Irect and latex agglutination tests-new approaches to improve their reliability and application potential for diagnosis of visceral leishmaniasis in the field

number: 
456
إنجليزية
Degree: 
Imprint: 
Medicine
Author: 
Muhanned Muhsin AL-Etby
Supervisor: 
Dr. Tarik I. Al-Jeboori
year: 
2000
Abstract:

In Iraq, visceral leishmaniasis is a protozoal disease that causes serious public health problem. The etiological diagnosis is difficult and not practical especially in the field settings. A limited number of the available serological methods for diagnosis and epidemiological studies are suitable for field application, however, the direct agglutination and the latex agglutination tests were proved convenient for the use under field conditions. In this study, the two diagnostic tools, used in Iraq, BMA & IFAT were not very efficient for the diagnosis of VL. BMA is not practical because of its low sensitivity (46.3%), while IFAT needs sophisticated equipment and high skills that not available in the endemic areas, besides its little sensitivity (83.8%) and sometimes interpretation of the results is difficult. And in order to increase the application potential of the direct aeslurination test for the detection of anti-leishmania antibodies in human sera. we developed an antigen based on stained and freeze-dneld Leishmania donovam promastigotes. The production of freeze-dried antigen was obtained when the parasite antigen incubated with 30% (v/v) Dimethylformaraide that resulted in ideal preservation of the structure as well as its antigenicity. The direct agglutination test was evaluated using freeze-dried antigen. The freeze-dried antigen remained fully active, even after storage at 56 CC for the study period (6 months). With a cutoff value of 1:800, the sensitivity was 100%-and the specificity was 99.3%, but with a cutoff value of 1:1600 the sensitivity and specificity were 96.3% and 100% respectively. The results were comparable when aqueous antigen was used. The test showed a high reproducibility with no day-to-day variation in the reading and excellent repeatability with no difference between the readings obtained with different antigen batches. Thus, the major advantages of the freeze-dried antigen are that the production of a large batch of this antigen allows reproducible and repeatable results over a long period of time, and it can be stored at ambient temperature, hence, it makes the test an excellent diagnostic tool for use in the field. There was no significant difference (p.O.OOl) in the results of direct agglutination test based on freeze-dried antigen when applied on filter paper blood elutes and serum samples. These findings further simplified the test since the filter paper blood sampling could avoid the patients the discomfort caused by venepuncture, as well as blood sample processing to obtain serum. The latex agglutination test, was improved by the use of antigens prepared by different methods from promastigotes (sonicated and trypsin-treated and sonicated )When sonicated promastigotes soluble antigen was used for the sensitization of latex particles, it gave the highest sensitivity (100%). while the highest specificity was obtained when the trypsin-treated sonicated promastigotes soluble antigen was used. The soluble antigens prepared from axenic amastigotes gave lower sensitivities compared with those prepared from promastigotes (92.2 - 100% vs 86.9 - 89.7%), results that might indicate a change in the antigenicity of the axenic amastigotes. Moreover, the shelf life of the sensitized latex particles was 4.5 months at 4°C.These results focus on the fact that promastigotes are preferred to be used as a source of soluble antigens. When latex agglutination test is used as a screening test, sonicated promastigotes soluble antigen must be used as it gave high sensitivity (100%). But when it is intended for the serodiagnosis (e.g. at the dispensary level) the trypsin-treated promastigotes soluble antigens shoud be used because it gave the highest specificity (97.4%). Finally the direct agglutination test using freeze-dried antigen was applied on a limited field study to confirm its potential as an epidemiological tool. The results have shown that this test could clearly distinguish sera from children living in endemic area (Al-Suwira) , children living in non-endemic area (Baghdad), as well as that of active Kala-azar cases based on their titer distribution.