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 cysteine 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% and 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

study on the effect of myrrh extract [commiphora molmol plant] on adult Fasciola worm in vitro

A new drug derived from botanical source myrrh was found to possess high therapeutic efficacy as a schistosomicidal, fasciolicidal and cestodicidal drug. However, no work was conducted to evaluate the effect of myrrh extract on Fasciola adult worms in vitro. To evaluate the fasciolicidal activity of myrrh extract at different concentrations and to detect the ultrastructural tegumental changes of adult Fasciola worms upon their exposure to different concentrations of myrrh extract using scanning electron microscopy [SEM]. Viable Fasciola worms were collected from the liver of slaughtered cows. They were transferred to a culture medium containing different concentrations of myrrh extract making the experimental groups. Two control groups were included; worms incubated in culture medium only and worms incubated in culture medium containing the drug solvent [cremophor EL]. Worms were observed at 1, 3 and 24 h after exposure and the number of dead worms was calculated, After 24 hours incubation, 2 adult worms from each group were processed for SEM. The present study indicated that myrrh extract had a rapid and severe effect on Fasciola worms in vitro, with widespread disruption to their tegument present after 24 h incubation in the drug concentrations used. The effect of myrrh extract was both time and dose dependent

Comparison of polymerase chain reaction, immunochromatographic assay and staining techniques in diagnosis of cryptosporiodiosis

Cryptosporidiosis represents a major health problem worldwide. In developed countries, massive outbreaks have been reported while in developing countries, it is associated with significant morbidity and mortality, especially among infants and children. Although the modified acid-fast technique is the commonly used slain for its detection, its sensitivity and specificity appeared to be rather low. The present study aimed at comparing the conventional diagnostic method with the recent techniques namely immunochromatographic [ICT] strip assay and multiplex allele specific polymerase chain reaction [MAS-PCR]. The second objective was to genotype the diagnosed isolates using MASPCR. Seventy six immunocompromised patients having acute or chronic diarrhea were selected from the attendance of the pediatrics, oncology and nephrology clinics in Suez Canal University Hospital. Cryptosporidiosis was diagnosed by Kinyoun acid fast stain, ICT strip assay and MAS-PCR. Samples proved positive for cryptosporidiosis were genotyped using MAS-PCR. Using MAS-PCR as Gold standard method, modified Kinyoun acid fast stain and ICT strip showed sensitivity [79 vs 89%], specificity [98 vs 100%], positive predictive value [94 vs 100%], negative predictive value [93 vs 100%] and diagnostic accuracy [88.5 vs 94.5%]. Using MAS-PCR for genotyping, C. parvum comprised the majority [68.4%] of cases while C. hominis was only 26.3%. Only one patient had mixed genotype infection. C. parvum infections were associated with low intensity of oocyst shedding while C hominis infections were with high intensity of oocyst shedding. The agreement between microscopy and MAS-PCR results proved that only 60% of positive cases identified as C. hominis [type 1] by MASPCR were positive by microscopy while, 92.3% of C. parvum [type II] positive cases by MAS-PCR were positive by microscopy. The agreement between ICT strip and MAS-PCR results proved that the strip identified 100% of positive cases of C. hominis [type I] and 84.6% of C. parvum [type II] positive cases by MAS-PCR. The ICT strip assay gave very good results regarding performance and came second to MASPCR in ranking which has an additional advantage due to its ability to genotype diagnosed isolates. The low sensitivity of staining method and high cost of MAS-PCR recommend the ICT strips for the wide use especially in field of diagnosis and in outbreaks where large number of tests needs to be performed in a short period of time

Effect of exogenous adminstration of anti-oxidants 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 antox for 4 weeks before infection [prophylactic strategy]. Mild pathological changes were detected on 13.4%, 19.9% and 86.8% of mice in Gs I, II and III, respectively. Moderate pathological changes were found in 29.9%, 26.6% and 6.6% of mice in Gs I, II and III, respectively. While, the majority of severe pathological changes were in Gs I and II [56.7% and 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 and 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

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 and 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

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 [NaNO[2]] in 0.6 mM, 0.8 mM and 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 NaNO[2] inhibited the growth and decreased viability of B. hominis with minimal lethal concentration dose 1 mM on the 4[th] 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 NaNO[2]. 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 micro M in cecum, 90.4 +/- 11.6 micro M in ileum, 60.1 +/- 4.7 micro M in blood and 63.6 +/- 7.3 micro M in stool elutes while, the mean levels of NO in control mice were 70.2 +/- 3.1 in cecum, 67.8 +/- 4.7 micro M in ileum, 30.9 +/- 4.2 micro M in blood and 28.1 +/- 2.9 micro M in stool elutes. The differences were statistically highly significant. NO-donor drugs proved useful in treatment and increase the host resistance to B. hominis

Detection of cyclospora cayetanensis infections among diarrheal children attending Suez Canal University Hospital

The objective of the present study is to detect the occurrence of Cyclospora cayetanensis among diarrheal children with or without immunosuppressant conditions. Stool samples were collected and examined for identification of the parasite using different concentration methods [Formaline ethely acetate, Potassium hydroxide and Sheather's floatation] and different stains [acid fast, modified Ziehl Neelsen, modified Kinyoun and modified safranin]. Confirmation of the results was performed using autofluorescence technique. Cyclospora oocysts were detected in 19.6% of 230 diarrheal children without immunosuppressant conditions and 34.6% of 230 diarrheal children with immunosuppressant conditions with significant difference between the two groups. Sheather's floatation method was significantly more sensitive than direct smear, FEA and KOH sedimentation methods [with sensitivities of 93.6%, 63.7%, 75.8% and 79.8%, respectively]. Additionally, the modified safranin was the best staining method as it was significantly more sensitive than the acid fast and the modified Ziehl Neelsen staining. On the other hand, no significant differences were found between the acid fast, the modified Ziehl Neelsen or the modified Kinyoun staining. Cyclosporiasis predominated in the age group 1-4 years compared to other age groups. There was a significant association between infection with Cyclospora and low socioeconomic level, living in rural areas and using tap water. Cyclospora was found as a sole parasite in 55 out of 124 [44.4%] infected children. Co-infection with other pathogenic parasites occurred in 69 cases; most commonly Cryptosporidium [30/124]. It was concluded that cyclosporiasis is common among diarrheal children especially if it is associated with immunosuppressant conditions. Feces examination for oocysts using Sheather's floatation and the modified safranin staining are recommended for diagnosis

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

Cyclospora organisms named C. cercopitheci, C. colobi and 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%] and 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 co-infection with primates Cyclospora species [C. cercopitheci, C. colobi and 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 and MAFT. The infection severity in symptomatic patients were identified by MZN and 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

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-l 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

Degree of symptoms versus copro-antigen levels in giardia lamblia infection

A total of 82 out-patients were examined for Giardia coproantigens and 12 neonate stool samples as control. ELISA had a sensitivity of 100% and specificity of 91.67%. ELISA [OD] 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 [OD] 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

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

Observation of acid fast C. cayetanensis 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 pm] spherical acid-fast C. cayetanensis oocysts and their identification 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

The probable relationship between toxoplasma gondii infection and cryptogenic epilepsy

Cryptogenic epilepsy is a group of epilepsy syndromes for which the etiology is unknown but the underlying brain disease is suspected. So the present work aims to study the probable relationship between Toxoplasma gondii and cryptogenic epilepsy. Fifty patient, were selected with cryptogenic epilepsy and 50 patients with known cause of epilepsy in comparision to 50 healthy as control group to investigate the sero-positivity rate for anti-To-Toxoplasma IgG antibody by ELISA. The sero-positiviry rate of anti-Toxoplasma IgG antibodies among cryptogenic epilepsy patients was [20%] and it was higher than healthy controls [2%] and known cause epilepsy patients [4%] with highly significant statistically difference [P< 0.01]. There might be a causal relationship between chronic toxoplasmosis and cryptogenic epilepsy

Biological variability of trichomonas vaginalis clinical isolates fom symptomatic and asymptomatic patients

The vaginal specimens of 60 women were cultured using modified thioglycolate medium. Thirteen samples were positive for Trichomonas vaginalis. The pathogenicity of these clinical isolates was investigated by intraperitoneal injection to mice. Additionally, their susceptibility to metronidazole was evaluated. All the clinical isolates produced infection in mice, but marked variations in pathogenicity and susceptibility were observed. Four out of 13 isolates produced mortality in mice, indicating a higher pathogenicity of the four isolates. Concerning metronidazole susceptibility, only one isolate was considered resistant and the other twelve showed different degrees of susceptibility

Possible mechanism[s] for cryptosporidial diarrhea: parasitologic and physiopathologic studies in experimental animals

The present work was planned to study the possible mechanism[s] of diarrhea in cryptosporidiosis Stool eluates and intestinal homogenates from naturally and experimentally infected animals together with purified sporozoites were examined utilizing using chamber to demonstrate their possible enterotoxic effects Besides the histopathological changes in experimentally infected mice were studied to assess the possible underlying mechanism[s] of diarrhea in cryptosporidiosis. Characterization of the enterotoxic activity associated with cryptosporidiosis may have a great impact on the development of effective strategies for its treatment. The results of the present study revealed that in intestinal cryptosporidiosis enterotoxic substances are secreted which likely induce diarrhea. They induce an increase in the transepithelial potential difference [DIsc] to reach its maximum after 15 minutes and then slowly decrease to reach the baseline after 55 minutes [for stool eluates] and 35 minutes [for intestinal homogenates] On the other hand the purified sporozoites showed an increase in DIsc after 9 minutes and then a decrease after that to become maintained at relatively high level. The enterotoxins were found to be time and dose dependent and heat labile. The osmotic gap showed that the mechanism of diarrhea is rather secretory. In experimentally infected mice, shedding of oocysts first appeared in stools 3 days postinoculation [PI], reaching a peak 9 days [PI] and disappeared 15 days PI. Although the infected mice showed mild to severe degree of intestinal inflammation [marked villous atrophy and crypts hyperplasia], the stool was semiformed. Besides, the present study showed marked validity of mouse as an in vivo model to study the pathophysiology of cryptosporidial diarrhea. This model is inexpensive and serves as a suitable alternative to neonatal calves for efficient oocyst propagation