Developing multiplex molecular diagnostics for tropical pathogens

Acute undifferentiated fevers are illnesses with generalised symptoms, making clinical diagnosis difficult. Diagnosis largely depends on laboratory tests, however, access to these tests can be limited in rural areas of low- and middle-income countries (LMICs) due to the remoteness of these areas as well as the high cost of the tests. This thesis explores the theme of diagnostics for acute undifferentiated fever (AUF) in rural South and Southeast Asia, making them more accessible and economical both for service providers/patients and as a surveillance tools. Three diagnostic tests were developed around this concept. Oligonucleotides for real-time PCR assays were designed to detect multiple targets from a single specimen. A rapid test based on CRISPR technology was developed as a point-of-care (POC) test that can be done outside laboratory settings. A protocol for a molecular detection of viruses was developed to evaluate using dried blood spots (DBS), a more convenient specimen type, on the automated BD MAX™ instrument. Lastly, the Chembio DPP® Fever Panel II Asia System, consisting of an antigen and IgM antibody cassette was evaluated both in the laboratory and at site using patient whole blood samples. This thesis is part of the South and Southeast Asia Community-based Trials Network (SEACTN) research programme which aims to investigate the morbidity and mortality in rural communities across South and Southeast Asia. The real-time reverse transcriptase (RT) PCR tests for whole blood and DBS will be used to detect pathogens in patient samples collected in SEACTN.