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.

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.

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.

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.