Evaluation of immunoperoxidase staining technique in the diagnosis of Acanthamoeba keratitis.

PURPOSE: We describe a simple procedure of Immunoperoxidase (IP) technique, using indigenously raised antibody, to screen corneal scrapings for Acanthamoeba cysts and trophozoites. This study sought to determine the utility of this test in the diagnosis of Acanthamoeba keratitis. METHODS: A high titre polyclonal antibody against a local clinical isolate (axenic) of Acanthamoeba species (trophozoite lysate antigen) was raised in rabbits and used for standardization of IP technique for corneal scrapings. Twenty two smears of corneal scrapings, collected from patients showing Acanthamoeba cysts in corneal scrapings stained with calcofluorwhite (pool-1) and patients showing no cysts in similar scrapings (pool-2), were coded and stained by IP technique by a masked technician. All 22 patients had also been tested for bacteria, fungus, and Acanthamoeba in their corneal scrapings by smears and cultures. IP stained smears were examined for organisms including cysts and trophozoites of Acanthamoeba and background staining by two observers masked to the results of other smears and cultures. The validity of the IP test in detection of Acanthamoeba cysts and trophozoites was measured by sensitivity, specificity, positive predictive value and negative predictive value in comparison (McNemar test for paired comparison) with calcofluor white staining and culture. RESULTS: Based on the readings of observer 1 and compared to calcofluor white staining, the IP test had a sensitivity of 100%, a specificity of 94%, positive predictive value of 80% and negative predictive value of 100%. When compared to culture, the values were 83%, 100%, 100% and 94% respectively. Trophozoites missed in calcofluor white stained smears, were detected in 2 out of 6 cases of culture-positive Acanthamoeba keratitis. The Kappa coefficient of interobserver agreement was determined as fair (30.4%). CONCLUSION: The immunoperoxidase technique is a simple and useful test in the diagnosis of Acanthamoeba keratitis. This can supplement the culture results.

Comparison between R-phycocyanin-labeled and R-phycoerythrin-labeled monoclonal antibody (Mab) probes for the detection of Entamoeba histolytica trophozoites.

A comparison between R-phycocyanin (R-PC)-labeled monoclonal antibody (MAb) probe and R-phycoerythrin (R-PE)-labeled MAb probe for the detection of the three standard reference strains of the cultured-derived Entamoeba histolytica trophozoites, namely HK-9, HM-1:IMSS, and HTH-56:MUTM were evaluated by using direct immunofluorescence antibody (DIFA) assay five times for each strain. Under the blue irradiation of the fluorescent microscope, both R-PC-labeled and R-PE-labeled MAb probes showed consistently greenish-yellow trophozoites and golden-orange trophozoites, respectively. The R-PE-labeled MAb probe stained the trophozoites more brightly and clearly than those stained by the R-PC-labeled MAb probe of the same Eh208C2-2MAb. When observed under the green irradiation, both probes showed the same intensity of brightly red color at the trophozoites of all three strains of E. histolytica. The sensitivity of both tests was 100%. Since this Eh208C2-2MAb could recognize specifically E. histolytica pyruvate:ferredoxin oxidoreductase (PFOR) enzyme, therefore, our two antibody probes would be valuable for use as a rapid, easy and sensitive test for diagnosis of invasive amebiasis. Further applications of these two probes directly onto the fecal sample spots and to more culture-derived strains of E. histolytica/E. dispar of known zymodemes in collaboration with the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDRB), Dhaka, Bangladesh, are under investigation.

Anti-66 kDa antileishmanial antibodies as specific immunodiagnostic probe for visceral leishmaniasis.

A 66 kDa plasma membrane associated molecule of promastigotes of Leishmania donovani (MHOM/IN/1978/UR6) was affinity purified under acidic conditions. Employing purified 66 kDa antigen in micro ELISA, 36 (97.3%) of the 37 patients of visceral leishmaniasis (bone marrow aspirates positive for Leishman Donovan bodies) had detectable levels of anti 66 kDa anti leishmanial antibodies. The sera of the patients confirmed to have visceral leishmaniasis had significantly (P < 0.001) higher optical density values (0.636 +/- 0.230) as compared to sera (OD 0.185 +/- 0.131) from patients clinically suspected to have visceral leishmaniasis (bone marrow aspirates negative for Leishman Donovan bodies). None of the 35 sera from apparently healthy subjects from non endemic area had anti 66 kDa antibodies. However, sera from one (8.3%) of the 12 healthy subjects, who was a first degree relative of a patient of visceral leishmaniasis and residing in an area endemic for visceral leishmaniasis, had anti 66 kDa antibodies. It is felt that detection of anti 66 kDa antibodies in a micro ELISA assay provides a highly sensitive and specific tool for confirming ongoing visceral leishmaniasis.

Detection of blood stage antigens of Plasmodium vivax by sandwich ELISA using pan-species monoclonal antibodies and polyclonal antibodies.

This paper reports an improved PcAb-McAb-ELISA test to detect blood stage Plasmodium vivax antigen in which the plates were coated with rabbit anti-P. cynomolgi polyclonal antibody to capture the antigens in test samples and two monoclonal antibodies, M26-32 and 3F9, were added together to react with the captured antigens. The coincidence rate with this test was 93% with microscopically confirmed P. vivax cases, 97% with normal samples, 95% with microscopically negative fever cases from nonendemic areas and 86% from endemic areas, respectively. The sensitivity was greater than 1 parasite/10(5) RBC.