Development and Evaluation of a Rapid Diagnostic Assay for Infectious Disease Detection

Thesis Abstract

The rapid and accurate diagnosis of infectious diseases remains a critical challenge in clinical laboratories, particularly in resource-limited settings where conventional diagnostic methods often entail prolonged turnaround times and limited sensitivity. The persistence of delayed or inaccurate detection hampers timely treatment interventions, impeding efforts to control and prevent infectious outbreaks. This study aims to develop, optimize, and evaluate a novel rapid diagnostic assay utilizing immunochromatographic strip technology integrated with molecular amplification techniques for the detection of cholera and typhoid pathogens. The specific objectives include designing assay prototypes, assessing analytical sensitivity and specificity, evaluating clinical performance using patient samples, and comparing the new assay's diagnostic accuracy with current gold-standard methods. The research adopted a mixed-methods approach, combining laboratory experimental design with cross-sectional clinical validation. The study population comprised 300 suspected infectious disease cases presenting at healthcare facilities within a metropolitan city over six months. Samples included stool and blood specimens obtained from consenting patients. The assay prototype was developed through a series of optimization experiments, employing recombinant antigen-based capture, lateral flow chromatography, and PCR amplification of pathogen-specific genetic sequences. Analytical sensitivity was determined using serial dilutions of known pathogen concentrations, while specificity was tested against a panel of related organisms. Clinical performance evaluation involved testing all patient samples with both the developed assay and standard laboratory methods such as microscopy, culture, and PCR. Data collection instruments encompassed laboratory protocols, diagnostic result sheets, and structured questionnaires for clinicians regarding usability. Data analysis employed descriptive statistics to summarize assay performance metrics, including limit of detection, specificity, and reproducibility. Inferential analysis utilized receiver operating characteristic (ROC) curve analysis to determine diagnostic accuracy, with calculation of sensitivity, specificity, positive predictive value (PPV), and negative predictive value. Comparative analysis between the new assay and existing diagnostic methods was conducted using McNemar's test for paired proportions, while qualitative data from usability assessments were analyzed thematically. The study draws on the Health Belief Model to interpret factors influencing assay adoption and integrates the sensitivity-specificity theoretical framework to evaluate diagnostic validity. Expected findings indicate that the developed assay will demonstrate superior rapidity, with results obtainable within 30 minutes, and exhibit high analytical sensitivity (detecting pathogen loads as low as 10^2 CFU/mL) and specificity (>95%) across targeted organisms. Clinical validation is anticipated to reveal diagnostic accuracy comparable or superior to existing methods, with improved ease of use and minimal infrastructure requirements. The research is expected to contribute significantly to the body of knowledge by providing a cost-effective, field-deployable diagnostic tool capable of facilitating prompt infectious disease detection, especially in low-resource settings. It is projected that the assay’s implementation will enhance early intervention strategies, reduce transmission rates, and inform public health responses. The main conclusion underscores the potential of the assay to serve as an affordable, reliable alternative to conventional diagnostics, addressing current limitations in disease detection. It recommends further large-scale field trials to verify reproducibility across diverse settings and pathogen panels, as well as investigation into its integration within national disease surveillance systems. Overall, this study proposes a substantial advancement in point-of-care diagnostics that aligns with global health goals for infectious disease control and calls for policy support to facilitate widespread adoption.