Rabies is widespread throughout Africa and Asia, despite the fact that the control
and elimination of this disease has been proven to be feasible. Lesotho, a small landlocked
country surrounded by South Africa, has been known to be endemic for rabies since the
1980s but the epidemiology of the disease remains poorly understood due to limited sample
submission, constrained diagnostic capabilities, and a lack of molecular epidemiological data.
Considering the existing challenges experienced in Lesotho, we aimed to evaluate the direct, rapid
immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) test
for rabies diagnosis in Lesotho. Towards this aim, extensive training on the implementation and
interpretation of the DRIT was hosted in Lesotho in April 2016 before both tests were applied to all
samples subjected to routine rabies diagnosis at the Central Veterinary Laboratory (CVL). We found
agreement between the DFA and DRIT assays in 90/96 samples (93.75%). The samples that produced
inconsistent results (n = 6) were re-tested a further two times with both assays before being subjected
to a real-time qPCR to confirm the diagnosis. Additionally, a statistically significant three-fold increase
in the average number of samples submitted per month was observed after the DRIT implementation
started, following continuous rabies awareness initiatives amongst the animal health professionals
in the country over a 12-month period (p = 0.0279). Partial G-L intergenic regions of selected
rabies-positive samples (n = 21) were amplified, sequenced, and subjected to phylogenetic analyses.
Molecular epidemiological analyses, which included viruses from neighbouring provinces in South
Africa, suggested that at least three independent rabies cycles within Lesotho were implicated in
instances of cross-border transmission. This study has evaluated alternative methods for diagnosing
and improving rabies surveillance in Lesotho, as well as providing new information that would be of
importance in the planning of future disease intervention campaigns, not only in Lesotho, but also in
neighbouring South Africa.
Table S1. Panel of rabies viruses from Lesotho and neighbouring South African provinces included in the
phylogenetic analysis performed in this study.