Detection of diarrhoeal pathogens in human faeces using an automated, robotic platform

Jex, Aaron R.; Stanley, Keith K.; Lo, William; Littman, Rachael; Verweij, Jaco J.; Campbell, Bronwyn E.; Nolan, Matthew J.; Pangasa, Aradhana; Stevens, Melita; Haydon, Shane R.; Gasser, Robin B.

doi:10.1016/j.mcp.2011.10.004

Cited by 27 publications

(17 citation statements)

References 48 publications

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“…The minimum amount of DNA detectable (i.e., 1 fg or 2.5 DNA copies) by MT-PCR was comparable to that of previous studies using the same platform (44,46), and the test was approximately 1,000 times more sensitive than conventional PCR (21).…”

Section: Discussionsupporting

confidence: 78%

“…Following the dispensing of each sample and the initiation of the assay, the following setup process and analysis were executed by the program Easy-Plex Assay Setup (AusDiagnostics), with the secondary amplification in MT-PCR and the melting curve analysis being semiautomated (44,48). A sample was recorded as test positive using the auto-call function of the Easy-Plex software (AusDiagnostics) if the amplicon produced a single melting curve which was within 1.5°C of the expected melting temperature, the height of the peak was higher than 0.2 dF/dT (where dF/dT is the derivative of fluorescence over temperature), and the peak width was Յ3.5°C.…”

Section: Mt-pcrmentioning

confidence: 99%

“…Although MT-PCR was originally developed to quantify gene transcription (40), MT-PCR has been applied to the sensitive and simultaneous detection of some fungi, such as Candida spp. (41,42), enteric pathogens of humans (43,44), gastrointestinal nematodes of sheep (45), and toxigenic cyanobacteria (46). As two genotypes of the T. orientalis complex (i.e., Chitose and Ikeda) are presently recognized to relate to clinical disease, there is a need to identify and differentiate each of them from nonpathogenic genotypes (i.e., Buffeli and type 5) of T. orientalis known to occur in southeast Australia (21).…”

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confidence: 99%

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Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

et al. 2015

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bOriental 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.T ick-borne diseases (TBDs) pose a major threat to livestock production worldwide and can have a significant impact on farming communities due to economic losses (1). Theileriosis is one of the important TBDs of cattle, sheep, and/or other ruminants, mainly in tropical and subtropical regions of the world (2). In cattle, East Coast fever (ECF) and Mediterranean/tropical theileriosis are due to Theileria parva and Theileria annulata, respectively, whereas oriental theileriosis is caused by Theileria orientalis. The prevalence of various forms of theileriosis in different parts of the world is dependent on the occurrence of suitable tick vectors for their transmission (3).Oriental theileriosis is caused by one or more genotypes of the T. orientalis complex and is transmitted by ixodid ticks, primarily Haemaphysalis spp. (4-6). Presently, 11 genotypes of T. orientalis complex (designated Chitose or type 1, Ikeda or type 2, Buffeli or type 3, types 4 to 8, and N-1 to N-3) have been identified using a number of molecular markers, including major piroplasm surface protein (MPSP) (7, 8), 23-kDa piroplasm membrane protein (p23) (9-11, 60), small-subunit (SSU) rRNA gene (8, 12, 13), and/or the first and second internal transcribed spacers of nuclear ribosomal DNA (ITS-1 and ITS-2, respectively) (12, 14). Of these genotypes, Ikeda and Chitose are recognized to be associated with clinical outbreaks of oriental theileriosis, mainly in the Asia-Pacific region (15-21). The major clin...

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Section: Discussionsupporting

confidence: 78%

Section: Mt-pcrmentioning

confidence: 99%

mentioning

confidence: 99%

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Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

et al. 2015

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“…and coinfecting diarrheal pathogens from human fecal genomic samples was described. The assay was rapid (taking Ͻ2 h, with ϳ5 to 10 min of technical work following the extraction of genomic DNA), and due to automation, data analysis required little molecular biological expertise, making it well suited to various diagnostic facilities and settings (e.g., hospitals or quarantine facilities) (210). Other tools, based on the Cryptosporidium oocyst wall protein (COWP) gene, have been used to amplify DNAs of C. parvum, C. hominis, Cryptosporidium meleagridis, and species and genotypes closely related to C. parvum (9,242).…”

Section: Fumagillin (20 Mg Three Times a Day For 14 Days) E Intestinmentioning

confidence: 99%

Enteric Protozoa in the Developed World: a Public Health Perspective

et al. 2012

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SUMMARY Several enteric protozoa cause severe morbidity and mortality in both humans and animals worldwide. In developed settings, enteric protozoa are often ignored as a cause of diarrheal illness due to better hygiene conditions, and as such, very little effort is used toward laboratory diagnosis. Although these protozoa contribute to the high burden of infectious diseases, estimates of their true prevalence are sometimes affected by the lack of sensitive diagnostic techniques to detect them in clinical and environmental specimens. Despite recent advances in the epidemiology, molecular biology, and treatment of protozoan illnesses, gaps in knowledge still exist, requiring further research. There is evidence that climate-related changes will contribute to their burden due to displacement of ecosystems and human and animal populations, increases in atmospheric temperature, flooding and other environmental conditions suitable for transmission, and the need for the reuse of alternative water sources to meet growing population needs. This review discusses the common enteric protozoa from a public health perspective, highlighting their epidemiology, modes of transmission, prevention, and control. It also discusses the potential impact of climate changes on their epidemiology and the issues surrounding waterborne transmission and suggests a multidisciplinary approach to their prevention and control.

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“…The small sample volume required and the potential number of additional DNA targets that can be quantified, in particular when working with fecal samples, makes this approach highly attractive for large-scale population screening for the presence of multiple urinary and intestinal microorganisms. 17,29,30 In this study, we also introduced arbitrary units (AU) of copy numbers of Schistosoma DNA as PCR output, which provides a more realistic view on the existing high linear range of intensity of Schistosoma infection than C t values. In addition, for more in depth comparison between different PCRs, further standardization, including converting DNA loads into egg counts, is needed.…”

Section: Discussionmentioning

confidence: 99%

Real-Time Polymerase Chain Reaction for Detection of Schistosoma DNA in Small-Volume Urine Samples Reflects Focal Distribution of Urogenital Schistosomiasis in Primary School Girls in KwaZulu Natal, South Africa

Pillay¹,

Taylor²,

Zulu³

et al. 2014

The American Society of Tropical Medicine and Hygiene

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Abstract. Schistosoma haematobium eggs and Schistosoma DNA levels were measured in urine samples from 708 girls recruited from 18 randomly sampled primary schools in South Africa. Microscopic analysis of two 10-mL urine subsamples collected on three consecutive days confirmed high day-to-day variation; 103 (14.5%) girls had positive results at all six examinations, and at least one positive sample was seen in 225 (31.8%) girls. Schistosoma-specific DNA, which was measured in a 200-μL urine subsample by using real-time polymerase chain reaction, was detected in 180 (25.4%) cases, and levels of DNA corresponded significantly with average urine egg excretion. In concordance with microscopic results, polymerase chain reaction results were significantly associated with history of gynecologic symptoms and confirmed highly focal distribution of urogenital schistosomiasis. Parasite-specific DNA detection has a sensitivity comparable to single urine microscopy and could be used as a standardized high-throughput procedure to assess distribution of urogenital schistosomiasis in relatively large study populations by using small sample volumes.

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Detection of diarrhoeal pathogens in human faeces using an automated, robotic platform

Cited by 27 publications

References 48 publications

Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

Enteric Protozoa in the Developed World: a Public Health Perspective

Real-Time Polymerase Chain Reaction for Detection of Schistosoma DNA in Small-Volume Urine Samples Reflects Focal Distribution of Urogenital Schistosomiasis in Primary School Girls in KwaZulu Natal, South Africa

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