Amphizoic amoebae: pathogenic free-living protozoa; review of the literature and review of cases in Thailand.

A large variety of species of free-living amoebae (FLA) caused an indefinite form of these protozoa. Non-fixed form, as indicated by amoeboid movement and possed the bacteria to survive in nature. Two species of pathogenic FLA: Naegleria fowleri and Acanthamoeba spp. were identified as the causative agents of Primary Amoebic Meningoencephalitis (PAM) and Granulomatous Amoebic Encephalitis (GAE) respectively. They were suggested to amphizoic protozoa, capable of living as parasites or as free-living and they were also considered to be distributed worldwide. These amoebae were detected in lakes, rivers and ponds. The first case of meningoencephalitis was observed in 1961 by Fowler Many cases were reported later on and the pathogenicity was tested by nasal inoculation of mice. In fact, quite a number of FLA were isolated but only a few species were pathogenic to humans. The three typical features which allow recognition of Naegleria spp. flagellate stage, round cyst and promitotic trophozoite. This promitosis distinguishes the Naegleria genus from Acanthamoeba spp. The disease caused by PAM usually occurs with acute onset, whereas chronic for GAE. The GAE cases mentioned are mostly in debilitated patients, chronic alcoholics or patients under treatment with immunosuppressive methods. About 6 cases of PAM were reported in Thailand during 1982-1997. Four cases of GAE were reported in 1994 and two isolated cases ofAcanthamoebafrom keratitis patients were reported in 2000. Finally one case of PAM and one case of GAE were reported in 2001. The surveys of FLA were set up to study the distribution of these pathogenic amoebae and determine the prevalence of amoebae in aquatic habitats of human environments. About 40% were identified as Acanthamoeba spp., 30% were Naegleria spp., 20% were Hartmanella and 10% were Vahlkampfia. Only 10% of Naegleria spp. belonged to Naegleria fowleri.

Early detection and identification of amphizoic amoebae from nasal exudates of a symptomatic case.

A man visited the Out Patient Department of the hospital for Tropical Diseases in February 2004 with low grade fever and severe headache for a week. He had the history of diving in a natural pond 2-3 days before the onset of the disease. A thick bloody mucous was observed from the nasal discharge. Fresh microscopic observation of the exudates in 0.85% sodium chloride revealed numerous active amoeba trophozoites. Two groups of the trophozoites were observed The first group was 10 micro sized amoeba with active directional movement by lobopodia and the second group was 15-30 micro sized amoeba with active multiprogressive movement by filopodia. Few flagellate forms were observed after exflagellation in distilled water and some polygonal cysts were also found. Giemsa' stain was used to differentiate the amoeba trophozoites from the leukocytes. It was concluded that this patient was infected by both Naegleria spp. and Acanthamoeba spp. This is the first report of double infection of free-living amoeba in a symptomatic and non-fatal patient.

Development of Isospora belli in Hct-8, Hep-2, human fibroblast, BEK and Vero culture cells.

The development of Isospora belli, a human coccidian parasite, was studied in different cell lines. Merozoites were observed in all kinds of cells, whereas sporogony was demonstrated only in Hct-8. This implied that not only the human cell line can be infected, but also some animal cell lines. Unizoites could be found in Vero cells. The merozoites were transferred to a new culture cell for three passages and maintained for two weeks, but no oocyst production was observed in any culture cells during cultivation.

The hematological status, plasma vitamin B12 and folic acid levels, and intestinal pathology in rats infected with Giardia lamblia.

The purpose of our study was to investigate the hematological status, vitamin B12 and folic acid absorption and intestinal pathology after Giardia lamblia infection in a rat model. Adult Wistar rats were assigned randomly to receive human giardia cysts orally in the amount of 5 x 10(5) or 1.0 x 10(6) cysts, or none in the controls. The results showed that all the rats injected with giardia cysts became infected. The cyst output in the infected rats varied considerably. In rats infected with 5.0 x 10(5) giardia cysts, the incubation period until cyst output was 10 days compared with 4 days in rats infected with the higher amount of 1.0 x 106 giardia cysts. The highest peaks for cysts output in these 2 groups were on days 4-33, which decreased gradually to days 40-58. The hematocrit and hemoglobin levels in the infected rats were statistically significantly lower than in the controls on days 16, 22, 33, and 37 post-infection (p < 0.05). A reverse relationship between giardia cyst output and hemoglobin concentration was found in the infected rats (p = 0.05). There were no significant differences in plasma vitamin B12 and folic acid levels between the infected rats and the control rats. No pathological changes were found in the small intestine of infected rats. These findings suggest that giardiasis did not affect the absorption of plasma vitamin B12 and folic acid but caused anemia in a rat model.

Comparison of Cryptosporidium parvum development in various cell lines for screening in vitro drug testing.

This study describes the development of Cryptosporidium parvum in MDCK, MA-104, Hep-2 and Vero cell lines. Differences in susceptibility, infectivity, and the methodology of excystation were determined. Various solutions were considered to determine the factors which enhanced the excystation (eg with and without sodium hypochlorite, trypsin or sodium taurocholate). It was shown that the sporozoites could be excysted in media either with or without trypsin and sodium taurocholate, but the number of sporozoites in the latter solution was less than the former one. Only oocysts digested by sodium hypochlorite and trypsin can enter the culture cells. Numerous meronts and oocysts were demonstrated and persisted for 9 days. Asexual stages were not observed in MA-104. Only few oocysts could be detected 1-3 days post-inoculation. There was a significant difference between the number of oocysts, which invaded MDCK, MA-104, and Hep-2 cells. MDCK gave the highest susceptibility to oocyst invasion among the three cell lines and asexual stages were also found. Among the 25 isolates, which had been cultivated, 23 isolates could infect MDCK and Hep-2. Only 2 isolates could not infect the MDCK cell. These 2 isolates could infect the Vero cell and yielded high numbers of trophozoites. Praziquantel (PZQ), doxycycline, and paromomycin (PRM) were tested on the infecting parasites. The drugs were added either with the inoculum or 24 hours after inoculation. None of them was effective, including PRM, which had been previously reported as effective.

Prevalence of intestinal parasitic infection among Thai people with mental handicaps.

The prevalence of intestinal parasitic infection was studied by stool examination in institutionalized and non-institutionalized Thai people with mental handicaps. It was found that the prevalence of infection was much higher in institutionalized (57.6%) than in non-institutionalized people (7.5%). The common parasites found in institutionalized people were Trichuris trichiura (29.7%), Entamoeba coli (23.1%), Giardia intestinalis (8.0%), Hymenolepis nana (7.8%), and Entamoeba histolytica/dispar (7.1%). Institutionalized mentally handicapped people should be considered as a high risk group for intestinal parasitic infection and a parasitic control measure should be emphasized.

Cyclospora cayetanensis: oocyst characteristics and excystation.

In Thailand in 1999-2000, Cyclospora oocysts from two HIV-infected patients and one patient with prolonged diarrhea were detected by formalin-ether concentration technique. Sporulation was performed by mixing stool samples in 2.5% potassium dichromate solution, sporulated oocysts were then treated with various solutions before mechanical rupturing in order to establish excystation, fewer than 10% of the sporulated oocysts could be excysted. Our techniques provided more details of the characteristic appearance of sporocysts and sporozoites within the oocysts (DMSO-modified acid-fast technique with our modification).