Proteomic approaches towards understanding amoebiasis

2-DGE: Two-dimensional gel electrophoresis, ABPs: Actin binding proteins, ALA: Amoebic liver abscess, Ap-A: Amoebapore A, BSA: bovine serum albumin, CaBPs: Calcium binding proteins, CP-A5: Cysteine proteinase A5, CRD: Carbohydrate recognition domain, EhADH3: E. histolytica alcohol dehydrogenase 3, EhCP: E. histolytica cysteine peptidase, EhLPPG: E. histolytica lipopeptidophosphoglycans, Gal/GalNAc: Galactose-N-acetylgalactosamine, GS: Glucose starvation, GTP: Guanosine triphosphate, HSP70: Heat shock protem-70, KERP1: Lysine glutamic acid-rich protein, LC-MS/MS: Liquid chromatography/tandem mass spectrometry, MS: Mass spectrometry, NAD: Nicotinamide adenine dinucleotide, NADP: Nicotinamide adenine dinucleotide phosphate, PAK: P21-activated kinase, PI3-K: PI3-kinase, PNT: Pyridine nucleotide transhydrogenase, TMKs: Transmembrane kinases, URE3-BP: Upstream regulatory element 3-binding protein

Contribution of multiplex real time PCR for detection and differentiation of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples of immunocompromised patients

Intestinal microsporidiosis is among the most frequent opportunistic diseases in immunocompromised patients. Routine diagnosis is generally performed by light microscopy of stained fecal samples. While unequivocal non-molecular species identification, important for cases management, is achievable only through electron microscopy. This study aimed to evaluate the contribution of multiplex real time PCR for simultaneous detection and differentiation of Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens of patients with immunosuppressive conditions. Stool samples were obtained from 78 immunocompromised patients suffering from diarrhea. The samples were screened for intestinal microsporidiosis by light microscopy using Weber's modified trichrome stain. The samples were subjected to multiplex real time PCR using Enterocytozoon bieneusi [E. bieneusi] primers and a probe specific on the internal transcribed spacer [ITS] sequence. Encephalitozoon intestinalis [E. intestinalis] primers and probe were specific for the small ribosomal subunit RNA gene sequence. Of 78 samples, 20 [25.6%] were detected positive by multiplex real time PCR. E. intestinalis was identified in 8 cases [40%], E. bieneusi in 7 [35%], and both species in 5 [25%]. Light microscopy detected a total of 22 samples [28.2%], 7 of which did not show the belt-like structure characteristic for microsporidial spores [empty-looking spores]. Compared to real time PCR, light microscopy had 75% sensitivity, 87.9% specificity, 68.2% PPV, 91.1% NPV and 84.6% accuracy in detection of microsporidia. No significant difference was found regarding the detection of E. intestinalis, E. bieneusi or both species by microscopy. Multiplex real time PCR proved to be more effective than classical trichrome stain for simultaneous identification and differentiation between E. bieneusi and E. intestinalis

Molecular diagnosis and genotyping of Dienlamoeba fragilis by polymerase chain reaction compared to microscopy and culture

The diagnosis of D. fragilis by microscopic identification of the parasite in stool is time consuming and relatively insensitive. To evaluate microscopy, culture and PCR for detection of D. fragilis in stool samples and to identify the genotypes of D. fragilis isolates among the study population. A total of 82 fresh human fecal samples were examined by microscopy using Wheatley's trichrome permanent staining, culture using the modified Boeck and Drbohlav medium and PCR targeting the small subunit [SSU] rRNA gene. Additionally, the existence of genetic variation among D. fragilis isolates [proved positive by PCR] was examined by restriction fragment length polymorphism analysis. PCR detected 25 isolates [30.5%], MBD culture detected 24 isolates [29.3%], while microscopy detected 8 isolates [9.8%]. Sensitivities of PCR, culture and microscopy were 92.6%, 88.9% and 29.6%, respectively. The agreement between PCR and culture results was substantial [KA=0.86]. PCR followed by RFLP analysis revealed the existence of two genetic variants among 25 D. fragilis isolates. Genotype I predominated in 23 [92%] samples, while the remaining two isolates corresponded to genotype II. It is recommended to use culture for routine diagnosis of D. fragilis in suspected gastrointestinal cases. Two genetic variants of D. fragilis existed in the Egyptian isolates

Efficacy of black seed oil from Nigella sativa against murine infection with cysts of Me49 strain of toxoplasma gondii

New therapies for toxoplasmosis are critically needed. Nigella sativa, commonly known as black seed or black cumin, has been known to include many medicinal properties. It has anti-inflammatory, and immuno-potentiating effects, antihelminthic and antiprotozoal activities. To study the effect of black seed oil [BSO] from Nigella sativa against Toxoplasma gondii Me49 strain in a murine model of infection. Two separate studies were performed, in which mice were orally inoculated with 10 or 20 T. gondii [Me49 strain] brain cysts. In each study, three groups of mice [35 each] were assigned to treatment with BSO for 2 weeks before T. gondii infection [BSO prophylactic], day 4 post infection [BSO therapeutic], or left untreated [infected untreated control]. The BSO effect on toxoplasmosis was evaluated by the assessment of [1] survival rate and brain cyst burden, [2] brain histopathological lesions and [3] immunohistochemical expression of inducible nitric oxide synthase [iNOS]. In infection induced by inoculation of 10, but not 20, cysts/mouse of the Me49 strain, BSO in prophylactic or therapeutic regimens significantly enhanced protection of infected mice against death [P = 0.01] and reduced brain cyst burdens at 5, 7 and 12 weeks post infection [PI] [P < 0.05] compared to the infected untreated control. The brains of BSO prophylactic or therapeutic groups showed milder meningitis, encephalitis and perivascular cuffing compared to the infected untreated control [P < 0.05]. Moreover, expression of iNOS was significantly enhanced in both BSO prophylactic and therapeutic groups compared to the untreated infected control. The BSO prophylactic group showed a significant enhanced expression of iNOS, selective to the brain endothelial cells, in the 1st week PI. Infection with 20 cysts was more aggressive, resulting in death of all untreated mice by day 35, and 26.7% and 20% protection respectively in BSO prophylactic and therapeutic groups. The estimated probabilities of survival were not significantly different among the 3 groups [P = 0.112]. BSO showed promising prophylactic and therapeutic effects on murine toxoplasmosis

Hydatid disease in Yemeni patients attending public and private hospitals in Sana'a City, Yemen

Hydatid disease is endemic and represents a major health problem in Yemen. The aim of this study is to determine the magnitude of the problem of hydatidosis in patients attending Public and Private Hospitals at Sana'a city, Yemen. 66 patients with hydatid disease were identified during the period from August 2006 to February 2007. Complete medical history for all CE patients were collected and analyzed. Among the 66 CE patients, 67% were females and 33% males. Liver was the most common involved organ. Single cyst was more frequently detected than multiple cysts and approximately 94% of the cysts were >/= 5 cm. Moreover, Public hospitals were the main source of patients with CE disease. Hydatidosis is still an endemic disease and an important health problem in Yemen which needs to be studied further. Therefore, accurate information on the distribution of the disease is the first step for the control and prevention of the disease. Moreover, it is crucial to investigate the role of different intermediate hosts and genotypes of E. granulosus in humans and animals

Detection of human cryplosporidium species in surface water sources in Ismailia using polymerase chain reaction

Gryptosporidium is a water-borne parasite that has caused several outbreaks ofgastrointestinal disease worldwide. Two species of Giyptosporidium are mainly found to cause disease in man, C. hominis which shows anthroponotic transmission, and C. parvum with zoonotic transmission. The present study aimed to verify the presence of human Cryplosporidium species in surface water sources in lsmailia using Polymerase Chain Reaction [PCR]. A total of 88 water samples were collected from Ismailia canal [24 samples], and from house taps [64 samples] at different seasons of the year. After filtration and concentration, the water concentrates were examined for Cryplosporidium oocysts by modified Zichi Necisen stain. Identification of C parvum and C. hominis was performed by multiplex allele specific polymerase chain reaction [MAS-PCR] followed by high resolution melting curve [HRM] analysis. The water samples were also subjected to physiochemical and microbiological analysis. Cryplosporidium oocysts were detected in 19[79.2%] of canal water samples and in only 2 [3.1 h] tap samples. Canal water was significantly associated with higher concentration of Cryplosporidium oucysts [50-450 oocysts/L] compared to tap water [20-30 oocysts/L]. C. parvum was the most common species detected in water samples [16 of 21 positive samples, 76.2%], while C. Hominis was detected in only one sample [4.8%] Summer showed the highest percentage of positivity with Cryplosporidium oocysts, then winter followed by autumn and spring. The presence of Cryplosporidium had significant association with the turbidity levels of water samples and their basic pH. Significant associations were also found with presence of total and fecal coliforms. Presence of Cryplosporidium oocysts as significantly associated with the grade >100- 25<103 CFU for both total and fecal coliforms. Canal water was significantly associated with higher concentration of oocysts/L. compared to tap water. C. parvum was found in both sources indicating zoonotic contamination of water

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

Role of contact lenses in acanthamoeba keratitis

The present work was planned to study the role of contact lenses in Acanthamoeba keratitis. The study included two groups, 100 subjects each [contact lens and non-contact lens users]; 50% of each group were suffering from keratitis, while the others were asymptomatic. Ocular samples [corneal scrapings or swabs], contact lenses and lens care systems [storage containers and solutions] were examined for the presence of acanthamoeba using cultureand staining techniques. The isolation of acanthamoeba was achieved by the cultivation of the previous samples on non-nutrient agar overlaid with E. coliat 37C. Acanthamoebae were identified according to their morphological characters and the negative flagellation test. Acanthamoeba was identified in cultures of different specimens obtained from 20 subjects out of 200. The majority of acanthamoeba positive cases [90%] were contact lens users. Acanthamoeba was isolated more frequently from patients with keratitis and was not isolated from any asymptomatic non-contact lens users. Cultivation of the contact lenses was associated with high rate of acanthamoeba detection compared with the other sources of samples. From the present study, it was concluded that acanthamoebae are expected to be found in the contact lenses, their storage containers and rinsing solutions, which may predispose tokeratitis in contact lens users. Precise disinfection of contact lenses, cleaning of lens storage containers and the use of sterile rinsing solutions have a primary importance to prevent acanthamoeba contamination of contact lens care system