Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR).

Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions.

Failure of pyrantel in treatment of human hookworm infections (Ancylostoma duodenale) in the Kimberley region of north west Australia.

A survey of 108 individuals from a coastal Aboriginal community in north Western Australia revealed that two species of gastrointestinal protozoan parasites (Giardia duodenalis--39.8%, Entamoeba coli--40.7%) and five gastrointestinal helminths (Hymenolepis nana--54.6%, Hookworm [Ancylostoma duodenale]--30.6%, Enterobius vermicularis--6.5%, Trichuris trichiura--2.8%, Strongyloides stercoralis 1.9%) were present. A total of 29 individuals infected with hookworm were offered treatment with either pyrantel pamoate at a single dose rate of 10 mg/kg body weight or albendazole (single 400 mg dose). Seven days after treatment stool samples were examined. Pyrantel had no significant effect against hookworm. In contrast, albendazole cleared hookworm infections completely and reduced the prevalence of Giardia. The former result suggests that locally A. duodenale is resistant to pyrantel and despite its relatively low cost and wide availability, should not be considered a drug of choice at this dose rate in the treatment of hookworm infections (A. duodenale) in endemic regions.

A high prevalence of Toxoplasma in Australian chickens.

A small survey was undertaken of commercially reared free-range chickens in Western Australia using serology and molecular detection. Eighteen out of 20 serum samples showed antibody responses with titers of 1:64 in 5 chickens and ≥ 1:128 in 13 chickens. DNA extracted from 22 out of 50 tissue samples, 10 brains and 12 spleens, were positive by nested PCR, and sequencing at the B1 locus on DNA from 3 brain and 3 spleen samples confirmed that 2 isolates were Toxoplasma gondii, Type I, and 4 Type II/III. The high prevalence of Toxoplasma infection found in commercial, free-range chickens raises public health issues with respect to both exposure in the workplace, during carcass processing, and subsequent transmission during food handling and/or consumption as food. The results of this study emphasize the need for more data on the incidence of Toxoplasma infection in domestic animals and humans in Australia.

Comparison of PCR and microscopy for detection of Cryptosporidium parvum in human fecal specimens: clinical trial.

PCR technology offers alternatives to conventional diagnosis of Cryptosporidium for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect Cryptosporidium but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different Cryptosporidium genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of Cryptosporidium infections.