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      Risk Profiling of Hookworm Infection and Intensity in Southern Lao People’s Democratic Republic Using Bayesian Models

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          Abstract

          Background

          Among the common soil-transmitted helminth infections, hookworm causes the highest burden. Previous research in the southern part of Lao People’s Democratic Republic (Lao PDR) revealed high prevalence rates of hookworm infection. The purpose of this study was to predict the spatial distribution of hookworm infection and intensity, and to investigate risk factors in the Champasack province, southern Lao PDR.

          Methodology

          A cross-sectional parasitological and questionnaire survey was conducted in 51 villages. Data on demography, socioeconomic status, water, sanitation, and behavior were combined with remotely sensed environmental data. Bayesian mixed effects logistic and negative binomial models were utilized to investigate risk factors and spatial distribution of hookworm infection and intensity, and to make predictions for non-surveyed locations.

          Principal Findings

          A total of 3,371 individuals were examined with duplicate Kato-Katz thick smears and revealed a hookworm prevalence of 48.8%. Most infections (91.7%) were of light intensity (1-1,999 eggs/g of stool). Lower hookworm infection levels were associated with higher socioeconomic status. The lowest infection levels were found in preschool-aged children. Overall, females were at lower risk of infection, but women aged 50 years and above harbored the heaviest hookworm infection intensities. Hookworm was widespread in Champasack province with little evidence for spatial clustering. Infection risk was somewhat lower in the lowlands, mostly along the western bank of the Mekong River, while infection intensity was homogeneous across the Champasack province.

          Conclusions/Significance

          Hookworm transmission seems to occur within, rather than between villages in Champasack province. We present spatial risk maps of hookworm infection and intensity, which suggest that control efforts should be intensified in the Champasack province, particularly in mountainous areas.

          Author Summary

          Hookworm is a parasitic worm that can infect the human intestine. Infections are common in low- and middle-income tropical and subtropical countries, particularly where sanitation conditions are poor. Control programs aim at preventing heavy infection intensities, which are associated with morbidity. The aim of this study was to investigate risk factors for hookworm infection and intensity, and to predict their distribution in the Champasack province, southern Lao People’s Democratic Republic. We found that almost half of the 3,371 people examined were infected with hookworms. Better-off people had a lower risk and intensity of infection. Females showed lower prevalence and intensity of hookworm infection than males, except women aged 50 years and above, who were the most heavily infected. Preschool-aged children had the lowest infection levels. Hookworm transmission appeared to occur within, rather than between villages. Hookworm infections are widespread in the Champasack province, with homogeneous infection intensity and somewhat lower infection risk west of the Mekong River, which offers higher living conditions. Hookworm control should be intensified in the Champasack province, particularly in the mountainous areas.

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          Most cited references 13

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          Impact of hookworm infection and deworming on anaemia in non-pregnant populations: a systematic review

          Objectives To summarise age- and intensity-stratified associations between human hookworm infection and anaemia and to quantify the impact of treatment with the benzimidazoles, albendazole and mebendazole, on haemoglobin and anaemia in non-pregnant populations. Methods Electronic databases (MEDLINE, EMBASE, PubMed) were searched for relevant studies published between 1980 and 2009, regardless of language, and researchers contacted about potential data. Haemoglobin concentration (Hb) was compared between uninfected individuals and individuals harbouring hookworm infections of different intensities, expressed as standardised mean differences (SMD) and 95% confidence intervals (CI). Meta-analysis of randomised control trials (RCTs) investigated the impact of treatment on Hb and anaemia. Results Twenty-three cross-sectional studies, six pre- and post-intervention studies and 14 trials were included. Among cross-sectional studies, moderate- and heavy-intensity hookworm infections were associated with lower Hb in school-aged children, while all levels of infection intensity were associated with lower Hb in adults. Among RCTs using albendazole, impact of treatment corresponded to a 1.89 g/l increase (95%CI: 0.13–3.63) in mean Hb while mebendazole had no impact. There was a positive impact of 2.37 g/l (95%CI: 1.33–3.50) on mean Hb when albendazole was co-administered with praziquantel, but no apparent additional benefit of treatment with benzimidazoles combined with iron supplementation. The mean impact of treatment with benzimidazoles alone on moderate anaemia was small (relative risk (RR) 0.87) with a larger effect when combined with praziquantel (RR 0.61). Conclusions Anaemia is most strongly associated with moderate and heavy hookworm infection. The impact of anthelmintic treatment is greatest when albendazole is co-administered with praziquantel.
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            High Prevalence of Ancylostoma ceylanicum Hookworm Infections in Humans, Cambodia, 2012

            Human infections with Necator americanus and Ancylostoma duodenale hookworms continue to be recognized as a leading cause of iron deficiency anemia and protein malnutrition in developing countries ( 1 ). On the basis of parasitologic surveys of fecal samples, hookworms are estimated to infect 576–740 million persons globally, and over half of the infections occur in Asia and the Pacific regions ( 2 ). Recent molecular-based epidemiologic surveys have shown Ancylostoma ceylanicum to be the second most common hookworm species infecting humans in Asia. In Thailand, Laos, and Malaysia, 6%–23% of persons positive for hookworm eggs were infected with A. ceylanicum helminths ( 3 – 6 ). There are an estimated 19–73 million A. ceylanicum hookworm–infected persons in regions where this zoonotic helminth is known to be endemic ( 7 ). Dogs and cats act as natural reservoirs for hookworm transmission to humans, and the prevalence of A. ceylanicum hookworms in these animals ranges from 24% to 92% in the Asia-Pacific region ( 6 , 8 – 10 ). Much like anthroponotic helminths, A. ceylanicum hookworms have the potential to produce clinical symptoms of ground itch (a pruritic papular hypersensitivity response caused by the entry of helminths into the skin), epigastric pain, diarrhea, and anemia in humans ( 11 – 15 ). However, despite these reports, relatively little is known about the clinical significance and infection dynamics of this zoonotic hookworm in humans, dogs, and cats. Differentiation of the genus of hookworms infecting humans is imperative because each species varies in its biology, life cycle, pathophysiology, and epidemiology, and these differences have key implications when assessing hookworm-associated illnesses and establishing control measures. The internal transcribed spacer (ITS) –1 and –2 regions and the 5.8S region have been used to detect and characterize hookworm infections directly from eggs in human and animal feces ( 6 , 10 , 16 , 17 ). In addition, sequencing of the cytochrome c oxidase subunit 1 (cox1) gene has been successfully used to establish intraspecies genetic differences of many strongylid nematodes, including hookworms ( 18 – 21 ). The aim of our study was to determine the prevalence, associated risk factors, and infection dynamics of hookworm species infection in humans and dogs living in a rural Cambodian village. To carry out this investigation, we used a combination of conventional parasitologic and molecular epidemiologic approaches. Materials and Methods Study Site and Sample Collection The study was conducted in May 2012 in Dong, a rural village in Rovieng District, Preah Vihear Province, Cambodia. Preah Vihear Province is located in northern Cambodia, bordering Thailand and Laos (13°47′N 104°58′E). The climate is tropical; temperatures are warm and hot all year round, and seasons alternate between dry and wet. Subsistence farming (rice, vegetables, and fish) constitutes the primary source of income for the community. Drinking water is sourced from wells, well pumps, and rain water tanks, and just over half of the households own a latrine. All household electricity is battery or generator powered. Approximately half the households feed semidomesticated, free-roaming community dogs. These dogs are allowed to defecate indiscriminately within the village or outside the homes of their owners. The study protocol was approved by the Ethics Committee of the Canton of Basel and Baselland, Switzerland, and the National Ethics Committee Health Research, Ministry of Health, Cambodia. Dong, the village selected for study, had previously been categorized as having endemic soil-transmitted helminths ( 22 ). According to the treatment guidelines of the Cambodian helminths control program, all children attending primary school in the village were administered albendazole (400 mg) and mebendazole (500 mg) twice a year. At completion of the study, all participants who were found positive for Strongyloides spp. were treated with ivermectin (200 μg/kg body weight), and participants infected with other soil-transmitted helminths were treated with albendazole (400 mg). A cross-section of 67 households was randomly selected from a list provided by the Dong village authority. A total of 218 persons from those households were enrolled in the study. Of the 218 persons, 99 (45.4%) were male. The average age of participants was 30.0 years (range 2–84); female participants were marginally older, on average, than male participants (30.3 vs. 29.8 years of age). On the first day of the study, informed consent was obtained from the enrolled participants, and questionnaires were administered during interviews. Interviews with children (i.e., participants 2–17 years of age) were conducted with the assistance of a parent or legal guardian. All study participants responded to a questionnaire covering demographics, dietary habits, personal hygiene, and level of household income and assets. Prelabeled stool containers were distributed to the 218 study participants for collection of feces on the second morning of the study. Fecal samples were collected from participants’ dogs (N = 94), when applicable. Samples (≈3–5 g) from dogs were collected directly from the rectum at time of the participant interview and placed into a sterile plastic container. If insufficient stool was obtained from a dog, the animal was confined within the owner’s property and resampled on the second morning of the study. All fecal samples were chilled immediately in a cool box and transported to a laboratory in Rovieng Health Center (Rovieng District, Preah Vihear Province) within 2 h after collection. After fecal samples arrived at the laboratory, a minimum of 2 g of each sample was placed into a 15-mL centrifuge tube containing 10% formaldehyde for parasitologic analysis, and 1–2 g of each sample was placed into a 15-mL centrifuge tube containing 2.5% potassium dichromate for molecular analysis. These samples were then shipped at room temperature to the School of Veterinary Science, University of Queensland, Gatton, Queensland, Australia, for further analysis. Parasitologic Procedures All fecal samples were examined by microscope. The relative intensity of hookworm infection, in eggs per gram, was determined by floatation, using a sodium nitrate solution (specific gravity 1.20) ( 23 ). DNA Extraction Genomic DNA was extracted directly from fecal samples by using the PowerSoil DNA Isolation Kit (Mo Bio, Carlsbad, CA, USA) according to the manufacturer’s instructions, with the exception that fecal samples were subjected to a 5-min disruption by using 0.5-mm Zirconia/Silica beads (BioSpec Products, Inc., Bartlesville, OK, USA) instead of the beads provided by the manufacturer. Final elution of DNA was made in 100 μL of elution buffer. The extracted DNA was stored at −20°C until required for PCR amplification. Molecular Characterization of Hookworm species in Humans PCR was conducted by using primers RTHW1F and RTHW1R ( 10 ) in 25-μL volumes; each final reaction contained 1× CoralLoad PCR Buffer (QIAGEN Pty Ltd, Hilden, Germany), 12.5 pmol of each primer, 0.5 U of HotStar Taq DNA Polymerase (QIAGEN), and 2 μL of DNA. The cycling conditions were the same as the published protocol ( 10 ) except for an initial denaturation of 5 min at 95°C. A positive control of N. americanus and A. ceylanicum hookworms and negative controls of distilled water were included in each run. PCR amplicons that were ≈380 bp in size, corresponding to Ancylostoma spp. hookworms, were purified by using the PureLink Quick PCR Purification Kit (Life Technologies, Carlsbad, CA, USA) and submitted to the University of Queensland Animal Genetics Laboratory, Gatton, for bidirectional DNA sequencing. Molecular Characterization of Hookworms species in Dogs PCR–restriction fragment length polymorphism (RFLP) characterization of hookworms from dogs was carried out as described ( 17 , 24 ). In brief, RTGHFI and RTABCR1 primers were used to amplify a 545-bp region of ITS-1, 5.8S, and ITS-2 of A. caninum, A. ceylanicum, and Uncinaria stenocephala hookworms. In a separate PCR, a 673-bp region of an A. braziliense hookworm was amplified by using RTGHF1 and a specific reverse primer, RTAYR1. Both PCR reactions consisted of 1× CoralLoad PCR Buffer (QIAGEN), 12.5 pmol of each primer, 0.2 μL of 20 mg/mL bovine serum albumin, 2 μL DNA, and 1 U of HotStar Taq Polymerase (QIAGEN) in a 25-μL reaction. The cycling conditions were as published ( 17 , 24 ), except for an initial denaturation time of 5 min at 95°C. Amplified PCR product (10 μL; RTGHF1/RTABCR1) was digested with HinFI and RsaI endonucleases in separate reactions at 37°C for 3 h. The RFLP patterns generated by each sample were then compared to the expected RFLP profiles for each hookworm species. PCR and DNA Sequencing of cox1 of A. ceylanicum Hookworm Samples from dogs and humans that were positive for A. ceylanicum hookworms were further characterized to a haplotype level by analysis of the mitochondrial gene (cox1). AceyCOX1F (5′-GCTTTTGGTATTGTA-AGACAG-3′) and AceyCOX1R (5′- CTAACAACATAATAAG-TATCATG-3′) were specifically designed to amplify a 377-bp region of the cox1 gene of A. ceylanicum hookworm. The PCR was carried out in 25-μL volumes, with each reaction containing 1× CoralLoad PCR Buffer, 12.5 pmol of each primer, 0.5 U of HotStar Taq DNA polymerase, and 2 μL of DNA. The cycling conditions were 95°C for 5 min, followed by 50 cycles at 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 7 min. A positive control of A. ceylanicum hookworm and a negative control of distilled water were included in the run. PCR-positive samples were purified by using the PureLink Quick PCR Purification Kit according to the manufacturer’s protocol. Bidirectional DNA sequencing was performed by the University of Queensland Animal Genetics Laboratory. Phylogenetic Analyses DNA sequences were analyzed by using the Finch TV version 1.4.0 trace viewer (Geospiza, Inc., Seattle, WA, USA) and aligned by using BioEdit version 7.2.0 (www.mbio.ncsu.edu/BioEdit/bioedit.html) together with the cox1 gene sequences from the following hookworm species: A. ceylanicum Malaysia isolates (GenBank accession nos. KC247727– KC247745, Pos Iskandar [Human] and Sg Bumbun [Human]); A. caninum and A. duodenale (GenBank accession nos. NC012309 and NC003415, respectively); and A. ceylanicum Thailand genotype (GenBank accession no. KF896595). Neighbor-joining analyses were conducted by using Tamura-Nei parameter distance estimates, and the tree was constructed by using Mega4.1 (www.megasoftware.net). Bootstrap analyses were conducted using 1,000 replicates. Statistical Analyses We used STATA version 12 (StataCorp LP, College Station, TX, USA) for data entry and statistical analyses. The prevalence of hookworm infection was calculated by using descriptive statistics for microscopy and molecular results. A univariate model was used to assess potential risk factors associated with hookworm infection; odds ratios and 95% CI were reported. The level of statistical significance was set at p<0.05. Factors that were significant in univariate analysis were evaluated by multivariate analysis, when applicable. Results Prevalence of Hookworm Infection The prevalence of hookworm infection among the 218 persons tested in Dong village was 26.6% (58/218) as determined by microscopic examination and 57.4% (124/218) as determined by PCR based on amplification of the partial ITS gene. Among dogs, 80.9% (76/94) were positive for hookworms by microscopic examination, and 95.7% (90/94) were positive by PCR based on amplification of the partial ITS gene. Molecular Characterization of Hookworm Species Of the 124 persons with positive samples, 64 (51.6%) harbored A. ceylanicum hookworms; 57 (89.0%) of these infections were single infections. An equal percentage of persons, 64 (51.6%), were infected with N. americanus hookworms, mostly as single infections (59/64 [92.2%]), and 4 (3.2%) persons were infected with A. duodenale hookworms (Table). Table Hookworm species found in humans and dogs, Dong village, Rovieng District, Preah Vihear Province, Cambodia, 2012* Infected host, hookworm species No. (%) positive Humans Necator americanus 59 (47.6) Ancylostoma ceylanicum 57 (46.0) A. duodenale 1 (0.8) N. americanus and A. ceylanicum 4 (3.2) A. ceylanicum and A. duodenale 2 (1.6) N. americanus and A. ceylanicum and A. duodenale 1 (0.8) N. americanus and A. duodenale 0 Total 124 (100.) Dogs A. ceylanicum 81 (90.0) A. caninum 5 (5.6) A. ceylanicum and A. caninum 3 (3.3) A. ceylanicum and N. americanus 1 (1.1) Total 90 (100.0) *The presence of hookworms was determined by PCR amplification of the internal transcribed spacer–1 and –2 regions and the 5.8S region and by DNA sequencing. Of the 90 dogs with positive samples, 85 (94.4%) were infected with A. ceylanicum hookworms, mostly (81/85 [95.3%]) as single infections, and 8 (8.9%) were infected with A. caninum hookworms. One dog was found to be shedding N. americanus eggs (Table). Phylogenetic Analysis of cox1 Gene of A. ceylanicum Of 68 human and 82 dog samples positive for hookworms, 28 (41.2%) and 65 (79.3%), respectively, were successfully amplified at the cox1 gene. Of these, 21 PCR-positive amplicons from human samples and 27 PCR-positive amplicons from dog samples were randomly selected for DNA sequencing and subsequent phylogenetic analysis. The phylogenetic tree distinctly separated into 3 clusters; the A. ceylanicum hookworm isolates grouped together and were genetically distinct from A. caninum hookworm isolates (GenBank accession nos. NC012309 and FJ483518) and A. duodenale hookworm isolates (GenBank accession nos. NC003415 and AJ417718). Within A. ceylanicum hookworm isolates, there was strong bootstrap support (100%) for the division of isolates from various geographic locations into 2 clades. The first clade comprised 4 human isolates, 1 from the current study in Cambodia (Human 19) and 3 previously reported human isolates from Malaysia (GenBank accession no. KC772445; Human [Sg Bumbun]; Human [Gurney] and Human [Pos Iskandar]). The second clade comprised a mix of isolates from humans (n = 20) and dogs (n = 27) from villages in Cambodia; humans (n = 5), dogs (n = 11), and cats (n = 2) from Malaysia ( 21 ); and 1 dog in Thailand (GenBank accession no. KF896595). For human- and dog-derived A. ceylanicum hookworms, representative DNA sequences at each cox1 haplotype were submitted to GenBank under accession nos. KF896596–KF896605 (see sequences marked with asterisks in the Technical Appendix Figure). Age-related Prevalence and Intensity of N. americanus and A. ceylanicum Hookworm Infections The prevalence of N. americanus hookworms peaked in persons 31–50 years of age, whereas the prevalence of A. ceylanicum hookworms peaked in persons 15–20 years age and again in persons 31–50 years of age (Figure). The highest egg intensities for single infections attributed to N. americanus and A. ceylanicum hookworms occurred in persons 21–30 years of age (Figure). Figure Prevalence and intensity (eggs per gram) of Necator americanus and Ancylostoma ceylanicum hookworm infections in humans of different ages in rural Dong village, Rovieng District, Preah Vihear Province, Cambodia, 2012. Risk Factors Associated with Hookworm Infection of Humans and Dogs The results of regression analysis showed an increased risk for hookworm infection in persons who did not wear shoes while defecating (odds ratio 6.0, 95% CI 1.1–28.6; p = 0.038). No significant associations were found between the prevalence and intensity of hookworms by age group, sex, household income, or dietary practices. No risk factors of significance were associated with hookworm infection in dogs. Discussion In this study, zoonotic ancylostomiasis caused by A. ceylanicum hookworms was found to be highly endemic among humans in Dong village, Preah Vihear Province, Cambodia; community dogs were the likely zoonotic reservoir. This finding is in stark contrast to the consistent finding by other molecular-based prevalence studies in the region that N. americanus is the predominant hookworm species in humans, followed by A. ceylanicum and A. duodenale hookworms ( 3 , 5 , 6 , 10 ). PCR proved a superior alternative to microscopy-based techniques for the detection of hookworms in fecal samples ( 25 , 26 ). In Dong village, the prevalence of A. ceylanicum hookworms matched that of their anthroponotic counterpart, N. americanus hookworms, and infections with A. ceylanicum hookworms substantially out-numbered those with A. duodenale hookworms. In addition, most infected persons harbored single-species hookworm infections; just over 10% of hookworm-positive persons had mixed-species infections. These results raise questions about the potential infection dynamics between hookworm species within individual hosts. Our study supports an earlier hypothesis ( 7 ) that anthroponotic hookworms may have a cross-protective role in expelling and preventing the subsequent establishment of A. ceylanicum hookworms via a T helper 2 cell response ( 27 ). The major immunologic action against incoming L3 larvae (infective filariform larvae) and L4 larvae (final larval stage within the intestine) is regulated by the infection itself ( 28 ). Thus, the presence of a stable and long-lived (3–6 years) infection with anthroponotic species ( 29 ) may play a role in providing an unsuitable environment for the establishment of incoming larvae of another closely related (albeit potentially shorter-lived and suboptimally host adapted) species—in this case, A. ceylanicum hookworms. Reduced burdens of anthroponotic hookworm species may also have the added advantage of easing density-dependent intraspecific competition for limited resources within the intestinal niche ( 30 ), leading to an opportunistic establishment of A. ceylanicum hookworms. Although data on the natural life span of A. ceylanicum hookworms in humans do not exist, infections in Dutch servicemen 5 months after their return from New Guinea ( 31 ) suggest that chronic infections with this hookworm may occur. The initiating or causal factor for the emergence of highly endemic levels of monospecific infections with A. ceylanicum hookworms in Dong village remains unclear. In this study, potential causal factors for human infection are likely related to the high levels of A. ceylanicum hookworm infections in community dogs. In rural Malaysia, close contact with community dogs and cats was shown often to be associated with human infection with A. ceylanicum hookworms ( 6 ). In Dong village, dogs were reported to defecate indiscriminately in environments shared with humans, leading to widespread environmental contamination with infective A. ceylanicum hookworm larvae. For humans, defecating while bare foot was shown to be the most significant risk factor for infection with both species of hookworms. Whether these factors, coupled with the administration of preventative chemotherapy, led to an increased opportunity for the A. ceylanicum hookworm to replace the niche of its anthroponotic competitors remains unanswered. Either way, integrated control programs aimed at combining chemotherapeutic interventions with improvements in community hygiene and animal health programs will aid in curbing this potentially opportunistic zoonosis. Molecular epidemiologic data gathered from characterization of the cox1 gene of A. ceylanicum hookworms strongly support previous findings ( 21 ) that A. ceylanicum hookworm isolates from humans and animals formed 2 genetically distinct groups, 1 comprising isolates specific to humans and the other comprising isolates from humans, dogs, and cats. Most A. ceylanicum hookworm isolates from humans in Dong village clustered within the zoonotic haplotype, confirming that transmission from dogs to humans has occurred. Genetic groups inferred by the cox1 gene of A. ceylanicum hookworms were found to be independent of geographic source. Whether the 2 primary haplotypes differ in biologic, epidemiologic, and pathophysiologic characteristic warrants further investigation. The transmission dynamics of A. ceylanicum hookworms in humans of different ages largely paralleled that of N. americanus hookworms: persons 21–30 years of age excreted the highest number of eggs. This highly unexpected finding has key implications. First, this finding suggests that the previous classification of A. ceylanicum as an abnormal and minor hookworm of humans ( 32 ) no longer stands. Second, monospecific infections of humans with <100 A. ceylanicum worms have been reported to cause anemia, even in well-nourished persons ( 14 , 33 ). Thus, attention must be directed to A. ceylanicum infection as a major cause of human illness in areas where this zoonosis is endemic. The zoonotic helminth A. ceylanicum can no longer be classified as an abnormal hookworm of humans. Although previous studies have reported this hookworm’s emergence as the second most common human hookworm species in Southeast Asia, our study demonstrates its ability to infect humans at prevalence and intensity levels at par with that of its anthroponotic competitor, the N. americanus hookworm. We hypothesize that expansion of preventative chemotherapy in the absence of concurrent hygiene and animal health programs is a potential causal factor for the emergence of this zoonosis. Attention must be directed to the effects of A. ceylanicum hookworm infections on human health, and a One Health approach should be adopted for the control of this zoonosis. Technical Appendix Phylogenetic tree of Ancylostoma ceylanicum hookworms from 21 humans and 27 dogs in Cambodia together with reference isolates from Malaysia and Thailand.
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              Epidemiological and Genetic Data Supporting the Transmission of Ancylostoma ceylanicum among Human and Domestic Animals

              Introduction Hookworms are one of the most common parasitic nematodes that inhabit the small intestine of humans and animals such as dogs and cats. The two primary species of hookworm infecting humans are Ancylostoma duodenale and Necator americanus [1], with A. duodenale occurring mainly in the Middle East, North Africa, India, Australia and Europe, whilst N. americanus in the Americas, Sub-Saharan Africa, East Asia and Southeast Asia [2]. The socioeconomic and public health impact of human hookworm infections are extensive, infecting an estimated 600 million people worldwide and resulting in up to 135,000 deaths annually [3]. Infection in human causes iron-deficiency anemia which may result in mental retardation and growth deficiencies, particularly in children [4], [5]. Canine and feline hookworm species are also able to cause zoonotic disease in humans. For example, cutaneous larva migrans (CLM) or ‘creeping eruptions’ is a hypersensitivity reaction caused by migrating nematode larvae, of which Ancylostoma braziliense is the most frequently implicated aetiological agent in humans [6], . Another canine hookworm, Ancylostoma caninum is the leading cause of human eosinophilic enteritis (EE) and an outbreak of 150 cases was reported between 1988 and 1992 in Australia [8]–[10]. Cases have also been reported in the United States [11], Egypt [12], the Philippines, South America and Israel [13]. Ancylostoma ceylanicum however, is the only species of animal hookworm known to produce patent infections in humans. This has been demonstrated both experimentally [14], [15] and naturally. Natural infections with A. ceylanicum have been reported in Dutch servicemen returning from West New Guinea, who suffered heavy infection with concurrent anemia [16], whilst light infections have been mostly reported from humans in the Philippines [17], Taiwan [18], Thailand [19] and India [20]. More recently, zoonotic ancylostomiasis caused by A. ceylanicum was reported in temple and rural communities in Thailand [21], [22] and rural communities in Laos PDR [23] using copro-molecular diagnostic tools. Although hookworm infection is common, especially in rural and remote areas in Malaysia [24], [25], information on the species of hookworms present in humans and domestic animals is currently lacking. Recently, the first data on species-specific hookworm infections was reported by our group [26]. This present study aims to explore the role of domestic animals, particularly dogs and cats as reservoirs for hookworm infection among communities living in rural and remote areas of Malaysia. Materials and Methods Study area The study was carried out for a period of two years (i.e., 2009 to 2011) in 8 villages located in remote areas of West Malaysia which were previously recognized as geohelminth-endemic areas [24]. These villages were selected because (i) the hookworm prevalence in these areas were known to be high and (ii) it is accessible by road for rapid transfer of samples to the laboratory. Details of the studied villages and the populations sampled have been described elsewhere [26]. Cats, dogs and poultry are the most common domestic animals. Some villagers had monkeys, rabbits and birds as pets. The majority of these domestic animals are left to roam freely. The villagers have very close contact with the dogs and cats, even sharing food from the same plate with these animals. Occasionally, these animals also slept, defecated indoors and accompanied the villages into the jungle to harvest jungle products. Consent, structured questionnaire and fecal sample collection The aim and procedures of the study were explained and an informed consent sheet was signed by the head of the household or a designated literate substitute. A structured questionnaire was administered to obtain information on the demography (i.e., age, gender, education attainment), socioeconomic (i.e., occupation, household income), behavioral (i.e., personal hygiene such as wearing shoes, defecation practices), as well as information on the environmental sanitation and living condition characteristics such as type of water supply, latrine system and domestic animal ownership or contact. This questionnaire was designed in English and translated to Bahasa Malaysia, which is the national language for Malaysia and is well understood by the participants. Next, a dry, clean and leak proof screw capped pre-labeled fecal container with the individual's name and code was handed out to all participants for the collection of fecal sample the next day. Their ability to recognize their names was also checked. The participants were also guided on how to collect the sample. For the domestic animals, the owners were informed to collect only fresh fecal samples from the defecation site of these animals. Participants who turned up with fecal samples the following day were honored with a small token of appreciation. Microscopic examination of fecal samples The fresh fecal samples were transported back to the Department of Parasitology, Faculty of Medicine, University of Malaya on the same day of collection, preserved in 2.5% potassium dichromate and kept at 4°C until later analysis. Samples were concentrated using formalin ethyl acetate as previously described by Cheesbrough [27] for the presence of hookworms and other intestinal parasites. Briefly, 1 to 2 g of fecal sample was mixed with 7 ml of formalin and 3 ml ethyl acetate, centrifuged, stained with 0.85% iodine and examined under light microscope. Samples which were microscopically positive for hookworm eggs from both humans and animals were further characterized using molecular procedures. Genomic DNA extraction Genomic DNA was extracted directly from positive fecal sample using PowerSoil DNA Isolation Kit (Mo Bio, cat. no. 12888-100, CA, USA) according to the manufacturer's instructions. Briefly, approximately 0.2 to 0.3 g of fecal pellet was added into the PowerBead Tube, incubated at 70°C for 10 minutes with the presence of cell lysis and disruption agent provided by the manufacturer. This were then subjected to homogenization and lysis procedure for complete cell lysis by mechanical shaking (vortexing) using MO BIO Vortex Adapter (MO BIO, cat. no. 13000-V1). The final DNA elution was made in 50 µl of elution buffer and stored at −20°C until required for PCR amplification. DNA amplification by PCR Hookworm DNA amplification A direct PCR assay was used for the DNA amplification of hookworm species in both human and animal samples. Forward primer NC1 (5′-ACG TCT GGT TCA GGG TTC TT-3′) and reverse primer NC2 (5′-TTA GTT TCT TTT CCT CCG CT-3′) [28] were used to amplify an approximately 310 and 420 bp region of the internal transcribed spacer-2 (ITS-2), 5.8S and 28S ribosomal RNA gene of N. americanus and Ancylostoma spp., respectively. Control samples without DNA (DNase free water, Sigma Cat. no. W4502) and with N. americanus and Ancylostoma spp. genomic DNA (positive control) were included in each PCR run. The PCR mix consisted of 10× PCR buffer, 2.5 mM dNTPs, 25 mM MgCl2, 10 pmol of each primer, 5 U of Taq polymerase and 6 µl of DNA template made to a final volume of 50 µl. The sample was heated to 94°C for 5 min, followed by 30 cycles of 94°C for 30 s (denaturing), 55°C for 30 s (annealing), 72°C for 30 s (extension) and a final extension at 72°C for 7 min. For human samples, an additional PCR assay (two-step semi-nested) was carried out to confirm mixed infection with N. americanus. Samples which produced fragments of approximately 310 and/or 420 bp in the first PCR were subjected to a second round of PCR to produce a 250 and/or 130 bp amplicon corresponding to N. americanus and Ancylostoma spp., respectively. In the second PCR reaction, 6 µl of each NC1-NC2 amplicon was transferred to a fresh tube containing the same PCR reaction buffer with the primer set NA (5′-ATGTGCACGTTATTCACT-3′) for N. americanus [29], AD1 (5′- CGA CTT TAG AAC GTT TCG GC-3′) for Ancylostoma spp. [30] with NC2 as a common reverse primer and amplified for another 35 cycles. Samples were heated to 94°C for 5 min, followed by 35 cycles of 94°C for 1 min (denaturing), 55°C for 1 min (annealing), 72°C for 1 min (extension) and a final extension at 72°C for 7 min. Cycling was performed in a MyCycler thermal cycler (Bio-Rad, Hercules, USA) for both amplifications. In order to avoid false negative results via microscopy, all samples found to be negative were also included in the molecular analysis. However, none of these samples were detected positive through PCR. Sequencing of PCR product The positive amplicons were then purified using the QIAquick Gel Extraction Kit (QIAgen, cat. no. 28104, Hilden, Germany) according to the manufacturer's instructions except that final elution of DNA was made in 30 µl of elution buffer instead of 50 µl. All purified amplicons were sequenced in both directions using the same primer sets as in the respective PCR assay with an ABI 3730XL sequencer (Bioneer Corporation, South Korea). Sequence chromatograms were viewed using Sequence Scanner version 1.0 program (Applied Biosystems, USA). Forward and reverse sequences were edited, manually aligned and the consensus sequence was created for each sample using the BioEdit Sequence Alignment Editor program [31]. Similarity searches were carried out using Basic Local Alignment Search Tool (BLAST) to the National Centre for Biotechnology Information (NCBI) reference sequences. All sequences generated in this study were deposited in GenBank, under the accession numbers HQ452515 to HQ452543, JF960362 to JF960403, JN120871 to JN120898 and JN164657 to JN165660. Statistical analysis The data entry and statistical analysis was carried out using the SPSS software (Statistical Package for the Social Sciences) program for Windows version 17 (SPSS, Chicago, IL, USA). Prevalence of hookworm infection in both human and animal samples was determined on the basis of microscopic examination. To describe data, mean and standard deviation for continuous variables and proportion categorical variables were computed. Crude associations of the binary outcome variable with each independent variable were assessed by Pearson's Chi-square (X2 ). A univariate model was used to assess potential associations between hookworm infection (outcome of interest) and the potential associated factor characteristics. The level of statistical significance was set at p RM 500 258 20 7.8 1 Presence of proper latrine system* No 341 40 11.7 2.06 (2.01–2.12) 0.015 Yes 293 18 6.1 1 Type of toilet facility None 407 51 12.5 2.12 (2.06–2.16) <0.001 Pour flush toilet 227 7 3.1 1 Defaecation places status Others (Bush, River) 406 48 11.8 2.10 (2.04–2.13) 0.002 Pour flush toilet 228 10 4.4 1 Wear shoes/sandals outside the house* No 245 45 18.4 2.18 (2.11–2.26) <0.001 Yes 389 13 3.3 1 Close contact with cats/dogs* Yes 517 55 10.6 4.52 (1.39–14.72) 0.006 No 117 3 2.6 1 Eat with hand without prior washing No 531 54 10.2 2.80 (1.90–7.92) 0.043 Yes 103 4 2.9 Water supply Untreated sources (river, mountain, well) 289 36 12.5 1.10 (1.02–1.13) 0.008 Treated sources (Government pipe) 345 22 6.4 1 Garbage disposal Indiscriminately 330 30 9.1 1.01 (0.59–1.74) 0.958 Collected 304 28 9.2 1 Reference group marked as OR = 1; 95% CI: 95% Confidence interval; Significant association. (p<0.05). *Variables were confirmed by multivariate analysis as significant predictors of hookworm infection. Molecular characterization of hookworms Out of 58 human samples found to be positive microscopically, 47 (81.0%) were successfully amplified, sequenced and recently published [26]. Although most hookworm-positive individuals were infected with Necator americanus, Ancylostoma ceylanicum constituted 12.8% of single infections and 10.6% mixed infections with N. americanus. Out of 65 microscopically positive animal samples, 50 (76.9%) samples were successfully amplified by PCR and sequenced (Table 3). Of these 50 sequences, 52.0% (26 of 50) were found to be 100% homologous to previously published sequences of A. caninum (GenBank accession number EU159416, AM850106), 46.0% (23 of 50) samples were 99% identical to A. ceylanicum (GenBank accession number DQ438080, DQ831520) and 2.0% (1 of 50) were 100% identical to A. braziliense (GenBank accession number DQ438062). All A. caninum isolates (26 isolates) were derived from dogs, while 17 dogs (73.9% of 23) and 6 (26.1% of 23) cats were shown to harbor a single A. ceylanicum infection. A single A. braziliense infection was isolated from cat (data not shown). 10.1371/journal.pntd.0001522.t003 Table 3 Hookworm species detected from human and animal samples as determined by PCR assays. Characteristics PCR and Sequencing n % Human (N = 47) N. americanus 36 76.6 A. ceylanicum 6 12.8 Mixed (both species) 5 10.6 Animals (N = 50) A. caninum * 26 52.0 A. ceylanicum ** 23 46.0 A. braziliense *** 1 2.0 *A. caninum were found in dogs; **A. ceylanicum were found in both cats and dogs; ***A. braziliense was found in cats. Discussion To the best of our knowledge, this is the first investigation of the molecular epidemiology of hookworm in both humans and animals living in the same endemic area in Malaysia. Based on microscopic examination of feces, the overall prevalence of hookworm was determined to be at 9.1% in humans and 61.9% in animals (71.1% in dogs and 37.9% in cats). In Malaysia, intestinal parasitic infections (IPIs) including hookworm infections are still a major public health problem, especially among the impoverished and underprivileged communities living in rural and remote areas. This was illustrated by a recent study which reported 73.2% of 716 people were infected with at least one type of soil-transmitted helminth (STH), with 12.8% being hookworm infection [24]. In the present study, factors significantly associated with hookworm infection included lack of provision of proper latrine system, walking barefooted and close contact with cats and dogs. Similar risk factors were also highlighted in previous local studies among rural communities where A. lumbricoides and T. trichiura infections were high [24], [32], [33]. Observations made in the current study noted that most residents did not have proper sanitary facilities at home. For households that did possess latrines, they were mainly utilized by adults and were poorly maintained. The filth and smell discouraged many from using it. Children were allowed to defecate indiscriminately around their houses. In some cases, even adults defecated indiscriminately in the bushes and nearby river at the back of their houses. The damp soil and lush vegetation associated with Malaysia's tropical climate form ideal conditions for the development, survival and transmission of hookworm larvae. These conducive environmental conditions coupled with outdoor defecation of humans and animals lead to widespread contamination of hookworm larvae in the soil. Percutaneous infection results when humans walk barefooted outdoors, which is a common practice in this community. Hence, like in previous studies [22], [33] it was not surprising that walking barefooted outdoors constituted a significantly higher risk of being infected with hookworm as compared to those who wore shoes or sandals. The higher rate of soil-transmitted helminth (STH) infection in this community especially T. trichiura and A. lumbricoides infections could be due to anthelminthic resistance and high re-infection rates. Currently, the recommended regime for the treatment of STH infections is either albendazole or mebendazole. Although both drugs are deemed broad spectrum anthelminthic agents, important therapeutic differences do exist which affect their uses in clinical practice [34]. While global reports of anthelminthic drug resistance are rare, evidences of emerging resistance do exist throughout the world [34]–[36] and has already been observed and reported in Malaysia [37], [38]. Another important problem encountered in the treatment management is the high re-infection rate occurring especially in highly endemic areas. Two studies in Malaysia have found that re-infection can occur as early as 2 months post treatment, by 4 months almost half of the treated population had been re-infected [37] and by 6 months the intensity of infections had returned to pre-treatment levels [38]. The findings that intensity of STH infections attained the pre-treatment levels by 6 months after treatment have also been reported in other regions of the world [35]. The role of companion animals as reservoirs for zoonotic diseases has been known as a significant health problem worldwide [39]. This current study once again recognizes and highlights the significant association of close contact between humans and animals especially dogs and cats, with regards to hookworm infections. Comparable results were also observed in other rural communities in Malaysia [24], [33] and endemic areas in Thailand [21], [22] and India [40]. In addition, the practice of inappropriate disposal of animal feces in public areas in the studied communities is a common habit among the villagers, leading to environmental contamination and thus increasing the risk of infection to other animals and human. The probable zoonotic risk of A. ceylanicum is of great concern to public health as the hookworm has been shown to be highly endemic among domestic animals in these communities with an overall prevalence of 61.9%. It is difficult to compare the current status of hookworm infection among animals in Malaysia since there is limited prior documented data. Even if data was available, the incrimination of a specific species is not possible as molecular tools were not employed in these previous studies. The only prevalence data on hookworm available was among stray dogs in Kuala Lumpur and Sarawak (East Malaysia) [41], [42] where a rate of more than 95% were reported. More recently, a survey of intestinal parasitic infections (IPIs) of dogs from temples in Thailand revealed that 58.9% of dogs were infected with hookworm [21]. Another survey of IPIs among dogs in rural India also found high prevalence of hookworm ranging from 93.0% to 98.0% [40], [43]. In our molecular analysis, N. americanus and A. ceylanicum were identified in human, while A. caninum, A. ceylanicum and A. braziliense were detected in animals. Nearly a quarter of hookworm-positive individuals were found to harbor infections with A. ceylanicum and 46.0% of hookworm-positive dogs and cats were found infected with this species. This finding implied that dogs and cats may act as the probable sources of infection to humans. The results was further strengthened by our epidemiological analysis which revealed that those individuals who had close contact with cats and dogs were 3 times more likely to be infected with hookworm. However, as the hookworm species were genetically characterized on the basis of its DNA sequences at the ITS-2, 5.8S and 28S of the ribosomal RNA gene, genetic differences among each species could not be excluded in the present study. The utilization of several and more polymorphic genetic marker than ITS-2 such as cytochrome c oxidase subunit 1 (cox1) gene should be taken into consideration in future study in order to determine if the same genotype of A. ceylanicum circulates between the human and animal hosts. In addition, examination of the extent of interspecific sequence differences subsequently would provide phylogentically informative characters to infer the evolutionary relationship of member of the species. The clinical impact of A. ceylanicum in humans remains to be investigated. However, in the present study, all the eleven infected individuals with A. ceylanicum were asymptomatic. Unfortunately, the clinical observation of creeping eruptions, and EE, and iron deficiency anemia were not investigated in this study. A recent study on zoonotic ancylostomiasis caused by A. ceylanicum in Thailand reported that infected individuals reported to suffer from poor health and abdominal pain [21].The epidemiological and clinical significance of A. ceylanicum remains largely unresolved due to the limited availability of published research data. Clinical signs reported ranged from asymptomatic light to heavy infections with anemia, lethargy and excessive hunger [16]. In an experimental infection with A. ceylanicum [14], [15], subjects developed clinical signs similar to those described from experimental infection with N. americanus and A. duodenale in human [44], [45]. Further studies investigating the epidemiology, transmission dynamics and clinical significance of A. ceylanicum in a community endemic for hookworm disease will be beneficial in unravelling the true significance of this zoonosis in humans. Besides A. ceylanicum, 46.0% of animals were harboring A. caninum and 2.0% with A. braziliense. Reports from other countries have highlighted that A. caninum is ubiquitously found in dogs and cats in tropical and sub-tropical climates in Asia and Australia [20], [21], [40]–[43], [46]–[48]. To date, there is no reported data on A. caninum among dogs in Malaysia. This study demonstrated that all of the eight A. caninum isolated were from dogs. This finding was in accordance with previous studies in other countries where A. caninum has been reported to be the predominant hookworm species in dogs in Thailand [21], India [40], Australia [46] and many other parts of the world [47]. Although A. caninum is regarded as an uncommon parasite of cats, its infection has been reported among cats in Australia [48]. However, none of the cats in this study was infected with A. caninum. In addition, BLAST result for one of the hookworm isolated from cat in the present study was 100% identical to a previously published sequence of A. braziliense. Malaysia is one of the few countries besides Australia to report on A. braziliense infections in the Asia-Pacific region [48]. In conclusion, this present study provided evidence based on the combination of epidemiological, conventional diagnostic and molecular tools that A. ceylanicum infection is common and that its transmission dynamic in endemic areas in Malaysia is heightened by the close contact between human and domestic animal (i.e., dogs and cats) populations. Supporting Information Checklist S1 STROBE checklist. (DOC) Click here for additional data file.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                30 March 2015
                March 2015
                : 9
                : 3
                Affiliations
                [1 ]Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Basel, Switzerland
                [2 ]University of Basel, Basel, Switzerland
                [3 ]National Institute of Public Health, Ministry of Health, Vientiane, Lao People’s Democratic Republic
                [4 ]Faculty of Basic Sciences, University of Health Sciences, Vientiane, Lao People’s Democratic Republic
                Jiangsu Institute of Parasitic Diseases, CHINA
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: PV SS JU PO. Performed the experiments: SS YV DB KA PO. Analyzed the data: AF PV. Wrote the paper: AF SS PO.

                Article
                PNTD-D-14-00296
                10.1371/journal.pntd.0003486
                4378892
                25822794

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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                Figures: 4, Tables: 3, Pages: 20
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                Funding
                This investigation was funded by the Swiss National Science Foundation and the Swiss Agency for Development and Cooperation (project no. NF3270B0-110020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article

                Infectious disease & Microbiology

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