Veerle Lejon

Chagas disease (CD), caused by Trypanosoma cruzi , is a significant public health challenge in Latin America, where an estimated 70% of infected individuals remain undiagnosed. Rapid diagnostic tests (RDTs) offer a promising solution for new screening strategies, yet their performance has not been systematically assessed in Mexican cohorts. Here, we evaluated the diagnostic performance of five RDTs: Chagas Rapid Test (Certum), Bio-Chagas Rapid Test (Mexlab), Bioline ^TM Chagas Ab (Abbott), Chagas Rapid Test (Amunet) and Chagas Sero K -SeT (Coris BioConcept). Performance was tested using 79 archived human serum samples from Yucatán. All tests were benchmarked against the diagnostic reference algorithm established by the Mexican Official Norm ( NOM ), where samples were characterised as positive if they were reactive to ≥ 2 conventional serological assays (Chagatest Lisado , Chagatest Recombinante v4.0 and Chagatest HAI). The RDTs demonstrated variable diagnostic sensitivity. The Bioline ^TM Chagas Ab test demonstrated the highest sensitivity (97.2%, 95% CI: 85.5 - 99.9; 35/36 positive samples detected) and almost perfect statistical agreement with the NOM reference algorithm (κ= 0.90; p < 0.0001). Similarly, the Chagas Sero K -SeT prototype showed a high sensitivity (91.7%, 95% CI: 77.5 – 98.2; 33/36 positive samples detected) and substantial concordance (κ= 0.87, p < 0.0001) with the NOM -based composite result. In contrast, the remaining RDTs demonstrated low sensitivities (Certum: 0.0%, 95% CI: 0.0–9.7; Mexlab: 5.6%, 95% CI: 0.7–18.7; Amunet: 33.3%, 95% CI: 18.6–51.0), limiting their suitability for CD diagnostic algorithms, at least in Yucatán. Specificity was consistently high across the RDTs, ranging from 93.0% to 100% (95% CIs: 80.9–100). These findings underscore the need to locally validate diagnostic tools. The Bioline ^TM Chagas Ab and Chagas Sero K -SeT tests met the WHO benchmarks for RDT performance and, if validated in larger cohorts, could strengthen the screening of T. cruzi infection and epidemiological surveillance in Mexico. Author summary: Chagas disease, caused by the parasite Trypanosoma cruzi , remains underdiagnosed in Mexico. Rapid diagnostic tests (RDTs) could help expand access to screening, particularly in settings where laboratory testing is difficult to implement. However, the performance of these tests can vary geographically and has not been systematically evaluated in Mexico. We evaluated four commercially available RDTs and one developmental prototype using archived serum samples from Yucatán, a region where Chagas disease is endemic. Results were compared with the diagnostic algorithm recommended by the Mexican Official Norm. The Bioline™ Chagas Ab test showed the highest sensitivity, detecting 35 of 36 positive samples, while the Chagas Sero K-SeT prototype detected 33. The other three commercial RDTs showed substantially lower sensitivity, detecting between 0 and 12 positive samples. Specificity was high across all five tests, ranging from 93.0% to 100%, indicating that most negative samples were correctly identified. However, the low sensitivity of three tests limits their usefulness for Chagas disease screening in Yucatán. Our findings highlight the importance of locally validating RDTs before their implementation. The Bioline ^TM Chagas Ab and Chagas Sero K -SeT tests show promise for Chagas disease screening in Mexico but require further evaluation using larger and geographically diverse cohorts.

Pigs are suspected to act as a reservoir for Trypanosoma brucei gambiense and may therefore contribute to the re-emergence of human African trypanosomiasis (HAT), despite ongoing efforts to eliminate transmission. To clarify the role of this species in HAT epidemiology and to better understand the dynamics of T. b. gambiense infection in blood, we experimentally infected pigs with this parasite. In this study, we compared nine infected pigs with eight control pigs over a period of more than ten months using parasitological, molecular, and serological methods. The results showed that each diagnostic method has limitations, in terms of sensitivity to detect the presence of T. b. gambiense , and specificity to differentiate infection caused by T. b. gambiense from infection caused by T. b. brucei, responsible for animal African trypanosomosis. Nevertheless, the overall analysis showed that pigs can remain infected with T. b. gambiense for at least ten months. Hence, pigs should be considered a potential long-term reservoir of T. b. gambiense that could compromise the sustainability of the current elimination process. As a result, there is a need to pursue investigations on this topic, considering possible co-infections with T. b. brucei . In the meantime, adapted measures should be envisaged to limit contact between pigs and tsetse flies and/or to clear potential pig infections with T. b. gambiense by curative treatment.

This study is aimed at evaluating the inclusion of miniature anion‐exchange centrifugation technique (mAECT) in the diagnosis and fexinidazole as treatment of gHAT in Central African Republic (CAR) after capacity building. A cross‐sectional pilot study was conducted during a mass diagnostic campaign from 16 September to 22 October 2022 in Nola‐Bilolo, a historic focus in CAR. The serological test, card agglutination test for trypanosomiasis (CATT), was performed on whole blood and diluted plasma to screen participants, followed by a confirmatory parasitological test by capillary tube centrifugation (CTC) and mAECT. Positive cases were treated mainly with fexinidazole. A total of 2070 participants were screened, out of an estimated population of 3584, representing a participation rate of 58%. The seroprevalence of HAT was 1.6% (CI 95%: 77%–99%) (33/2070) by the CATT plasma end‐dilution titre ≥ 1:16. Blood from serological subjects was examined microscopically after concentration by CTC and mAECT was positive for trypanosomes in 48.48% (CI 95%: 31%–66%) (16/33) and 87.88% (CI 95%: 77%–99%) (29/33) of cases, respectively. The prevalence of microscopically confirmed HAT was 0.77% (CI 95%: 0.40%–1.15%) and 1.4% (CI 95%: 1%–2%) by CTC and mAECT, respectively. Twenty‐one (72.41%) patients were treated with fexinidazole with a 0% interruption rate. The introduction of fexinidazole (2021) and mAECT (2022) likely contributed to the rise in reported cases (from 45 in 2021 to 111 in 2024) and improved screening coverage in the study area, reflecting enhanced case detection and access to care. There is a necessity to establish diagnostic quality assurance and to reinforce the other control measures including vector control.

Background: Passive screening of gambiense human African trypanosomiasis (HAT) is based on rapid diagnostic tests (RDT), but sensitivity of the currently commercialised RDTs has hardly been assessed prospectively. In view of the increasing importance of remote testing for HAT, the diagnostic performance of reference laboratory tests also needs further documentation. Methodology/Principal Findings: The study is registered in ClinicalTrials.Gov under identifier NCT03356665. Clinical suspects in 29 health facilities in DR Congo were screened consecutively between October 2017 and December 2020 with 3 HAT RDTs, including HAT Sero K-SeT, an RDT that is nowadays still commercialised. HAT RDT positives were examined parasitologically and their dried blood spots tested in trypanolysis, indirect ELISA/T.b. gambiense, LAMP Trypanosoma brucei Detection Kit and m18S and TgsGp qPCR. Association of clinical signs with HAT, and sensitivity, specificity, and predictive values of the screening and reference laboratory tests were estimated using parasitology as the gold standard. Trypanosomes were detected in 42/3113 study participants. Logistic regression revealed that sleep disruption, enlarged lymph nodes, psychiatric problems, recurrent fever not responding to anti-malarials and motor disorders were significantly associated with HAT (p < 0.05, odds 3.0-10.6). Together, the RDTs detected 253/3113 seropositives. Sensitivity and specificity of HAT Sero K-SeT were respectively 100% (42/42; 95% CI 91.6-100%) and 93.9% (2882/3071; 95% CI 92.9-94.6%). Specificities of the reference laboratory tests were ≥ 91.6%, except for LAMP. Sensitivity of ELISA/T.b. gambiense and trypanolysis were 93.9% (31/33; 95% CI 80.4-98.9) and 84.9% (28/33; 95% CI 69.1-93.4), and were ≤ 63.6% for LAMP, m18S and TgsGp qPCR. Conclusions/Significance: Compared to the WHO’s target product profiles for gambiense HAT RDTs, the HAT Sero K-SeT RDT had ideal sensitivity but its specificity was on the borderline of minimally acceptable. Sub-optimal sensitivities of trypanolysis and to a lesser extent, indirect ELISA/T.b. gambiense when applied on DBS, were confirmed. Molecular tests for remote testing need to be improved and evaluated further.

Human African Trypanosomiasis (HAT) is a severe disease endemic to Sub-Saharan Africa caused by the parasite Trypanosoma brucei . Diagnosis of the chronic form of HAT relies heavily on serological screening tests that detect specific antibodies. Trypanosoma brucei parasites rely on the antigenic variation of their variant surface glycoproteins (VSG) to evade host antibody recognition. Although VSG is the primary target of host antibodies, studies of the antibody response to VSG have been limited by low-throughput approaches that allow for the analysis of only a few VSG proteins at a time, primarily in animal models. Here, we use phage immunoprecipitation sequencing (PhIP-seq) to characterize the antibody response to VSG in HAT patient serum. Using a comprehensive T7 bacteriophage display library containing peptides representing 12,108 unique T. brucei VSGs, our analysis revealed significant differences in the anti-VSG antibody between the acute and chronic forms of HAT. Our results show that, in chronic HAT, the top lobe of the VSG is the primary interface of antibody interactions. By characterizing the targets of anti-VSG immunity, we also identified a potential bias in the CATT, the most common test used for HAT screening. Finally, we were able to identify sets of seroprevalent VSG peptides for each form of the disease that successfully distinguish between infected and uninfected individuals. Our results demonstrate the utility of characterizing host antibody responses against African trypanosomes, which can reveal unique features of parasite biology while also uncovering new diagnostic targets.

Background: Passive screening of gambiense human African trypanosomiasis (HAT) is based on rapid diagnostic tests (RDT), but sensitivity of the currently commercialised RDTs has hardly been assessed prospectively. In view of the increasing importance of remote testing for HAT, the diagnostic performance of reference laboratory tests also needs further documentation. Methodology/Principal Findings: The study is registered in ClinicalTrials.Gov under identifier NCT03356665 . Clinical suspects in 29 health facilities in DR Congo were screened consecutively between October 2017 and December 2020 with 3 HAT RDTs, including HAT Sero K -SeT, an RDT that is nowadays still commercialised. HAT RDT positives were examined parasitologically and their dried blood spots tested in trypanolysis, indirect ELISA/ T.b. gambiense , LAMP Trypanosoma brucei Detection Kit and m18S and TgsGp qPCR. Association of clinical signs with HAT, and sensitivity, specificity, and predictive values of the screening and reference laboratory tests were estimated using parasitology as the gold standard. Trypanosomes were detected in 42/3113 study participants. Logistic regression revealed that sleep disruption, enlarged lymph nodes, psychiatric problems, recurrent fever not responding to anti-malarials and motor disorders were significantly associated with HAT ( p 99% sensitivity of the target product profile established by WHO, but its specificity was on the limit of minimally acceptable, set by WHO at >95%. Antibody detection with trypanolysis and/or indirect ELISA/ T.b. gambiense is already routinely implemented, but the use of dried blood spots seems to reduce the tests’ sensitivity. LAMP Trypanosoma brucei Detection Kit, m18S and TgsGp qPCR were not reliable. These results confirm other study data from Guinea and Côte d’Ivoire. The diagnostic performance of newly emerging molecular tests, should be further evaluated.

Background Coordinated efforts in the control of gambiense human African trypanosomiasis (gHAT) have significantly reduced its endemicity. WHO targets interruption of transmission by 2030. However, challenges remain, including low sensitivity of the current parasitological confirmation tests, leaving a potential human reservoir untreated. Acoziborole, a single-dose oral treatment, effective in both disease stages with a good safety profile, offers the potential of treatment of parasitologically negative gHAT seropositive subjects, which could improve diagnostic sensitivity. The STROGHAT study aims to evaluate whether this approach can lead to elimination of T.b. gambiense from its human reservoir, and to provide further safety data on acoziborole. It also includes a costing analysis and a prospective evaluation of the performance of the screening and diagnostic tests used. Methods STROGHAT is a one-arm epidemiological study, with a nested phase IIIb, one-arm, open label, non-randomized, multicentre clinical trial. It will be implemented over four years in the endemic region of the Equateur North, in the Democratic Republic of Congo. For the first three years, parasitologically negative gHAT seropositive subjects will be treated with acoziborole, while parasitologically confirmed cases will receive standard of care. Individual follow-up needs and accurate prevalence estimate will be based on immunological and molecular tests performed for all gHAT screening test seropositive subjects at a reference laboratory. In the fourth year, standard procedures will resume, and a prevalence survey will assess whether interruption of transmission has been achieved. Discussion The manuscript outlines the study background, objectives and methods while discussing its strengths and challenges. If successful, the STROGHAT study will provide critical evidence on the effectiveness, safety and feasibility of the new strategy, and inform future elimination strategies. Clinical trial registration NCT06356974. Date of registration: April 4, 2024.

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

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. Method: During active screening, venous blood samples from 1095 individuals from Côte d’Ivoire and Guinea were tested consecutively with commercial (Bioline HAT 2.0, HAT Sero-K-SeT, CATT) and prototype (HAT Sero-K-SeT 2.0, DCN) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis, indirect ELISA) and molecular laboratory tests (conventional PCR, SHERLOCK, Trypanozoon S²-RT-qPCR, SNP RT-qPCR). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Results: One gHAT case was diagnosed. Overall test specificities (n=1094) were: CATT 98.9% (98.1-99.4%); HAT Sero-K-SeT 86.7% (84.5-88.5%); Bioline HAT 2.0 82.1% (79.7-84.2%); DCN HAT RDT 78.2% (75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Bioline HAT 2.0 and HAT Sero-K-Set 2.0. 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 laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular 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 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.