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Published on: November 7, 2018
Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a
Jie Liu1, Furqan Kabir2, Jainaba Manneh3
1Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA.
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.
Insights
New molecular diagnostic tests accurately detect childhood diarrhea pathogens. These tests show high sensitivity and specificity, correlating pathogen load with disease severity, improving infectious disease diagnostics globally.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Childhood diarrhea is caused by diverse, difficult-to-assay pathogens.
- Molecular diagnostics offer a standardized approach for pathogen detection and quantification.
- Developing and validating molecular tests is crucial for accurate diagnosis.
Purpose of the Study:
- To develop and assess molecular diagnostic tests for 15 enteropathogens.
- To evaluate the performance of these tests across multiple global laboratories.
- To determine the association between pathogen load and diarrheal disease.
Main Methods:
- Developed and validated molecular tests (PCR-Luminex, real-time PCR, TaqMan array card) for 15 enteropathogens.
- Assessed analytical and clinical performance against traditional methods (culture, ELISA, PCR) using stool specimens.
- Utilized univariate logistic regression to analyze pathogen quantities (Cq values) and their association with diarrhea.
Main Results:
- Molecular tests demonstrated excellent analytical and clinical performance across five international laboratories.
- Compared to comparator methods, molecular techniques exhibited higher sensitivity (20-85%) but comparable specificity (median 97.3%).
- Higher pathogen loads, measured by quantification cycle (Cq), significantly correlated with increased odds of diarrhea for key pathogens like Shigella spp. and enteropathogenic E. coli.
Conclusions:
- Molecular diagnostic tests are implementable globally with high fidelity.
- These techniques offer sensitive pathogen detection and enable quantification to establish diarrheal disease associations, even in mixed infections.
- The study provides unprecedented insights into the infectious causes of diarrhea through precise molecular measurements.
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