Preparation and Development of An Immunochromatographic Test for early Detection of Canine Visceral Leishmaniasis: A Preliminary Study
-
Akram Azambakhtiar
,
Sedigheh Nabian
,
Mehdi Mohebali
,
Mohammad Taheri
,
Zabihollah Zarei
,
Ahmad Hosseini-Safa
Abstract
Background: Canine visceral leishmaniasis (CVL), caused by Leishmania infantum (L. infantum), is a serious parasitic disease. Domestic dogs in endemic regions act as primary reservoirs for the parasite. Early diagnosis, control, and regular screening of dogs are essential for effective disease management. This study aims to develop a practical, low-cost immunochromatographic test (ICT) for detecting specific anti-Leishmania antibodies in domestic dogs.
Methods: Overall, 93 canine serum samples were collected from endemic and non-endemic areas of CVL in Iran. Rabbit anti-canine antibodies were conjugated with gold nanoparticles, and strips were coated with Leishmania antigens. A drop of serum was added to each strip, and a positive result was indicated by two red lines. The validity of ICT for the detection of CVL in the field was determined with comparing to direct agglutination test (DAT) as gold serological test on 40 sera with anti-Leishmania antibodies at titers ≥1:320 considered as positive control as well as 53 sera with no anti-Leishmania antibodies including 10 collected from healthy dogs and 43 from other infectious diseases considered as negative control sera.
Results: A sensitivity of 82.5% (CI 95%.78.1-86.9) and specificity of 90.5% (CI 95%. 87.7-92.5) were found at a 1:320 cut off titer when DAT confirmed cases were compared with negative control. The agreement (0. 871) was found between ICT and DAT using kappa analysis.
Conclusion: A relatively good agreement was found between ICT and DAT. Further researches on test validation with larger populations in endemic and non-endemic areas of CVL, is recommended.
1. Kato H. Epidemiology of Leishmaniasis: Risk factors for its pathology and infection. Parasitol Int. 2025;105:102999.
2. Yadav P, Azam M, Ramesh V, et al. Unusual observations in leishmaniasis; an overview. Pathogens. 2023;12(2):297.
3. Mohebali M, Edrissian G, Akhoundi B, et al. Visceral leishmaniasis in Iran: An update on epidemiological features from 2013 to 2022. Iran J Parasitol. 2023;18(3):279-93.
4. Farooq I, Singh R, Selvapandiyan A, et al. The burden of visceral leishmaniasis: need of review, innovations, and solutions. Challenges and Solutions Against Visceral Leishmaniasis. 2024;28:1-17.
5. Grifferty G, Shirley H, McGloin J, et al. Vulnerabilities to and the socioeconomic and psychosocial impacts of the leishmaniases: A Review. Res Rep Trop Med. 2021; 12:135-151.
6. Morales-Yuste M, Martin-Sanchez J, Corpas-Lopez V. Canine leishmaniasis: Update on epidemiology, diagnosis, treatment, and prevention. Vet Sci. 2022;9(8):387.
7. Mohebali M, Moradi-Asl E, Rassi Y. Geographic distribution and spatial analysis of Leishmania infantum infection in domestic and wild animal reservoir hosts of zoonotic visceral leishmaniasis in Iran: A systematic review. J Vector Borne Dis. 2018;55(3):173-83.
8. Vilas-Boas DF, Nakasone EKN, Gonçalves AAM, et al. Global distribution of canine visceral leishmaniasis and the role of the dog in the epidemiology of the disease. Pathogens. 2024;13(6):455.
9. Van J, Diro E. Visceral leishmaniasis: recent advances in diagnostics and treatment regimens. Infect Dis Clin North Am. 2019;33(1):79-99.
10. Cosma C, Maia C, Khan N, et al. Leishmaniasis in humans and animals: A one health approach for surveillance, prevention and control in a changing world. Trop Med Infect Dis. 2024;9(11):258.
11. Sunyoto T, Boelaert M, Meheus F. Understanding the economic impact of leishmaniasis on households in endemic countries: a systematic review. Expert Rev Anti Infect Ther. 2019;17(1):57-69.
12. Okwor I, Uzonna J. Social and economic burden of human leishmaniasis. Am J Trop Med Hyg. 2016;94(3):489-93.
13. Mohebali M, Edrisian GH, Nadim A, et al. Application of direct agglutination test (DAT) for the diagnosis and seroepidemiological studies of visceral leishmaniasis in Iran. Iran J Parasitol. 2006; (1):15-25.
14. Mohebali M, Keshavarz H, Shirmohammad S, et al. The diagnostic accuracy of direct agglutination test for serodiagnosis of human visceral leishmaniasis: a systematic review with meta-analysis. BMC Infect Dis. 2020;20(1):946.
15. Harith A, Slappendel R, Reiter I, et al. Application of a direct agglutination test for detection of specific anti-Leishmania antibodies in the canine reservoir. J Clin Microbiol. 1989;27(10):2252-7.
16. Heidari S, Hajjaran H, Kazemi B, 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.
17. Malmasi A, Janitabar S, Mohebali M, et al. Seroepidemiologic survey of canine visceral leishmaniasis in Tehran and Alborz Provinces of Iran. J Arthropod Borne Dis. 2014;8(2):132-8.
18. Gao C, Yang Y, Shi F, et al. Development of an immunochromatographic test for diagnosis of visceral leishmaniasis based on detection of a circulating antigen. PLoS Negl Trop Dis. 2015;9(6):e0003902.
19. Hosseini Farash BR, Mohebali M, Kazemi B, et al. Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts. Sci Rep. 2022;12(1):10426.
20. Ejazi SA, Bhattacharyya A, Choudhury ST, et al. Immunoproteomic identification and characterization of Leishmania membrane proteins as non-invasive diagnostic candidates for clinical visceral leishmaniasis. Sci Rep. 2018;8(1):12110.
21. Hosseini Farash BR, Mohebali M, Kazemi B, et al. Cloning of K26 hydrophilic antigen from Iranian strain of Leishmania infantum. Iran J Public Health. 2017;46(10):1359-65.
22. Roberts T, Keddie SH, Rattanavong S, et al. Accuracy of the direct agglutination test for diagnosis of visceral leishmaniasis: a systematic review and meta-analysis. BMC Infect Dis. 2023;23(1):782.
23. Harith A, Kolk A, Kager P, et al. A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis. Trans R Soc Trop Med Hyg. 1986;80(4):583-6.
24. Isnard A, Shio MT, Olivier M. Impact of Leishmania metalloprotease GP63 on macrophage signaling. Front Cell Infect Microbiol. 2012;2:72.
25. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5.
26. Chan A, Ayala JM, Alvarez F, et al. The role of Leishmania GP63 in the modulation of innate inflammatory response to Leishmania major infection. PLoS One. 2021;16(12):e0262158.
27. Azambakhtiar A, Nabian S, Mohebali M, et al. Gelatin Zymography of Major Proteases in Exosomes of Leishmania major Promastigotes. Iran J Parasitol. 2023;18(4):546-53.
28. Bernadina W, De Luna R, Oliva G, et al. An immunodiffusion assay for the detection of canine leishmaniasis due to infection with Leishmania infantum. Vet Parasitol. 1997;73(3-4):207-13.
29. Cao-Milán R, Liz-Marzán LM. Gold nanoparticle conjugates: recent advances toward clinical applications. Expert Opin Drug Deliv. 2014;11(5):741-52.
30. Manisha Vaish MV, Singh O, Jaya Chakravarty JC, et al. rK39 antigen for the diagnosis of visceral leishmaniasis by using human saliva. Am J Trop Med Hyg. 2012;86(4):598-600.
31. Tyagi H, Kushwaha A, Kumar A, et al. A facile pH controlled citrate-based reduction method for gold nanoparticle synthesis at room temperature. Nanoscale Res Lett. 2016;11(1):362.
32. Daraban Bocaneti F, Ivanescu LM, Miron L, et al. An overview on leishmaniasis in Romania: Diagnosis and therapeutics. Trop Med Infect Dis. 2022;7(11):334.
33. Singh S, Singh V, Abhishek K. Visceral Leishmaniasis as a public health problem in India: action toward its elimination. Visceral Leishmaniasis and Post-kala-azar Dermal Leishmaniasis: CRC Press. 2025.
34. Bhunia GS, Shit PK, Bhunia GS, et al. Introduction of visceral leishmaniasis (kala-azar). Spatial Mapping and Modelling for Kala-azar Disease. 2020;1-18.
35. Reimão JQ, Coser EM, Lee MR, et al. Laboratory diagnosis of cutaneous and visceral leishmaniasis: current and future methods. Microorganisms. 2020;8(11):1632.
36. Akhoundi B, Mohebali M, Babakhan L, et al. Rapid detection of human Leishmania infantum infection: A comparative field study using the fast agglutination screening test and the direct agglutination test. Travel Med Infect Dis. 2010;8(5):305-10.
37. Sarkari B, Rezaei Z, Mohebali M. Immunodiagnosis of visceral leishmaniasis: current status and challenges: a review article. Iran J Parasitol. 2018;13(3):331-41.
38. Babakhan L, Mohebali M, Akhoundi B, et al. Rapid detection of Leishmania infantum infection in dogs: a comparative study using fast agglutination screening test (FAST) and direct agglutination test (DAT) in Iran. Parasitol Res. 2009;105(3):717-20.
39. Guay-Vincent MM, Matte C, Berthiaume AM, et al. Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates. PLoS Pathog. 2022;18(10):e1010640.
40. Mohebali M, Taran M, Zarei Z. Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination. Vet Parasitol. 2004;121 (3-4):239-45.
41. Campino L, Maia C. The role of reservoirs: canine leishmaniasis. Drug Resistance in Leishmania Parasites: Consequences, Molecular Mechanisms and Possible Treatments. 2018:59-83.
42. Momčilović S, Cantacessi C, Arsić-Arsenijević V, et al. Rapid diagnosis of parasitic diseases: current scenario and future needs. Clin Microbiol Infect. 2019;25(3):290-309.
43. Karimi Kakh M, Golchin M, Kazemi Arababadi M, et al. Application of the Leishmania infantum 21‐kDa recombinant protein for the development of an immunochromatographic test. Parasite Immunol. 2020;42(10):e12770.
44. Mohammadiha A, Haghighi A, Mohebali M, et al. Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. Vet Parasitol. 2013;192(1-3):83-90.
45. Piarroux R, Gambarelli F, Dumon H, et al. Comparison of PCR with direct examination of bone marrow aspiration, myeloculture, and serology for diagnosis of visceral leishmaniasis in immunocompromised patients. J Clin Microbiol. 1994;32(3):746-9.
2. Yadav P, Azam M, Ramesh V, et al. Unusual observations in leishmaniasis; an overview. Pathogens. 2023;12(2):297.
3. Mohebali M, Edrissian G, Akhoundi B, et al. Visceral leishmaniasis in Iran: An update on epidemiological features from 2013 to 2022. Iran J Parasitol. 2023;18(3):279-93.
4. Farooq I, Singh R, Selvapandiyan A, et al. The burden of visceral leishmaniasis: need of review, innovations, and solutions. Challenges and Solutions Against Visceral Leishmaniasis. 2024;28:1-17.
5. Grifferty G, Shirley H, McGloin J, et al. Vulnerabilities to and the socioeconomic and psychosocial impacts of the leishmaniases: A Review. Res Rep Trop Med. 2021; 12:135-151.
6. Morales-Yuste M, Martin-Sanchez J, Corpas-Lopez V. Canine leishmaniasis: Update on epidemiology, diagnosis, treatment, and prevention. Vet Sci. 2022;9(8):387.
7. Mohebali M, Moradi-Asl E, Rassi Y. Geographic distribution and spatial analysis of Leishmania infantum infection in domestic and wild animal reservoir hosts of zoonotic visceral leishmaniasis in Iran: A systematic review. J Vector Borne Dis. 2018;55(3):173-83.
8. Vilas-Boas DF, Nakasone EKN, Gonçalves AAM, et al. Global distribution of canine visceral leishmaniasis and the role of the dog in the epidemiology of the disease. Pathogens. 2024;13(6):455.
9. Van J, Diro E. Visceral leishmaniasis: recent advances in diagnostics and treatment regimens. Infect Dis Clin North Am. 2019;33(1):79-99.
10. Cosma C, Maia C, Khan N, et al. Leishmaniasis in humans and animals: A one health approach for surveillance, prevention and control in a changing world. Trop Med Infect Dis. 2024;9(11):258.
11. Sunyoto T, Boelaert M, Meheus F. Understanding the economic impact of leishmaniasis on households in endemic countries: a systematic review. Expert Rev Anti Infect Ther. 2019;17(1):57-69.
12. Okwor I, Uzonna J. Social and economic burden of human leishmaniasis. Am J Trop Med Hyg. 2016;94(3):489-93.
13. Mohebali M, Edrisian GH, Nadim A, et al. Application of direct agglutination test (DAT) for the diagnosis and seroepidemiological studies of visceral leishmaniasis in Iran. Iran J Parasitol. 2006; (1):15-25.
14. Mohebali M, Keshavarz H, Shirmohammad S, et al. The diagnostic accuracy of direct agglutination test for serodiagnosis of human visceral leishmaniasis: a systematic review with meta-analysis. BMC Infect Dis. 2020;20(1):946.
15. Harith A, Slappendel R, Reiter I, et al. Application of a direct agglutination test for detection of specific anti-Leishmania antibodies in the canine reservoir. J Clin Microbiol. 1989;27(10):2252-7.
16. Heidari S, Hajjaran H, Kazemi B, 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.
17. Malmasi A, Janitabar S, Mohebali M, et al. Seroepidemiologic survey of canine visceral leishmaniasis in Tehran and Alborz Provinces of Iran. J Arthropod Borne Dis. 2014;8(2):132-8.
18. Gao C, Yang Y, Shi F, et al. Development of an immunochromatographic test for diagnosis of visceral leishmaniasis based on detection of a circulating antigen. PLoS Negl Trop Dis. 2015;9(6):e0003902.
19. Hosseini Farash BR, Mohebali M, Kazemi B, et al. Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts. Sci Rep. 2022;12(1):10426.
20. Ejazi SA, Bhattacharyya A, Choudhury ST, et al. Immunoproteomic identification and characterization of Leishmania membrane proteins as non-invasive diagnostic candidates for clinical visceral leishmaniasis. Sci Rep. 2018;8(1):12110.
21. Hosseini Farash BR, Mohebali M, Kazemi B, et al. Cloning of K26 hydrophilic antigen from Iranian strain of Leishmania infantum. Iran J Public Health. 2017;46(10):1359-65.
22. Roberts T, Keddie SH, Rattanavong S, et al. Accuracy of the direct agglutination test for diagnosis of visceral leishmaniasis: a systematic review and meta-analysis. BMC Infect Dis. 2023;23(1):782.
23. Harith A, Kolk A, Kager P, et al. A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis. Trans R Soc Trop Med Hyg. 1986;80(4):583-6.
24. Isnard A, Shio MT, Olivier M. Impact of Leishmania metalloprotease GP63 on macrophage signaling. Front Cell Infect Microbiol. 2012;2:72.
25. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5.
26. Chan A, Ayala JM, Alvarez F, et al. The role of Leishmania GP63 in the modulation of innate inflammatory response to Leishmania major infection. PLoS One. 2021;16(12):e0262158.
27. Azambakhtiar A, Nabian S, Mohebali M, et al. Gelatin Zymography of Major Proteases in Exosomes of Leishmania major Promastigotes. Iran J Parasitol. 2023;18(4):546-53.
28. Bernadina W, De Luna R, Oliva G, et al. An immunodiffusion assay for the detection of canine leishmaniasis due to infection with Leishmania infantum. Vet Parasitol. 1997;73(3-4):207-13.
29. Cao-Milán R, Liz-Marzán LM. Gold nanoparticle conjugates: recent advances toward clinical applications. Expert Opin Drug Deliv. 2014;11(5):741-52.
30. Manisha Vaish MV, Singh O, Jaya Chakravarty JC, et al. rK39 antigen for the diagnosis of visceral leishmaniasis by using human saliva. Am J Trop Med Hyg. 2012;86(4):598-600.
31. Tyagi H, Kushwaha A, Kumar A, et al. A facile pH controlled citrate-based reduction method for gold nanoparticle synthesis at room temperature. Nanoscale Res Lett. 2016;11(1):362.
32. Daraban Bocaneti F, Ivanescu LM, Miron L, et al. An overview on leishmaniasis in Romania: Diagnosis and therapeutics. Trop Med Infect Dis. 2022;7(11):334.
33. Singh S, Singh V, Abhishek K. Visceral Leishmaniasis as a public health problem in India: action toward its elimination. Visceral Leishmaniasis and Post-kala-azar Dermal Leishmaniasis: CRC Press. 2025.
34. Bhunia GS, Shit PK, Bhunia GS, et al. Introduction of visceral leishmaniasis (kala-azar). Spatial Mapping and Modelling for Kala-azar Disease. 2020;1-18.
35. Reimão JQ, Coser EM, Lee MR, et al. Laboratory diagnosis of cutaneous and visceral leishmaniasis: current and future methods. Microorganisms. 2020;8(11):1632.
36. Akhoundi B, Mohebali M, Babakhan L, et al. Rapid detection of human Leishmania infantum infection: A comparative field study using the fast agglutination screening test and the direct agglutination test. Travel Med Infect Dis. 2010;8(5):305-10.
37. Sarkari B, Rezaei Z, Mohebali M. Immunodiagnosis of visceral leishmaniasis: current status and challenges: a review article. Iran J Parasitol. 2018;13(3):331-41.
38. Babakhan L, Mohebali M, Akhoundi B, et al. Rapid detection of Leishmania infantum infection in dogs: a comparative study using fast agglutination screening test (FAST) and direct agglutination test (DAT) in Iran. Parasitol Res. 2009;105(3):717-20.
39. Guay-Vincent MM, Matte C, Berthiaume AM, et al. Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates. PLoS Pathog. 2022;18(10):e1010640.
40. Mohebali M, Taran M, Zarei Z. Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination. Vet Parasitol. 2004;121 (3-4):239-45.
41. Campino L, Maia C. The role of reservoirs: canine leishmaniasis. Drug Resistance in Leishmania Parasites: Consequences, Molecular Mechanisms and Possible Treatments. 2018:59-83.
42. Momčilović S, Cantacessi C, Arsić-Arsenijević V, et al. Rapid diagnosis of parasitic diseases: current scenario and future needs. Clin Microbiol Infect. 2019;25(3):290-309.
43. Karimi Kakh M, Golchin M, Kazemi Arababadi M, et al. Application of the Leishmania infantum 21‐kDa recombinant protein for the development of an immunochromatographic test. Parasite Immunol. 2020;42(10):e12770.
44. Mohammadiha A, Haghighi A, Mohebali M, et al. Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. Vet Parasitol. 2013;192(1-3):83-90.
45. Piarroux R, Gambarelli F, Dumon H, et al. Comparison of PCR with direct examination of bone marrow aspiration, myeloculture, and serology for diagnosis of visceral leishmaniasis in immunocompromised patients. J Clin Microbiol. 1994;32(3):746-9.
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| Issue | Vol 20 No 3 (2025) | |
| Section | Original Article(s) | |
| Keywords | ||
| Visceral leishmaniasis Dog Immunochromatographic test Direct agglutination test | ||
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How to Cite
1.
Azambakhtiar A, Nabian S, Mohebali M, Taheri M, Zarei Z, Hosseini-Safa A. Preparation and Development of An Immunochromatographic Test for early Detection of Canine Visceral Leishmaniasis: A Preliminary Study. Iran J Parasitol. 2025;20(3):417-424.
