Abdelhafeiz Mahamoud1, Yousif Awad1, Hussam Ali Osman1, Atif El Agib2, Rubens Riscala Madi3, Saul J Semiao-Santos4, Abdallah El Harith1. 1. 1Laboratory of Biomedical Research, Ahfad University for Women, P.O. Box 167, Omdurman, Sudan. 2. 2Tropical Medicine Research Institute, P.O. Box 1304, Khartoum, Sudan. 3. 3Post-graduate Program in Health and Environment, University of Tiradentes, Aracaju, Brazil. 4. 4Department of Medicine and Nursing, University of Tiradentes, Sergipe, Brazil.
Abstract
PURPOSE: Based on world-wide evaluation, the direct agglutination test (DAT) is now generally acknowledged as one of the leading diagnostics for visceral leishmaniasis (VL). To enhance more routine and mass application, but simultaneously ensure safety to both user and environment, further improvements need to be introduced. METHODOLOGY: In the current format, a two-sixfold titre decrease was observed due to using formaldehyde as an antigen preservative in DAT. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3 % (wt/vol) for conserving promastigote status after β-mercaptoethanol (β-ME) treatment and repeating exposure of the parasite to the fixative after Coomassie Brilliant Blue staining. RESULTS: Microbial contamination was not observed in any of the antigen aliquots preserved in 0.05 % (wt/vol) sodium dichloroisocyanurate (chlorine) instead of formaldehyde for 6 months or longer. By excluding formaldehyde, restoring the normal antibody level, prior to treatment of sera with β-ME only minimally influenced the test outcome. A comparable successful reduction in non-specific agglutination, as with β-ME, was achieved by incorporating urea (0.3 % wt/vol) in the improved DAT procedure (P=0.646; T=23.0). As with the current procedure, the improved equivalent (formaldehyde and β-ME free) showed good reliability for VL detection (VL - Fr=52.39, W=0.70, P<0.001; and non-VL - Fr=65.97, W=0.83, P<0.001). A much lower cut-off (titre 1 : 400 versus 1 : 3200) for VL diagnosis can be adopted if urea is integrated in the improved procedure. CONCLUSIONS: By introducing the modifications mentioned, we think we have succeeded to a reasonable degree in increasing the DAT potential for VL control.
PURPOSE: Based on world-wide evaluation, the direct agglutination test (DAT) is now generally acknowledged as one of the leading diagnostics for visceral leishmaniasis (VL). To enhance more routine and mass application, but simultaneously ensure safety to both user and environment, further improvements need to be introduced. METHODOLOGY: In the current format, a two-sixfold titre decrease was observed due to using formaldehyde as an antigen preservative in DAT. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3 % (wt/vol) for conserving promastigote status after β-mercaptoethanol (β-ME) treatment and repeating exposure of the parasite to the fixative after Coomassie Brilliant Blue staining. RESULTS: Microbial contamination was not observed in any of the antigen aliquots preserved in 0.05 % (wt/vol) sodium dichloroisocyanurate (chlorine) instead of formaldehyde for 6 months or longer. By excluding formaldehyde, restoring the normal antibody level, prior to treatment of sera with β-ME only minimally influenced the test outcome. A comparable successful reduction in non-specific agglutination, as with β-ME, was achieved by incorporating urea (0.3 % wt/vol) in the improved DAT procedure (P=0.646; T=23.0). As with the current procedure, the improved equivalent (formaldehyde and β-ME free) showed good reliability for VL detection (VL - Fr=52.39, W=0.70, P<0.001; and non-VL - Fr=65.97, W=0.83, P<0.001). A much lower cut-off (titre 1 : 400 versus 1 : 3200) for VL diagnosis can be adopted if urea is integrated in the improved procedure. CONCLUSIONS: By introducing the modifications mentioned, we think we have succeeded to a reasonable degree in increasing the DAT potential for VL control.
Entities:
Keywords:
direct agglutination test; environment; formaldehyde; toxicity; user; β-mercaptoethanol
Authors: Abdallah El Harith; Abdelhafeiz Mahamoud; Yousif Awad; Durria Mansour; Elfadil Mustafa Abass; Atif El Agib; Rubens Riscala Madi; Saul J Semiao-Santos; Hussam Ali Osman Journal: Open Forum Infect Dis Date: 2019-05-15 Impact factor: 3.835