Ethical approvals for the study were obtained from Enugu State Ministry of Health Research Ethics Committee (Ref No: MH/MSD/REC21/782), and the University of Nigeria, Nsukka Institutional Animal Care and Use Committee (IACUC). Informed consent was obtained from all human participants before sample collection.

Blood samples were collected in EDTA-coated sample bottles from hospital diagnostic laboratories (for human samples) and abattoirs (for animal samples) from two states in Southern Nigeria (Delta and Enugu States). After collection, samples were transported in ice packs (2–8 °C) to the laboratory. Upon arrival, all samples were processed within few hours. Serum was extracted by centrifugation at 3000 rpm for 10 minutes and aliquoted into sterile, labeled vials. Serum samples not analyzed immediately were stored at 4 °C to preserve sample integrity.

The serum samples were first screened for HAT with ELISA using plates coated with native antigens. The samples that tested positive on ELISA at 1:40 and 1:200 serum dilution were then screened with CATT kit (obtained from the Institute of Tropical Medicine, Belgium) at 1:4, 1:8, 1:16, 1:32, 1:64 serum dilutions. Due to low availability of RDT kits, 11 human samples and 5 animal samples that tested positive on both ELISA (1:200 dilution) and CATT (1:64 dilution) following the flowchart ( Figure 1 ) were further tested with the RDT kit (Abbott Bioline HAT 2.0), which was obtained from the Federal Ministry of Health.

The observed failure of the RDT kit to detect any of the ELISA/CATT seropositive human samples whereas it was able to detect two seropositive animal samples is both surprising and unexpected. This raises important concerns regarding the sensitivity and specificity of the RDT in this geographic location, suggesting potential limitations in its sensitivity and diagnostic reliability for Human African Trypanosomiasis in Nigeria. It has been demonstrated that Trypanosoma brucei gambiense VSGs and immune-reactivity tests are clearly correlated with geographical origin, and Nigerian samples have exhibited variable responses to immunologic/serologic tests as the targeted Variable Antigen Type (VAT) for these serologic tests are known to be absent in isolates from certain geographical locations such as Nigeria and Cameroon (11, 12). It is becoming apparent that variations in local strains, as indicated by the genetic diversity of Trypanosoma species across different geographical regions, may compromise the effectiveness of serologic tests generally, including RDTs (11). These serologic tools, which were developed and validated elsewhere, may not reliably detect Nigeria’s local parasite strains, thereby increasing the risk of false negatives (7–9). This RDT kit has previously been shown to perform poorly with unreliable results in passive surveillance studies in Nigeria (15). However, it is still being used for case detection and surveillance by the health authorities. This could contribute largely to the failure to detect cases in Nigeria, leading to the misleading prevalence reports/data recorded by WHO for policy implementation (6). Hence, there is a need to take into consideration the geographic diversity of Trypanosoma strains in the development of diagnostic tools.

The RDT kits are specifically intended for use in the diagnosis of human Trypanosoma brucei gambiense infections, and are being used by the Federal Ministry of Health for HAT surveillance in Nigeria, though with variable results in different regions (16). The usual concern for HAT RDTs in many endemic areas is the occurrence of false positive reactions (low specificity), which are believed to be associated with cross-reacting antibodies in malaria co-endemic areas (7, 17). In high-prevalence settings, a test with low specificity will produce more false positives. Hence, increasing specificity ensures that positive results are more likely to be true cases. This improves the positive predictive value (PPV) which is the likelihood that a person who tests positive actually has the disease. It is widely believed that RDTs using recombinant antigens have higher specificity than those with native antigens (9, 18). Hence, the use of recombinant antigens in the Abbot Bioline HAT 2.0 RDT kit was supposed to improve specificity from previous RDTs. However, the findings in this report show that this may compromise sensitivity in some cases, especially in low-prevalence settings where a higher sensitivity is more desirable to ensure that all positive cases are detected. This is particularly important when disease elimination is targeted, as is the case for HAT. Nigeria has low HAT prevalence, and the use of a low-sensitive surveillance tool like this RDT kit portends great dangers for false negatives, thereby increasing the risk of missed infections, continued disease transmission, and the possibility of disease/epidemic re-emergence. This is in addition to the well-established absence of some VATs used in HAT serologic tests like RDTs in isolates from certain geographical locations such as Nigeria and Cameroon (11, 12).

While indigenous researchers have reported HAT case detection in Nigeria using several serologic, molecular, and biological means (2, 19, 20), these reports are hardly validated by the health authorities for appropriate disease reporting. This is because WHO largely relies on a particular serologic test (trypanolysis) for the validation of detected cases before official acceptance for reporting, despite the identified drawbacks of these serologic tests with regards to VSG and VAT geographical variability (10, 14). It is interesting to note that even the WHO-recorded case of 2016 was also negative in a trypanolysis test (3). Although these serologic tests have been acclaimed to be specific for the detection of human infection by T. brucei gambiense, Ilboudo et al., 2022 had demonstrated using pigs that the VATs used in the trypanolysis test is not specific for Tbg infection (14). Similarly, Matovu et al. (18) reported that serological tests designed for gambiense HAT can detect antibodies in cattle, further supporting the possibility of cross-reactivity or non-specific binding in animal hosts (18). The positive test results in animal samples obtained in this study raises important questions about the specificity of these diagnostic tools. One may argue that it is possible these positive animal samples were infected with Tbg. But the question remains as to why the RDT kit is not detecting the human samples that were also positive on ELISA and CATT like the animal samples. On the other hand, the animal samples were positive in the invariant surface glycoprotein (ISG) test line of the RDT (test line 1), and it is possible that this antigen is not specific to human or Tbg infection. It is also possible that the RDT was detecting ISG from other animal trypanosomes in the animal samples. However, these RDTs were designed to specifically detect Tbg infection in human samples. This particularly highlights the need to investigate the specificity of antigens used in these serologic tests for human Trypanosoma infections (14).

The use of this RDT for HAT surveillance may significantly underestimate the burden of HAT in Nigeria because failure to detect seropositive human samples and the detection of animal samples in this study location indicates significant shortcomings in the sensitivity and specificity of the RDT kit. Hence, there is a need to validate diagnostic tools in various geographical regions before field deployment. Although this study used a small number of samples with the RDT kit, the observations are consistent with the results previously reported that indicated the unreliability of the RDT kit in passive HAT surveillance in Nigeria (15).