTs. Thus, the present study aimed to evaluate LM and an RDT, combining both P. falciparum histidine-rich protein-2 (PfHRP-2) and Plasmodium lactate dehydrogenase (pLDH), for falciparum malaria diagnosis and survey in a malaria-endemic area during the transmission season against nested polymerase chain reaction (PCR) as the reference method. Methods: A household-based, cross-sectional malaria survey was conducted in Mawza District, a malaria-endemic area in Taiz governorate. A total of 488 participants were screened using LM and PfHRP-2/pLDH RDT. Positive samples (160) and randomly selected negative samples (52) by both RDT and LM were further analysed using 18S rRNA-based nested PCR. Results: The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the RDT were 96.0 % (95 % confidence interval (CI): 90.9-98.3), 56.0 % (95 % CI: 44.7-66.8), 76.3 % (95 % CI: 69.0-82.3), and 90.4 % (95 % CI: 78.8-96.8), respectively. On the other hand, LM showed sensitivity of 37.6 % (95 % CI: 29.6-46.3), specificity of 97.6 % (95 % CI: 91.7-99.7), PPV of 95.9 % (95 % CI: 86.3-98.9), and NPV of 51.3 % (95 % CI: 43.2-59.2). The sensitivity of LM dropped to 8.5 % for detecting asymptomatic malaria. Malaria prevalence was 32.8 % (32.1 and 37.5 % for ≥10 and <10 years, respectively) with the RDT compared with 10.7 % (10.8 and 9.4 % for age groups of ≥10 and <10 years, respectively) with LM. Among asymptomatic malaria individuals, LM and RDT-based prevalence rates were 1.6 and 25.6 %, respectively. However, rates of 88.2 and 94.1 % of infection with P. falciparum were found among patients who reported fever in the 48 h prior to the survey by LM and PfHRP-2/pLDH RDT, respectively. Conclusions: The PfHRP-2/pLDH RDT shows high sensitivity for the survey of falciparum malaria even for asymptomatic malaria cases. Although the RDT had high sensitivity, its high false-positivity rate limits its utility as a single diagnostic tool for clinical diagnosis of malaria. On the other hand, low sensitivity of LM indicates that a high proportion of malaria cases is missed, underestimating the true prevalence of malaria in the community. Higher NPV of PfHRP-2/pLDH RDT than LM can give a straightforward exclusion of malaria among febrile patients, helping to avoid unnecessary presumptive treatments

Does vitamin A supplementation protect schoolchildren from acquiring soil-transmitted helminthiasis? A randomized controlled trial

Background Despite the intensive global efforts to control intestinal parasitic infections, the prevalence of soil-transmitted helminth (STH) infections is still very high in many developing countries particularly among children in rural areas. Methods A randomized, double-blind, placebo-controlled trial was conducted on 250 Aboriginal schoolchildren in Malaysia to investigate the effects of a single high-dose of vitamin A supplementation (200 000 IU) on STH reinfection. The effect of the supplement was assessed at 3 and 6 months after receiving interventions; after a complete 3-day deworming course of 400 mg/daily of albendazole tablets. Results Almost all children (98.6%) were infected with at least one STH species. The overall prevalence of ascariasis, trichuriasis and hookworm infection was 67.8%, 95.5% and 13.4%, respectively. Reinfection rates of Ascaris, Trichuris and hookworm were high; at 6 months, assessment reached 80% of the prevalence reported before treatment. There were no significant differences in the reinfection rates and intensities of STH between vitamin A supplemented-children and those who received placebo at 3 and 6 months (p > 0.05). Conclusions Vitamin A supplementation showed no protective effect against STH reinfection and this could be due to the high endemicity of STH in this community. Long-term interventions to reduce poverty will help significantly in reducing this continuing problem and there is no doubt that reducing intestinal parasitic infection would have a positive impact on the health, nutrition and education of these children