Molecular epidemiology of giardiasis among Orang Asli in Malaysia: application of the triosephosphate isomerase gene

Anuar et al. BMC Infectious Diseases 2014, 14:78 http://www.biomedcentral.com/1471-2334/14/78 RESEARCH ARTICLE Open Access Molecular epidemiology of giardiasis among Orang Asli in Malaysia: application of the triosephosphate isomerase gene Tengku Shahrul Anuar1, Siti Nor Azreen2, Fatmah Md Salleh2 and Norhayati Moktar2* Abstract Background: Giardia duodenalis is a flagellate parasite which has been considered the most common protozoa infecting human worldwide. Molecular characterization of G. duodenalis isolates have revealed the existence of eight groups (Assemblage A to H) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals. Methods: This cross-sectional study was conducted to identify assemblage’s related risk factors of G. duodenalis among Orang Asli in Malaysia. Stool samples were collected from 611 individuals aged between 2 and 74 years old of whom 266 were males and 345 were females. Socioeconomic data were collected through a pre-tested questionnaire. All stool samples were processed with formalin-ether sedimentation and Wheatley’s trichrome staining techniques for the primary identification of G. duodenalis. Molecular identification was carried out by the amplification of a triosephosphate isomerase gene using nested-PCR assay. Results: Sixty-two samples (10.2%) were identified as assemblage A and 36 (5.9%) were assemblage B. Risk analysis based on the detected assemblages using univariate and logistic regression analyses identified subjects who have close contact with household pets i.e. dogs and cats (OR = 2.60; 95% CI = 1.42, 4.78; P = 0.002) was found to be significant predictor for assemblage A. On the other hand, there were three significant risk factors caused by assemblage B: (i) children ≤15 years old (OR = 2.33; 95% CI = 1.11, 4.87; P = 0.025), (ii) consuming raw vegetables (OR = 2.82; 95% CI = 1.27, 6.26; P = 0.011) and (iii) the presence of other family members infected with giardiasis (OR = 6.31; 95% CI = 2.99, 13.31; P < 0.001). Conclusions: The present study highlighted that G. duodenalis infection among Orang Asli was caused by both assemblages with significant high prevalence of assemblage A. Therefore, taking precaution after having contact with household pets and their stool, screening and treating infected individuals, awareness on the importance of good health practices and washing vegetables are the practical intervention ways in preventing giardiasis in Orang Asli community. Keywords: Giardia duodenalis, Assemblage, Risk factors, Orang Asli, Malaysia * Correspondence: 2 Department of Parasitology and Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia Full list of author information is available at the end of the article © 2014 Anuar et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Anuar et al. BMC Infectious Diseases 2014, 14:78 http://www.biomedcentral.com/1471-2334/14/78 Background Giardia is a genus of intestinal flagellates that infects a wide range of vertebrate hosts. The genus currently comprises six species, namely Giardia agilis in amphibians, Giardia ardeae and Giardia psittaci in birds, Giardia microti and Giardia muris in rodents and Giardia duodenalis in mammals. These species are distinguished on the basis of the morphology and ultrastructure of their trophozoite [1]. Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia) is the only species within the genus Giardia that infects humans, although it is also found in other mammals including pets and livestock [2]. The infection has a global distribution, with an estimated 2.8 × 108 cases per year, represents the most common gastrointestinal parasitic infection of humans in developed countries [3]. In Asia, Africa and Latin America, about 200 million people have symptomatic giardiasis with some 500,000 new cases reported each year [4]. There are four characteristics of G. duodenalis that influence the epidemiology of infection: (i) the infective dose is about 10 to 100 cysts in humans; (ii) cysts are immediately infectious when excreted in stool and can be transmitted by human-to-human or animal-toanimal contact; (iii) cysts are remarkably stable and can survive for weeks to months in the environment and (iv) environmental contamination can lead to the contamination of drinking water and food [5,6]. A considerable amount of data has shown that G. duodenalis should be considered a species complex whose members show little variation in their morphology yet can be assigned to at least eight distinct assemblages (A to H) based on genetic analyses [7,8]. The analysis of more than a thousand human isolates from different geographical locations, examined by PCR amplification of DNA extracted directly from stool has demonstrated that in almost all cases, only G. duodenalis assemblages A and B are associated with human infections [5]. The prevalence of each assemblage varies considerably from country to country; assemblage B seems more common overall, but no strong conclusions can be drawn from current data. The remaining assemblages (C to G) are likely to be host specific, as assemblages C and D have been identified in dogs, cats, coyotes and wolves; assemblage E in cattle, sheep, goats, pigs, water buffaloes and muflons; assemblage F in cats and assemblage G in rats. In Malaysia, giardiasis is an endemic disease and is associated with malnutrition among children in the rural areas resulting in stunting, wasting and vitamin A deficiency [9,10]. The prevalence of giardiasis varies between 0.2 to 20% [11-13]. Most of the epidemiological studies detected G. duodenalis on the basis of microscopic examination without employing molecular approach. Data on genotypes of G. duodenalis up to the assemblage level remains scarce. In a previous genotyping study using SSU rRNA locus, one specimen was identified as assemblage A Page 2 of 12 in 42 specimens and the rest were assemblage B [14]. In a study on immunocompromised patients, assemblage A was identified in four of the microscopy-positive specimens using triosephosphate isomerase (tpi) gene [15]. Assemblage A was also isolated from environmental samples including recreational lake water and water bodies in a zoo [16,17]. In addition, genotyping study was conducted on animals and assemblages A and E were detected among goats [18]. Recent study conducted by Huey et al. [19] based on multilocus analysis revealed that 42% of the Orang Asli isolates belong (...truncated)