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. 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 ß-mercaptoethanol (ß-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 ß-ME- and urea-modified DAT versions. The specificity of the newly developed SDS-DAT was assessed against that of ß-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.

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. 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.

User and environment friendly direct agglutination test for the sero-diagnosis of visceral leishmaniasis: exclusion of formaldehyde and ß-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. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3 % (wt/vol) for conserving promastigote status after ß-mercaptoethanol (ß-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 ß-ME only minimally influenced the test outcome. A comparable successful reduction in non-specific agglutination, as with ß-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 ß-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.

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 (0.5-18 years) from Umrimta (central Sudan). A significant observation during this 11-year period of uninterrupted monitoring using a standard liquid direct agglutination test (LQ-DAT) version was the exclusive VL occurrence (100%) in the child and adolescent populations of Umrimta when compared with other endemic areas (27.3%-48.0%). Among 12 child and adolescent suspects who initially tested marginal in the standard LQ-DAT, 6 scored unequivocally positive readings both in an improved LQ-DAT version (based on an autochthonous Leishmania donovani strain) and rK28 VL reference test. None of the 4 (2.5%) VL adult suspects (≥19years) referred had positive outcomes in the improved LQ-DAT version or the VL reference freeze-dried direct agglutination and rK28 tests. Further incorporation of antigens derived from autochthonous L. donovani strains from Umrimta (central Sudan) or Gedaref (eastern Sudan) in LQ-DAT significantly increased the agglutination titer levels in the respective VL homologous sera (p=0.0263 T=505 and p=0.2814T=219), suggesting possible antigenic variation within the predominant Sudanese L. donovani complex. Additional research is required to determine characteristics other than the serologically-based ones reported for the L. donovani strain involved.

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%). When challenged with sera of individuals with non-VL conditions, including leukaemia and African trypanosomiasis, the specificity of the beta-ME ELISA was 100% (158/158), compared to 98.8% (156/158) for DAT. In an endemic population (n=145) manifesting a clinical suspicion of VL, results obtained with the beta-ME ELISA were highly concordant with those of DAT, both in the seropositive (65/68=95.6%) and seronegative (77/80=96.3%) groups. Furthermore, the incorporated intact antigen demonstrated higher sensitivity in ELISA (16/18=88.9%) than the water-soluble equivalent (13/18=72.2%). The stability of the formaldehyde-fixed antigen (2 months at 4 degrees C) in beta-ME ELISA, as well as the option for direct testing of whole-blood samples and visual reading of results (within 2 h, compared to 18 h for DAT), advocate the simultaneous application of the technique with DAT for confirmation of VL in laboratories with limited facilities.

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=0.945) and stored under similar conditions, but were significantly different (P=0.019) from those that were FF and kept continuously at the optimum temperature for storage (4-8 degrees C). Taking the parasitological result as the gold standard and using a pre-established titre of 1 : 3200 as the DAT cut-off, the GP antigen revealed a sensitivity (91/105, 86.7 %) and specificity (187/203, 92.1 %) comparable to that of FD antigen (92/105, 87.6 %, and 188/203, 92.6 %, respectively) and FF antigen (94/105, 89.5 %, and 188/203, 92.6 %, respectively). At a titre range of 1 : 400-1 : 800, statistically determined as the optimum cut-off for the three antigens, sensitivities of 92.4, 90.5 and 96.2 % and specificities of 90.6, 90.1 and 88.7 % were achieved for the GP, FD and FF antigens, respectively, at a peripheral hospital. Regardless of the antigen preparation used, DAT results obtained in the peripheral hospital were highly reproducible in the central laboratory in Omdurman (weighted kappa: GP=0.957, FD=0.979 and FF=0.936). With a diagnostic reliability comparable to formaldehyde fixation and stability under ambient conditions similar to freeze drying, glycerol preservation, by virtue of its high potential for reproduction, meets the requirements for the management of VL in developing countries.

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. In this study, a 3.4-year-old batch (of more than 90 test batches produced to date) was chosen to validate the diagnostic performance of this test against microscopy, FD-DAT, and rK39 in 96 VL and 42 non-VL serum samples. Relatively higher sensitivity (95/96, 99.0%) was recorded for the LQ-DAT than for the FD-DAT (92/96, 95.8%) and rK39 (76/96, 79.2%), probably because of the use of the endemic autochthonous Leishmania donovani isolate as the antigen. Experience with the LQ-DAT, its low cost of production, ease of providing this test, and diagnostic reliability compared with the FD-DAT suggest that widescale implementation of the LQ-DAT can contribute to sustainable VL control in Sudan.

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). Overall, rKLO8- and rK39 ELISA were most sensitive in immunocompetent patients from all endemic regions (96-100%) and the sensitivity was reduced to 81.8% in HIV co-infected patients from France. Sera of patients from India demonstrated significantly higher antibody responses to rKLO8 and rK39 compared with sera from Sudan (p<0.0001) and France (p<0.0037). Further, some Indian and Sudanese patients reacted better with rKLO8 than rK39. Sensitivity of DAT (ITMA-DAT) was high in Sudan (94%) and India (92.3%) but low in France being 88.5% and 54.5% for VL and VL/HIV patients, respectively. In contrast, rapid tests displayed high sensitivity only in patients from India (96.2%) but not Sudan (64-88%) and France (73.1-88.5% and 63.6-81.8% in VL and VL/HIV patients, respectively). While the sensitivity varied, all tests showed high specificity in Sudan (96.7-100%) and India (96.6%).Heterogeneity of Leishmania parasites which is common in many endemic regions complicates the diagnosis of visceral leishmaniasis. Therefore, tests based on homologous Leishmania antigens are required for particular endemic regions to detect cases which are difficult to be diagnosed with currently available tests.

Performance of an indigenous ß-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 ß-mercaptoethanol-modified promastigote antigen (ß-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 ß-ME-Ag, and 1 : 160 for REF-Ag, a sensitivity and a specificity of 100 % were estimated for ß-ME-Ag in comparison with 96.6 % and 100 %, respectively, for REF-Ag. Overall, levels of agglutination titres recorded for the two antigens were highly concordant (Cohen's κ = 0.879) in both the CVL and non-CVL groups. Based on current results, and ease experienced in processing the antigen and reading the test outcome, we recommend incorporation of ß-ME-Ag in DAT for confirmation or exclusion of suspected CVL in dogs.

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 0.4-1.8×10(7) ml(-1), and yields of 0.5-3.4×10(7) (P = 0.527) and 0.4-2.4×10(7) (P = 0.062) were recorded for two LIT medium variants containing HP or HS as supplement instead of FCS. Significantly, higher promastigote yields of 1.3-4.9×10(7) ml(-1) were demonstrated when LIT medium was supplemented with HP of blood group O but not A, B, AB or equally pooled ABO (P = 0.007-0.020). Matching (P = 0.56) strong positive (1 : 10 2400 to ≥1 : 262 144 00) and weak negative (1 : 5-1 : 160) direct agglutination test (DAT) titres, respectively, were demonstrated in 24 visceral leishmaniasis (VL) and 45 non-VL sera for both standard LIT-FCS and alternative LIT-HP derived antigens. Our findings indicate strong potential for sustainable production of promastigotes for important diagnostic procedures such as DAT in the VL affected areas.

A ß-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 ß-mercaptoethanol (ß-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]: 86.3-100%), slightly lower sensitivity was demonstrated for the newly developed ß-ME-ELISA (93.5%, 29/31; 95% CI: 77.2-98.9%). Sensitivity was higher for ß-ME-ELISA compared with TRYP-ELISA (87.1%, 27/31; 95% CI: 69.2-95.8%) in serum samples from dogs with CVL. When tested with sera from 37 healthy dogs and from 45 dogs with clinical conditions other than CVL, a specificity of 97.6% (80/82; 95% CI: 90.1-99.6%) was estimated for ß-ME-ELISA as compared to 100% (82/82; 95% CI: 94.4-100%) and 95.1% (78/82; 95% CI: 87.3-98.4%) for DAT and TRYP-ELISA, respectively. Observed agreement was 94.0% (95% CI: 88.7-97.1%) between DAT and ß-ME-ELISA (κ = 0.879; 95% CI: 0.803-0.956) and 87.4% (95% CI: 80.8-92.1%) between DAT and TRYP-ELISA (κ = 0.743; 95% CI: 0.636-0.851). Current results advocate application of the new ß-ME-ELISA for diagnosis of CVL at the laboratory level and confirmation of results obtained with the DAT in field studies.

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

BACKGROUND: A ß-mercaptoethnol (ß-ME)-treated promastigote antigen of L. donovani was successfully employed in direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL). OBJECTIVE: The ß-ME-treated antigen was further incorporated into an enzyme-linked immunosorbent assay set-up (ß-ME ELISA) and evaluated for VL diagnosis against outcome of reference freeze-dried DAT (FD-DAT) and rK39 strip test (RKT) commercial kits. METHODS: Two-hundred and ninety-two sera from patients with high VL suspicion of whom 105 had confirmed L. donovani infection were tested. RESULTS: Relatively higher sensitivities of 93.3% (95% CI: 88.4-98.2) and 92.4% (95% CI: 87.3-97.5) were determined for ß-ME ELISA and FD-DAT as compared to 83.8% (95% CI: 76.7-90.8) for RKT. Of 73 VL sera that scored maximum absorbance values (>0.81) in ß-ME ELISA, 66 (90.4%) tested at the highest agglutination titres (>1:51200) in FD-DAT as did 56 (76.7%) also at comparable reaction intensities (3 + colour intensity) in RKT. Compared with FD-DAT (94.7%, 95% CI: 91.5-97.9) or RKT (93.0%, 95% CI: 89.3-96.6), lower specificity was estimated for ß-ME ELISA (90.4%, 95% CI: 86.1-94.6). Based both on positive and negative microscopy for L. donovani in organ aspirates of all VL suspects enrolled (292), significantly higher correlation (p<0.01, 0.919) was established between ß-ME ELISA and FD-DAT than between ß-ME ELISA and RKT (p<0.01, 0.824). Taking into calculation the combined estimates of sensitivity, specificity, positive and negative predictive values, higher agreement (94.8%) was determined between total performance of ß-ME ELISA and FD-DAT than between that of ß-ME ELISA and RKT (90.7%). CONCLUSION: Based on results and merits discussed, we recommend application of this ß-ME ELISA both for diagnosis of VL at laboratory level and confirmation of results obtained with DAT or RKT in the field.

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. For comparison on semi-qualitative grounds with DAT, the RKT was applied to aliquots from positive samples that were two-fold serially diluted in saline to determine, as for the DAT, the end-point reaction in RKT. RESULTS: While qualitatively both RKT and DAT demonstrated comparable reliability for VL detection (sensitivity = 96% and specificity = 98.7% or 99.3%), no significant correlation (r = 0.13) could be established between intensities of their positive reactions in 25 cases studied. A negative correlation was further determined in those 25 VL cases between the positive intensities of the RKT and antibody levels measured semi-quantitatively with the same procedure (r = -0.36) or the DAT (r = -0.30). Irrespective of the low, moderate or high antibody levels measured with RKT (< or = 1:8 and 1:16-1:32 > or = 1:256) or DAT (< or = 1:25,600 and 1:51,200- 1:409,600 > or = 1:3,276,800) in patients with confirmed or unconfirmed VL infection, exclusively strong positive intensities were obtained with RKT. CONCLUSION: For further optimizing diagnosis and simultaneously assessing magnitude of immune response to L. donovani infection in Sudanese patients, the combined application of RKT and DAT is recommended.

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 (/=1:25,600), 86 (73.5%) scored maximum (0.81 to >/=1.35) and 30 (25.6%) medium (0.27 to 0.80) beta-ME ELISA absorbance values. VL diagnosis was established for 142 (44.1%) patients in the VL-symptomatic group (n = 322), based on positive microscopy for Leishmania donovani in lymph node aspirates or positive DAT (titer, >/=1:3,200). Of the 125 sera from the symptomatic patients for whom microscopy was positive for VL, 111 (88.8%) had comparable positive DAT and beta-ME ELISA readings. In all 17 sera from the symptomatic DAT-positive patients for whom leishmaniasis was not established by microscopy but who responded favorably to antileishmanial therapy, absorbance values (>/=0.27) indicative of VL were obtained by beta-ME ELISA. Of 197 symptomatic patients for whom microscopy was negative for VL, 172 (87.3%) tested negative in beta-ME ELISA and 180 (91.4%) in DAT. Based on the high reliability demonstrated here for VL detection, beta-ME ELISA fulfills the requirement of confirming DAT results in patients manifesting suspected VL.

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. For all non-VL patients, a maximum DAT titre of 1/800 was recorded for either of the two antigens. For all VL patients, in comparison with the freeze-dried, the glycerol-preserved antigen always had equal or higher titre; in 16 of the 24 VL sera tested, the latter antigen scored three- to sixfold higher titres. As this glycerol preservation method is economical and easy to perform, it has better potential for wider-scale application than freeze-drying.