AMEBIASIS AND COMPARISON OF MICROSCOPY: MATERIALS AND METHODS

22 Dec
2009

Study Areas
The study was carried out in the foothills of Mount Kilimanjaro in Tanzania. Residents in this area are mainly engaged in coffee and banana farming. The main source of water is microbial-contaminated furrow water from rivers flowing from the mountain springs. The Kilimanjaro area is considered an endemic area for amebiasis in the tropics. Other diseases in this area from the records of health centers include malaria, upper respiratory infections, diarrheal diseases, and intestinal worms. Three villages (Rundugai, Mabogini, and Mvuleni) with an average population of 5,000 and one health center were selected for active cases (people who were not sick/no symptoms of infection). Three hospitals (Kibosho, Kilimanjaro Christian Medical College, and Kibongoto) were included in this study for passive cases (people who had symptoms of amoebal infection). Information relating to population structure; disease cases; possible sources of amoebal infection; personal hygiene; history of amebiasis; and the source of drinking, cooking, and washing water was obtained from the health center serving the area.

Data on Amoebal Infection in Hospitals

Data on amoebal infection in various hospitals in Kilimanjaro during January 1999 to June 2001 were collected and analyzed as percent rate of infection. The amoebal infection diagnosis in these hospitals was based on the microscopic examination. On average, 500 patients under and above five years old were examined for amoebal infection.
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Collection of Stool Samples

The study population was divided into groups: 1)616 active cases included individuals who were requested to report to their health center for routine examination of intestinal parasites, and 2) 226 passive cases included individuals who reported in three hospitals with diarrheal problems. The morn¬ing stool samples were collected in special stool containers. All stool samples were labeled and brought to the Kilimanjaro Christian Medical College laboratory in cool boxes for examination within 24 hours of collection. For each stool sample, color, consistency, and presence of blood or mucus were recorded. A pool of 842 stool samples was used for diagnosis of E. histolytica and E. dispar by microscopic examination or monoclonal ELISA kits (TechLab, Corporate Research Park, Blacks-burg, VA). Each sample was divided into four parts and used for: 1) microscopic examination, 2) Entamoeba Test—E. histolytica plus E. dispar, 3) E. histolytica test, and 4) possible repeat test.

Microscopic Examination

Lugol’s iodine was added to the stool smear and covered with a cover slip and examined within five- to 15 minutes using the 100X objective. Detailed processing and examination procedures were as described by Bailey. During examination, all observed parasites were recorded. canadian pharmacy generic viagra

Entamoeba Test Versus E. Histolytica II Test

The Entamoeba Test and the E. histolytica II Test (gold standard) were used. These two tests are based on a monoclonal antibody against galactose adhesions distinct epitopes of E. histolytica or E. dispar, which do not cross-react serologically and are used for the rapid detection of the parasites in stools. The Entamoeba Test (sensitivity and specificity of 87.7% and 98.3%, respectively) is designed to detect but not differentiate the antigens of E. histolytica and E. dispar in stool. The E. histolytica II Test (sensitivity and specificity of 96.9% and 100%, respectively) is designed to detect specifically E. histolytica in stool. The Entamoeba Test was performed on all the stool specimens. The E. hisolytica II Test was performed only on those specimens which were positive with the Entamoeba Test. As specified earlier, a total of 616 (active cases) and 226 (passive cases) stool samples (less than 24 hours old) were analyzed.

Each stool sample was thoroughly mixed prior to performing the assay. This included vortexing of the stool sample prior to transfer to the diluent, and complete mixing of the diluted stool sample prior to transfer to the microwell. The diluent was formulated to stabilize the adhesin in the stool sample and minimize degradation. The diluted stool sampie was stored at 2-8°C until the test was performed. It has been found that under these conditions, the stool sample remained positive when tested daily over a period of five days.

Preparation of the Stool for Monoclonal ELISA

One vial for each sample to be tested was set up. A 0.4-ml diluent was added to each vial labeled directly on the side and vortexed to ensure adequate mixing. For formed stools 0.15-0.20 g and for liquid stools, 0.4 ml were used. The stools were vortexed before being transferred to their respective labeled vials. The vials were vortexed for 10 seconds and stored at 2-8°C until ELISA was performed. The specimens were vortexed again before transferring the diluted specimen to the wells.

The test procedure for monoclonal ELISA was performed as described in the TechLab manual. A test sample was considered positive if it had an obvious yellow color when compared to the negative control well. A test sample was considered negative if the reaction was colorless. If the yellow was not distinct, the test was repeated. buy kamagra tablets

STATISTICAL ANALYSIS

To compare the percentage differences among various groups, the mean percent rate of infections was calculated. To compare the microscopic test and ELISA tests, the sensitivity and specificity, positive predictive value (PPV) and negative predictive values (NPV) were computed—assuming that the ELISA test can adequately serve as a gold standard, although there is no specific ELISA test for E. dispar alone. It can be determined from the results of both ELISA tests on a stool sample. So the total number of those with E. dispar is the number which test positive on the Entamoeba Test minus the number which test positive on the E. histolytica-ll Test. Since the examination by microscopy cannot distinguish between the two amoeba, therefore, the total number of positive and negative microscopic diagnosis is the same for E. histolytica infection. Using these facts, the sensitivity and specificity, PPV, and NPV for E. histolytica and E. dispar have been computed using a dichotomous approach.

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