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

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.

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

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.

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

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.

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

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.

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

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.

Identification of an area predominantly endemic for childhood and adolescent visceral leishmaniasis in central Sudan.

Although widely spread throughout Sudan, visceral leishmaniasis (VL) is predominantly endemic in the Gedaref, southern Blue-Nile, and Umrimta areas located in the eastern, southern, and central regions, respectively. Regardless of form (endemic or epidemic), VL occurrence follows similar patterns as all ages and both sexes are affected. From January 2005 to May 2016, we received a total of 563 patients with high suspicion for VL from various endemic areas; 159 were children and adolescents (0.

Local Production of a Liquid Direct Agglutination Test as a Sustainable Measure for Control of Visceral Leishmaniasis in Sudan.

A prerequisite for the control of visceral leishmaniasis (VL) is the accessibility to reference diagnostics. The high price of the freeze-dried direct agglutination test (FD-DAT) and the short shelf-life time of the rK39 strip test (rK39) have limited the application of these tests in Sudan. An original liquid DAT (LQ-DAT) with high reproducibility compared with the FD-DAT and rK39 has been routinely produced in our laboratory since 1999.

Heterogeneity of Leishmania donovani parasites complicates diagnosis of visceral leishmaniasis: comparison of different serological tests in three endemic regions.

Diagnostic tests for visceral leishmaniasis that are based on antigens of a single Leishmania strain can have low diagnostic performance in regions where heterologous parasites predominate. The aim of this study was to investigate and compare the performance of five serological tests, based on different Leishmania antigens, in three endemic countries for visceral leishmaniasis. A total number of 231 sera of symptomatic and asymptomatic cases and controls from three endemic regions of visceral leishmaniasis in East Sudan, North India and South France were evaluated by following serological tests: rKLO8- and rK39 ELISA, DAT (ITMA-DAT) and two rapid tests of rK39 (IT LEISH) and rKE16 (Signal-KA).

Performance of an indigenous β-mercaptoethanol-modified antigen in comparison with a commercial reference in direct agglutination test for detection of canine visceral leishmaniasis.

We compared the performance of a locally produced β-mercaptoethanol-modified promastigote antigen (β-ME-Ag) of an indigenous Leishmania infantum strain against that of a trypsinized Leishmania donovani reference (REF-Ag) in the direct agglutination test (DAT) for detection of canine visceral leishmaniasis (CVL). One hundred and fifty-one serum samples collected from dogs belonging to four groups with different conditions were included. At a DAT titre of 1 : 320, statistically determined as optimal cut-off value for β-ME-Ag, and 1 : 160 for REF-Ag, a sensitivity and a specificity of 100 % were estimated for β-ME-Ag in comparison with 96.

Successful substitution of fetal calf serum by human plasma for bulk cultivation of Leishmania donovani promastigotes.

The potential of human plasma (HP) or serum (HS) as a replacement for fetal calf serum (FCS) was evaluated in a liver infusion tryptose (LIT) medium for bulk cultivation of Leishmania donovani promastigotes. The promastigote yield with the LIT-FCS standard medium was 0.4-1.

A β-mercaptoethanol-modified enzyme-linked immunosorbent assay for diagnosis of canine visceral leishmaniasis.

Two immunoglobulin G enzyme-linked immunosorbent assay (ELISA) versions using whole promastigotes of Leishmania infantum (syn. Leishmania chagasi) treated either with β-mercaptoethanol (β-ME-ELISA) or trypsin (TRYP-ELISA) as antigens were developed for the diagnosis of canine visceral leishmaniasis (CVL). By comparison with the direct agglutination test (DAT; 100%, 31/31; 95% confidence interval [CI]: 86.

Validation of a β-ME ELISA for detection of anti Leishmania donovani antibodies in Eastern Sudan.

Background: A β-mercaptoethnol (β-ME)-treated promastigote antigen of L. donovani was successfully employed in direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL).

Objective: The β-ME-treated antigen was further incorporated into an enzyme-linked immunosorbent assay set-up (β-ME ELISA) and evaluated for VL diagnosis against outcome of reference freeze-dried DAT (FD-DAT) and rK39 strip test (RKT) commercial kits.

Qualitative and semi-quantitative comparison of an rK39 strip test and direct agglutination test for detection of anti-Leishmania donovani antibodies in the Sudan.

Background: Until now, the comparison of the rK39 strip test (RKT) and direct agglutination test (DAT) for detection of visceral leishmaniasis (VL) is exclusively based on either positive or negative qualification of the reaction outcome.

Objective: In this study, we compared the diagnostic performance of RKT and DAT for VL both qualitatively and semi-quantitatively.

Methods: For comparison based on semi-quantitative grounds, the execution of RKT and DAT was according to the standard procedures.

Use of a newly developed beta-mercaptoethanol enzyme-linked immunosorbent assay to diagnose visceral leishmaniasis in patients in eastern Sudan.

Corroboration of serology results is essential for restricting the risk of inappropriate antileishmanial prescription. A direct agglutination test (DAT) and a recently developed beta-mercaptoethanol-modified enzyme-linked immunosorbent assay (beta-ME ELISA) based on the use of antigen prepared as described for the DAT were applied to 416 sera from two Sudanese populations with and without clinical evidence of visceral leishmaniasis (VL). Of 285 sera with the lowest antileishmanial DAT titers ( View Article and Find Full Text PDF

Evaluation of a glycerol-preserved antigen in the direct agglutination test for diagnosis of visceral leishmaniasis at rural level in eastern Sudan.

Three-hundred and eight patients with suspected visceral leishmaniasis (VL) were received at Doka Hospital (eastern Sudan) during the period September 2004 to October 2005. The sensitivity and specificity of a glycerol-preserved (GP) antigen for VL diagnosis was assessed against the results of repeated lymph node aspiration and readings from a direct agglutination test (DAT) employing standard formaldehyde-fixed (FF) or freeze-dried (FD) antigen. Despite 13 months of storage at ambient temperature (28-47 degrees C), the GP antigen mean titres obtained from these 308 patients were no different from those that were FD (P=0.

Beta-mercaptoethanol-modified ELISA for diagnosis of visceral leishmaniasis.

Following antigen preparation procedures similar to those of the direct agglutination test (DAT), an IgG ELISA employing intact beta-mercaptoethanol (beta-ME)-treated Leishmania donovani promastigotes was developed. The performance of the beta-ME ELISA thus developed was assessed in patients with confirmed visceral leishmaniasis (VL), revealing slightly lower sensitivity (39/40=97.5%) than that of the DAT (40/40=100%).

Use of glycerol as an alternative to freeze-drying for long-term preservation of antigen for the direct agglutination test.

The potential of glycerol for long-term preservation of the direct agglutination test (DAT) antigen was evaluated at a fluctuating laboratory temperature of 25-37 degrees C and at constant temperatures of 37 and 45 degrees C for a period of 222 days. DAT titres recorded for the three antigen aliquots preserved in 50% (v/v) glycerol and stored at 25-37, 37 or 45 degrees C at 11 time intervals were within the same range of the control antigen kept at 4 degrees C. Performance of the glycerol-preserved antigen stored at 45 degrees C was compared with that of a freeze-dried version on 24 visceral leishmaniasis (VL) and 54 non-VL patients.