User and environment friendly direct agglutination test for the sero-diagnosis of visceral leishmaniasis: exclusion of formaldehyde and β-mercaptoethanol in test execution

Mahamoud, Abdelhafeiz; Awad, Yousif; Osman, Hussam A.; Agib, Atif el; Madi, Rubens Riscala; Semião-Santos, Saul J.; Harith, Abdallah el

doi:10.1099/jmm.0.000858

Cited by 4 publications

(10 citation statements)

References 17 publications

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“…As previously reported, an L. donovani strain isolated from a VL patient from the Gedarif area in Eastern Sudan was used as a source of the antigen. [2][3][4] Irrespective of form, the promastigotes retained the Coommassie Brilliant Blue stain despite repeated washing with the citratesaline solution (0.056 M sodium citrate and 0.15 M sodium chloride). The produced antigen was then preserved in citratesaline solution supplemented with 1.2% (w/v) citrate-saline/ formaldehyde (CSF) at a promastigote concentration of 1.6 × 10 7 /mL to simultaneously preserve promastigote morphology and evade auto-agglutination.…”

Section: Methodsmentioning

confidence: 99%

“…In total, 47 blood spots collected onto filter paper and 18 serum samples from patients with VL from our serum bank were included. [2][3][4] Thirty other blood and serum samples also from our serum bank from suspects with negative lymph node aspirates, negative LQ-DAT, and FD-DAT results were used as negative controls.…”

Section: Methodsmentioning

confidence: 99%

“…To ensure complete miscibility, the gelatin/saline mixture was heated up to 80°C. 4 To reduce nonspecific agglutination, the gelatin diluent was cooled down to room temperature and supplemented with urea (0.3% wt/vol) instead of β-ME (0.8% v/). 4 Execution of the test was the same as for LQ-DAT using V-shaped well microtiter plates.…”

Section: Methodsmentioning

confidence: 99%

“…1 However, in the course of laboratory and field application during the past 25-28 years in Sudan, both tests revealed several drawbacks that negatively impacted their application on a routine basis. [2][3][4][5] The limited shelf-life (±30 days) of the rK39 at ambient temperatures and high application cost of the freezedried direct agglutination test (FD-DAT) (±$32.0/vial vs average income in Sudan of $70.0 a month) were the most important shortcomings. 2 The strict temperature range for the test kit (5-35°C) limited the use of the FD-DAT in Sudan, particularly during the hot season (April-October) when the mid-day temperatures of ±40°C are considered normal almost throughout the whole country.…”

Section: Introductionmentioning

confidence: 99%

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Modifications in a Reference Freeze-Dried Direct Agglutination Test to Improve Visceral Leishmaniasis Detection

Harith

Awad

Mahamoud

et al. 2020

The American Journal of Tropical Medicine and Hygiene

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Currently, a significantly lower temperature (35°C) than initially established (56°C) is indicated as the maximum temperature storage for the commercial reference visceral leishmaniasis (VL) freeze-dried direct agglutination test (FD-DAT). Despite an approximately 50% loss in the number of promastigotes in an FD-DAT batch that expired 7 years earlier, the promastigotes maintained a similar morphology to the equivalent valid batch implying most likely that auto-agglutination, rather than aging, is the main reason for expiry. The substitution of normal saline which was initially recommended for reconstitution, by citrate-saline/formaldehyde (CSF) as an anti-clumping/preservative agent resulted in restoration of validity comparable with that of the freeze-dried original or the liquid direct agglutination test (LQ-DAT) version (Friedman ANOVA test = 1.0588; P = 0.5890). Following a similar reconstitution procedure as for the 7-year expired antigen, using significantly lower promastigote concentration (1.4 × 10 7 /mL) than in the non-expired (9.0 × 10 7 /mL), good reliability for VL detection and stability at 4°C (> 12 months) were achieved. In comparison with the original version using normal saline ($32.0/vial), the cost-effectiveness of the FD-DAT was appreciably improved by the CSF incorporation and lowering of promastigote concentration per unit suspension medium ($12.8/vial). With diagnostic reliability comparable with the full-out titration used, FD-DAT procedure based on single sample dilution at the VL cutoff (1:3,200) permitted the use of significantly smaller antigen volumes (0.1 mL vs. > 1.5 mL), therefore contributing to a further reduction in the application cost. The successful replacement of β-mercaptoethanol (β-ME) by urea (T = 21.00; P = 0.0868) provided the required safety for the test procedure similar to the widely applied LQ-DAT.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Modifications in a Reference Freeze-Dried Direct Agglutination Test to Improve Visceral Leishmaniasis Detection

Harith

Awad

Mahamoud

et al. 2020

The American Journal of Tropical Medicine and Hygiene

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“…To further facilitate LQ-DAT application in routine and mass screening of VL but simultaneously ensure safety for the user and environment, formaldehyde and β-ME were successfully excluded without compromising the reliability of the test for VL detection (Figure 1) [27]. By further incorporating chlorine and urea, subsequently replacing the 2 aforementioned pollutants, LQ-DAT demonstrated efficiency comparable to the in currently used freeze-dried or liquid versions (Friedman ANOVA test: Fr = 0.3250; P = .8500) [27], thus providing the required safety for routine diagnosis and for mass screening programs.…”

Section: Local Production and Optimization Of Lq-datmentioning

confidence: 99%

Are We Now Well Prepared for Another Major Visceral Leishmaniasis Epidemic in Sudan?

Harith

Mahamoud

Awad

et al. 2019

Open Forum Infectious Diseases

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To minimize the chance for future visceral leishmaniasis (VL) epidemics such as the 1988–1991 epidemic in Sudan, several VL detection tools have been introduced. There are many VL diagnostics with excellent sensitivities, specificities, and ease of use reported. However, additional test characteristics should be considered for use in the detection of future VL epidemics. The potential for local production or uninterrupted availability, low production and application costs, and stability at ≥45°C are of the utmost importance. Of the antibody-, antigen-, or DNA-based methods introduced, only a liquid direct agglutination test (LQ-DAT) remains in routine use. The LQ-DAT test may be the ideal diagnostic for detection of VL epidemics due to its low cost ($0.50/patient), stability under frequent and long-duration electric failures, and high level of reproducibility. The improved reliability for VL detection achieved locally through incorporating autochthonous L. donovani strains in antigen processing and precluding toxicants in test execution provides optimal sensitivity and safety for routine and mass application.

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Haematological malignancies as disorders negatively impacting specificities of the direct agglutination and rapid rK39 strip tests as reference diagnostics for visceral leishmanisis

Nail

Ahmed

Osman

et al. 2023

Access Microbiology

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Introduction. During several years of work in Sudan, we occasionally had been confronted with patients who presented clinical features highly suggestive of visceral leishmaniasis (VL) however direct agglutination test (DAT) readings that were either at the high negative or low positive titre range. Inquiries on the fate of those particular patients revealed mortality, undetermined diagnosis or that in some of them leukaemia was finally diagnosed. Gap statement. Investigate as to what extent haematological malignancies (HMs) interfere with VL diagnosis. Aim. Evaluate specificity of DAT version newly developed in this study wherein sodium dodecyle sulphate (SDS) was incorporated as a test sample denaturant in comparison with a standard reference wherein β-mercaptoethanol (β-ME) was used in test execution. Methodology. Seventy plasma samples from patients with HMs were collected and tested in a primary DAT version (P-DAT). The results obtained were compared with those of the rK39 strip test as VL reference diagnostic. HM samples revealing titres higher than the start dilution (1 : 100) in P-DAT were further tested in a β-ME- and urea-modified DAT versions. The specificity of the newly developed SDS-DAT was assessed against that of β-ME-DAT and rK39 strip tests as current reference diagnostics for VL. Results. Seven out of 70 patients with HMs scored positive outcomes (titre ≥1 : 3200) in P-DAT and four in the reference rK39 strip test. Of the seven that tested positive in P-DAT or four in the reference rK39, none reacted at titre >1 : 100 in the SDS-DAT. Significant reduction in non-specific agglutination reactions was achieved as a result in respect to the HM plasma samples (P value <0.05). Conclusion. To establish desired specificity for VL diagnosis in respect to HMs and subsequently minimize or avoid serious side effects due to unjustified anti-leishmanials prescription the combined application of the SDS-DAT here described and an improved version of the rK39 for confirmation is recommended.

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User and environment friendly direct agglutination test for the sero-diagnosis of visceral leishmaniasis: exclusion of formaldehyde and β-mercaptoethanol in test execution

Cited by 4 publications

References 17 publications

Modifications in a Reference Freeze-Dried Direct Agglutination Test to Improve Visceral Leishmaniasis Detection

Modifications in a Reference Freeze-Dried Direct Agglutination Test to Improve Visceral Leishmaniasis Detection

Are We Now Well Prepared for Another Major Visceral Leishmaniasis Epidemic in Sudan?

Haematological malignancies as disorders negatively impacting specificities of the direct agglutination and rapid rK39 strip tests as reference diagnostics for visceral leishmanisis

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