Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 121 | 2026
Research Articles

Evaluation of a new rapid diagnostic test, based on the chimeric protein Q5, for the diagnosis of human and canine forms of visceral leishmaniasis

Wagner José Tenório dos Santos1,+, Natalia Rocha Nadaes1, Allana Kelly Oliveira Dutra1, Adalucia da Silva2, Hemilly Rayanne Ferreira da Silva2, Artur Leonel de Castro Neto2, Virgínia Maria Barros de Lorena2, Valeria Rêgo Alves Pereira2, Allana Maria de Souza Pereira2, Maria Edileuza Felinto de Brito2, Milena de Paiva Cavalcanti2, Zulma Maria Medeiros2, Kamily Fagundes Pussi3, Herintha Coeto Neitzke-Abreu3, Valeria Marçal Felix de Lima4, Carlos Henrique Nery Costa5, Keila Gisele Azevedo F dos Santos1, Edimilson Domingos da Silva1, Osvaldo Pompilio de Melo Neto2,+

1Oswaldo Cruz-Fiocruz, Instituto de Tecnologia em Imunobiológicos, Bio-Manguinhos, Rio de Janeiro, RJ, Brasil
2Fundação Oswaldo Cruz-Fiocruz, Instituto Aggeu Magalhães, Recife, PE, Brasil
3Universidade Federal de Grande Dourados, Dourados, MS, Brasil
4Universidade Estadual Paulista, Araçatuba, SP, Brasil
5Universidade Federal do Piauí, Teresina, PI, Brasil

DOI: 10.1590/0074-02760250126
346 views 454 downloads
Print Email PDF Supplementary data
ABSTRACT

BACKGROUND Visceral leishmaniasis (VL), caused by Leishmania infantum, is the most severe form of leishmaniasis, prevalent in many countries, but still with limitations in diagnosis for both human and canine hosts. Serological assays based on recombinant proteins are the most efficient diagnostic alternatives, with the rapid diagnostic test (RDT) being the most cost-effective. The previously described chimeric Q5 is a recombinant protein derived from three native L. infantum antigens, which is potentially useful for both human and canine VL diagnosis, through preliminary enzyme-linked immunosorbent assay (ELISA), but which was not evaluated within an RDT setting.
OBJECTIVES To evaluate the diagnostic performance of the chimeric recombinant protein Q5 in both ELISA and RDT formats for the detection of human and canine VL, and to compare its performance with RDTs based on Lci2 and Lci13 antigens.
METHODS Here, we first expanded the Q5 evaluation through ELISA with a larger set of human and canine VL-positive sera from multiple origins. We confirmed a sensitivity ranging between 80% and 90% for the human VL and greater than 90% with the canine VL sera. A new RDT-Q5 was then set up and tested with multiple batches of human and canine sera.
FINDINGS An improved performance was seen for the human VL diagnosis (94% sensitivity), but it was reduced with canine sera (86% sensitivity). Specificity values for both the ELISA-Q5 and RDT-Q5 were generally greater than 95%, with limited (8%) or no false-positive results with human sera from individuals with cutaneous leishmaniasis (CL) and Chagas disease (CD), respectively. The RDT-Q5 performance was compared with RDTs based on two other recombinant proteins, Lci2 and Lci13, tested respectively for the human and canine VL diagnosis.
MAIN CONCLUSIONS Despite an equivalent performance for the human VL diagnosis, the RDT-Lci2 led to a much greater incidence of false-positive results with the CL and CD sera. In contrast, no setup for the RDT-Lci13 was effective with the canine sera. Our results confirm the RDT-Q5 as an efficient alternative for the VL diagnosis in the field, particularly for the human form of the disease.

key words: RDT ELISA visceral leishmaniasis recombinant proteins
REFERENCES
01. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet. 2018; 392(10151): 951-70.
02. Sasidharan S, Saudagar P. Leishmaniasis: where are we and where are we heading? Parasitol Res. 2021; 120(5): 1541-54.
03. Scarpini S, Dondi A, Totaro C, Biagi C, Melchionda F, Zama D, et al. Visceral leishmaniasis: epidemiology, diagnosis, and treatment regimens in different geographical areas with a focus on pediatrics. Microorganisms. 2022; 10(10): 1887.
04. Houghton RL, Benson DR, Reynolds L, McNeill P, Sleath P, Lodes M, et al. Multiepitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in patients with treated or untreated Chagas’ disease. J Infect Dis. 2000; 181: 325-30.
05. Elmahallawy EK, Sampedro MA, Rodriguez-Granger J, Hoyos- Mallecot Y, Agil A, Mari JMN, et al. Diagnosis of leishmaniasis. J Infect Dev Ctries. 2014; 8(8): 961-72.
06. Travi BL, Cordeiro-da-Silva A, Dantas-Torres F, Miró G. Canine visceral leishmaniasis: diagnosis and management of the reservoir living among us. PLoS Negl Trop Dis. 2018; 12(1): 1-13.
07. Thakur S, Joshi J, Kaur S. Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods. J Parasit Dis. 2020; 44(2): 253-72.
08. Mann S, Frasca K, Scherrer S, Henao-Martínez AF, Newman S, Ramanan P, et al. A review of leishmaniasis: current knowledge and future directions. Curr Trop Med Reports. 2021; 8(2): 121-32.
09. Morales-Yuste M, Martín-Sánchez J, Corpas-Lopez V. Canine leishmaniasis: update on epidemiology, diagnosis, treatment, and prevention. Vet Sci. 2022; 9(8): 1-20.
10. Singh OP, Sundar S. Developments in diagnosis of visceral leishmaniasis in the elimination era. J Parasitol Res. 2015; 2015: 239469.
11. Duthie MS, Lison A, Courtenay O. Advances toward diagnostic tools for managing zoonotic visceral leishmaniasis. Trends Parasitol. 2018; 34(10): 881-90.
12. van Griensven J, Diro E. Visceral leishmaniasis: recent advances in diagnostics and treatment regimens. Infect Dis Clin North Am. 2019; 33(1): 79-99.
13. Cunningham J, Hasker E, Das P, El Safi S, Goto H, Mondal D, et al. A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis. Clin Infect Dis. 2012; 55(10): 1312-9.
14. Bangert M, Flores-Chávez MD, Llanes-Acevedo IP, Arcones C, Chicharro C, García E, et al. Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain. PLoS Negl Trop Dis. 2018; 12(3): 1-12.
15. do Vale INPC, Saliba JW, Fonseca GSF, Peruhype-Magalhães V, de Araújo FF, Pascoal-Xavier MA, et al. Laboratorial algorithm for serological diagnosis of visceral leishmaniasis using rK39-ICT, DATLPC and FC-Simplex IgG1. J Immunol Methods. 2020; 480: 112765.
16. Pattabhi S, Whittle J, Mohamath R, El-Safi S, Moulton GG, Guderian JA, et al. Design, development and evaluation of rK28-based point-of-care tests for improving rapid diagnosis of visceral leishmaniasis. PLoS Negl Trop Dis. 2010; 4(9): e822.
17. Mukhtar M, Abdoun A, Ahmed AE, Ghalib H, Reed SG, Boelaert M, et al. Diagnostic accuracy of rK28-based immunochromatographic rapid diagnostic tests for visceral leishmaniasis: A prospective clinical cohort study in Sudan. Trans R Soc Trop Med Hyg. 2015; 109(9): 594-600.
18. Dhom-Lemos L, Viana AG, Cunha JLR, Cardoso MS, Mendes TAO, Pinheiro GRG, et al. Leishmania infantum recombinant kinesin degenerated derived repeat (rKDDR): a novel potential antigen for serodiagnosis of visceral leishmaniasis. PLoS One. 2019; 14(1): 1-17.
19. Boarino A, Scalone A, Gradoni L, Ferroglio E, Vitale F, Zanatta R, et al. Development of recombinant chimeric antigen expressing immunodominant B Epitopes of Leishmania infantum for serodiagnosis of visceral leishmaniasis. Clin Vaccine Immunol. 2005; 12(5): 647-53.
20. Grimaldi G, Teva A, Ferreira AL, dos Santos CB, Pinto IS, De- Azevedo CT, et al. Evaluation of a novel chromatographic immunoassay based on Dual-Path Platform technology (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis. Trans R Soc Trop Med Hyg. 2012; 106(1): 54-9.
21. Fraga DBM, Da Silva ED, Pacheco LV, Borja LS, de Oliveira IQ, Coura-Vital W, et al. A multicentric evaluation of the recombinant Leishmania infantum antigen-based immunochromatographic assay for the serodiagnosis of canine visceral leishmaniasis. Parasit Vectors. 2014; 7: 136.
22. Laurenti MD, de Santana LMV, Tomokane TY, De Lucca HRL, Aschar M, Souza CSF, et al. Comparative evaluation of the DPP® CVL rapid test for canine serodiagnosis in area of visceral leishmaniasis. Vet Parasitol. 2014; 205(3-4): 444-50.
23. Peixoto HM, de Oliveira MRF, Romero GAS. Serological diagnosis of canine visceral leishmaniasis in Brazil: systematic review and meta-analysis. Trop Med Int Health. 2015: 334-52.
24. Figueiredo FB, de Vasconcelos TCB, Madeira MF, Menezes RC, Maia-Elkhoury ANS, Marcelino AP, et al. Validation of the Dualpath Platform chromatographic immunoassay (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis. Mem Inst Oswaldo Cruz. 2018; 113(11): 1-7.
25. de Jesus MS, Cruz JVA, Coelho LB, Borja LS, da Silva ED, Solcà MS, et al. Early detection and persistent positivity of anti-Leishmania antibodies using a recombinant protein-based ELISA in naturally infected dogs in Brazil. Parasit Vectors. 2021; 14(1): 1-9.
26. Faria AR, Veloso LC, Coura-Vital W, Reis AB, Damasceno LM, Gazzinelli RT, et al. Novel recombinant multiepitope proteins for the diagnosis of asymptomatic Leishmania infantum-infected dogs. PLoS Negl Trop Dis. 2015; 9(1): 13-6.
27. Faria AR, Pires SF, Reis AB, Coura-Vital W, da Silveira JAG, de Sousa GM, et al. Canine visceral leishmaniasis follow-up: a new anti-IgG serological test more sensitive than ITS-1 conventional PCR. Vet Parasitol. 2017; 248: 62-7.
28. Santos TTO, Ramos FF, Gonçalves IAP, Tavares GSV, Ludolf F, Bandeira RS, et al. Potential of recombinant LiHyQ, a novel Leishmania infantum protein, for the diagnosis of canine visceral leishmaniasis and as a diagnostic and prognostic marker for human leishmaniasis and human immunodeficiency virus co-infection: a preliminary study. Acta Trop. 2021; 224: 106126.
29. Hober S, Nord K, Linhult M. Protein A chromatography for antibody purification. J Chromatogr B Anal Technol Biomed Life Sci. 2007; 848(1): 40-7.
30. Oliveira GGS, Magalhães FB, Teixeira MCA, Pereira AM, Pinheiro CGM, Santos LR, et al. Characterization of novel Leishmania infantum recombinant proteins encoded by genes from five families with distinct capacities for serodiagnosis of canine and human visceral leishmaniasis. Am J Trop Med Hyg. 2011; 85(6): 1025-34.
31. Magalhães FB, Castro Neto AL, Nascimento MB, Santos WJT, Medeiros ZM, Lima Neto ASAS, et al. Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis. PLoS One. 2017; 12(9): e0184867.
32. Santos WJT, Tavares DHC, Castro Neto AL, Nascimento MB, Dhalia R, Albuquerque AL, et al. Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. PLoS Negl Trop Dis. 2020; 14(7): 1-21.
33. Silva ED, Silva AAO, Santos EF, Leony LM, Freitas NEM, Daltro RT, et al. Development of a new lateral flow assay based on IBMP- 8.1 and IBMP-8.4 chimeric antigens to diagnose Chagas disease. Biomed Res Int. 2020; 2020: 1803515.
34. de Araújo Paz LF, da Silva A, da Silva HRF, Cavalcanti MP, de Lima VMF, Beltrão MROC, et al. Diagnostic potential for the detection of canine visceral leishmaniasis of an ELISA assay based on the Q5 recombinant protein: a large-scale and comparative evaluation using canine sera with a positive diagnosis from the Dual-Path-Platform (DPP) test. Vet Sci. 2023; 10(10): 608.
35. Anfossi L, Di Nardo F, Profiti M, Nogarol C, Cavalera S, Baggiani C, et al. A versatile and sensitive lateral flow immunoassay for the rapid diagnosis of visceral leishmaniasis. Anal Bioanal Chem. 2018; 410(17): 4123-34.
36. Cota GF, de Sousa MR, Demarqui FN, Rabello A. The diagnostic accuracy of serologic and molecular methods for detecting visceral leishmaniasis in HIV infected patients: meta-analysis. PLoS Negl Trop Dis. 2012; 6: e1665.
37. Lindoso JAL, Moreira CHV, Cunha MA, Queiroz IT. Visceral leishmaniasis and HIV coinfection: current perspectives. HIV AIDS (Auckl). 2018; 10: 193-201.
38. Freire ML, de Assis TM, Oliveira E, de Avelar DM, Siqueira IC, Barral A, et al. Performance of serological tests available in Brazil for the diagnosis of human visceral leishmaniasis. PLoS Negl Trop Dis. 2019; 13(7): 1-12.
39. Lage DP, Martins VT, Duarte MC, Costa LE, Garde E, Dimer LM, et al. A new Leishmania-specific hypothetical protein and its non-described specific B cell conformational epitope applied in the serodiagnosis of canine visceral leishmaniasis. Parasitol Res. 2016; 115(4): 1649-58.
40. de Souza ACA, de Castro RB, dos Santos YL, Pavione NRT, Agripino JM, Bahia MT, et al. High performance of ELISA test using recombinant rLiNTPDase2 from Leishmania infantum: a phase II diagnosis of canine visceral leishmaniasis. Acta Trop. 2020; 209: 105535.
41. Heidari S, Hajjaran H, Kazemi B, Gharechahi J, Mohebali M, Ranjbar MM, et al. Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis. Exp Parasitol. 2021; 222: 108065.
42. Jameie F, Dalimi A, Pirestani M, Mohebali M. Development of a multi-epitope recombinant protein for the diagnosis of human visceral leishmaniasis. Iran J Parasitol. 2021; 16(1): 1-10.
43. Siqueira WF, Viana AG, Cunha JLR, Rosa LM, Bueno LL, Bartholomeu DC, et al. The increased presence of repetitive motifs in the KDDR-plus recombinant protein, a kinesin-derived antigen from Leishmania infantum, improves the diagnostic performance of serological tests for human and canine visceral leishmaniasis. PLoS Negl Trop Dis. 2021; 15(9): e0009759.
44. Soto M, Requena JM, Quijada L, Alonso C. Multicomponent chimeric antigen for serodiagnosis of canine visceral leishmaniasis. J Clin Microbiol. 1998; 36(1): 58-63.
45. Faria AR, Costa MM, Giusta MS, Grimaldi G, Penido MLO, Gazzinelli RT, et al. High-throughput analysis of synthetic peptides for the immunodiagnosis of canine visceral leishmaniasis. PLoS Negl Trop Dis. 2011; 13; 5(9): e1310.
46. Braz LMA, Tahmasebi R, Hefford PM, Lindoso JAL. Visceral leishmaniasis diagnosis: a rapid test is a must at the hospital bedside. Clinics. 2020; 75: 1-2.
47. Ramos REM, Santos WJT, Magalhães FB, Diniz GTN, Costa CHN, Netoi OPDM, et al. Assessment of a recombinant protein from Leishmania infantum as a novel tool for visceral leishmaniasis (VL) diagnosis in VL/HIV co-infection cases. PLoS One. 2021; 16: 1-10.
48. Laurenti MD, Rossi CN, da Matta VLR, Tomokane TY, Corbett CEP, Secundino NFC, et al. Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector. Vet Parasitol. 2013; 196(3-4): 296-300.
49. Porrozzi R, Santos Da Costa MV, Teva A, Falqueto A, Ferreira AL, Dos Santos CD, et al. Comparative evaluation of enzymelinked immunosorbent assays based on crude and recombinant leishmanial antigens for serodiagnosis of symptomatic and asymptomatic Leishmania infantum visceral infections in dogs. Clin Vaccine Immunol. 2007; 14(5): 544-8.
50. Gunasekera M, Narine M, Ashton M, Esfandiari J. Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum. J Immunol Methods. 2015; 424: 7-13.

Financial support: FACEPE, CNPq, FIOTEC.
+ Corresponding authors: wagner.tenorio@bio.fiocruz.br| ORCID https://orcid.org/0000-0001-7605-4687; osvaldo.pompilio@fiocruz.br | ORCID https://orcid.org/0000-0001-5402-7346
Received 19 May 2025
Accepted 24 February 2026

HOW TO CITE
dos Santos WJT, Nadaes NR, Dutra AKO, da Silva A, da Silva HRF, de Castro Neto AL, et al. Evaluation of a new rapid diagnostic test, based on the chimeric protein Q5, for the diagnosis of human and canine forms of visceral leishmaniasis. Mem Inst Oswaldo Cruz. 2026; 121: e250126.

HANDLING EDITOR
Alexandre J da Silva | ORCID https://orcid.org/0000-0002-0865-6421

Our Location

Memórias do Instituto Oswaldo Cruz

Av. Brasil 4365, Castelo Mourisco 
sala 201, Manguinhos, 21040-900 
Rio de Janeiro, RJ, Brazil

Tel.: +55-21-2562-1222

This email address is being protected from spambots. You need JavaScript enabled to view it.

Support Program

logo iocb

01 news 18 01

faperj   cnpq capes

Site Map

Member Login