Background
Human African trypanosomiasis (HAT) occurs in two forms, caused by Trypanosoma brucei gambiense in West and Central Africa and Trypanosoma brucei rhodesiense in Eastern and Southern Africa. Although substantial progress toward elimination has been achieved in endemic regions, T. brucei infections continue to pose diagnostic challenges in non-endemic countries in people arriving from affected areas. South Africa is not endemic for HAT and all detected cases are imported or travel-associated. Reference laboratories therefore play a critical role in confirmatory diagnosis.

Methods
We conducted a retrospective laboratory-based review of samples submitted for trypanosomiasis testing at the National Institute for Communicable Diseases (NICD) between 2016 and 2025, evaluating diagnostic outcomes, specimen characteristics, and referral patterns. All specimens referred for suspected or confirmatory T. brucei infection were included, and demographic, referral and laboratory data were analysed.

Results
A total of 38 patients with submitted samples were included; 69% were male and 29% female, with a median age of 45 years (range 12–87). Whole blood (81.8%) and cerebrospinal fluid (CSF) (10.9%) were the most frequently submitted specimen types. Whole blood demonstrated a positivity rate of 23.6%, while none of the CSF samples tested positive. A real-time PCR for detection of Trypanosoma brucei species complemented microscopy and enabled analysis of additional specimen types; notably, one case was diagnosed from serum using PCR. Most submissions (92%) originated from the private health sector, primarily in Gauteng Province, of which 25% tested positive. Confirmed cases reported travel or residence in Zambia, Malawi, Tanzania, and the Democratic Republic of the Congo.

Conclusions
This review highlights the importance of centralised reference laboratory services for detecting imported HAT in non-endemic countries. Expanded molecular diagnostic capacity and sustained clinical awareness remain essential for timely detection. Implementation of multiplex PCR capable of distinguishing T. brucei subspecies, currently under development at the PRL, will enhance surveillance sensitivity and support ongoing elimination efforts in the African region.