Sensitivity and specifi city of HAT Sero-K-SeT, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by Trypanosoma brucei gambiense: a case-control study

Background Human African trypanosomiasis (HAT) is a life-threatening infection aff ecting rural populations in sub- Saharan Africa. Large-scale population screening by antibody detection with the Card Agglutination Test for Trypanosomiasis (CATT)/Trypanosoma brucei (T b) gambiense helped reduce the number of reported cases of gambiense HAT to fewer than 10 000 in 2011. Because low case numbers lead to decreased cost-eff ectiveness of such active screening, we aimed to assess diagnostic accuracy of a rapid serodiagnostic test (HAT Sero-K-SeT) applicable in primary health-care centres. Methods In our case-control study, we assessed participants older than 11 years who presented for HAT Sero-K-SeT and CATT/T b gambiense at primary care centres or to mobile teams (and existing patients with confi rmed disease status at these centres) in Bandundu Province, DR Congo. We defi ned cases as patients with trypanosomes that had been identifi ed in lymph node aspirate, blood, or cerebrospinal fl uid. During screening, we recruited controls without previous history of HAT or detectable trypanosomes in blood or lymph who resided in the same area as the cases. We assessed diagnostic accuracy of three antibody detection tests for gambiense HAT: HAT Sero-K-SeT and CATT/T b gambiense (done with venous blood at the primary care centres) and immune trypanolysis (done with plasma at the Institute of Tropical Medicine, Antwerp, Belgium). Findings Between June 6, 2012, and Feb 25, 2013, we included 134 cases and 356 controls. HAT Sero-K-SeT had a sensitivity of 0·985 (132 true positives, 95% CI 0·947–0·996) and a specifi city of 0·986 (351 true negatives, 0·968–0·994), which did not diff er signifi cantly from CATT/T b gambiense (sensitivity 95% CI 0·955, 95% CI 0·906–0·979 [128 true positives] and specifi city 0·972, 0·949–0·985 [346 true negatives]) or immune trypanolysis (sensitivity 0·985, 0·947–0·996 [132 true positives] and specifi city 0·980, 0·960–0·990 [349 true negatives]). Interpretation The diagnostic accuracy of HAT Sero-K-SeT is adequate for T b gambiense antibody detection in local health centres and could be used for active screening whenever a cold chain and electricity supply are unavailable and CATT/T b gambiense cannot be done

Correction: A lineage-specific rapid diagnostic test (Chagas Sero K-SeT) identifies Brazilian Trypanosoma cruzi II/V/VI reservoir hosts among diverse mammalian orders.

[This corrects the article DOI: 10.1371/journal.pone.0227828.]

A lineage-specific rapid diagnostic test (Chagas Sero K-SeT) identifies Brazilian Trypanosoma cruzi II/V/VI reservoir hosts among diverse mammalian orders.

Trypanosoma cruzi, the protozoan agent of Chagas disease in the Americas, is comprised of six genetic lineages (TcI-TcVI) and a possible seventh (TcBat, related to TcI). Identification of T. cruzi lineages infecting reservoir mammalian species is fundamental to resolving transmission cycles. However, this is hindered by the limited sensitivity and technical complexity of parasite isolation and genotyping. An alternative approach is serology using T. cruzi lineage-specific epitopes, such as those of the trypomastigote small surface antigen (TSSA). For surveillance of T. cruzi lineage infections in mammal species from diverse Brazilian regions, we apply a novel rapid diagnostic test (RDT, Chagas Sero K-SeT), which incorporates the TSSA peptide epitope specific to TcII/V/VI (TSSApep-II/V/VI) and Protein G detection of antibodies. Chagas Sero K-SeT RDT results with sera from experimentally infected mice, from tamarin primates (Leontopithecus spp.) and from canines (Canis familiaris) were concordant with corresponding TSSApep-II/V/VI ELISAs. The Chagas Sero K-Set detected TcII/V/VI infections in Leontopithecus spp. from the Atlantic forest (n = 46), in C. familiaris (n = 16) and Thrichomys laurentius (n = 2) from Caatinga biome and Chiroptera (n = 1) from Acre, Amazonia. The Chagas Sero K-SeT RDT is directly applicable to TcII/V/VI-specific serological surveillance of T. cruzi infection in several different mammalian Orders. It can replace ELISAs and provides efficient, point-of-sampling, low-cost detection of TcII/V/VI infections, with at least equivalent sensitivity, although some mammals may be difficult to trap, and, not unexpectedly, Chagas Sero K-SeT could not recognise feline IgG. Knowledge of sylvatic hosts of T. cruzi can be expanded, new reservoir species discovered, and the ecology of transmission cycles clarified, particularly with adaptation to further mammalian Orders

Assessing antibody decline after chemotherapy of early chronic Chagas disease patients

Background: Chagas disease remains a significant public health problem in Latin America. There are only two chemotherapy drugs, nifurtimox and benznidazole, and both may have severe side effects. After complete chemotherapy of acute cases, seropositive diagnosis may revert to negative. However, there are no definitive parasitological or serological biomarkers of cure. Methods: Following a pilot study with seven Bolivian migrants to Spain, we tested 71 serum samples from chronic patients (mean age 12.6 years) inhabiting the Argentine Chaco region. Benznidazole chemotherapy (5–8 mg/kg day, twice daily for 60 days) was administered during 2011–2016. Subsequently, pre-and post-chemotherapy serum samples were analysed in pairs by IgG1 and IgG ELISA using two different antigens and Chagas Sero K-SeT rapid diagnostic tests (RDT). Molecular diagnosis by kDNA-PCR was applied to post-treatment samples. Results: Pilot data demonstrated IgG1 antibody decline in three of seven patients from Bolivia 1 year post-treatment. All Argentine patients in 2017 (averaging 5 years post-treatment), except one, were positive by conventional serology. All were kDNA-PCR-negative. Most (91.5%) pre-treatment samples were positive by the Chagas Sero K-SeT RDT, confirming the predominance of TcII/V/VI. IgG1 and IgG of Argentine patients showed significant decline in antibody titres post-chemotherapy, with either lysate (IgG, P = 0.0001, IgG1, P = 0.0001) or TcII/V/VI peptide antigen (IgG, P = 0.0001, IgG1, P = 0.0001). IgG1 decline was more discriminative than IgG. Antibody decline after treatment was also detected by the RDT. Incomplete treatment was associated with high IgG1 post-treatment titres against lysate (P = 0.013), as were IgG post-treatment titres to TcII/V/VI peptide (P = 0.0001). High pre-treatment IgG1 with lysate was associated with Qom ethnicity (P = 0.045). No associations were found between gender, age, body mass index and pre- or post-treatment antibody titres. Conclusions: We show that following chemotherapy of early chronic Chagas disease, significant decline in IgG1 antibody suggests cure, whereas sustained or increased IgG1 is a potential indicator of treatment failure. Due to restricted sensitivity, IgG1 should not be used as a diagnostic marker but has promise, with further development, as a biomarker of cure. Graphical abstract: We show that following chemotherapy of early chronic Chagas disease, a significant decline in IgG1 antibody suggests cure, whereas sustained or increased IgG1 is a potential indicator of treatment failure. Due to restricted sensitivity, IgG1 should not be used as a diagnostic marker but has promise, with further development, as a biomarker of cure.Fil: Murphy, Niamh. University of London; Reino UnidoFil: Cardinal, Marta Victoria. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. Laboratorio de Eco-Epidemiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Bhattacharyya, Tapan. University of London; Reino UnidoFil: Enriquez, Gustavo Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Macchiaverna, Natalia Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Alvedro, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Freilij, Héctor. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Martinez de Salazar, Pablo. Barcelona Institute For Global Health; EspañaFil: Molina, Israel. Barcelona Institute For Global Health; EspañaFil: Mertens, Pascal. No especifíca;Fil: Gilleman, Quentin. No especifíca;Fil: Gurtler, Ricardo Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Miles, Michael A.. University of London; Reino Unid

Lineage-specific rapid diagnostic tests can resolve Trypanosoma cruzi TcII/V/VI ecological and epidemiological associations in the Argentine Chaco.

BACKGROUND: Trypanosoma cruzi, the protozoan agent of Chagas disease, is comprised of at least 6 genetic lineages (TcI-TcVI). Their geographical distribution, clinical associations and reservoir hosts are not fully elucidated, as genotyping is hampered due to the difficulty in isolating representative populations of organisms. Lineage-specific serological techniques may address these issues. METHODS: Trypanosoma cruzi lineage-specific serological assays were performed on human, canine, feline and armadillo sera from the Gran Chaco in northern Argentina, a region of ongoing transmission. Synthetic peptides representing lineage-specific epitopes of the trypomastigote small surface antigen (TSSA) were used in ELISA, and the TcII/V/VI shared epitope peptide (TSSApep-II/V/VI) was used in the Chagas Sero K-SeT rapid diagnostic test (RDT). RESULTS: Chagas Sero K-SeT RDT, using Protein G to detect human and canine IgG, was at least as sensitive as TSSApep-II/V/VI ELISA using specific secondary antibodies. For sera from humans TSSApep-II/V/VI seroprevalence by Chagas Sero K-SeT was 273/393 (69.5%), for dogs 48/73 (65.8%) and for armadillos 1/7 (14.3%); by ELISA for cats 5/19 (26.3%). The seroprevalence for humans was similar to that for Bolivian patients, amongst whom we previously observed an association of TSSApep-II/V/VI seropositivity with severity of cardiomyopathy. In humans, prevalence of TSSApep-II/V/VI recognition was associated with locality, and with increasing and decreasing age within the Qom and Creole populations, respectively. For dogs TSSApep-II/V/VI recognition was associated with being born before community-wide insecticide spraying (P = 0.05) and with Qom household (P < 0.001). CONCLUSIONS: We show here that Chagas Sero K-SeT RDT can replace ELISA for TSSApep-II/V/VI serology of humans and dogs; for humans there were statistically significant associations between a positive Chagas Sero K-SeT RDT and being resident in Area IV, and for dogs association with Qom household or with being born before the mass spraying campaign; we also show that with cats the TcII/V/VI epitope can be detected by ELISA. We assessed the lineage distribution in an unprecedented 83% of the human T. cruzi-seropositive population. These results form the basis for more detailed studies, enabling rapid in-the-field surveillance of the distribution and clustering of these lineages among humans and mammalian reservoirs of T. cruzi infection

Severity of Chagasic Cardiomyopathy Is Associated With Response to a Novel Rapid Diagnostic Test for Trypanosoma cruzi TcII/V/VI.

Background: Trypanosoma cruzi causes Chagas disease in the Americas. The outcome of infection ranges from lifelong asymptomatic status to severe disease. Relationship between T. cruzi lineage (TcI-TcVI) infection history and prognosis is not understood. We previously described peptide-based lineage-specific enzyme-linked immunosorbent assay (ELISA) with trypomastigote small surface antigen (TSSA). Methods: A novel rapid diagnostic test (RDT; Chagas Sero K-SeT) that incorporates a peptide that corresponds to the TSSA II/V/VI common epitope was developed and validated by comparison with ELISA. Patients from Bolivia and Peru, including individuals with varying cardiac pathology, and matched mothers and neonates, were then tested using Chagas Sero K-SeT. Results: Chagas Sero K-SeT and ELISA results, with a Bolivian subset of cardiac patients, mothers, and neonates, were in accord. In adult chronic infections (n = 121), comparison of severity class A (no evidence of Chagas cardiomyopathy) with class B (electrocardiogram suggestive of Chagas cardiomyopathy) and class C/D (decreased left ventricular ejection fraction; moderate/severe Chagas cardiomyopathy) revealed a statistically significant increase in Chagas Sero K-SeT reactivity with increasing severity (χ2 for trend, 7.39; P = .007). In Peru, Chagas Sero K-SeT detected the sporadic TcII/V/VI infections. Conclusions: We developed a low cost RDT that can replace ELISA for identification of TSSA II/V/VI immunoglobulin G. Most importantly, we show that response to this RDT is associated with severity of Chagas cardiomyopathy and thus may have prognostic value. Repeated challenge with T. cruzi infection may both exacerbate disease progression and boost the immune response to the TSSApep-II/V/VI epitope

Visceral Leishmaniasis IgG1 Rapid Monitoring of Cure vs. Relapse, and Potential for Diagnosis of Post Kala-Azar Dermal Leishmaniasis.

Background: There is a recognized need for an improved diagnostic test to assess post-chemotherapeutic treatment outcome in visceral leishmaniasis (VL) and to diagnose post kala-azar dermal leishmaniasis (PKDL). We previously demonstrated by ELISA and a prototype novel rapid diagnostic test (RDT), that high anti-Leishmania IgG1 is associated with post-treatment relapse versus cure in VL. Methodology: Here, we further evaluate this novel, low-cost RDT, named VL Sero K-SeT, and ELISA for monitoring IgG1 levels in VL patients after treatment. IgG1 levels against L. donovani lysate were determined. We applied these assays to Indian sera from cured VL at 6 months post treatment as well as to relapse and PKDL patients. Sudanese sera from pre- and post-treatment and relapse were also tested. Results: Of 104 paired Indian sera taken before and after treatment for VL, when deemed clinically cured, 81 (77.9%) were positive by VL Sero K-SeT before treatment; by 6 months, 68 of these 81 (84.0%) had a negative or reduced RDT test line intensity. ELISAs differed in positivity rate between pre- and post-treatment (p = 0.0162). Twenty eight of 33 (84.8%) Indian samples taken at diagnosis of relapse were RDT positive. A comparison of Indian VL Sero K-SeT data from patients deemed cured and relapsed confirmed that there was a significant difference (p < 0.0001) in positivity rate for the two groups using this RDT. Ten of 17 (58.8%) Sudanese sera went from positive to negative or decreased VL Sero K-SeT at the end of 11-30 days of treatment. Forty nine of 63 (77.8%) PKDL samples from India were positive by VL Sero K-SeT. Conclusion: We have further shown the relevance of IgG1 in determining clinical status in VL patients. A positive VL Sero K-SeT may also be helpful in supporting diagnosis of PKDL. With further refinement, such as the use of specific antigens, the VL Sero K-SeT and/or IgG1 ELISA may be adjuncts to current VL control programmes

Sensitivity and specificity of HAT Sero-K-SeT, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by Trypanosoma brucei gambiense: a case-control study

Background: Human African trypanosomiasis (HAT) is a life-threatening infection affecting rural populations in sub-Saharan Africa. Large-scale population screening by antibody detection with the Card Agglutination Test for Trypanosomiasis (CATT)/Trypanosoma brucei (T b) gambiense helped reduce the number of reported cases of gambiense HAT to fewer than 10 000 in 2011. Because low case numbers lead to decreased cost-effectiveness of such active screening, we aimed to assess diagnostic accuracy of a rapid serodiagnostic test (HAT Sero-K-SeT) applicable in primary health-care centres. Methods: In our case-control study, we assessed participants older than 11 years who presented for HAT Sero-K-SeT and CATT/T b gambiense at primary care centres or to mobile teams (and existing patients with confirmed disease status at these centres) in Bandundu Province, DR Congo. We defined cases as patients with trypanosomes that had been identified in lymph node aspirate, blood, or cerebrospinal fluid. During screening, we recruited controls without previous history of HAT or detectable trypanosomes in blood or lymph who resided in the same area as the cases. We assessed diagnostic accuracy of three antibody detection tests for gambiense HAT: HAT Sero-K-SeT and CATT/T b gambiense (done with venous blood at the primary care centres) and immune trypanolysis (done with plasma at the Institute of Tropical Medicine, Antwerp, Belgium). Findings: Between June 6, 2012, and Feb 25, 2013, we included 134 cases and 356 controls. HAT Sero-K-SeT had a sensitivity of 0·985 (132 true positives, 95% CI 0·947–0·996) and a specificity of 0·986 (351 true negatives, 0·968–0·994), which did not differ significantly from CATT/T b gambiense (sensitivity 95% CI 0·955, 95% CI 0·906–0·979 [128 true positives] and specificity 0·972, 0·949–0·985 [346 true negatives]) or immune trypanolysis (sensitivity 0·985, 0·947–0·996 [132 true positives] and specificity 0·980, 0·960–0·990 [349 true negatives]). Interpretation: The diagnostic accuracy of HAT Sero-K-SeT is adequate for T b gambiense antibody detection in local health centres and could be used for active screening whenever a cold chain and electricity supply are unavailable and CATT/T b gambiense cannot be done. Funding: European Commission FP7 (NIDIAG, Grant Agreement 260260) and WHO Control of Neglected Tropical Diseases, Innovative and Intensified Disease Management