AusDiagnostics Pty. Ltd.

Alexandria, Australia

AusDiagnostics Pty. Ltd.

Alexandria, Australia

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PubMed | University of New England of Australia and AusDiagnostics Pty. Ltd.
Type: Journal Article | Journal: Veterinary parasitology | Year: 2014

The specific diagnosis of gastrointestinal nematode infections in ruminants is routinely based on larval culture technique and on the morphological identification of developed third-stage larvae. However, research on the ecology and developmental requirements of different species suggests that environmental conditions (e.g., temperature and humidity) for optimal development to occur vary between the different species. Thus, employing a common culture protocol for all species will favour the development of certain species over others and can cause a biased result in particular when species proportions in a mixed infection are to be determined. Furthermore, the morphological identification of L3 larvae is complicated by a lack of distinctive, obvious features that would allow the identification of all key species. In the present paper we review in detail the potential limitations of larval culture technique and morphological identification and provide account to some modern molecular alternatives to the specific diagnosis of gastrointestinal nematode infection in ruminants.


Lau A.,Center for Infectious Diseases and Microbiology | Lau A.,Westmead Millennium Institute | Halliday C.,Center for Infectious Diseases and Microbiology | Halliday C.,West Health Institute | And 6 more authors.
Journal of Clinical Microbiology | Year: 2010

We applied multiplex-tandem PCR (MT-PCR) to 255 EDTA whole-blood specimens, 29 serum specimens, and 24 plasma specimens from 109 patients with Candida bloodstream infection (candidemia) to determine whether a diagnosis could be expedited in comparison with the time to diagnosis by the use of standard blood culture. Overall, the MT-PCR performed better than blood culture with DNA extracted from whole blood from 52/74 (70%) patients, accelerating the time to detection (blood culture flagging) and determination of the pathogenic species (by use of the API 32C system [bioMérieux, Marcy l'Etoile, France]) by up to 4 days (mean, 2.2 days; range, 0.5 to 8 days). Candida DNA was detected more often in serum (71%) and plasma (75%) than in whole blood (54%), although relatively small numbers of serum and plasma specimens were tested. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay with whole blood were 75%, 97%, 95%, and 85%, respectively. Fungal DNA was not detected by MT-PCR in 6/24 (25%) wholeblood samples drawn simultaneously with the positive blood culture sample. MT-PCR performed better with whole-blood specimens stored at -20° C (75%) and when DNA was extracted within 1 week of sampling (66%). The molecular and culture identification results correlated for 61 of 66 patients (92%); one discrepant result was due to misidentification by culture. All but one sample from 53 patients who were at high risk of candidemia but did not have proven disease were negative by MT-PCR. The results demonstrate the good potential of MT-PCR to detect candidemia, to provide Candida species identification prior to blood culture positivity, and to provide improved sensitivity when applied to with serum and plasma specimens. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Stark D.,St Vincents Hospital | Stark D.,University of Technology, Sydney | Al-Qassab S.E.,University of Technology, Sydney | Barratt J.L.N.,St Vincents Hospital | And 9 more authors.
Journal of Clinical Microbiology | Year: 2011

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites. Copyright © 2011, American Society for Microbiology. All Rights Reserved.


Roeber F.,University of Melbourne | Roeber F.,AusDiagnostics Pty. Ltd. | Jex A.R.,University of Melbourne | Gasser R.B.,University of Melbourne | Gasser R.B.,Institute of Tropical Medicine
Methods in Molecular Biology | Year: 2014

The diagnosis of gastrointestinal nematode infections in small ruminants is central to studying the biology and epidemiology of these parasites and underpins their control. Traditional methods of diagnosis are inaccurate, time-consuming and laborious. Here, we describe a step-by-step protocol for the molecular-based diagnosis of infections by real-time PCR. © Springer Science+Business Media New York 2015.


Lau A.,University of Sydney | Stanley K.,AusDiagnostics Pty. Ltd. | Sorrell T.,University of Sydney
Methods in Molecular Biology | Year: 2013

Multiplex, real-time PCR has become an invaluable tool for the rapid identi fication of pathogens in clinical specimens enabling earlier and more targeted management of antimicrobial therapy. In this chapter, we describe the methodology behind a novel multiplex-tandem PCR (MT-PCR) platform designed for the rapid identi fication of up to 18 fungal pathogens in blood cultures, primary isolation plates, and whole blood, serum, and plasma. © Springer Science+Business Media New York 2013.


Jex A.R.,University of Melbourne | Stanley K.K.,AusDiagnostics Pty Ltd | Lo W.,AusDiagnostics Pty Ltd | Littman R.,University of Melbourne | And 7 more authors.
Molecular and Cellular Probes | Year: 2012

Infectious diarrhoeal diseases represent a major socio-economic burden to humans, and are linked to a range of pathogens, including viruses, bacteria and protists. The accurate detection of such pathogens is central to control. However, detection often relies on methods that have limited diagnostic sensitivity and specificity. Here, we assessed an automated, robotic platform for the simultaneous detection of eight major pathogens associated with infectious diarrhoea. Genomic DNA samples (n=167) from faeces from humans with diarrhoea and diagnosed as cryptosporidiosis, and 100 uninfected control subjects, were tested for adenovirus 40/41, norovirus, Clostridium difficile, Campylobacter, Salmonella, Shigella, Cryptosporidium and Giardia by multiplexed-tandem PCR, and also characterized by single-strand conformation polymorphism analysis (SSCP) and selective sequencing. All 167 samples tested positive for Cryptosporidium, five for adenovirus 40/41, four for Campylobacter, three for C. difficile and seven for Shigella spp., with no false positive results for any assay. The automated PCR exhibited a high sensitivity, with <10 individual pathogens being readily detected. The robotic detection platform assessed here represents a sensitive, high-throughput tool for key pathogens linked to infectious diarrhoea in humans. This platform requires little molecular biological expertise and is well suited to various diagnostic facilities and settings. © 2011 Elsevier Ltd.


Perera P.K.,University of Melbourne | Gasser R.B.,University of Melbourne | Firestone S.M.,University of Melbourne | Smith L.,AusDiagnostics Pty. Ltd. | And 2 more authors.
Journal of Clinical Microbiology | Year: 2015

Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


Roeber F.,University of Melbourne | Jex A.R.,University of Melbourne | Campbell A.J.D.,University of Melbourne | Nielsen R.,Veterinary Health Research Pty. Ltd. | And 3 more authors.
International Journal for Parasitology | Year: 2012

The accurate diagnosis of strongylid nematode infections is central to investigating their epidemiology and for parasite control. To overcome major limitations in sensitivity or specificity of traditional methods, including faecal egg count (FEC) and/or larval culture (LC), we evaluated and established a semi-automated, high throughput multiplexed-tandem PCR (MT-PCR) platform for the diagnosis of gastrointestinal strongylid nematode infections in sheep, and established its diagnostic sensitivity (100%) and specificity (87.5%) based on the testing of 100 faecal DNA samples from helminth-free sheep and 30 samples from sheep with infections confirmed by necropsy. Subsequently, the platform was employed to test 219 faecal samples from sheep with naturally acquired infections from various geographical localities within Australia and the results compared with those from conventional LC using 139 of the 219 samples. The results obtained using both MT-PCR and LC correlated significantly for most nematodes examined, but revealed that Oesophagostomum venulosum and Chabertia ovina (parasites of the large intestine) were significantly under-represented in the LC results. The results showed that Trichostrongylus spp. (87%), Teladorsagia circumcincta (80%) and Haemonchus contortus (67%) had the highest prevalences, followed by O. venulosum (51%) and C. ovina (12%). The molecular-diagnostic platform established can be used for species- or genus-specific diagnosis of patent nematode infections within 24. h (compared with 7-10. days for LC), and is a sensitive and cost effective tool for routine application in research and service laboratories. © 2012.


PubMed | University of Melbourne and AusDiagnostics Pty.
Type: Journal Article | Journal: Journal of clinical microbiology | Year: 2014

Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region.

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