Study of factors affecting growth of Leishmania in cultures.

The rate of growth of Leishmania major and L. infantum in El-On's culture media supplemented with human, dog, rat and avian blood was studied in vitro. Rabbit blood was used as a control. The effect of culture with these types of blood on the infectivity of both Leishmania strains to albino mice was also studied. The results showed that a good yield of both L. major and L. infantum parasites can be obtained in culture by using avian blood as substitute for rabbit blood in El-ON's medium. In addition, rat blood gave good results with L. infantum. The morphological forms of L. major and L. infantum on all types of blood supplemented media: elongated promastigotes, spindle promastigotes, paramastigotes and amastigoes were present all through the culture period with variable percentages. The infectivity to experimental animals was not affected by culture of both Leishmania strains on rabbit, human, rat, dog as well as avian blood supplemented media.

Quantitative inhibition ELISA for diagnosis of schistosomiasis using human IgG labeled with fluorescein and anti-fluorescein/peroxidase.

Usually mouse monoclonal antibodies are used in inhibition assays for antibody determination. Interference may occur in these inhibition assays due to presence of naturally occurring anti-mouse antibodies in some human serum samples. To avoid such interference, human IgG isolated from a pool of serum samples of S. mansoni patients and highly positive for IgG against S. mansoni soluble egg antigen (SEA) was used in inhibition ELISA for diagnosis of S. mansoni infection. The assay was based on inhibition of binding of human IgG labeled with fluorescein to S. mansoni SEA coating microtitration plates by tested serum samples. Plates were washed and labeled human IgG reacted with SEA was linked to peroxidase enzyme by incubation with anti-fluorescein/peroxidase conjugate. The assay showed 90% sensitivity and 96.3% specificity. The level of inhibition in ELISA showed highly significant positive correlation with stool egg output (Kandall's tau b = 0.512, P < 0.001). To make the assay quantitative, serial dilutions of the highly positive human serum pool, used for preparation of human IgG, were applied in each plate and concentration of anti-SEA antibodies in serum samples tested was calculated from a 4-parameters logistic curve equation. The highly positive serum pool used as a standard was considered to contain one million arbitrary units of immunoglobulins against S. mansoni SEA. Human IgG is expected to be more practical in inhibition assays than mouse monoclonal antibodies due to elimination of interference caused by naturally occurring human anti-mouse antibodies. Also, large amount of human IgG could be purfied from remnants of serum samples highly positive for the proposed antibodies. A higher specificity and sensitivity could be obtained if IgG is isolated by affinity purification instead of ammonium sulphate precipitation. In conclusion, human IgG isolated from highly positive serum samples could be used in sensitive and specific diagnostic antibody determination inhibition assays for diagnosis of infectious and autoimmune diseases.

Studies on the liver fluke Fasciola in Egypt: I--Morphological and morphometrical studies.

A comparative scanning electron microscopy and morphometrical study of the two geographically isolated species of Fasciola (F. heptica European isolate and F. gigantica from Egypt) were studied in order to clarify their genetic relationships and specific identification. Although the present study has revealed that most of the diagnostic morphological and morphometrical criteria in the two species are highly variable, the position of the ventral sucker relative to the whole body length produced the most significant differentiating criterion, in addition to the presence of markedly larger tegumental papillae on the ventral surface of F. gigiantica.

Diagnosis of symptomatic and asymptomatic Trichomonas vaginalis infection by applying one tube nested PCR to vaginal discharge.

One tube nested-PCR targeting a species-specific Tv-E650 repeat family of T. vaginalis genome, was applied to vaginal discharge specimen to diagnose symptomatic and asymptomatic trichomoniasis. The test was compared with axenic culture examination, wet mount preparation and Papanicolaou stained smears. Four-hundred and fifty cases symptomatic and symptomatic were collected over two years. Out of 290 symptomatic women with cervicovaginitis and 160 asymptomatic women, 35 were culture positive for trichomoniasis. All culture positive specimens were PCR-positive giving a single product at 290 bp by agarose gel electrophoresis and recording 100% sensitivity, similar to culture examination. Among the 35 culture positive specimens, 12 were positive by wet mount and 21 were positive by Pap smear giving a 34.2% and 60% sensitivity, respectively. The standard and boiling DNA extraction methods were equally reliable but the latter was more simple, rapid and cheap. No specimens negative by PCR for trichomoniasis were positive by culture, wet mount or Pap smear. Moreover, specimens from cases with cervicovaginitis of non-trichomonal origin were negative by PCR. All samples of extracted DNA of T. vaginalis from positive culture tubes, used as positive controls, were also PCR positive at the expected product in gel, while no PCR product was detected with the negative DNA control including Chylamidia trachomatis and human DNA. It was found that the one-tube nested-PCR targeting the Tv-E650 repeat family of T. vaginalis is a simple, rapid sensitive and specific accurate method for diagnosis of symptomatic and asymptomatic vaginal trichomoniasis when applied to vaginal discharge.

Characterization of Egyptian isolates of Trichomonas vaginalis I. Isoenzyme patterns.

Three T. vaginalis isolates from Egypt were compared for their isoenzyme electrophoretic patterns on cellulose acetate. The enzymes studied were: glucose-6-dehydrogenase (G6PD); malate dehydrogenase (MDH); phosphoglucomutase (PGM); glucose phosphate isomerase (GPI); malic enzyme (ME); hexokinase (HK) and lactate dehydrogenase (LDH). The three isolates shared the same isoenzyme banding patterns of MDH; GPI; HK and LDH. Two of these isolates were similar in their banding patterns of G6PD, PGM and different from those of the third isolate. The latter was similar to one of the two isolates and different from the other in the ME isoenzyme patterns.

Characterization of Egyptian isolates of Trichomonas vaginalis: I. Serotyping.

Antitrichomonal hyperimmune sera against T. vaginalis stocks isolated from Egyptian female patients were employed for serological differentiation of somatic and soluble antigens in the Ouchterlony gel double immunodiffusion technique. It was concluded that soluble trichomonal antigens present in association with living flagellates are stock--specific reacting with some, but not all the antitrichomonal hyperimmune sera, while those present in association with dead parasites are common antigens reacting with all the sera. Three stocks, E1, E2 and E3 could be differentiated into two strains using their stock--specific antigens. The somatic antigens of six trichomonal stocks reacted with all the hyperimmune sera denoting common antigenic make up.

Opportunistic parasitic infections in immunocompromised hosts.

Parasitological and serological examination was done for 111 cases with various types of malignancies under immunosuppressive therapy and another 20 apparently healthy individuals as a control group to determine the prevalence of opportunistic parasitic infections among immunocompromised patients. Single examination showed that 74 (66.7%) harboured infection with different parasites: Strongyloides stercoralis infection was found in 4 (3.64%) cases; 3 cases (2.7%) had Pneumocystis carinii infection. No Cryptosporidium oocysts were detected; IFAT for toxoplasmosis was positive in 40 cases (36%) with titres ranging from 1/16 - 1/256 but IFAT-IgM was negative. The control group did not show any parasitic infection except that IFAT was positive in 4 out of 20 (20%) with titres ranging from 1/16 to 1/128 but IFAT-IgM was also negative. Although Toxoplasma infection was higher among patients the difference was insignificant. Generally the percentages recorded for the different parasites were found to be within the expected prevalence. One case report of concomitant opportunistic Pneumonocytis and Toxoplasma infection is reviewed.