Savanat Tharavanij

Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens

A murine monoclonal antibody (MAb) (Eh208C2-2) raised against crude lysate of the pathogenic HM-1:IMSS strain of Entamoeba histolytica was used in an enzyme-linked immunosorbent assay for the detection of E. histolytica antigens in faecal specimens. The detection limit of the assay was 110 and 280 amoebae/ml of the HM-1:IMSS and HK-9 strains in phosphate-buffered saline, respectively. The assay was applied to single stool samples from 3 groups of individuals comprising 40 patients whose stools were positive for E. histolytica trophozoites and/or cysts (group I), 48 patients whose stools were negative for E. histolytica but positive for other parasites (group II), and 36 parasitologically-negative healthy controls (group III). Positivity rates of 77.5%, 2.1% and 2.7% were found in samples from groups I, II and III respectively. Specificity, positive and negative predictive values, and efficiency of the assay were 97.6%, 93.9%, 90.1% and 91.1% respectively. When group I samples were further divided into a trophozoite-positive subgroup IA (13 samples) and a cyst-positive subgroup IB (27 samples), the positive rates were 100% and 66.7%, respectively (P < 0.025).

Diagnosis of Plasmodium vivax malaria using a specific deoxyribonucleic acid probe

A deoxyribonucleic acid (DNA) probe which specifically distinguishes Plasmodium vivax from P. falciparum malaria has been derived from a P. vivax genomic DNA library. This probe, VPL101, consists of 3.2 kilobase pairs and does not hybridize with up to 6 micrograms of human or P. falciparum DNA. VPL101 contains at least two copies of a 205 base pair repeat sequence. The subcloned repeat probe, VPL101/5, reacted with 73 of 76 microscopically diagnosed P. vivax samples but not with any of 17 human DNA samples or any of 8 P. falciparum DNA samples from cultured parasites. It was possible to detect P. vivax in mixed infections in which only P. falciparum parasites were identifiable by microscopy. This P. vivax DNA probe provides a useful epidemiological tool for malaria control programmes.

Monoclonal antibody-based immunohistochemical demonstration of Entamoeba histolytica in liver tissues of experimentally infected hamster (Mesocricetus auratus)

Histopathological changes and the presence of Entamoeba histolytica trophozoites was sequentially followed after intrahepatic inoculation of the parasites in 42 hamsters, 35 of which received no treatment whereas the remaining seven were treated with metronidazole. The liver tissues were examined for amoebic trophozoites by a monoclonal antibody (mAb)-based immunofluorescence assay (IFA), a mAb-based immunoperoxidase (IPx) and H & E staining. The number of hamsters developing abscesses was increased with time and was highest on day 30. Cellular infiltration with inflammatory cells and glycogen depletion were observed as early as day 5, followed thereafter by more intense inflammation of portal canals, periportal fibrosis, bile duct proliferation and hepatocyte degeneration. In 7 metronidazole-treated hamsters, no obvious pathological damage was seen. In a group of seven hamsters each, both IPx and IFA were positive in 3, 3, 4, 5 and 4 hamsters and in 3, 4, 3, 3 and 5 hamsters on days 5, 10, 15, 20 and 30, respectively. In 18 control hamsters, IPx, IFA and H & E were all negative. If the result from H & E was used as a gold standard, agreement between H & E and IFA and H & E and IPx were 91.4%, and 88.6%, respectively. Sensitivity and specificity were 93.8% and 89.5%, respectively for IFA, and 93.8% and 84.2%, respectively for IPx.

Antibody against a ring-infected erythrocyte surface antigen in cerebral and non-cerebral malaria patients

An indirect fluorescent antibody test for glutaraldehyde-fixed, ring-infected erythrocyte surface antigen was performed on admission sera from 45 patients with complicated cerebral Plasmodium falciparum malaria, 33 with uncomplicated cerebral malaria, 91 non-cerebral malaria patients, and 53 blood donors from a non-malarious area. 14 (31%), 28 (85%), 64 (70%), and 1 (2%), respectively, had titres greater than or equal to 1/25, considered as positive. The seropositive rate and the geometric mean reciprocal titre of patients with complicated cerebral malaria were significantly lower than those of uncomplicated and non-cerebral patients, particularly in the 6-14 and 15-29 year age groups. Compared with non-cerebral patients, lower seropositive rates for patients with complicated cerebral malaria were demonstrated only in those who had been ill 4 or more days before admission; whereas lower rates for patients with complications, when compared with rates in those with uncomplicated cerebral malaria, occurred irrespective of the duration of illness.

Mouse IgE response to an allergen from Entamoeba histolytica.

Attempts were made to demonstrate the presence of allergenic components in an extract of axenically cultured Entamoeba histolytica (HK 9). Various strains of mice were immunized i.p. with a low dose of the extract mixed with Al(OH)3 and boosted with the same mixture 4 wk later. A high titer of IgE antibody response was demonstrated in BALB/c, DBA/2 (H-2d), CBA (H-2k), and SWM strains but not in C3H/He (H-2k) strain. The extract was fractionated by gel filtration with Sephadex G-200 and then by DEAE-cellulose chromatography with a gradient buffer with progressive increase in molarity. Fractions having the strongest activity to stimulate the IgE antibody response in the mouse were subjected to SDS polyacrylamide gel electrophoresis. From the results of gel filtration and polyacrylamide gel electrophoresis, the molecular weight on the partially purified allergen was estimated to be 25,000 to 27,000 daltons.

A specific S-antigen of Plasmodium falciparum is expressed in a proportion of primary isolates in Brazil, Thailand and Papua New Guinea

The expression by Plasmodium falciparum of a specific S-antigen has been examined in primary isolates in different regions of the world using a monoclonal antibody that recognizes an epitope within a known repeated amino acid sequence. The epitope was expressed by a small proportion of primary isolates in each of Brazil, Thailand and Papua New Guinea, demonstrating that this S-antigen gene is widespread. The data are consistent with the possibility that the occurrence of P. falciparum strains expressing a particular S-antigen is periodic, related to the duration of immunity against that antigen in a given human population.