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Volume 7, Issue 1

Validation of an improved reference freeze-dried direct agglutination test for detecting leishmaniasis in the canine reservoir Open Access

Abstract

Proper identification and management of post-kala-azar dermal leishmaniasis (PKDL) and canine leishmaniasis (CanL) cases are among the prerequisites to the effective control of visceral leishmaniasis worldwide. Unlike PKDL, CanL still awaits effective improvement because of its cryptic nature, absence of parasites in lesions or lymph nodes and not complete sensitivity of some diagnostic tools in use. Because of the need for certain skills and equipment, both the liquid direct agglutination test and freeze-dried direct agglutination test (FD-DAT) versions are, in comparison with the indirect immunofluorescence antibody test (IFAT) or enzyme-linked immunosorbent assay (ELISA), practical and feasible diagnostic alternatives.

Validate the performance of an improved FD-DAT to suit routine and large-scale applications in CanL endemic areas.

Introducing citrate-saline formaldehyde (CSF) as an anti-clumping agent to replace normal saline for antigen reconstitution and drastically, however, eligibly lower the concentration of promastigotes (1.4×10) in comparison with the original FD-DAT reference (5×10 ml). To ensure optimal safety, -mercaptoethanol was replaced by urea or SDS as a serum-reducing agent.

By improving the procedure for reconstitution of FD-DAT antigen with CSF, a 150% reduction in the test application cost was achieved. Expired test batches (±4 years earlier) were successfully revitalized to full validity. As compared to the 48 h shelf-life time for the original, an FD-DAT batch reconstituted here with CSF maintained stability for ±12 months.

The highly concordant results with IFAT and ELISA (one-way ANOVA test, =0.142, homogeneity of variances =0.009) as routine CanL diagnostics further motivate the application of the improved FD-DAT for the detection of the disease in endemic areas.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): anti-clumping agent , canine leishmaniasis , citrate-saline formaldehyde , detection , freeze-dried direct agglutination , improvement , SDS and urea
Funding
This study was supported by the:
  • Ahfad University for Women
    • Principle Award Recipient: Abdallahel Harith
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. The Microbiology Society waived the open access fees for this article.
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2025-01-06
2025-01-17
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/content/journal/acmi/10.1099/acmi.0.000890.v4
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Validation of an improved reference freeze-dried direct agglutination test for detecting leishmaniasis in the canine reservoir
Access Microbiology 7, 000890.v4 (2025); https://doi.org/10.1099/acmi.0.000890.v4
/content/journal/acmi/10.1099/acmi.0.000890.v4
/content/journal/acmi/10.1099/acmi.0.000890.v4
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