Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study.

BACKGROUND: Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease. METHODS: We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. FINDINGS: The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043). INTERPRETATION: Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. FUNDING: Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.

A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens.

The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes.

Giardia intestinalis assemblages A and B infections in Nepal.

Giardia intestinalis is comprised of two major genotypes, A and B, which may vary in their propensity to cause disease. We tested for the presence of these two genotypes in stool samples from patients with gastrointestinal symptoms in Nepal. A total of 1,096 clinical specimens were screened by microscopy, and 45 samples with G. intestinalis were identified. Giardia infection was confirmed in 35 of 45 samples by a Giardia specific real-time polymerase chain reaction (PCR) assay. Genotyping of the Giardia PCR product by restriction fragment length polymorphism indicated that 74% (26 of 35) were assemblage B, 20% (7 of 35) were assemblage A, and 6% (2 of 35) were mixed assemblages.