Yi YanFollow

Date of Award

January 2025

Document Type

Open Access Thesis

Degree Name

Master of Public Health (MPH)

Department

School of Public Health

First Advisor

Serap Aksoy

Abstract

Trypanosomiasis, caused by protozoan parasites of the Trypanosoma genus, remains a significant health and veterinary burden in sub-Saharan Africa, with Human African Trypanosomiasis (HAT) and Animal African Trypanosomiasis (AAT) posing ongoing threats to underserved populations. Despite advances in therapeutics, the lack of sensitive and scalable diagnostic tools continues to limit early detection, effective disease management, and evaluation of emerging vaccine candidates. Current diagnostic methods such as light microscopy and serological tests frequently fail to detect low levels of parasitemia during the early stages of infection, thereby delaying intervention and increasing the risk of disease progression and transmission.

Our study mainly investigated two molecular detection platforms, luciferase-based assays and quantitative polymerase chain reaction (qPCR), for their sensitivity and reliability in detecting Trypanosoma brucei brucei parasites at low densities. Initial experiments using luciferase-expressing transgenic parasites demonstrated significant limitations, including signal saturation at high parasite densities, poor linearity, and unreliable quantification below 1×105 parasites/mL. These issues were exacerbated when applied to infected mouse blood samples, where biological variability and host-derived interference further reduced assay accuracy. Conversely, qPCR assays targeting the Tb177 satellite DNA sequence exhibited markedly improved performance. Standard curves generated from both cultured procyclic forms (PCF) and infected mouse blood-derived bloodstream form (BSF) samples consistently demonstrated high linearity (R2 > 0.95), wide dynamic range, and detection sensitivity down to 3×10-8 ng/reaction of parasite DNA. Manual phenol–chloroform extraction also proved more reliable than column-based extraction, possibly due to better preservation of small circular DNA molecules. However, calculated parasite densities in test samples were consistently lower than expected, particularly in BSF samples, suggesting underestimation in extrapolated results. Furthermore, no parasite DNA was detected in blood collected on days 1 and 2 post-infection, likely reflecting the transitional phase between PCF and BSF during early host adaptation.

However, a key limitation identified was the dependence on matched biological sources for both test and standard curve samples. Future work will need to assess whether a generalized standard curve can be validated across independent sample sets. Overall, these findings underscore the challenges in early trypanosome detection and support the development of qPCR as a more sensitive and practical diagnostic tool for research and vaccine evaluation. This study contributes to the foundation for improving early diagnosis and disease monitoring in trypanosomiasis control programs, with implications for both human and animal health.

Comments

This is an Open Access Thesis.

Download
Open Access

This Article is Open Access

Share

COinS