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Alexandria, Australia

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.,SydPath | Stark D.,University of Technology, Sydney | Al-Qassab S.E.,University of Technology, Sydney | Barratt J.L.N.,SydPath | 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.

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.

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.

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.

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