Evaluation of anti-gal enzyme-linked immunosorbent assay for the diagnosis of Indian post-kala-azar dermal leishmaniasis.

BACKGROUND: Elimination of kala azar from India is challenging as there are potential reservoirs of Leishmania donovani in patients with post-kala-azar dermal leishmaniasis (PKDL). The vast repertoire of carbohydrate moieties on L. donovani is known to elicit specific and strong humoral responses in patients with kala azar. AIM: The present study was undertaken to evaluate the diagnostic performances of anti-gal antibodies using enzyme-linked immunosorbent assay for successful serological diagnosis of PKDL in Indian patients and to differentiate cases of past cured visceral leishmaniasis infections. METHODS: We developed Gal enzyme-linked immunosorbent assay to measure specific anti-gal IgG isotype in the sera of 71 Indian patients with PKDL. The diagnostic efficacy of the newly developed assay was evaluated for precision, sensitivity and accuracy. RESULTS: Gal2 enzyme-linked immunosorbent assay revealed three-fold increased anti-gal titers in 71 patients with active PKDL compared to controls. Subclass enzyme-linked immunosorbent assay analysis further revealed enhanced IgG2 and IgG3 anti-gal titers in patients with PKDL compared to control subjects. The rank order for specificity and sensitivity for IgG subclasses was IgG3>IgG2>IgG4>IgG1. The area under the curve values of 0.98 and 0.99 were obtained for IgG and IgG3 Gal2 enzyme-linked immunosorbent assays respectively. Overall sensitivity and specificity were 95.7% (95% CI: 88.1-99.1) and 98.1% (95% confidence interval: 90.1-99.9), and 98.5% (95% CI: 92.4-99.9) and 98.1% (95% CI: 90.1-99.9), respectively. Intra-assay coefficient of variation was 1.5% and inter-assay coefficient of variation was 11.7%. LIMITATIONS: The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. CONCLUSION: Taken together, anti-gal titers detected through Gal2 enzyme-linked immunosorbent assay can serve as an effective diagnostic tool in disease elimination setting and help in better case management in endemic districts.

Glycoproteins in circulating immune complexes are biomarkers of patients with Indian PKDL: A study from endemic districts of West Bengal, India.

BACKGROUND: Post Kala Azar Dermal Leishmaniasis (PKDL) occurs as dermal consequence of previous Visceral Leishmaniasis (VL) infection and serves as an important reservoir for transmission of VL. Diagnosis of PKDL is often challenging for its symptomatic resemblance to other co-endemic diseases like Leprosy or Vitiligo. Parasitological examination by slit-skin smear and culture are the standard methods but lack high sensitivity. Thus, for efficient control of VL, reliable diagnostic and prognostic assay of PKDL are required. OBJECTIVE: Previously, glycoproteins (9-OAcSA) have been reported as promising biomarkers of Indian VL patients. However, till date, the status of glycans in Indian PKDL patients remains unexplored. Accordingly, in this study, the glyco-profile of PKDL Circulating Immune Complexes (CICs) as compared to other cross diseases like Vitiligo and Leprosyhas been investigated. Further, a novel Glyco CIC assay has been developed for efficient Indian PKDL patient diagnosis. METHODS/PRINCIPAL FINDING: In the present study, 90 PKDL patients were enrolled from 3 VL endemic districts of West Bengal during 2015-16. Glycosylation profile of isolated CICs from sera of PKDL patients were initially analyzed through gradient SDS gel electrophoresis followed by PAS silver double staining, which revealed the presence of several glycan rich PKDL specific proteins of varying molecular weights. To further characterize the glyco-profile of acid dissociated affinity purified immuno-reactive antigens present in the CICs, glycosylation was demonstrated in these purified CIC antigens by DIG glycan differentiation kit with or without glycosidase as well as neuraminidase treatment. Diagnostic evaluation of the newly developed colorimetric Glyco CIC assay through Receiver Operating Characteristic (ROC) curve analysis revealed excellent (0.99) AUC value as compared to other conventional serodiagnostic assays like PEG CIC, Parasite ELISA (IgG and IgM). Additionally, longitudinal monitoring of 18 PKDL patients further revealed its good prognostic utility. CONCLUSION: These results highlight the glycosylation status of CICs among Indian PKDL patients present in all the studied endemic districts of West Bengal. These PKDL biomarkers were completely absent in cross diseases like Vitiligo and Leprosy. Further, the newly developed Glyco CIC assay had an improved sensitivity of 95.6%, specificity of 99.3%, NPV of 97.1% and PPV of 98.9%.

Identification and glycobiological characterization of circulating immune complexes in patients with visceral leishmaniasis and post kala azar dermal leishmaniasis.

Here, we investigated the quantitative and qualitative differences in antibody classes and subclasses in serum immune complexes (ICs) of Visceral Leishmaniasis (VL), Post Kala-azar Dermal Leishmaniasis (PKDL) and different cross reactive diseases like Malaria, Leprosy, Vitiligo as compared to control subjects. IC levels were measured through a newly developed PEG ELISA, using L. donovani promastigote membrane antigen coated plate. Antibody classes and subclasses were identified using polyspecific sera and monoclonal antibodies, respectively. ICs were purified using polyethylene glycol (PEG) precipitation. Conditional logistic regression showed an association between IgG1-containing ICs and increased risk of PKDL (OR = 75, P < 0.05) and an association of IgG-containing ICs with VL (OR = 621, P = 0.001). PEG ELISA demonstrated almost 13-15 fold higher IgG containing ICs titers in VL as compared to control (P < 0.001). The assay further established a significant (P < 0.05) difference in the IgG containing ICs titers between VL and PKDL. The isolated ICs were further analyzed by subjecting them to one-dimensional PAGE and subsequently stained with combination of periodic acid schiff (PAS) with silver. A differential banding pattern between VL and PKDL was obtained. Four distinct bands with carbohydrate rich glycoconjugates were identified in PKDL ICs, which were absent in VL and control group. It suggests the scope for developing a novel differential diagnostic assay.

The rK39 immunochromatic dipstick testing: a study for K39 seroprevalence in dogs and human leishmaniasis patients for possible animal reservoir of cutaneous and visceral leishmaniasis in endemic focus of Satluj river valley of Himachal Pradesh (India).

BACKGROUND: The newly recognized endemic focus of leishmaniasis in Satluj river valley of Himachal Pradesh (India) has both localized cutaneous leishmaniasis (LCL) and visceral leishmaniasis (VL) predominantly caused by Leishmania donovani. Rapid rK39 immunochromatographic dipstick test detects circulating antibodies to recombinant K39 antigen of L. donovani-infantum complex and is highly specific/sensitive in diagnosing symptomatic or asymptomatic infection in humans and dogs. METHODS: The sera from two VL patients and 13 LCL patients, and 31 dogs were subjected to rK39 immunochromatographic dipstick testing with an aim to identify possible animal reservoir for leishmaniasis in this endemic focus. RESULTS AND CONCLUSION: The positive rapid rK39 immunochromatographic dipstick test in 100% VL and 31.8% LCL patients, and 6.5% dogs suggests that both VL and LCL in this focus are apparently being caused by L. donovani-infantum and that reservoir infection is perhaps being chiefly maintained in asymptomatic dogs. However, it needs corroborative evidence in the form of in-vitro parasite cultivation and/or PCR studies for confirmation. A more elaborate study is recommended.

Adenosine deaminase activity in sera of patients with visceral leishmaniasis in India.

BACKGROUND: Serum adenosine deaminase (ADA) activity increases in diseases where cellular immunity is involved. Since cell-mediated immune responses play a paramount role in the pathogenesis and healing of the visceral leishmaniasis, therefore, the present study was undertaken to evaluate the serum ADA activity in different pathological conditions. METHODS: Adenosine deaminase was determined in sera of active visceral leishmaniasis (VL) patients at diagnosis and at posttreatment (n=22), healthy controls (n=15), patients with malaria (n=10), leprosy (n=10) and tuberculosis (n=10). RESULTS: Serum levels of ADA were significantly higher in active VL patients as compared to controls and patients with other diseases. ADA levels were also raised in patients with malaria, though not significantly as compared with active VL patients. Sera from VL patients at posttreatment showed significantly decreased ADA levels over sera from patients at diagnosis. CONCLUSIONS: The results therefore suggest that ADA is involved in the pathogenesis and could be used as a clinical marker in the diagnosis of visceral leishmaniasis.

An indirect fluorescent antibody (IFA) test for the serodiagnosis of Kala-Azar.

The IFA test developed using Leishmania donovani promastigote and amastigote antigens showed very high antibody titre in clinically and parasitologically established cases (30) of kala-azar, the geometrical mean reciprocal titre (GMRT) being 870 +/- 5.4 and 5370 +/- 1.80 respectively with the two antigens. In contrast, the GMRT of normal subjects of endemic area was only 12.44 +/- 6.19 and 80.35 +/- 1.66 respectively with these antigens. None of the subjects from non-endemic area suffering with other parasitic diseases, such as malaria, filaria, amoebiasis, leprosy or tuberculosis (20 cases each) gave a positive response to any of the antigen. The test holds promise in the diagnosis of Kala-azar.

Serum tumor necrosis factor levels and disease dissemination in leprosy and leishmaniasis.

It has been suggested that tumor necrosis factor alpha (TNF alpha) may serve as an important antigen-independent host defense mechanism against parasitic organisms. Sera from 66 patients with leishmaniasis and 68 patients with leprosy, all from Ethiopia, were tested for TNF alpha using an enzyme-linked immunoassay. Sera from patients with the multi-parasitic/bacillary type of disease (visceral or diffuse cutaneous leishmaniasis and lepromatous leprosy), known to be associated with absent or low specific T cell response, contained significantly higher TNF alpha titers than those of patients with pauci-parasitic/bacillary disease (localized cutaneous leishmaniasis and nonlepromatous leprosy). High titers of TNF alpha in the absence of a functioning T cell response do not appear to confer resistance against Leishmania aethiopica and Mycobacterium leprae.

Serum interferon activity of patients with leishmaniasis

Serum from a significant proportion of 29 cutaneous and 12 mucocutaneous leishmaniasis patients exhibited interferon activity in a cytopathic assay: positive tests were obtained for 24.1% and 41.7% of the patients, respectively. Similar positive frequencies were observed in other parasitic diseases (schistosomiasis, 12.5%; toxoplasmosis, 20%; Chagas' disease, 16%; leprosy, 12.5%; tuberculosis, 30%). In contrast, none of the 44 serum samples from American visceral leishmaniasis (AVL) patients had interferon activity during the active stage of the disease. However, 13% of the samples obtained from patients recovered from AVL were positive

Serum interferon activity of patients with leishmaniasis.

Serum from a significant proportion of 29 cutaneous and 12 mucocutaneous leishmaniasis patients exhibited interferon activity in a cytopathic assay: positive tests were obtained for 24.1% and 41.7% of the patients, respectively. Similar positive frequencies were observed in other parasitic diseases (schistosomiasis, 12.5%; toxoplasmosis, 20%; Chagas' disease, 16%; leprosy, 12.5%; tuberculosis, 30%). In contrast, none of the 44 serum samples from American visceral leishmaniasis (AVL) patients had interferon activity during the active stage of the disease. However, 13% of the samples obtained from patients recovered from AVL were positive.