Rebecca J. Traub

Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community.

Giardia duodenalis isolates recovered from humans and dogs living in the same locality in a remote tea-growing community of northeast India were characterized at 3 different loci; the SSU-rDNA, elongation factor 1-alpha (ef1-alpha) and triose phosphate isomerase (tpi) gene. Phylogenetic analysis of the SSU-rDNA and efl-alpha genes provided poor genetic resolution of the isolates within various assemblages, stressing the importance of using multiple loci when inferring genotypes to Giardia. Analysis of the tpi gene provided better genetic resolution and placed canine Giardia isolates within the genetic groupings of human isolates (Assemblages A and B). Further evidence for zoonotic transmission was supported by epidemiological data showing a highly significant association between the prevalence of Giardia in humans and presence of a Giardia-positive dog in the same household (odds ratio 3.01, 95% CI, 1.11, 8.39, P = 0.0000).

Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites.

ABSTRACT Little is known of the prevalence of Cryptosporidium and Giardia parasites in sheep and the genotypes that they harbor, although potentially sheep may contribute significantly to contamination of watersheds. In the present study, conducted in Western Australia, a total of 1,647 sheep fecal samples were screened for the presence of Cryptosporidium and Giardia spp. using microscopy, and a subset (n = 500) were screened by PCR and genotyped. Analysis revealed that although both parasites were detected in a high proportion of samples by PCR (44% and 26% for Giardia and Cryptosporidium spp., respectively), with the exception of one Cryptosporidium hominis isolate, the majority of isolates genotyped are not commonly found in humans. These results suggest that the public health risk of sheep-derived Cryptosporidium and Giardia spp. in catchment areas and effluent may be overestimated and warrant further investigation.

Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers

Blastocystis is an enteric protist and one of the most frequently reported parasitic infections in humans and a variety of animal hosts. It has also been reported in numerous parasite surveys of animals in zoological gardens and in particular in non-human primate species. PCR-based methods capable of the direct detection of Blastocystis in faeces were used to detect Blastocystis from various hosts, including non-human primates, Australian native fauna, elephants and giraffes, as well as their keepers from a Western Australian zoo. Additional faecal samples were also collected from elephants and giraffes from four other zoos in Amsterdam (The Netherlands), Antwerp (Belgium), Melbourne and Werribee (Australia). Information regarding the general health and lifestyle of the human volunteers were obtained by questionnaire. Overall, 42% and 63% of animals and zoo-keepers sampled from the Western Australian zoo were positive for Blastocystis, respectively. The occurrence of Blastocystis in elephants and giraffes from other cities was similar. This is the first report of Blastocystis found in the elephant, giraffe, quokka, southern hairy nosed wombat and western grey kangaroo. Three novel and what appear to be highly host-specific subtypes (STs) of Blastocystis in the elephant, giraffe and quokka are also described. These findings indicate that further exploration of the genetic diversity of Blastocystis is crucial. Most zoo-keepers at the Perth Zoo were harbouring Blastocystis. Four of these zoo-keeper isolates were identical to the isolates from the southern hairy nosed wombat and five primate species.

Direct characterization of Blastocystis from faeces by PCR and evidence of zoonotic potential

In vitro propagation followed by PCR, and a PCR-based method capable of the direct detection of Blastocystis in faeces were utilized to detect Blastocystis from various hosts in Australia, including primates and their handlers from the Perth Zoo. In addition, Blastocystis isolates from dogs and humans living in a localized endemic community in Thailand were also characterized genetically. PCR-based detection directly from faeces was shown to be more sensitive compared with in vitro culture for the detection of Blastocystis. Moreover, phylogenetic analysis of Blastocystis isolates amplified utilizing in vitro techniques prior to PCR revealed that this method favoured the preferential amplification of Blastocystis subtype 5 over subtype 1. This study is the first to provide molecular-based evidence supporting the zoonotic potential of Blastocystis in dogs, possums and primates in a natural setting.

A New PCR-Based Approach Indicates the Range of Clonorchis sinensis Now Extends to Central Thailand

Differentiation of the fish-borne trematodes belonging to the Opisthorchiidae, Heterophyidae and Lecithodendriidae is important from a clinical and epidemiological perspective, yet it is impossible to do using conventional coprological techniques, as the eggs are morphologically similar. Epidemiological investigation therefore currently relies on morphological examination of adult worms following expulsion chemotherapy. A PCR test capable of amplifying a segment of the internal transcribed spacer region of ribosomal DNA for the opisthorchiid and heterophyid flukes eggs taken directly from faeces was developed and evaluated in a rural community in central Thailand. The lowest quantity of DNA that could be amplified from individual adults of Opisthorchis viverrini, Clonorchis sinensis and Haplorchis taichui was estimated at 0.6 pg, 0.8 pg and 3 pg, respectively. The PCR was capable of detecting mixed infection with the aforementioned species of flukes under experimental conditions. A total of 11.6% of individuals in rural communities in Sanamchaikaet district, central Thailand, were positive for ‘Opisthorchis-like’ eggs in their faeces using conventional parasitological detection techniques. In comparison to microscopy, the PCR yielded a sensitivity and specificity of 71.0% and 76.7%, respectively. Analysis of the microscopy-positive PCR products revealed 64% and 23% of individuals to be infected with O. viverrini and C. sinensis, respectively. The remaining 13% (three individuals) were identified as eggs of Didymozoidae, presumably being passed mechanically in the faeces following the ingestion of infected fishes. An immediate finding of this study is the identification and first report of a C. sinensis–endemic community in central Thailand. This extends the known range of this liver fluke in Southeast Asia. The PCR developed herein provides an important tool for the specific identification of liver and intestinal fluke species for future epidemiological surveys.

Epidemiological and genetic data supporting the transmission of Ancylostoma ceylanicum among human and domestic animals

Background Currently, information on species-specific hookworm infection is unavailable in Malaysia and is restricted worldwide due to limited application of molecular diagnostic tools. Given the importance of accurate identification of hookworms, this study was conducted as part of an ongoing molecular epidemiological investigation aimed at providing the first documented data on species-specific hookworm infection, associated risk factors and the role of domestic animals as reservoirs for hookworm infections in endemic communities of Malaysia. Methods/Findings A total of 634 human and 105 domestic canine and feline fecal samples were randomly collected. The overall prevalence of hookworm in humans and animals determined via microscopy was 9.1% (95% CI = 7.0–11.7%) and 61.9% (95% CI = 51.2–71.2%), respectively. Multivariate analysis indicated that participants without the provision of proper latrine systems (OR = 3.5; 95% CI = 1.53–8.00; p = 0.003), walking barefooted (OR = 5.6; 95% CI = 2.91–10.73; p<0.001) and in close contact with pets or livestock (OR = 2.9; 95% CI = 1.19–7.15; p = 0.009) were more likely to be infected with hookworms. Molecular analysis revealed that while 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. As for cats and dogs, 52.0% were positive for A. ceylanicum, 46.0% for Ancylostoma caninum and 2.0% for Ancylostoma braziliense and all were single infections. 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 of human and domestic animal (i.e., dogs and cats) populations.

PCR-based coprodiagnostic tools reveal dogs as reservoirs of zoonotic ancylostomiasis caused by Ancylostoma ceylanicum in temple communities in Bangkok

A survey of gastrointestinal parasites of dogs and humans from temple communities in Bangkok revealed that 58% of dogs and 3.4% of humans, among those sampled, were infected with hookworms utilising faecal flotation techniques and microscopy. A previously established polymerase chain reaction (PCR)-RFLP approach was utilised to determine the species of hookworms infecting dogs found positive for hookworm eggs. Single infections with Ancylostoma ceylanicum and Ancylostoma caninum were recorded in 77% and 9% of hookworm positive dogs, respectively and mixed infections with both species of Ancylostoma were recorded in 14% of dogs. A single-step PCR for the multiplex detection of Ancylostoma species and Necator americanus DNA in human faeces was developed and applied to characterise the species of hookworms in microscopy positive individuals. Single infection with N. americanus was recorded in five and A.ceylanicum infection in two, out of seven individuals positive for hookworm. This study demonstrates that humans are at risk of acquiring infection with A. ceylanicum in communities where this species of hookworm is endemic in dogs.

The prevalence, intensities and risk factors associated with geohelminth infection in tea‐growing communities of Assam, India

Objective  To determine the prevalence, intensity and associated risk factors for infection with Ascaris, hookworms and Trichuris in three tea‐growing communities in Assam, India.

Determining the zoonotic significance of Giardia and Cryptosporidium in Australian dogs and cats

In a recent study of intestinal parasites in dogs and cats in Australia, Giardia was found to be the most prevalent parasite in dogs. The aim of the current study through the use of molecular tools was to determine the zoonotic significance of the Giardia and Cryptosporidium isolates recovered from dogs and cats during the Australian study. Of the isolates successfully amplified all but one of the Giardia from dogs was either Assemblage C and/or D, with one Assemblage A. Of the cat samples amplified all but one were Assemblage F, with one Assemblage D. We hypothesize that the lack of zoonotic Giardia Assemblages recovered is a result of their being a low prevalence of Giardia in the human population. The Cryptosporidium recovered from dogs and cats was determined to be C. canis and C. felis, respectively, a finding which supports growing evidence that Cryptosporidium in companion animals is of limited public health significance to healthy people.

Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

Background Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy. Methodology/Principal Findings Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections. Conclusions/Significance Multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and Giardia compared to microscopy, especially in samples exhibiting polyparasitism. The superior performance of multiplex PCR to detect polyparasitism and more accurately determine infection intensity suggests that it is a more appropriate technique for use in epidemiologic studies and for monitoring large-scale intervention trials.