Saul J. Semião-Santos

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.

Development of a Dipstick Assay for Detection of Leishmania-Specific Canine Antibodies

ABSTRACT A dipstick assay, based on Leishmania infantum antigen, for the rapid detection of Leishmania-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the direct agglutination test (DAT) and the fast agglutination screening test (FAST). In the present study the dipstick test had a sensitivity of 99.2% and a specificity of 87.9%. The DAT had a sensitivity of 97.7% and a specificity of 95.2%, whereas the FAST had also a sensitivity of 97.7% and a specificity of 93.0%. High degrees of agreement were observed between the dipstick test and DAT (93.7%; κ value, 0.86), between the dipstick test and FAST (91.8%; κ value, 0.82), and between the DAT and FAST (95.2%; κ value, 0.90). The high sensitivity and ease of performance make the dipstick test very suitable for surveillance surveys.

Seroepidemiology of canine leishmaniosis in Évora (southern Portugal): 20-year trends.

BackgroundCanine leishmaniosis (CanL) is an endemic zoonosis in the southern regions of Europe. This paper reports the trend in CanL seroprevalence in the municipality of Évora (southern Portugal), where the disease is endemic, over a period of 20 years. The work comprises three different studies that were conducted in the years of 1990 (n = 3,614), 1999 (n = 3,563) and 2010 (n = 1,485 dogs). Blood samples were collected during the anti-rabies vaccination campaigns. Anti-Leishmania antibodies were detected with the direct agglutination test (DAT).FindingsThe total percentages of DAT seropositive dogs were 3.9% (in 1990), 9.4% (in 1999) and 5.6% (in 2010). The overall seroprevalence was significantly higher in 1999 compared to 1990, but in 2010 a significant decrease was found in comparison with 1999. However, compared to 1990 the overall seroprevalence was still significantly higher in 2010. From 1990 to 2010 seroprevalence has switched from significantly lower to higher in the rural areas. Relatively few dogs showed clinical signs of overt disease (0.8% to 2.0%) with lymphadenopathy, onychogryphosis and skin involvement as most frequently observed. Gender associated differences in seroprevalence were not found, and most commonly seropositive dogs were working or stray animals. The mean age of seropositive dogs was significantly higher than seronegative dogs in all three sampling rounds.ConclusionsA high proportion of dogs, which are apparently healthy, yet seropositive, may remain an important factor in limiting the outcome of zoonotic leishmaniosis control efforts.

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.

Allergy to grass pollen: mapping of Dactylis glomerata and Phleum pratense allergens for dogs by two-dimensional immunoblotting

Introduction Much less is known about grass-pollen allergens to dogs, when compared with humans. Genetic-based patterns might play an important role in sensitization profiles, conditioning the success of allergen-specific immunotherapy. Aim Mapping of Dactylis glomerata (D. glomerata) and Phleum pratense (P. pratense) allergens for grass pollen-sensitized atopic dogs, for better understanding how individual allergograms may influence the response to grass-pollen immunotherapy. Material and methods To identify D. glomerata and P. pratense allergoms for dogs, 15 individuals allergic to grass pollen and sensitized to D. glomerata and P. pratense were selected. D. glomerata and P. pratense proteomes were separated by isoelectric focusing (IEF), one-dimensional (1-D) and two-dimensional (2-D) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were blotted onto Polyvinylidene difluoride (PVDF) membranes and allergens were identified by patient sera IgE in Western Blotting (WB). Results In D. glomerata, 17 allergens were identified from IEF and 11 from 1-D SDS-PAGE, while from P. pratense, 18 and 6 allergens were identified, respectively. From 2-D SDS-PAGE 13 spots were identified from D. glomerata and 27 from P. pratense. Conclusions Several similarities were found between dog and human D. glomerata and P. pratense sensitization profiles but no relationship between clinical signs and a specific pattern of allergen recognition was observed. Similarities were found in each patient pattern of sensitization between D. glomerata and P. pratense, also suggesting cross-reactive phenomena. Further molecular epidemiology approach is needed to understand the role of the sensitization pattern in allergen-specific immunotherapy effectiveness in grass-pollen allergic dogs.

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.

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. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3 % (wt/vol) for conserving promastigote status after &bgr;‐mercaptoethanol (&bgr;‐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 &bgr;‐ME only minimally influenced the test outcome. A comparable successful reduction in non‐specific agglutination, as with &bgr;‐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 &bgr;‐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.

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]: 86.3–100%), slightly lower sensitivity was demonstrated for the newly developed β-ME-ELISA (93.5%, 29/31; 95% CI: 77.2–98.9%). Sensitivity was higher for β-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 β-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 β-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 β-ME-ELISA for diagnosis of CVL at the laboratory level and confirmation of results obtained with the DAT in field studies.