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Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality
Maureen H Diaz1, Jessica L Waller1, M Jordan Theodore2
1Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Insights
Child Health and Mortality Prevention Surveillance (CHAMPS) developed multiplex real-time PCR TaqMan Array Cards (TACs) to identify infectious agents causing child deaths. This advanced diagnostic tool enhances infectious disease surveillance in affected regions.
Area of Science:
- Medical Laboratory Science
- Infectious Disease Epidemiology
- Molecular Diagnostics
Background:
- Child mortality remains a critical public health issue in sub-Saharan Africa and South Asia.
- Identifying infectious agents is crucial for developing effective prevention and treatment strategies.
- Existing diagnostic methods may not be sufficient for comprehensive surveillance of diverse pathogens.
Purpose of the Study:
- To develop and implement advanced molecular diagnostic tools for infectious disease surveillance.
- To create multiplex real-time PCR panels for identifying a wide range of pathogens in pediatric deaths.
- To optimize laboratory workflows for high-throughput testing of postmortem specimens.
Main Methods:
- Development of custom TaqMan Array Cards (TACs) with over 100 real-time PCR targets.
- Configuration of multiplex panels for bacteria, viruses, parasites, and fungi, tailored to regional significance.
- Optimization of nucleic acid extraction, quality assurance, and data management protocols for molecular testing.
Main Results:
- Successful implementation of multiplex TACs capable of detecting >100 infectious agents per card.
- Enhanced sensitivity and efficiency in pathogen detection through multiplex real-time PCR on a microfluidic platform.
- Established comprehensive molecular testing workflow for high-quality data in surveillance.
Conclusions:
- Multiplex TACs significantly improve the utility of molecular technology for infectious disease diagnostics and surveillance.
- The developed protocols ensure robust and reliable identification of pathogens contributing to child mortality.
- This approach provides valuable data for public health interventions and disease prevention efforts.
Abstract:
Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30-45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.
