Comparison of gdh polymerase chain reaction-restriction fragment length polymorphism and tpi assemblage-specific primers for characterization of Giardia intestinalis in children.

Background: Giardia is a diarrheagenic eukaryotic parasite that consists of at least eight morphologically identical but genetically distinct genotypes. Human giardiasis is caused mainly by A and B assemblages. Aim and objectives: The study aimed to compare the performance of gdh polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tpi assemblage-specific primers in genotyping of G. intestinalis. Materials and Methods: Stool samples of 315 children were microscopically screened for G. intestinalis. Positive samples were genotyped using tpi assemblage-specific primers and gdh semi-nested PCR-RFLP techniques. Results: The prevalence of Giardia was 18.1%. The detected genotypes using tpi and gdh approaches were assemblage A (15.8% vs. 12.7%) and assemblage B (36.8% vs. 74.5%) as single infections and mixed assemblages A and B (47.4% vs. 12.7%). The two approaches showed a moderate agreement (kappa index = 0.413, P < 0.001). PCR-RFLP of gdh gene revealed that sub-assemblages BIII and BIV were equally detected (30.9% each). The remaining samples were equally divided between sub-assemblage AII, mixed BIII and BIV, and mixed AII and BIII (12.7% each). A significant association was detected between the retrieved sub-assemblages and the presence of symptoms. Conclusions: Although both approaches confirmed the predominance of assemblage B, the use of assemblage-specific primers is more effective in elucidating the true picture of mixed assemblage infection.

Levels of Serum Ferritin and Hepcidin in Patients with Uncomplicated Falciparum Malaria in Hodeidah, Yemen: Considerations for Assessing Iron Status.

Understanding the key regulator of iron homeostasis is critical to the improvement of iron supplementation practices in malaria-endemic areas. This study aimed to determine iron indices and hepcidin (HEPC) level in patients infected with Plasmodium falciparum compared to apparently healthy, malaria-negative subjects in Hodeidah, Yemen. The study included 70 Plasmodium falciparum-infected and 20 malaria-negative adults. Blood films were examined for detection and estimation of parasitemia. Hemoglobin (Hb) level was measured using an automated hematology analyzer. Serum iron and total iron binding capacity (TIBC) were determined by spectrophotometric methods. Levels of serum ferritin (FER) and HEPC were measured by enzyme-linked immunosorbent assays. Data were stratified by sex and age. Comparable Hb levels were found in P. falciparum-infected patients and malaria-negative subjects in each sex and age group (p > 0.05). Compared to their malaria-negative counterparts, disturbed iron homeostasis in patients was evidenced by the significantly lower serum iron levels in females (p = 0.007) and those aged <25 years (p = 0.02) and the significantly higher TIBC in males (p = 0.008). Levels of serum FER and HEPC were significantly elevated in P. falciparum-infected patients compared to the corresponding malaria-negative participants (p < 0.001). Serum FER correlated positively with parasite density (p = 0.004). In conclusion, patients with uncomplicated P. falciparum in Hodeidah display elevated levels of serum HEPC and FER. Hemoglobin level may not reflect the disturbed iron homeostasis in these patients. The combined measurement of iron indices and HEPC provides comprehensive information on the iron status so that the right intervention can be chosen.

Expression of microRNA-223 and microRNA-146b in serum and liver tissue of mice infected with Schistosoma mansoni.

MicroRNAs (miRNAs) play regulatory roles in several diseases. In schistosomiasis, the main pathological changes are caused by the granulomatous reaction induced by egg deposition. We aimed to study the changes in host miRNA-223 and miRNA-146b expression in relation to egg deposition and development of hepatic pathology in murine schistosomiasis mansoni. Blood and liver tissue samples were collected from non-infected mice (group I), S. mansoni-infected mice at the 4th, 8th, and 12th weeks post-infection (p.i.) (groups II-IV), and 4 weeks after praziquantel treatment (group V). The collected samples were processed for RNA extraction, reverse transcription, and real-time PCR analysis of miRNA-223 and miRNA-146b. miRNAs' relative expression was estimated by the ΔΔCt method. Liver tissue samples were examined for egg count estimation and histopathological evaluation. Results revealed that miRNA-223 was significantly downregulated in liver tissues 8 and 12 weeks p.i., whereas miRNA-146b expression increased gradually with the progression of infection with a significantly higher level at week 12 p.i. compared to week 4 p.i. Serum expression levels nearly followed the same pattern as the tissue levels. The dysregulated expression of miRNAs correlated with liver egg counts and was more obvious with the demonstration of chronic granulomas, fibrous transformation, and distorted hepatic architecture 12 weeks p.i. Restoration of normal expression levels was observed 4 weeks after treatment. Collectively, these findings provide new insights for in-depth understanding of host-parasite interaction in schistosomiasis and pave a new way for monitoring the progress of hepatic pathology before and after treatment.

Detection of Giardia intestinalis assemblages A and B among children from three villages in the West Delta region, Egypt using assemblage specific primers.

Giardia intestinalis is a common diarrheagenic parasite infecting children globally. It has been classified into eight morphologically identical but genetically distinct genotypes. Human infection is mainly associated with A and B assemblages with variable geographical distribution. The present work aimed to study the epidemiology of assemblages A and B in children inhabiting different areas in Lower Egypt. Stool samples were collected from 315 children and examined microscopically for parasitic infections. Giardia positive samples were genotyped using tpi assemblage specific primers. The prevalence of Giardia was 18.1% among the examined children. Mixed assemblages A and B was more common (47.4%) than single assemblage B (36.8%) or A (15.8%). The distribution of different genotypes was significantly associated with the residence area, animal contact, and handwashing habits. A non-significant association was observed between Giardia assemblages and the clinical manifestations. Assemblage B is the predominant genotype among Egyptian children. The distribution of different Giardia assemblages is strongly associated with the studied area and the habits of its people.

microRNA-155 Expression and Butyrylcholinesterase Activity in the Liver Tissue of Mice Infected with Toxoplasma gondii (Avirulent and Virulent Strains).

PURPOSE: Toxoplasma gondii is an apicomplexan parasite that exhibits distinct strain-related virulence patterns in mice. It can induce hepatic inflammation. The present study investigated MicroRNA-155 (miRNA-155) expression and butyrylcholinesterase (BChE) activity in the liver tissue of mice infected with virulent and avirulent strains of T. gondii. METHODS: Mice groups included: Group (A), uninfected controls; Group (B), infected with T. gondii avirulent strain (ME-49) and euthanized 7, 27, 47, or 67 days post-infection (pi); Group (C), infected by T. gondii virulent strain (RH) and euthanized 7 days pi; and Group (D), infected by T. gondii virulent strain (RH), treated 24 h pi with sulfamethoxazole-trimethoprim (150 mg/Kg/day and 30 mg/Kg/day, respectively) and euthanized 5, 10, or 20 days pi. miRNA-155 expression was estimated in the liver tissue using the reverse transcription real-time polymerase chain reaction and the ΔΔCt method. BChE activity was estimated in liver homogenates by Ellman's colorimetric method. Liver sections were examined histopathologically. RESULTS: revealed a significant elevation in miRNA-155 expression and a significant reduction of BChE activity in all the infected untreated groups compared to the uninfected mice. In group B, the maximum upregulation of miRNA-155 expression and the least reduction in BChE activity were detected 7 days pi. In group D, complete restoration of normal levels occurred 20 days pi. Liver sections showed distinct histopathological patterns with detection of intracellular tachyzoites in group B. CONCLUSION: miRNA-155 and BChE play a role in regulating host-parasite interaction in toxoplasmosis and may contribute to the pathogenesis of T. gondii induced hepatic damage.

Temporal expression of Toxoplasma stage-specific genes in brain tissue: coincidence with parasitological and histopathological findings in mice models.

In the intermediate hosts, tachyzoites of T. gondii predominate in the acute stage while bradyzoites persist inside tissue cysts with the potential for reactivation. The two stages exhibit different metabolic and antigenic characters. The present study aimed to investigate temporal expression of Toxoplasma SAG1 and BAG1 genes in the brain tissue and the coincident parasitological and histopathological findings in mice models of toxoplasmosis. The study included group A: mice infected with RH strain and sacrificed 7 days post-infection (p.i.); group B: mice infected with RH strain and treated with sulfamethoxazole-trimethoprim (30 mg/kg/day and 150 mg/kg/day respectively) 24 h p.i. until sacrificed at days 5, 10, or 20 post-treatment; group C: mice infected with ME-49 strain and sacrificed at days 7, 27, 47, or 67 p.i; and group D: mice infected with ME-49 strain and received dexamethasone daily starting at day 68 p.i. and scarified at days 6 or 10 post-treatment. All mice were inspected daily for abnormal physical signs. Peritoneal exudate and brain homogenate were examined for detection of Toxoplasma stages. Brain sections were examined histopathologically. SAG1 and BAG1 gene expression was evaluated using reverse transcription real-time polymerase chain reaction and the ΔΔCt method. Results revealed that marked BAG1 upregulation is consistent with detection of Toxoplasma cysts and degenerative changes while predominance of tachyzoites and inflammatory infiltrate is compatible with SAG1 upregulation. The study sheds light on the potential for using stage-specific gene expression pattern as markers for evaluation of toxoplasmosis disease progression in clinical settings.

Irritable bowel syndrome in Egyptian patients: plausible risk factors and association with intestinal protozoa.

The pathogenesis of irritable bowel syndrome (IBS) is not yet clear. Our study suggested parasitic infection and other plausible risk factors among Egyptian IBS patients. We studied 40 IBS patients diagnosed according to Rome III criteria (Group I), 40 with other gastrointestinal symptoms (Group II) and 40 healthy controls (Group III). Stool samples were examined using direct wet smear, sedimentation technique, trichrome stain and immune-chromatographic tests for Cryptosporidium parvum. IBS patients displayed a significantly greater percentage of Blastocystis hominis infection (45%) than non-IBS patients (20%) and healthy controls (10%). Dientamoeba fragilis was identified in two IBS patients. Detection of B. hominis was independent of demographic characters, IBS subtype, Helicobacter pylori infection or medications, but with a positive association with a history of antibiotic intake with IBS.

Cryptosporidiosis and other intestinal parasites in 3 Yemeni orphanages: prevalence, risk, and morbidity.

The epidemiological pattern of cryptosporidiosis and other intestinal parasites were studied among 3 orphanages in Sana'a City. A total of 292 orphans (225 males & 67 females), aged 4-20 years old were recruited. A questionnaire of demographic information, health status and other relevant information was filled out for each one. Fresh stool specimens were processed by wet mount preparation, formalin-ether concentration and MZL stain. Among 292 orphans, the overall parasitic rate was 62.7%. C. parvum showed highest prevalence (24%) followed by G. lamblia (16.8%), E. histolytica /E.dispar (13%), and then E. coli (18.5%). Hymenolepis nana was in 7.2% of children but only 3 cases of Ascaris lumbricoides. The risk of C. parvum was significantly higher in children < or = 10 years of age compared to older ones (OR = 3.86). The risk of other parasites did not differ significantly with orphans' age. Males showed 2.634 risk of C. parvum and 1.657 risk of G. lamblia compared to females with statistically significant difference. Bad personal hygiene was associated with higher risk of cryptosporidiosis (OR = 2.634, CI = 1.483-4.677) but the risk of other parasites was not significant. Diarrhea and abdominal pain were recorded in the three orphans, but without significance associated with parasites, either overall or for individual species but, A. lumbricoides was detected in 3 diarrheic children.

Use of a non-mercury containing fixative for diagnosis of giardiasis.

In the present study, methanol was used instead of Schaudinn's fluid for preparation of permanently stained faecal smears. The efficacy of this method to detect Giardia lamblia trophozoites and cysts were compared to merthiolate iodine formalin (MIF) direct smear method and MIF concentration (MIFC) technique. Fresh faecal samples were collected from school students with gastrointestinal symptoms. In 50 Giardia positive faecal samples, trophozoites were detected alone in 9 samples and in association with cysts in 24 ones. A paired comparison of the frequency of detection of trophozoites and cysts by various methods was performed using McNemar statistical test. The results revealed that trichrome staining of methanol fixed smears was the most sensitive technique for trophozoite detection followed by MIF direct smear method (p < 0.05). For cysts, trichrome staining and MIFC had nearly equal sensitivity and were more sensitive than MIF direct smear method but with no statistically significant difference.

Alteration in circulating soluble Fas level in Toxoplasma seropositive pregnant women.

The role of sFas in human toxoplasmosis was designed, included 23 pregnant women serologically positive for anti-Toxoplasma antibodies, and ten sero-negative pregnant females constituted the control group. Antibody titer was assessed by indirect haemagglutination test. The presence of specific IgM antibodies was determined by comparing antibody titer before and after serum treatment with 2-mercapto-ethanol. sFas was quantified in sera of cases and controls by enzyme linked immunosorbent assay. Antibody titers. ranged from 1/160 to 1/640. All cases were negative for specific IgM antibodies indicating that they had passed the acute stage of infection Statistical analysis revealed significant elevation in sFas level in cases compared to controls. The sFas role in establishment of a stable host parasite interaction in toxoplasmosis was discussed.

Levels of growth hormone, albumin and ferritin in human fascioliasis and schistosomiasis.

This study assessed levels of growth hormone, albumin and ferritin in human fascioliasis and schistosomiasis. Forty three patients and ten healthy parasite free subjects were included as controls. All were subjected to clinical, parasitological, and haematological examinations. Serum growth hormone (GH) levels were measured by an immunoenzymometric assay. Statistical analysis revealed significant elevation in GH levels in acute and chronic fascioliasis patients compared to controls. Serum albumin was significantly low in schistosomiasis patients compared to controls. Non-significant changes in serum ferritin were in all patients. The results were critically discussed on the light of the work done before.

Influence of the antioxidant drug (Antox) on experimental giardiasis and microsporidiosis.

The effect of antioxidant (Antox) on Giardia lamblia and Microsporidium sp. in rats and mice respectively was studied. Parasitologic effect was assessed by the mean parasitic count in infected animals' stool treated and non-treated, and infection intensity in stained section. Biochemical by measuring activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO) levels and cytokine induced neutrophil chemoattractant-1 (CINC-1) in intestinal homogenates in these animals as shown by cell injury, lipid peroxidation and neutrophil infiltrations. The present results showed that Antox significantly exacerbated G. lamblia and Microsporidium sp. This was manifested by a significant increase in number of G. lamblia cysts and trophozoites in stool and intestinal sections of treated infected rats. Also, microsporidian spores were significantly higher in stool of treated infected mice and infection intensity increased in the intestinal sections. The biochemical study showed a significantly higher degree of cell injury, lipid peroxidation and intestinal neutrophils accumulation in non-treated infected animals whether with G. lamblia or microsporidia. The changes reduced after treatment in giardiasis but none in microsporidiosis. The results were tabulated photographed, and critically discussed.

The serum gastrin level in patients with schistosomiasis and fascioliasis.

The study assessed changes in fasting serum gastrin level in early schistosomiasis and chronic fascioliasis in 52 patients. Forty patients had either schistosomiasis or fascioliasis alone. Twelve patients had combined infection with both parasitosis. Also, ten healthy parasite free individuals were selected as a control. All were clinically examined and subjected to stool analysis, liver function tests and sero-diagnosis for H. pylori. Serum gastrin was measured by radioimmunoassay and showed significant elevation in all patients (with schistosomiasis, fascioliasis or both parasites) compared to controls. A significantly higher serum gastrin level was detected among patients with combined infection relative to either with schistosomiasis or fascioliasis. Alkaline phosphatase activity increased significantly in all patients as compared to control. The activity of the enzyme had significant positive correlation with egg counts in patients with either parasite and with serum gastrin level in those with combined infection. The results were discussed.