Levels of Genetic Variants Among Symptomatic Blastocystis Subtypes and their Relationship to Mucosal Immune Surveillance in the Precancerous Colons of Experimentally Infected Rats.

PURPOSE: The relationship between the genetic diversity of Blastocystis and immune surveillance in precancerous colons with blastocystosis is still under investigation. This study aimed to identify the genetic Blastocystis variants among 54 symptomatic human isolates and their relationship to mucosal immune surveillance in the precancerous polyps of experimentally infected rats. METHODS: Polymerase chain reaction and high-resolution melting (PCR/HRM) curves discriminated human symptomatic Blastocystis isolates into subtypes (STs)/intrasubtypes, which were orally administered to rats to induce experimental infection. Then, the mucosal immune responses of the infected colons were evaluated in relation to polyp formation through immunostaining to identify mucus MUC2 and determine mucosal immune cell (goblet, lymphocyte and mast) counts, secretory IgA levels and parasitic intestinal invasion. RESULTS: ST1, ST3, and ST4 were found in 18.5% (10/54), 54.7% (29/54), and 27.8% (15/54) of the samples, respectively. Then, the HRM curve discriminated ST3 into the wild, mutant, and heterozygous [17/54 (31.5%), 5/54 (9.3%), and 7/54 (12.9%)] intrasubtypes. ST1 and ST4 had no genetic variations. Precancerous polyps were detected in the colons of 40.5% of the infected rats. ST1 constituted 14.7% of these cases, while the wild, mutant, and heterozygous intrasubtypes of ST3 showed polyps in 12.9%, 5.5%, and 5.5% of cases, respectively. Only 1.9% of the polyps were related to ST4. MUC2 showed weak immunostaining in 44.5% of the infected colons, and 38.9% were polyp inducers. Low goblet cell numbers and high interepithelial lymphocyte counts were significantly associated with polyp formation, particularly with ST1 and wild ST3. Among the polyp inducers, high numbers of mast cells were detected in wild ST3 and ST4, while a low number was found with heterozygous ST3. The level of secretory IgA was low in polyp-inducing STs. Most of the results were statistically significant. CONCLUSION: Immunosurveillance showed a potential relationship between ST1 and the ST3 intrasubtypes and precancerous polyps. This relationship may provide insight into the prevention and/or development of new immunotherapeutic strategies to combat colorectal cancer.

Identification of a Cyclospora cayetanensis Oocyst Antigens and Their Validity in the Detection of Immunogenic Patterns of Cyclosporiasis Patients.

INTRODUCTION: The diagnosis of cyclosporiasis is currently based on the microscopic detection of oocysts, which may provide invalid results. The availability of simple, objective immunological screening tests would facilitate epidemiological studies of cyclosporiasis. Therefore, the present study aimed to identify the antigens of Cyclospora cayetanensis oocysts and their validity in serodiagnosis. METHODS: According to parasitological and molecular diagnoses, three study groups were specified. Group (G) I included 30 patients with cyclosporiasis, GII included 12 patients with other parasitic infections, and GIII included 16 healthy subjects. SDS-PAGE was used to analyse C. cayetanensis antigens, and the validity of western blotting and enzyme-linked immunosorbent assays (ELISAs) was then assessed amongst the sera of all study groups. RESULTS: The C. cayetanensis antigenic profile showed eight characteristic bands with molecular weights ranging from 14 to 175 kDa. Western blot analysis of sera revealed 93.3% (28/30 of GI) and 92.8% (26/28 of GII and III) sensitivity and specificity, respectively, dividing the patients in GI into four subgroups. The most frequent diagnostic bands (71.4% of GI sera) showed weights of 26-28 kDa, followed by 71 kDa (53.6%). ELISA sensitivity was 90% (27/30), and specificity was 78.6%. Validation showed perfect agreement between the PCR and western blot results, and ELISA presented substantial agreement with both the PCR and western blot results. CONCLUSIONS: Our findings suggest the existence of high immunogenic diversity in C. cayetanensis and indicate that the 26-28 kDa immunogenic groups may potentially be used as a diagnostic marker of cyclosporiasis. Due to the high validity of ELISA, it might be the test of choice for the routine serodiagnosis of cyclosporiasis.

Antiprotozoal activity of magnesium oxide (MgO) nanoparticles against Cyclospora cayetanensis oocysts.

Outbreaks of Cyclospora cayetanensis infection have been linked to consumption of food and water contaminated by oocysts that can survive both physical and chemical disinfectants. Magnesium oxide (MgO) nanoparticles (NPs) can be potentially used in food as bactericides. In this study, C. cayetanensis pre- and post-sporulated oocysts were exposed to MgO NPs with different doses ranging from 1.25-25 mg/ml. With comparison to control, the antiprotozoal activity of MgO NPs was evaluated by identifying the median effective concentration dose (EC50), lethal concentration dose (LC90), microscopically changes on treated oocysts and rates of sporulation. Among pre- and post-sporulated oocysts, MgO NPs ≥ EC50 was observed after 24 h at concentrations 10 and 12.5 mg/ml, respectively, while ≥ LC90 was observed after 24 h, 48 h and 72 h at concentrations 15, 12.5 and 10 mg/ml, respectively. MgO NPs treated oocysts showed abnormal morphological changes such as an increase in size, wall injury, deposition of vacuolated homogenous particles in the cytoplasm, evacuation of oocyst's contents, and collapse. Sporocysts of treated oocysts were noticed to be peripherally shifted. Sporulation failure of treated oocysts achieved ≥90% after 24 h and 72 h of incubation with 15 and 12.5 mg/ml, respectively, while it was 10.1% among non-treated. All the differences were statistically significant. Our results demonstrated that MgO NPs has a significant anti-Cyclospora effect on both unsporulated and sporulated oocysts, especially considering that it could be biologically synthesized, that way it can be used safely as a preventive agent in food and water disinfectant treatment.

Nested PCR targeting intergenic spacer (IGS) in genotyping of Giardia duodenalis isolated from symptomatic and asymptomatic infected Egyptian school children.

Distinct sequences of Giardia duodenalis assemblages raised the hypothesis that certain assemblages may contribute to its clinical outcome. However, sequences analysis is time consuming, expensive, and needs many manual operations. Nested PCR targeting intergenic spacer (IGS) region was applied successfully to genotype G. duodenalis. This study aimed to identify the prevalence of G. duodenalis assemblages among giardiasis school children and its relation to the presence of symptoms using nested IGS/PCR. Of 65 microscopically confirmed Giardia-positive samples, 65 samples were genotyped proving high sensitivity (92.3%) of IGS/PCR. Negative IGS/PCR samples were also negative for ß-giardin gene. Subassemblage AI was the commonest with 66.6% (20/30) among asymptomatic children compared to 53.3% (16/30) of symptomatic, while assemblage B was found in 40% (12/30) of symptomatic compared to 20% (6/30) of asymptomatic. The difference was significant. AII was only found in asymptomatic with 13.4% (4/30), while mixed infections (AI&B) were recorded only in 6.6% (2/30) of symptomatic group. A significant relation was found between younger children susceptibility for AI and B infections as presented in 77.7 (12/16) and 83.3% (10/12) of symptomatic, respectively, and 80 (16/80) and 33.4% (2/4) of asymptomatic, respectively. Significant relations were found between AI with intermittent diarrhea and B with chronic. A significant relation was found between assemblage distributions and heavy infection intensity. In conclusion, higher incidence of assemblage B among symptomatic children compared to asymptomatic could denote its possible pathogenic potential.

Wide genetic variations at 18S ribosomal RNA locus of Cyclospora cayetanensis isolated from Egyptian patients using high resolution melting curve.

A variable clinical picture of cyclosporiasis including gastrointestinal tract (GIT) symptomatic or asymptomatic beside extraintestinal consequences suggests a possibility of heterogenicity of Cyclospora cayetanensis. The present work aimed to explore the possibility of genetic variation of C. cayetanensis using high-resolution melting (HRM) curve of polymerase chain reaction (PCR) amplified 18S rRNA genes. DNAs extracted from the stool samples of 70 cyclosporiasis patients were amplified and scanned by PCR/HRM curve. The results showed that there are four different genotypic profiles of C. cayetanensis with presence of mixed ones. Although Tm of all profiles was within the same range, they were discerned by plotting of the temperature-shifted florescence difference between normalized melting curves (dF/dT). Genotypic profile I was found alone in 40 % of patients and mixed with genotypic profile II and/or III in 25.7 % of patients, followed by genotypic profile II in 14.3 % then genotypic profile III and IV (10 % each). A significant relation was found between genotypic profiles and GIT symptomatic status as profile I and profile II were mostly detected in patients with acute GIT symptoms without or with chronic illness, respectively, while profile IV cases only were GIT asymptomatic. Statistical significance relations between genotypic profiles and age, gender, residence and oocyst shape index were determined. In conclusion, PCR/HRM proved a wide variation on C. cayetanensis genes that could be reflected on its pathogenic effects and explaining the variability of the clinical manifestations presented by cyclosporiasis patients.

Predominance of Giardia lamblia assemblage A among iron deficiency anaemic pre-school Egyptian children.

Intestinal parasites and nutritional deficiency can coexist and influence each other. This study aimed to clarify the association between Giardia genotypes and presence of iron deficiency anaemia (IDA) among pre-school Egyptian children. Two groups (IDA and non-anaemic) of giardiasis children (44/group) were selected according to their recovery response after treatment of giardiasis. Each group included 24 and 20 gastrointestinal symptomatic and asymptomatic, respectively. Giardia human genotypes were performed by intergenic spacer (IGS) gene based polymerase chain reaction (PCR) with high-resolution melting curve (HRM). PCR/HRM proved that Tms of assemblage A and B ranged from 79.31 ± 0.29 to 84.77 ± 0.31. In IDA patients, assemblages A and B were found among 40/44 (90.9 %) and 4/44 (9.1 %), respectively, while in non-anaemic patients, assemblages A and B were found in 10/44 (22.7 %) and 32/44 (72.7 %), respectively, beside two (4.6 %) cases had mixed infection. The difference was statistically significant. No significant relation was found between symptomatic or asymptomatic assemblages and IDA as assemblage A was found in 21/24 (87.5 %) and 19/20 (95 %) of symptomatic and asymptomatic, respectively, while 3/24 (12.5 %) and 1/20 (5 %) of assemblage B were symptomatic was asymptomatic, respectively. A significant relation was found between assemblage A subtypes distribution among IDA patients as AI and AII were detected on 23 (52.3 %) and 16 (36.4 %) of patients, respectively, while one case (2.3 %) had mixed infection. In conclusion, assemblage A is predominant among IDA giardiasis children suggesting its role in enhancing the occurrence of IDA while B has a protective role.

HLA class II alleles: susceptibility or resistance to cystic echinococcosis in Yemeni patients.

This is the first study dealing with the association between HLA alleles and cystic echinococcosis (CE) in Yemeni patients. The present study aimed to detect the association of HLA-DRB(1) alleles and susceptibility or resistance to CE in Yemeni patients by HLA-DRB(1) typing; first by HLA-DRB(1) amplification using PCR then using the allele-specific probing technique based on the reverse hybridization principle. This case-control study was carried out on 66 unrelated patients with confirmed CE and 66 apparently healthy individuals. The association of class II HLA-DRB alleles was examined in the patients with CE and compared with control subjects. Frequency of HLA-DR16 allele was 18.2% among patients and was statistically significant (higher) than in the control group [3%; odds ratio (OR) = 6.5, chi (2) = 7.1, P = 0.011]. Frequencies of HLA-DR1, DR8, and DR52 alleles were decreased in the patient group (0.0%, 0.0%, and 56%, respectively) than in the control group (19.7%, 9.1%, and 74.2%, respectively) (OR = 0.0, 0.0, 0.443 and P < 0.0001, 0.04, 0.05, respectively). HLA-DR16 allele was found to be statistically positively associated with the occurrence of isolated hepatic CE, single cysts, and cysts >5 cm in size. In contrast, HLA-DR1 and DR52 alleles were found to be statistically negatively associated with the occurrence of isolated hepatic CE. This study demonstrates that susceptibility to CE in Yemeni patients is statistically significantly associated with the HLA-DR16 allele and resistance to CE is statistically significantly associated with HLA-DR1, DR8, and DR52 alleles. Thus, this study has identified that carriers of HLA-DR16 are at high risk for CE, so appropriate preventive measures and quick and careful treatment should be applied to those patients.

Genetic diversity of Dientamoeba fragilis isolates of irritable bowel syndrome patients by high-resolution melting-curve (HRM) analysis.

Dientamoeba fragilis is a parasite that has been recognized as a causative agent of gastrointestinal symptoms. The search for genetic variation in D. fragilis based on the small-subunit (SSU) rRNA gene using restriction fragment length polymorphism was found not useful for molecular epidemiology. In this study, genetic variability of different clinical isolates of D. fragilis was explored by high-resolution melting curve (HRM) following polymerase chain reaction (PCR) in a one-step closed-tube method. Thirty fecal samples from irritable bowel syndrome (IBS) patients having D. fragilis trophozoites and negative for other organisms were involved in this study. According to the type of diarrhea, eight patients had acute, 14 patients had chronic intermittent, and eight patients had diarrhea alternating with constipation. HRM proved that four profiles (subtypes) were present as detecting by scanning mutation. One of these profiles (profile 1) was predominant (50%). Profile 2 was present on 20%. Profiles 3 and 4 were present on 16.7% and 13.4%, respectively. No mixed profiles were detected among the samples. The melting curves characterized by T(m)1=77.17+/-0.29 degrees C in profile 1, T(m)1=77.37+/-1.45 degrees C in profile 2, T(m)1=74.24+/-0.08 degrees C and T(m)2=79.64+/-0.09 degrees C in profile 3, and T(m)1=75.51 +/- 0.09 degrees C and T(m)=79.42 +/- 0.09 degrees C in profile 4. The relation between these profiles and types of diarrhea proved that the majority of patients having profile 1 (73.4%) and profile 4 (75%) had chronic intermittent diarrhea. All of the patients having profile 2 had acute diarrhea while all of the patients having profile 3 had diarrhea alternating with constipation. Although profile 1 was detected among all types of diarrhea, it was corresponding to 11/14 of patients with chronic intermittent diarrhea. All the differences were clinically and statistically significant. In conclusion, HRM following PCR was proved as a wide variation on D. fragilis genotypes that could be related to the characters of diarrhea among IBS patients. As the differences in HRM reflect different sequences of SSU RNA gene, thus, another study for identifying the sequences of these isolates (profiles) will be done and published later.

Antiparasitic activity of cystine protease inhibitor E-64 against Giardia lamblia excystation in vitro and in vivo.

In this work, the therapeutic effect of E-64, a broad spectrum cystine protease inhibitor against Giardia lamblia excystation was studied in vitro and in vivo. Purification of cysts from heavily infected human faecal samples followed by excystation and axenic cultivation of the emerging trophozoites in TYI-S-33 medium were done. In vivo, the response was evaluated experimentally through counting oocysts out-put every other day until the infection eradicated from the stools of infected E-64 treated mice compared to untreated. Also, the histopathological examination of the small intestine was compared between both of the infected groups. In the present study G. lamblia cysts incubated with E 64 in vitro completely failed in excystation in 90% while trophozoites released on 10% (partially excysted on 5% and completely excysted on 5%) compared to 90 % completely excysted on other non incubated (without E-64) of cysts beside, the trophozoites didn't release on 10% (partially excysted on 5% & completely non-excysted on 5%). In vivo, the evaluation of the therapeutic response proved that the decreasing in the oocysts out-put counting every other day until the infection eradicated from the stools of infected treated mice was very marked in comparison to untreated mice. The differences were statistically significant. The histopathological examination of the small intestine of infected non treated group proved that all the different pathological grades were found while in infected E-64 treated group, only grade I was detected. So, E-64 showed a good therapeutic effect which raises its use in the treatment of human giardiasis

Evaluation of the nitric oxide activity against Blastocystis hominis in vitro and in vivo.

The effect of exogenous nitric oxide (NO) on growth, viability and ultra-structural of B. hominis was assessed in vitro by sodium nitrite (NaNO2) in 0.6 mM, 0.8 mM & 1 mM concentrations. The viability of B. hominis was identified using neutral red stain. The role of NO as an endogenous oxidant was assessed by identifying its level in cecum tissue, ileum tissue, blood and stool elutes of mice infected with B. hominis symptomatic human isolates using reactive nitrogen assay compared to control. In vitro study revealed that NaNO2 inhibited the growth and decreased viability of B. hominis with minimal lethal concentra-tion dose 1 mM on the 4th day while, minimal effects were detected with 0.6 and 0.8 mM. Transmission electron microscopy study proved that apoptotic-like features were observed in growing axenic culture of B. hominis upon exposure to NaNO2. These changes were not only found on the vacuolar (central body) form but also they were detected on granular, multi-vacuolar and cyst forms. In vivo study proved that high levels of NO were found in infected mice compared to low changes in control group. The high levels were in cecum tissue particularly. The mean levels of NO among infected mice were 211.8 +/- 20.7 microM in cecum, 90.4 +/- 11.6 microM in ileum, 60.1 +/- 4.7 microM in blood and 63.6 +/- 7.3 microM in stool elutes while, the mean levels of NO in control mice were 70.2 +/- 3.1 in cecum, 67.8 +/- 4.7 microM in ileum, 30.9 +/- 4.2 microM in blood and 28.1 +/- 2.9 microM in stool elutes. The differences were statistically highly significant. NO-donor drugs proved useful in treatment and increase the host resistance to B. hominis.

Pathophysiological variability of different genotypes of human Blastocystis hominis Egyptian isolates in experimentally infected rats.

The genotyping of Blastocystis hominis clinical isolates obtained from 28 gastrointestinal symptomatic patients and 16 asymptomatic individuals were identified by polymerase chain reaction using sequenced-tagged site (STS) primers. Then, pathophysiological variability between different B. hominis genotypes was evaluated in experimentally infected rats. Only four B. hominis subtypes (1, 2, 3, and 4) were detected (18.2%, 9.1%, 54.5%, and 18.2%, respectively) in human isolates. In symptomatic isolates, subtypes 1, 3, and 4 were detected in 8 (28.6%), 16 (57.1%), and 4 (14.3%) patients, respectively. In asymptomatic isolates, subtypes 2, 3, and 4 were identified in 4 (25%), 8 (50%), and 4 (25%), respectively. Subtype 3 was the commonest in humans. Different degrees of pathological changes were found among infected rats by symptomatic subtypes compared with asymptomatic subtypes. The moderate and severe degrees of pathological changes were found only in symptomatic subtypes infected rats while mild degree was found only in asymptomatic subtypes infected rats. Only subtype 1 induced mortality rate with 25% among infected rats. On evaluation of the intestinal cell permeability in the Ussing chamber, a prominent increase in short circuit current (DeltaIsc) was found in symptomatic subtype 1 compared to symptomatic subtypes 3 and 4 infected rats. Minimal effects were found in the asymptomatic and control groups. The results proved that subtype 1 was clinically and statistically highly relevant to the pathogenicity of B. hominis while subtype 2 was irrelevant. Also, the results suggest the presence of pathogenic and nonpathogenic strains among subtypes 3 and 4.

Infectivity of Trichomonas vaginalis pseudocysts inoculated intra-vaginally in mice.

In addition to the trophozoite, pseudocyst is another morphological form which is recently identified among genitourinary trichomonads. Although, this pseudocyst is competent to divide, its role in Trichomonas life cycle has not yet been confirmed. In this study the ability of intra-vaginally inoculated T. vaginalis pseudocysts to induce trichomoniasis in infected mice was evaluated in comparison to the trophozoites. Pseudocysts formation was induced by using thermal-freezing cycle method. The infectivity of the pseudocysts was proved by the presence of T. vaginalis parasite in mice's vaginal washes inoculated in vitro. SEM proved that the pseudocysts withstood on vaginal tissue for 72 hours post infection without any morphological changes. Although the histopathological studies using H & E, PAS and cathepsin D stain proved that there were no differences could be found between trophozoites and pseudocysts in onset of infection, but the pseudocyst had higher infectivity and invasive effects than the trophozoite. So, T. vaginalis pseudocyst is an active form that can induce trichomoniasis.

Comparison between Trichmonas vaginalis symptomatic and asymptomatic isolates effects in intravaginally infected rats.

In this study, histopathological and immunohistochemical changes of the posterior vaginal fornix's and upper portion of the vagina were compared on rats infected with symptomatic and asymptomatic human isolates. Eighteen symptomatic and asymptomatic female isolates were used (nine/ each). Two groups of infected female rats were included in this study (3 rats /isolate). The results showed that there were no differences between symptomatic and asymptomatic isolates in histopathological changes; T. vaginalis of both isolates adhered to PAS epithelial cells at the surface and traversed under these cells. Both isolates were PAS and cathepsn D positive. By scanning electron microscopy many of T. vaginalis of the isolates adhered to microvilli of the epithelium cells in the same manner. Transmission electron microscopy proved that both isolates used the pseudopodia to adhere to the vagina upper part cells. The experimental infections did not differentiate between symptomatic and asymptomatic human isolates regarding histopathological and immunohistochemical changes.

Effect of exogenous adminstration of antioxidants on Blastocystis hominis in vivo.

The effect of exogenous administration of antioxidant (Anttox) on the course of B. hominis in experimentally infected mice was studied. B. hominis isolates were obtained from 10 gastrointestinal symptomatic adult patients. Three groups of 30 infected mice (3/isolate) were used. GI was untreated infected, GII was treated by antox for 4 weeks after infection diagnosis (treatment strategy), and GIII antox treated by for 4 weeks before infection (prophylactic strategy). Mild pathological changes were detected on 13.4%, 19.9% & 86.8% of mice in Gs I, II & III, respectively. Moderate pathological changes were found in 29.9%, 26.6% & 6.6% of mice in Gs I, II & III, respectively. While, the majority of severe pathological changes were in Gs I & II (56.7% & 53.5%) as compared to GIII (6.6%). Meanwhile, 86.8% of mice in GIII had B. hominis forms > 10/high power field compared to 3.3% in Gs I & II, respectively. Although 19.8% of mice in GII were positive for B. hominis by direct smear, no growth resulted in vitro and all the forms were non-viable by using neutral red stain. All the differences were statistically significant. So, antioxidant exacerbated B. hominis intensity but it decreased the pathological changes.

Molecular identification of Cycospora spp. using multiplex PCR from diarrheic children compared to others conventional methods.

Cyclospora organisms named C. cercopitheci, C. colobi & C. papionis were identified in stool samples from several primates. They were morphologically indistinguishable from C. cayetanensis but genetically different. In the present work, Cyclospora infection was diagnosed among 140 diarrheic children with three conventional diagnostic methods and was confirmed using nested PCR. The possibility of infection with not only C. cayetanensis but the three Cyclospora species of primates was identified by multiplex PCR among all cyclosporiasis patients diagnosed by different methods. The results showed that Cyclospora was detected in 25 (17.8%), 31 (22.2%), 32 (22.9%) & 35 (25%) patients using modified Kinyoun stain, auto-fluorescent characteristics, sporulation process of the oocysts and nested PCR respectively. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value and Kappa test were calculated in relation to nested PCR. The single nucleotide polymorphisms (SNPs) in the 18S rRNA gene of C. cayetanensis were identified in 35 cyclosporiasis patients and only one patient had the possibility of human coinfection with primates Cyclospora species (C. cercopitheci, C. colobi & C. papionis) and C. cayetanensis by appearance of a 361-bp. Scanning electron microscopy proved no morphological differences could be detected among Cyclospora oocysts isolated from this patient.

Real-time PCR and flow cytometry in detection of Cyclospora oocysts in fecal samples of symptomatic and asymptomatic pediatrics patients.

The magnitude of Cyclospora oocysts excretion in relation to infection intensity among cyclosporiasis patients was assessed using flow cytometry and quantitative real-time PCR (RT-PCR). Oocysts from stool samples of 25 (14.8%) gastro-intestinal symptomatic pediatrics patients (169) and of 10 (2.8%) asymptomatic gastrointestinal ones (350) were identified by modified Ziehl-Neelsen (MZN) and modified Acid Fast Trichrome (MAFT) staining methods and confirmed by its auto-fluorescent characterizations. Also, 10 infants with negative stool samples were selected as controls. The intensity of infection was calculated as number of oocysts/200 microscopic filed with immersion 400. Flow cytometry and RT-PCR assessed relation between symptoms and oocysts excretions compared to MZN & MAFT. The infection severity in symptomatic patients were identified by MZN & MAFT as mild (16%), moderate (24%) and severe (60%) All asymptomatic patients had mild infection. Flow cytometry was done for stool samples and 100% Cyclospora oocysts were in symptomatic and asymptomatic patients. None was detected in controls. RT-PCR was done for stools with both a species-specific primer set and dual fluorescent labeled Cyclospora cayetanensis hybridization probe by unique regions of 18S rRNA gene sequence. DNA of C. cayetanensis was in 100% of symptomatic and asymptomatic patients and in 20% of controls. In repetitive examination of stools Cyclospora oocysts were neither detected by staining nor flow cytometry. Based on oocysts counts, no differences were found between flow cytometry and RT-PCR in compared to staining methods.

Comparison between secretory leukocytic protease inhibitor and reactive nitrogen intermediates levels in cervicovaginal secretions from symptomatic and asymptomatic trichomoniasis Egyptians patients.

Although trichomoniasis is one of the most widespread sexually transmitted diseases, limited information is known about the host and parasite factors which cause symptomatic versus asymptomatic infections. Both of Secretory Leukocytic Protease Inhibitor (SLPI) and Reactive Nitrogen Intermediates (RNI) are major effectors in the innate immune response against infection. This study aimed to compare the level of SLPI and RNI in relation to the vaginal complains among trichomoniasis Egyptian patients. Two groups of trichomonas infected patients were included. Group I included 30 symptomatic patients distributed in three equal subgroups mild, moderate and severe accordiing to degree of symptoms and Group II included 10 asymptomatic patients. Besides, control Group III included 10 healthy females. Cervicovaginal levels of SLPI & RNI were determined in all patients. The mean level of SLPI was less in symptomatic patients (187.75+/-11.61 ng/ml) than in asymptomatic ones (361.18+/-53 ng/ml), with statistically significant difference. Mean level of SLPI was markedly lower in severe symptomatic patients (173.97+/--4.64 ng/ml) when compared with moderate (188.60+/-2.47 ng/ml) and mild (200.69+/-3.01 ng/ml) subgroups respectively. This difference was statistically significant. In controversy, mean levels of RNI in symptomatic patients were significantly higher (39.4+/-7.15 microM) than asymptomatic (38.89+/-6.49 microM). The mean level of RNI was significantly low in severe symptomatic (30.07+/-1.79 microM) than moderate (41.83+/-1.01 microM) and mild (46.30+/-2.02 microM) symptomatic subgroups. This difference was statistically significant. Both of SLPI & RNI levels returned to normal levels in 93.4% & 80% of symptomatic patients respectively one week after metronidazole therapeutic course.

INF-gamma, IL-5 and IgE profiles in chronic schistosomiasis mansoni Egyptian patients with or without hepatitis C infection.

The immune response against clinical forms of chronic schistosomiasis mansoni patients with or without HCV infection was evaluated by assays the serum levels of IFN-gamma and IL-5 for estimate the cell mediated immunity and IgE level to estimate the humoral immunity. This study included three patient groups. G.I included 25 patients with intestinal schistosomiasis, G.II included 15 patients with hepatosplenic schistosomiasis and G.III included 40 patients hepatosplenic schistosomiasis co-infected with HCV. Control G.IV included 15 healthy persons with matched age and sex. The intestinal group had high IFN-gamma (92%), normal level of IL-5 and IgE. The immune response was mainly 100% Th-1 response. The hepatosplenic patients had high IFN-gamma (26.7%), IL-5 (86.7%) and IgE (73.3%). The immune response was 73.4% Th-0, 13.3% Th-1 and 13.3% Th-2. The co-infected group had high IFN-gamma (62.7%), IL-5 (100%) and IgE (92.5%). The immune response was 62.5% Th-0 and 37.5% Th-2 immunity. The shift to Th-0 and Th-2 immunity as well as associated depression of Th-1 in mixed group of patients may be playing a role in the persistence and severity of both diseases. Such immunity defects add to decrease challenge against HCV clearance.

Cyclospora cayetanensis oocysts in sputum of a patient with active pulmonary tuberculosis, case report in Ismailia, Egypt.

Observation of acid fast C. cavetanensis oocysts were proved in a sputum sample of a 45 years-old male HIV negative patient who was admitted to Chest Hospital due to loss of weight, cough with expectoration of purulent sputum and dyspnea. The radiological picture suggested active pulmonary tuberculosis (TB). Sputum samples which were positive for acid fast bacilli as proved by Ziehl-Neelsen stain technique showed large (8-10 microm) spherical acid-fast C. cayetanensis oocysts and their identify was confirmed by molecular techniques (Nested PCR). The patient was successfully treated for TB since 4 years. However, this was the second time to report C. cayetanensis oocysts in human sputum. The first one was in Argentina. So, C. cayetanensis is a new respiratory system pathogen which must be considered in the differential diagnosis.

Degree of symptoms versus copro-antigen levels in Giardia lamblia infection.

A total of 82 out-patients were examined for Giardia copro-antigens and 12 neonate stool samples as control. ELISA had a sensitivity of 100% and specificity of 91.67%. ELISA (O.D.) had neither significant correlation to Giardia cyst count, to stool consistency or presence of blood, mucus or fat in stool, nor to age but positive correlation to the severity of diarrhoea, colic, nausea, anorexia, weight loss, distension and fatigue. Giardia cyst count was higher in cases with loose stool, while ELISA (O.D.) correlated positively with symptoms except constipation and vomiting. The different in clinical outcome of giardiasis can be attributed, partially to strain differences and host resistance.