Eiichi Okuzawa

Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction

Abstract An attempt to identify cysts of Entamoeba histolytica and E. dispar in human stool was conducted by polymerase chain reaction (PCR) using two sets of primers (p11 plus p12 and p13 plus p14) specific for either species of ameba. The cysts in stool specimens obtained from 12 infected individuals were concentrated, freeze-thawed, and treated with Triton X-100 before their examination by PCR. The results of PCR on the cysts were generally consistent with data obtained by PCR on ameba trophozoites hatched from the cysts, by zymodeme analysis, and by enzyme-linked immunosorbent assay (ELISA) and with clinical findings. This PCR was negative for the stool containing large numbers of cysts of either E. coli, E. hartmanni, or Giardia lamblia as well as for the stool specimens obtained from uninfected individuals. The ameba cyst in stool processed using the present method was effective for the PCR analysis even after 1 month of storage at 4°C. The present PCR was sensitive enough to detect ten cysts of either of the amebae.

Purification and identification of major soluble 40-kDa antigenic protein from Entamoeba histolytica: its application for serodiagnosis of asymptomatic amebiasis

One of the major soluble antigenic proteins of Entamoeba histolytica was purified to homogeneity and identified on a molecular basis. Its recombinant protein was expressed in Escherichia coli as a fusion protein with Shistosoma japonicum glutathione S-transferase. Apparent molecular weight of the purified antigenic protein was estimated to be 40-kDa and molecular-based analysis indicated that the purified protein was NADP+-dependent alcohol dehydrogenase (EhADH1). The application of the purified protein for the serodiagnosis of amebiasis was evaluated using an enzyme-linked immunosorbent assay applied to sera obtained from patients with amebiasis and healthy human controls. The purified protein was well recognized by the sera from asymptomatic amebiasis humans (22/22, 100%), whereas, it was less recognized by the sera from symptomatic amebiasis patients (5/16, 31%) with amebic colitis or liver abscess. To confirm the antigenicity of EhADH1, the recombinant glutathione S-transferase-EhADH1 fusion protein was also evaluated by the enzyme-linked immunosorbent assay using the same sera. The recombinant protein was also recognized by the sera from asymptomatic amebiasis humans (14/22, 64%) and less recognized by the sera from symptomatic amebiasis patients (2/16, 13%). These results suggest that the purified protein is applicable antigen for serodiagnostic screening of asymptomatic amebiasis humans.

Prevalence of Intestinal Parasites among Japanese Residents in Developing Countries

We examined fecal specimens of Japanese residents in developing countries in order to know the prevalence of intestinal parasites in the group. One fecal specimen was collected from each 981 (in 1995) and 1275 (in 1996) Japanese living in Asia, the Middle East, Europe, Africa and Latin America. The specimens were fixed with 10% formalin in each area, and were examined in Japan by concentration method (formalin-ether sedimentation) to find protozoan cysts or helminth eggs. The infection rate of intestinal parasites was 3.0% in 1995 and 2.4% in 1996. The rate was high in Africa (1995: 5.7%, 1996: 4.7%) and Asia (1995: 3.8%, 1996: 3.0%). Regarding to the species of the parasites, Giardia lamblia (17 cases), Trichuris trichiura (14) and Ascaris lumbricoides (11) were detected frequently. Additionally, 7 cases of Heterophyes heterophyes infection were found in Asia and the Middle East. The infection rate was higher in adults than that in children, and a positive relationship between the infection rate and duration of stay was observed. Among the Japanese infected with intestinal parasites, abdominal symptoms such as diarrhea or abdominal pain were common (36.8%). It is also noteworthy that 28.1% of the Japanese infected had a history of gastric diseases such as gastric ulcer. Although the infection rate of intestinal parasites among Japanese residents in developing countries was low, compared to that of the natives in the countries, the rate is still higher than that in Japanese living in the home country. It is necessary to continue preventive measures such as health education in order to eradicate intestinal parasitic infections from this group.