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.

Sero-epidemiological study of kala-azar in a village of Varanasi district, India.

OBJECTIVE: To evaluate five kala-azar serological tests for field use. METHOD: Serological survey in Pandit Ka Purva village in Varanasi district, India, using Sia water test, aldehyde test, direct agglutination test (DAT), micro-enzyme-linked immunosorbent assay (ELISA) and dot-ELISA. RESULTS: The total population of the village was 518, 67 of whom showed typical clinical and parasitological features of kala-azar, including seven who died. The age distribution of kala-azar cases showed significant differences, being highest among the 45-54-year age group. The disease was more prevalent among males. Serum samples were collected from 498 persons (96% of total population) including 67 kala-azar cases and 40 disease controls (malaria, TB, leprosy, typhoid). Ten 10 serum samples from healthy controls living in endemic area were also collected. The test sensitivities were: Sia water test, 85.0%; aldehyde test, 62.7%; DAT, 94.0%; micro-ELISA, 91.0% and dot-ELISA, 97.0%. The test specificities were: Sia water test 92.5%, aldehyde test, 93.2%, DAT, 96.7; micro-ELISA, 97.6% and dot-ELISA, 98.4%. CONCLUSION: The dot-ELISA is highly sensitive and specific, cheap, and easy to interpret with the naked eye, making it a powerful screening test for the surveillance and diagnosis of Indian kala-azar at field level.

A hag mutant of Moraxella catarrhalis strain O35E is deficient in hemagglutination, autoagglutination, and immunoglobulin D-binding activities.

Previous studies correlated the presence of a 200-kDa protein on the surface of Moraxella catarrhalis with the ability of this organism to agglutinate human erythrocytes (M. Fitzgerald, R. Mulcahy, S. Murphy, C. Keane, D. Coakley, and T. Scott, FEMS Immunol. Med. Microbiol. 18:209-216, 1997). In the present study, the gene encoding the 200-kDa protein (designated Hag) of M. catarrhalis strain O35E was subjected to nucleotide sequence analysis and then was inactivated by insertional mutagenesis. The isogenic hag mutant was unable to agglutinate human erythrocytes and lost its ability to autoagglutinate but was still attached at wild-type levels to several human epithelial cell lines. The hag mutation also eliminated the ability of this mutant strain to bind human immunoglobulin D. The presence of the Hag protein on the M. catarrhalis cell surface, as well as that of the UspA1 and UspA2 proteins (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367-4377, 1997), was investigated by transmission electron and cryoimmunoelectron microscopy. Wild-type M. catarrhalis strain O35E possessed a dense layer of surface projections, whereas an isogenic uspA1 uspA2 hag triple mutant version of this strain did not possess any detectable surface projections. Examination of a uspA1 uspA2 double mutant that expressed the Hag protein revealed the presence of a relatively sparse layer of surface projections, similar to those seen on a uspA2 hag mutant that expressed UspA1. In contrast, a uspA1 hag mutant that expressed UspA2 formed a very dense layer of relatively short surface projections. These results indicate that the surface-exposed Hag protein and UspA1 and UspA2 have the potential to interact both with each other and directly with host defense systems.

The use of direct agglutination test (DAT) in serological diagnosis of Ethiopian cutaneous leishmaniasis.

Leishmania aethiopica (L.a.) is the main species of Leishmania that causes Ethiopian cutaneous leishmaniasis (ECL). The routine diagnosis of ECL depends on parasitological examination of smear, culture or biopsy. In this study, DAT was set-up and evaluated for its diagnostic performance using defined sera of 45 ECL patients, 18 visceral leishmaniasis (VL) patients, 12 patients with other diseases, and 37 normal controls. The test was also evaluated in 64 patients clinically diagnosed as ECL, leprosy, or other skin diseases. Using L.a. derived antigen, the sensitivity and specificity of the test was determined to be 90.5% and 91.8% respectively. However, using antigen derived from a non-homologous strain, only 4 sera of 21 active ECL patients were positive. Eighteen sera of VL patients were positive irrespective of the different antigen sources. The data show that DAT can be a useful addition to the diagnosis of ECL.

Mycobacterium leprae particle agglutination in diagnosis and monitoring of treatment of leprosy.

IgM antibody levels against PGL-1 antigen were measured by M. leprae particle agglutination (MLPA) in 156 untreated leprosy patients. The seropositivity rate was much higher in newly untreated MB patients (84.7%) than in PB patients (19.7%). The mean MLPA titers in MB and PB declined significantly after 1 month of MDT (p < 0.001). Seropositivities in control serum specimens were 11.3 per cent in active pulmonary tuberculosis patients, 2.6 per cent in dermatologic patients and 4.4 per cent in a healthy population, in low titers. The study confirms that, anti PGL-1 assay using MLPA is a sensitive and specific diagnostic tool for the diagnosis of leprosy especially MB patients. Additionally, it provides an alternative tool in monitoring leprosy patients under MDT.

Evaluation of direct agglutination test (DAT) as an immunodiagnostic tool for diagnosis of visceral leishmaniasis in Nepal.

Before field application of the direct agglutination test (DAT) for leishmaniasis, it was assessed as a diagnostic tool. Fifteen confirmed visceral leishmaniasis cases (bone marrow aspiration positive), 120 tuberculosis, 58 leprosy, 15 malaria, 26 intestinal parasitic infection cases, 24 endemic healthy controls from adjacent to the study area, and 18 controls from Kathmandu (who had never visited the VL endemic areas) were tested for anti-leishmanial antibody agglutination titers. Two of the tuberculosis cases were positive for anti-leishmanial agglutinating antibodies at 1:800. All the visceral leishmaniasis confirmed cases were reactive to anti-leishmanial antibody at > or = 1:3,200. Other specimens were negative for serology. The sensitivity of the direct agglutination test was 100% and the specificity was 99.2%. The direct agglutination test had positive and negative predictive values of 100% and 99.2% respectively. The direct agglutination test has been found to be simple, rapid, reliable, economic, safe and adaptable to micro-techniques using microtiter plates. It is specific and sensitive. The direct agglutination test is simple enough for it to be performed in a field laboratory.

Anti-PGL-1 antibody levels in Thai leprosy patients.

Phenolic glycolipid - 1 (PGL-1) is a Mycobacterium leprae specific cell wall component. It is an immunodominant antigen and can induce a strong humoral immune response. IgM antibody levels against PGL-1 were measured in Thai leprosy patients between October 1992-April 1994 by a commercially available M. leprae particle agglutination test (MLPA). The percentage of seropositivity was much higher in newly untreated multibacillary (MB) patients (83.9%) than in paucibacillary (PB) patients (17.8%). Antibody levels in the MB group varied in the range 32-8,192, whereas they varied in the range 32-256 in the PB group. Patients being treated with multidrug therapy (MDT) were 68.3% and 19.4% seropositive in the MB and PB groups, respectively. Seropositivities in control serum specimens were 11.3% in active pulmonary tuberculosis patients, 2.6% in dermatologic patients and 4.4% in a healthy population. In conclusion, the anti-PGL-1 assay using MLPA appears to be a sensitive and specific diagnostic tool for the diagnosis of MB patients. Additionally, it may provide an alternative to the BI determination in monitoring MB patients under MDT, and also in the surveillance of such patients after MDT.

A serologic Mycobacteria leprae gelatin particle agglutination (MLPA) in the diagnosis of leprosy: comparison with conventional enzyme-linked immunoassay and bacterial index.

A gelatin particle agglutination test (MLPA) for the detection of anti-phenolic glycolipid-1 (PGL-1) antibodies was compared with the slit skin smear method in the diagnosis of leprosy. MLPA and BI tests showed a good agreement rate of 88.1% and MLPA and ELISA tests showed an excellent agreement rate 96.2%. This MLPA test is simple and reliable, it will be very convenient for the medical practitioners, it would be of great benefit for leprosy patient as well because this test would look like a routine blood examination compared with slit skin smear method which is widely known diagnostic tool for leprosy.

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.

Long-term evaluation of immune status in leprosy patients undergoing multiple drug therapy.

A long-term survey of leprosy patients of all clinical types, starting at the time of diagnosis, was carried out to monitor clinical, bacteriological and immunological parameters at regular intervals during multiple drug therapy (MDT). The patients were assigned to two groups for treatment following WHO guidelines: paucibacillary (PB) and multibacillary (MB). Immunoglobulin levels, specific antibodies, skin-test responses to different soluble mycobacterial antigens (new tuberculins), and in vitro proliferative responses to mitogens and to antigens were measured during treatment, as were clinical changes, the bacterial index, and clinical improvement. No exact relations between disease activity and IgM antibody levels, both IgM immunoglobulin and specific IgM antibody to a species-specific antigen (ND-O-BSA), could be seen for MB patients. Changes in in vitro cell-mediated immunity and skin-test response seemed to be more directly related to the bacterial load and could reflect the improvement of bacteriological and clinical parameters during MDT.

Evaluation of modified lepro-agglutination as screening test for leprosy.

One hundred thirty-three leprosy sera (83 multibacillary (MB) and 50 paucibacillary (PB) cases) were screened by lepro-agglutination (LA) and M.leprae particle agglutination (MLPA) tests. Larger number of MB sera were positive by LA (86.75%) than by MLPA (45.12%) test. Thirty-seven of the 45 MB sera negative by MLPA test were positive by LA test. The reverse was true in three out of 11 MB sera. PB sera showed positivity of 16% in LA test and 24% in MLPA test. All the 55 sera from normal healthy individuals and 18 VDRL positive sera from syphilis patients were found to be negative by LA test.

An epidemiological study of leprosy infection by serology and polymerase chain reaction.

A population-based study has been carried out in two adjacent villages in a highly leprosy-endemic area of South Sulawesi, Indonesia. The prevalence of clinical leprosy was 10.0 per 1000 inhabitants. A total of 1015 serum samples and 1228 nasal swab specimens were collected. IgM antibodies in blood to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae were demonstrated by the gelatin particle agglutination test (MLPA) and by indirect ELISA (IgM-PGL). IgG antibodies to PGL-I (IgG-PGL) and lipoarabinomannan-B (IgG-LAM) were measured by indirect ELISA. The presence of M. leprae in nasal swab specimens was established by a polymerase chain reaction (PCR). The seropositivity rates in the population were 32% for MLPA, 30.8% for IgM-PGL, 6.7% for IgG-PGL, and 11.6% for IgG-LAM. Seropositivity rates for MLPA and IgM-PGL were highest in the younger age groups. There was no difference in seropositivity in any of the tests between household contacts of leprosy patients and noncontacts. The seropositivity rates in the MLPA and IgM-PGL were not randomly distributed among all households. The presence of M. leprae by PCR was demonstrated in 7.8% of the nasal swab specimens. No correlation was found between the results of the PCR and serology. This study indicates that M. leprae is widespread in the population, and that in endemic areas many individuals carry M. leprae in their nasal cavities without having obvious symptoms of leprosy.

Comparative assessment of the leprosy antibody absorption test, Mycobacterium leprae extract enzyme-linked immunosorbent assay, and gelatin particle agglutination test for serodiagnosis of lepromatous leprosy.

A comparative assessment of three serological methods for leprosy diagnosis (the fluorescent leprosy antibody absorption [FLA-ABS] test, the Mycobacterium leprae soluble-extract enzyme-linked immunosorbent assay [ELISA], and the M. leprae particle agglutination [MLPA] test) was carried out. The objective was to identify their performance in clinical and epidemiological diagnosis of leprosy. The study group included 45 lepromatous leprosy patients under treatment. Specificity was > 95% for all three assays, and sensitivity was 95, 58, and 74% for the FLA-ABS test, the MLPA test, and the ELISA, respectively. The only cross-reactivity for M. tuberculosis-infected patients was with the soluble-extract ELISA. Although the FLA-ABS test displayed the highest specificity and sensitivity values, it can only be used in well-developed laboratories, and the patient's clinical and epidemiological background must be considered when results are interpreted because the test remains positive after therapeutic success and could be positive for some household contacts. The MLPA test is easier to perform and interpret, and it is adequate for small laboratories and epidemiological studies intended to detect active untreated or irregularly treated leprosy cases. Therefore, the FLA-ABS and MLPA tests are complementary, and both should be used for serodiagnosis of leprosy.

Application of filter paper method for collection of blood for MLPA test.

The results of MLPA test using serum and filter paper eluate have been compared in this paper. Testing 64 patient samples at 1:32 dilution, 31 were negative by both serum and eluate, 20 were positive by both, six were positive only by serum and one was positive only eluate. In six other cases eluate gave equivocal results while serum result was clearly positive. Some eluates negative at 1:32 dilution gave weak positive agglutination at 1:16 dilution.

Microtiter particle agglutination test for diagnosis of leprosy.

The results of studying the microtiter particle agglutination (MPA) test for detecting anti-Mycobacterium leprae antibodies in blood sera are presented. The serodiagnostic test is based on the agglutination of colored polyacrolein latex microparticles (PAMP) conjugated with 3,6-di-O-methyl-D-glucose (DMG). Sera from 45 leprosy patients (LL, BL), 34 leprosy contacts, and 148 control subjects were investigated by the MPA test. A correlation between the anti-M. leprae antibodies and the bacterial load was found. In many long-treated leprosy patients increased titers of anti-DMG antibodies were observed, which might be due to specific polyneuritis in them. Four contacts of leprosy patients were also positive in the MPA test. "Nonleprosy" sera did not react in the test. The method proposed proved to be of high specificity and sensitivity for the serological diagnosis of leprosy. The rapidity, simplicity, and visual assessment of the results allow the method to be used in the field for epidemiological studies of leprosy contacts and the general population in leprosy-endemic areas.