Validation of in-house liquid direct agglutination test antigen: the potential diagnostic test in visceral Leishimaniasis endemic areas of Northwest Ethiopia.

BACKGROUND: Visceral leishmaniasis in Ethiopia is a re-emerging threat to public health, with increased geographical distribution and number of cases. It is a fatal disease without early diagnosis and treatment; thus, the availability of affordable diagnostic tools is crucial. However, due to delays caused by import regulations, procurement and late delivery of imported test kits, accessibility remains a problem in the control program. Therefore, we aimed to produce and evaluate the performance of an in-house liquid (AQ) direct agglutination test (DAT) antigen. RESULT: The AQ-DAT was produced at the Armauer Hansen Research Institute, using Leishmania donovani strain (MHOM/ET/67/L82). Sera from 272 participants; 110 microscopically confirmed cases of VL, 76 apparently healthy and 86 patients who had infectious disease other than VL were tested with AQ-DAT, and standard kits: Freeze-dried DAT (FD-DAT) and rK39. Taking microscopy as a gold standard; the sensitivity and specificity of the AQ-DAT were 97.3 and 98.8%, respectively. It had high degrees of agreement (k > 0.8), with a significant (P < 0.05) correlation compared to microscopy, FD-DAT, and rK39. CONCLUSION: Although further standardization is required, the in-house AQ-DAT could improve diagnostic accessibility, minimize intermittent stock outs and strengthen the national VL control program.

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%.

Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis.

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani, is difficult, as the dermal lesions are of several types and resemble those caused by other skin diseases, especially leprosy. Since the disease generally appears very late after the clinical cure of kala-azar in India, it is also difficult to correlate PKDL with a previous exposure to L. donovani. Very few attempts have been made so far to diagnose PKDL serologically, and the diagnostic methods vary in their sensitivities and specificities. Diagnosis of PKDL through sophisticated PCR methods, although highly sensitive, has limited practical use. We have developed a serodiagnostic method using an enzyme-linked immunosorbent assay to detect specific immunoglobulin (Ig) isotypes and IgG subclass antibodies in the sera of Indian PKDL patients. Our assay, which uses L. donovani promastigote membrane antigens, was 100% sensitive for the detection of IgG and 96.7% specific for the detection of IgG and IgG1. Optical density values for individual patients, however, demonstrated wide variations. Western blot analysis based on IgG reactivity could differentiate patients with PKDL from control subjects, which included patients with leprosy, patients from areas where kala-azar is endemic, and healthy subjects, by the detection of polypeptides of 67, 72, and 120 kDa. The recognition patterns of the majority of serum samples from patients with PKDL were also distinct from those of the serum samples from patients with visceral leishmaniasis (VL), at least for a 31-kDa polypeptide. To further differentiate patients with PKDL from those with active and cured VL, we analyzed the specific titers of the Ig isotypes and IgG subclasses. High levels of IgG, IgG1, IgG2, and IgG3 antibodies significantly differentiated patients with PKDL from patients cured of VL. The absence of antileishmanial IgE and IgG4 in patients with PKDL differentiated these patients from those with active VL. These results imply intrinsic differences in the antibodies generated in the sera from patients with PKDL and VL.

Characterization of immunoglobulin G and its subclass response to Indian kala-azar infection before and after chemotherapy.

Serologic parameters of kala-azar were evaluated by Western blot analysis. Sera from kala-azar patients with confirmed diagnoses were screened for immunoglobulin G (IgG) and IgG subclass-specific reactivity against Leishmania donovani membrane antigen (LAg). Heterogeneous LAg-specific IgG reactivity with numerous proteins with molecular masses ranging from 18 to 190 kDa was observed. Though the individual band patterns were varied, seven polypeptides of approximately 31, 34, 51, 63, 72, 91, and 120 kDa were immunoreactive with all the sera tested from kala-azar patients. The band patterns of the immunoblots of sera from patients after treatment and clinical cure with sodium antimony gluconate revealed a decrease in the frequency of the bands. Still, recognition of the 63- and 120-kDa bands was 100%, and the 55- and 91-kDa fractions were recognized in 93% of the sera from cured individuals. Among the IgG subclasses, IgG1 reacted with the greatest number of polypeptides. The 63-kDa protein was again detected by all of the IgG subclasses of all the sera tested. Other fractions recognized by the subclasses of more than 70% of the serum samples included those of 47, 51, 55, and 78 kDa. Following treatment, 63- and 51-kDa bands were the most reactive with the IgG subclasses. LAg-associated cross-reaction with other reference human antisera revealed a mild reactivity of the 63-kDa polypeptide with some of the serum samples from leprosy, malaria, typhoid, tuberculosis, and healthy controls. Western blot analysis of LAg entrapped in liposomes, strong vaccine candidates against experimental visceral leishmaniasis, revealed a more restricted band pattern. The 63-kDa fraction revealed by all pre- and posttreatment sera showed almost negligible levels of cross-reaction with sera from patients with other diseases or from healthy controls. These observations provide insight into induced immunity during kala-azar infection for future application.

Evaluation of enzyme-linked immunosorbent assay for diagnosis of post-kala-azar dermal leishmaniasis with crude or recombinant k39 antigen.

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.

Western blot analysis of humoral immune response to Leishmania donovani antigens in patients with post-kala-azar dermal leishmaniasis.

Sera from 32 Indian patients with post-kala-azar dermal leishmaniasis (PKDL) were examined for antibodies by immunoblot analysis using an antigen extract of Leishmania donovani. The study revealed that the humoral immune response in PKDL patients was quite distinct compared to that in kala-azar patients. Antibodies to 3 antigens of L. donovani (molecular sizes 110, 65 and 38-42 kDa) were predominant in a majority (78%) of PKDL patients. The most important finding was the consistent recognition of 2 parasite antigens (of 110 and 65 kDa) by PKDL sera; antibodies to the 110-kDa antigen were detectable in 97% of cases, while antibodies to the 65-kDa antigen were detectable in 100% of cases that were examined. None of the 18 cases of leprosy, 10 of vitiligo, or the 30 healthy persons included in the study showed antibodies to these 2 antigens. Thus Western blot analysis provided a highly sensitive test for PKDL patients. Further, it led to the identification of 2 parasite antigens (110 and 65 kDa) that elicit an antibody response in 97-100% of PKDL patients. Purified or recombinant versions of these proteins deserve consideration as potential target antigens in development of simpler, highly specific and sensitive serodiagnostic tests for PKDL.

Diagnostic and prognostic potential of a competitive enzyme-linked immunosorbent assay for leishmaniasis in India.

A Leishmania donovani species-specific monoclonal antibody (monoclonal antibody D2) was evaluated for its diagnostic and prognostic potential by a competitive enzyme-linked immunosorbent assay (C-ELISA) in sera from Indian patients with visceral leishmaniasis (VL) and seven patients with post-kala-azar dermal leishmaniasis (PKDL). These results were compared with those obtained by microscopy with Giemsa-stained tissue smears and a direct enzyme-linked immunosorbent assay (direct ELISA) with crude parasite antigen. Of 121 patients with clinically diagnosed VL examined, 103 (85.1%) were positive and 11 (9.1%) were negative by all three methods. An additional 7 (5.8%) who were negative by microscopy were positive by both C-ELISA and direct ELISA. Seven PKDL patients were also examined and were found to be positive by all three methods. Analysis of the chemotherapeutic response to sodium antimony gluconate of these 110 serologically positive VL patients showed that 57 (51.8%) were drug responsive and 53 (48.2%) were drug resistant. The C-ELISA with sera from 20 longitudinally monitored VL patients before and after chemotherapy showed a significant decrease in percent inhibition of monoclonal antibody D2 in drug-responsive patients. However, in drug-unresponsive patients, the percent inhibition of D2 was unchanged or was slightly increased. Our results therefore indicate (i) the applicability of L. donovani species-specific monoclonal antibody D2 for sensitive and specific serodiagnosis by C-ELISA, (ii) that the C-ELISA is more sensitive than microscopy, especially for early diagnosis, (iii) that L. donovani is still the main causative agent of VL, irrespective of the chemotherapeutic response, and (iv) that the C-ELISA can be used to evaluate the success of drug treatment.

Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.

Visceral leishmaniasis, or kala-azar, a fatal tropical disease, remains problematic, as early diagnosis is difficult and treatment often results in drug resistance and relapse. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA), using leishmanial membrane antigenic extracts (LAg) to detect specific antibody responses in 25 untreated Indian visceral leishmaniasis patients. To investigate the pathogenetic significance of isotype markers in kala-azar, relative levels of specific immunoglobulin G (IgG), IgM, IgA, IgE, and IgG subclasses were analyzed under clinically established diseased conditions. Since LAg showed higher sensitivity for specific IgG than lysate, the immunoglobulin isotype responses were evaluated, with LAg as antigen. Compared to 60 controls, which included patients with malaria, tuberculosis, leprosy, and typhoid and healthy subjects, visceral leishmaniasis patients showed significantly higher IgG (100% sensitivity, 85% specificity), IgM (48% sensitivity, 100% specificity), and IgE (44% sensitivity, 98.3% specificity) responses. Low levels of IgA in visceral leishmaniasis patients contrasted with a 13-fold-higher reactivity in sera from patients with leprosy. Among IgG subclasses, IgG1, -3, and -4 responses were significantly higher in visceral leishmaniasis patients than in the controls. IgG2 response, however, was significantly higher (twofold) in leprosy than even visceral leishmaniasis patients. The rank orders for sensitivity (IgG = IgG1 = IgG3 = IgG4 > IgG2 > IgM > IgE > IgA) and specificity (IgM = IgG3 > IgE > IgG4 > IgG2 > IgG > IgG1 > IgA) for LAg-specific antibody responses suggest the potentiality of IgG3 as a diagnostic marker for visceral leishmaniasis.

Immunoblot analysis of the antibody response to antigens of Leishmania donovani in Indian kala-azar.

When infected with Leishmania donovani, patients develop specific antibodies that constitute the basis of serodiagnosis. Using immunoblot analysis, we examined the antibody response to antigens of L. donovani in 35 kala-azar (KA) patients and 67 controls. Sera from KA patients recognised numerous antigens with molecular weights ranging from 14-110 kDa. Antigens of 40 kDa, 55 kDa, 65 kDa, 70 kDa and 82 kDa were recognised most frequently. All KA patients produce an antibody response to one or more of these antigens. The majority (83%) of KA cases recognised at least four of these five parasite antigens. The 70 kDa antigen showed the greatest sensitivity for Indian KA, and produced a positive reaction in 94% of patients. This antigen gave 10% false-positive reactions in controls comprising patients with related diseases (i.e. tuberculosis, leprosy and malaria) and in healthy controls. Data indicated that the 70 kDa antigen may include a member of the heat shock protein 70 family. Studies with four clinical isolates of L. donovani showed that the 70 kDa component was expressed in all the strains examined. Immunoblot assay (Western blotting) provided a sensitive diagnostic test for KA patients, and identified the 70 kDa parasite antigen that is promising as a potential target antigen for the development of less complex serodiagnostic assays for KA.

Is PGL-1 also present in Leishmania donovani promastigotes?

A soluble antigen complex (SAC) derived from the ruptured promastigotes of Leishmania donovani parasites (LD-SAC) was used for complement fixation test (CFT) in leprosy Cases of tuberculoid and borderline tuberculoid leprosy, post-kala azar dermal leishmaniasis (TT, BT, PKDL) and control sera gave negative CFT. Smear-positive cases of borderline (BB, BL) and lepromatous (LL) leprosy and drug-resisting cases of pulmonary tuberculosis gave positive CFT; smear-negative cases of LL leprosy sera also gave positive CFT. Sera of smear-negative inactive LL patients contained only PGL-1 and PDIM antigens for a long time after they become inactive. Therefore, the positive CFT in inactive LL makes us suspect whether PGL-1 is present in LD promastigotes.

Evanescent wave fibre optic sensor for detection of L. donovani specific antibodies in sera of kala azar patients.

An easy-to-use technique for detection of antibodies specific for the parasite L. donovani in human serum sample has been developed. The method is based on an evanescent wave generated from a tapered configuration of decladded optical fibre and does not require any volumetric measurement. Tapered fibres are immobilized with the purified cell surface protein of L. donovani by covalent bonding. Treated fibres are incubated with the patient serum for 10 min followed by incubation with goat anti human IgG tagged FITC. Fluorescent intensity from the fibre has been shown to be proportional to L. donovani specific antibodies present in the test sera. Direct readings can be obtained after signal enhancement through a photomultiplier tube within 5 min. The system, when tested on 12 positive sera, did not show any false negative result. Also, no false positive result was obtained with serum samples of patients infected with leprosy, tuberculosis, typhoid and malaria, showing the specificity of the sensor and efficacy of the technique.

Reactivity of various leishmanial antigens in a direct agglutination test and their value in differentiating post-kala azar dermal leishmaniasis from leprosy and other skin conditions.

A direct agglutination test (DAT) for the detection of post-kala azar dermal leishmaniasis (PKDL) was evaluated in conditions that simulate the disease clinically or immunologically. A reference strain of Leishmania donovani (LEM 1399), and antigen preparations from Leishmania isolates from Bangladeshi patients with post-kala azar dermal leishmaniasis or visceral leishmaniasis were used. A titre of at least 51,200 was obtained in tests of patients with PKDL with all three antigens, whereas a maximum titre of 1600 was recorded in patients with cutaneous leishmaniasis, mucocutaneous leishmaniasis or leprosy. Antigens from dermal isolates of L. tropica (LV 140) and L. braziliensis (LV 65) yielded titres of 1600-6400 in patients with PKDL. The lowest titre recorded in 70 patients tested with the homologous PKDL antigen was 409,600. In patients with leprosy, cutaneous leishmaniasis, syphilis, onchocerciasis, tuberculosis, blastomycosis or vitiligo, titres ranged from 100 to 1600. Tha DAT is better than current parasitological and histopathological methods for the diagnosis of PKDL in areas in which leprosy is co-endemic.

Evaluation of antibody responses in Indian kala-azar by immunoblot.

When infected with Leishmania species, patients develop specific antibodies that constitute the basis of serodiagnosis. using Western blot analysis we studied the specificity of anti-leishmania donovani antibodies in patients with visceral leishmaniasis, healthy subjects living in an endemic and non-endemic areas, and patients of other infectious diseases like malaria, leprosy, tuberculosis and tropical splenomegaly. Sera from patients with kala-azar recognised numerous antigens that had a molecular weight of 150 KD, 145 KD, 120 KD, 92 KD, 87 KD, 72 KD, 65 KD, 56 KD, 50 KD, 40 KD, 26 KD, 21 KD, 14 KD, AND 12 KD. The 150, 145, 120, 92, 87, 81, 65, 25, 21, 14, and 12 KD antigens had the greatest specificity for kala-azar sera while the bands of molecular weights 72, 56, 50, and 40 KD were found to be cross reactive with sera of patients of other diseases.

Leishmania donovani: isolation of a concanavalin-A specific antigen and its evaluation for serodiagnosis of visceral leishmaniasis.

A glycoconjugate antigen of 27-39 kDa was isolated from a cell-free extract of Leishmania donovani by affinity chromatography using a Concanavalin-A sepharose-4B column and eluted with 0.5 M alpha-methylmannoside. The antigen was recognized specifically by sera from kala-azar (visceral leishmaniasis) patients and did not react with sera from tuberculosis, leprosy or malaria patients. The antigen may therefore be useful in developing a serodiagnostic assay for visceral leishmaniasis.

Mapping of a visceral leishmaniasis-specific immunodominant B-cell epitope of Leishmania donovani Hsp70.

We have shown that a member of the 70-kDa heat shock protein (Hsp70) family is a major target of the humoral immune response during Leishmania donovani infection. A recombinant fusion protein was recognized by sera from 92% (35 of 38) of patients with visceral leishmaniasis, including representatives from each of the major regions where it is endemic. Serological analysis of recombinant Hsp70, expressed by a series of deletion constructs, identified the carboxy-terminal region as the immunodominant site. This region, which is the most evolutionarily divergent part of the molecule, was recognized by all sera from patients with visceral leishmaniasis which exhibited an anti-Hsp70 response. Purified recombinant L. donovani Hsp70 was not recognized by sera from patients with cutaneous leishmaniasis, Chagas' disease, leprosy, malaria, or schistosomiasis. To determine the regions involved in antibody recognition, a series of overlapping peptides were synthesized on polyethylene pins by the Pepscan method, and a hexamer, EADDRA, was identified by the visceral leishmaniasis serum samples as an immunodominant B-cell epitope.

[Application of monoclonal antibodies against Leishmania donovani, II. Detection of circulating antigen in sera of visceral leishmaniasis before and after treatment].

Monoclonal antibodies L12F7 against the target antigen of L. donovani promastigotes were used for the detection of circulating antigen (CAg) from sera of visceral leishmaniasis patients. The results showed that of 118 serum samples from visceral leishmaniasis tested, 105 were positive (88.9%). 55 normal serum samples were negative. No cross reaction with sera from patients with vivax malaria, schistosomiasis, leprosy and brucellosis, was found. The authors suggested that this assay may be used as a sensitive and new serodiagnostic test for detecting existing infection of visceral leishmaniasis for epidemiological survey and for assessment of cure after effective treatment.

Trypsin-treated and coomassie blue-stained epimastigote antigen in a microagglutination test for Chagas' disease.

A microagglutination test using trypsin-treated and Coomassie blue-stained Trypanosoma cruzi epimastigote antigen was adapted for the diagnosis of Chagas' disease. When incorporated in the test, 2-mercaptoethanol treatment of chagasic sera had no influence on antibody titer. In contrast, titers in sera from patients with visceral leishmaniasis, African trypanosomiasis, and autoimmune disorders, subjected to similar treatment, showed remarkable decline. Accordingly, a lower cut-off point for Chagas' disease serological negativity could be taken resulting in a higher sensitivity (95.6%); the specificity was 94.7%. Similar specificities were obtained with Leishmania donovani chagasi and L. d. donovani antigens applied to homologous visceral leishmaniasis and heterologous Chagas' sera. Of 316 nonchagasic sera, only 3 with leptospirosis and 1 with leprosy showed seropositive titers prior to and after 2-mercaptoethanol treatment.

Identification of specific and cross-reactive antigens of Leishmania donovani chagasi by human infection sera.

Cloned Leishmania donovani chagasi (Ldc) promastigotes were analyzed by SDS-PAGE separation and immunoblotting with human infection sera. The patterns of antigen reactivity were compared by using sera from individuals with Ldc, Leishmania mexicana amazonensis (Lma), Trypanosoma cruzi, Mycobacterium tuberculosis, or Mycobacterium leprae infections. Sera from individuals with these infections recognized Ldc antigens in several m.w. ranges. Reactivity was due to recognition of Ldc molecules and not to Ldc culture medium components, as shown by comparing Ldc promastigotes grown in the presence or absence of fetal bovine serum (FBS), by immunoblotting of FBS, and by [35S]methionine labeling. The major findings of the study were as follows. Immunoblots with Ldc promastigotes could be used to distinguish individuals with Ldc infections from those with Lma infections. Persons with Ldc infections had antibodies to a Ldc antigen of approximately 32 to 35 kd not recognized by persons with Lma infections. Individuals cured of acute Ldc infection did not develop antibodies that differed in specificity to those present during their acute phase of infection. Ldc antigens in the 62 to 66 kd region were recognized by all individuals with Ldc or Lma infections but were not recognized by individuals in the other disease groups or by control sera. This region was found to contain at least four distinct bands, one of which appeared to be glycosylated as indicated by periodic acid-Schiff staining and concanavalin A labeling; an apparently nonglycosylated protein of 62 to 63 kd was eluted from SDS-PAGE gels and was used to diagnose Ldc infection by the ELISA. Whereas crude Ldc antigen gave false positive results with T. cruzi and mycobacteria infection sera, the eluted 62 to 63 kd protein was 100% specific and sensitive in the diagnosis of Ldc infection.