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Abstract Background Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future “screen and treat” strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. Methods During active screening, venous blood samples from 1095 individuals from Côte d’Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. Results One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1–99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5–88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7–84.2%); DCN HAT RDT 78.2% (95% CI: 75.7–80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9–80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7–100% (n = 399) and 93.0–100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. Conclusions Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based “screen and treat” strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. Trial registration The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022. Graphical Abstract

Abstract Background Passive diagnosis of human African trypanosomiasis (HAT) at the health facility level is a major component of HAT control in Guinea. We examined which clinical signs and symptoms are associated with HAT, and assessed the performance of selected clinical presentations, of rapid diagnostic tests (RDT), and of reference laboratory tests on dried blood spots (DBS) for diagnosing HAT in Guinea. Method The study took place in 14 health facilities in Guinea, where 2345 clinical suspects were tested with RDTs (HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT). Seropositives underwent parasitological examination (reference test) to confirm HAT and their DBS were tested in indirect enzyme-linked immunoassay (ELISA)/Trypanosoma brucei gambiense, trypanolysis, Loopamp Trypanosoma brucei Detection kit (LAMP) and m18S quantitative PCR (qPCR). Multivariable regression analysis assessed association of clinical presentation with HAT. Sensitivity, specificity, positive and negative predictive values of key clinical presentations, of the RDTs and of the DBS tests for HAT diagnosis were determined. Results The HAT prevalence, as confirmed parasitologically, was 2.0% (48/2345, 95% CI: 1.5–2.7%). Odds ratios (OR) for HAT were increased for participants with swollen lymph nodes (OR = 96.7, 95% CI: 20.7–452.0), important weight loss (OR = 20.4, 95% CI: 7.05–58.9), severe itching (OR = 45.9, 95% CI: 7.3–288.7) or motor disorders (OR = 4.5, 95% CI: 0.89–22.5). Presence of at least one of these clinical presentations was 75.6% (95% CI: 73.8–77.4%) specific and 97.9% (95% CI: 88.9–99.9%) sensitive for HAT. HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT were respectively 97.5% (95% CI: 96.8–98.1%), 99.4% (95% CI: 99.0–99.7%) and 97.9% (95% CI: 97.2–98.4%) specific, and 100% (95% CI: 92.5–100.0%), 59.6% (95% CI: 44.3–73.3%) and 93.8% (95% CI: 82.8–98.7%) sensitive for HAT. The RDT’s positive and negative predictive values ranged from 45.2–66.7% and 99.2–100% respectively. All DBS tests had specificities ≥ 92.9%. While LAMP and m18S qPCR sensitivities were below 50%, trypanolysis and ELISA/T.b. gambiense had sensitivities of 85.3% (95% CI: 68.9–95.0%) and 67.6% (95% CI: 49.5–82.6%). Conclusions Presence of swollen lymph nodes, important weight loss, severe itching or motor disorders are simple but accurate clinical criteria for HAT referral in HAT endemic areas in Guinea. Diagnostic performances of HAT Sero-K-Set and SD Bioline HAT are sufficient for referring positives to microscopy. Trypanolysis on DBS may discriminate HAT patients from false RDT positives. Trial registration The trial was registered under NCT03356665 in clinicaltrials.gov (November 29, 2017, retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03356665 ) Graphical Abstract