Nanolitre real-time PCR detection of bacterial, parasitic, and viral agents from patients with diarrhoea in Nunavut, Canada

Abstract

BackgroundLittle is known about the microbiology of diarrhoeal disease in Canada's Arctic regions. There are a number of limitations of conventional microbiology testing techniques for diarrhoeal pathogens, and these may be further compromised in the Arctic, given the often long distances for specimen transport.ObjectiveTo develop a novel multiple-target nanolitre real-time reverse transcriptase (RT)-PCR platform to simultaneously test diarrhoeal specimens collected from residents of the Qikiqtani (Baffin Island) Region of Nunavut, Canada, for a wide range of bacterial, parasitic and viral agents.Study design/methodsDiarrhoeal stool samples submitted for bacterial culture to Qikiqtani General Hospital in Nunavut over an 18-month period were tested with a multiple-target nanolitre real-time PCR panel for major diarrhoeal pathogens including 8 bacterial, 6 viral and 2 parasitic targets.ResultsAmong 86 stool specimens tested by PCR, a total of 50 pathogens were detected with 1 or more pathogens found in 40 (46.5%) stool specimens. The organisms detected comprised 17 Cryptosporidium spp., 5 Clostridium difficile with toxin B, 6 Campylobacter spp., 6 Salmonella spp., 4 astroviruses, 3 noroviruses, 1 rotavirus, 1 Shigella spp. and 1 Giardia spp. The frequency of detection by PCR and bacterial culture was similar for Salmonella spp., but discrepant for Campylobacter spp., as Campylobacter was detected by culture from only 1/86 specimens. Similarly, Cryptosporidium spp. was detected in multiple samples by PCR but was not detected by microscopy or enzyme immunoassay.Conclusions Cryptosporidium spp., Campylobacter spp. and Clostridium difficile may be relatively common but possibly under-recognised pathogens in this region. Further study is needed to determine the regional epidemiology and clinical significance of these organisms. This method appears to be a useful tool for gastrointestinal pathogen research and may also be helpful for clinical diagnostics and outbreak investigation in remote regions where the yield of routine testing may be compromised.

Multiplex Polymerase Chain Reaction Tests for Detection of Pathogens Associated with Gastroenteritis

“…This technology is a high-throughput quantitative real-time reverse transcriptase (RT)-PCR platform that can perform over 3,000 separate PCR reactions in parallel in 33 nanolitre volumes in through-holes (similar to wells on a microtitre plate). A multiple-target nanolitre realtime PCR panel was developed for 16 major diarrhoeal pathogens by Goldfarb DM et al 42 . This panel detects:

8 bacteria:

Enterohemorrhagic Escherichia coli (EHEC), via detection of stx 1, stx 2, E. coli O157

Salmonella spp.

Shigella spp.

Campylobacter spp.

Yersinia entercolitica

Clostridium difficile, Clostridium difficile tcd B

Listeria monocytogenes

Vibrio parahaemolyticus

6 viruses:

Norovirus group 1

Norovirus group 2

Rotaviruses

Astroviruses

Adenoviruses 40/4

Sapoviruses

and 2 parasites:

Giardia lamblia

Cryptosporidium spp.

…”

Acute Enteric Infections Polymerase Chain Reaction Assay in Pediatric Practice: Opportunities and Challenges

“…Сравнительные исследования, проведенные разными авторами, так же как и наши данные, доказывают, что ПЦР значительно эффектив-нее для выявления любого возбудителя диареи, чем традиционные бактериологические методы [2,5,9,12,14]. Особенно диагностическое пре-восходство ПЦР значимо в выявлении таких возбудителей ОКИ, как Campylobacter spp., E. coli и Clostridium difficile.…”

“…По мнению исследователей, занимавшихся подобными сравнениями, титул «золотого стандарта» прямой этиологической диагностики ОКИ переходит от биологических методов к ПЦР. Методом ПЦР в настоящее время проводят исследования по эпидемио-логии ОКИ в разных регионах мира [1,2,5,7,8,9,10,11], изучают распространенность бес-симптомного носительства возбудителей ОКИ и закономерности его перехода в острое заболе-вание [6,7]. Сходство симптомов ОКИ, вызван-ных различными возбудителями, определяет целесо образность использования ПЦР имен-но в мультиплексной форме, то есть с набором праймеров различной специфичности.…”

Molecular diagnosis of infectious diarrhea: focus on enteric protozoa

“…It is also possible that co-pathogenicity occurs in areas of high exposure. In either case, more information than presence or absence of a given pathogen is needed to MS2 RNA [68,73] McAuliffe et al Not included [72] Taniuchi et al PhHV DNA [70] Goldfarb et al Disease Network continue to apply broad pathogen coverage to determine which infections and possible co-infections are associated with disease. These diarrheal models will inform clinicians in the developing world about the clinical relevance of a patient's pathobiota.…”

“…A representative set of multiplexed PCR assays for pathogen detection in stool that include the major enteric protozoa is briefly described in TABLE 1. These assays typically display comparable sensitivity and specificity in clinical samples to corresponding singleplex assays and significantly improved efficacy relative to conventional approaches [39, [69][70][71][72][73][74][75].…”