ABSTRACT
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
Subject(s)
Feces/parasitology , Enterocytozoon , Encephalitozoon , Polymerase Chain Reaction/methods , Diagnosis, DifferentialABSTRACT
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
ABSTRACT
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
Subject(s)
Humans , Male , Female , Diagnostic Techniques and Procedures , Polymerase Chain Reaction/methods , Immunoassay/methods , Comparative Study , Electrophoresis, Agar GelABSTRACT
Zoonotic cutaneous leishmaniasis [ZCL] is endemic in North Sinai Governorate. It is a parasitic disease in which the immune system is implicated in its pathogenesis. The erythrocyte lipid peroxidation [ELPO], erythrocyte reduced glutathione [ERGT], glutathione peroxidase [GTPo] and serum vitamin C [SVC] were estimated in 19 patients with ZCL and 16 controls. The ELPO and ERGT were significantly higher in the patient group, while the GTPo and SVC were relatively lower. These results showed that the oxidative stress has a role in active ZCL, probably induces the endogen anti-oxidant system. That is to say, the oxidant-anti-oxidant balance system changes to the oxidative side with marked increase of erythrocyte lipid peroxidation and erythrocyte reduced glutathione