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

Cysteine proteases inhibitors [phenyl vinyl sulfone and valproic acid] in treatment of schistosomiasis mansoni-infected mice: an experimental study to evaluate their role in comparison to praziquantel

The emerged resistance to praziquantel [PZQ] in treatment of schistosomiasis necessitates the search for novel drugs. Cysteine proteases inhibitors [CPIs] have shown promising results in either parasitic infections or non-parasitic diseases. The study aimed to evaluate the therapeutic effects of two CPIs: phenyl vinyl sulfone [PVS] and valproic acid [VA] in comparison to PZQ in S. mansoni-experimentally infected mice. Swiss albino mice were experimentally infected with S. mansoni cercariae. The mice were divided into 4 groups [25 mice each], and G1 mice were not treated and used as control group. Mice of G2, G3 and G4 were treated by the evaluated drugs [PVS, VA and PZQ, respectively] at the end of the 6[th] week post infection [PI]. The evaluating parameters were 1] fecal egg count, 2] worm burden, 3] tissue egg count, 4] oogram pattern and 5] hepatic granuloma number and size. The results showed that by the end of 10[th] week PI, PZQ was the most effective drug resulting in decrease worm burden in the portal vein, increase proportion of dead eggs in the oogram pattern, decrease in the hepatic egg count and decrease in granuloma numbers. On the other hand, the granuloma diameter was smallest in PVS treated group compared to the other groups. CPIs have a relative fair favorable therapeutic outcome on schistosomiasis mansoni with the advantage of being novel drugs with no therapeutic resistance, especially PVS which showed an added specific anti-immunopathological effect reflected by the small hepatic granuloma size

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

Identification of antigenic epitopes of strongyloides stercoralis filariform larval antigen for immunodiagnosis of strongyloidiasis using western blot

Microscopic diagnosis of strongyloidiasis depending on detection of larvae in fecal samples is not sensitive, especially in asymptornatic patients, and development of reliable serological methods is imperative. Western blot [WB] technique showed promising results for the reactivity analysis in several parasitic infections. The objective of the present study is to identify relevant proteins of S. stercoralis filariform larvae [L3] using WB and a panel of serum samples for immunodiagnosis of strongyloidiasis. Material and S. stercoralis L3 were cultured from fecal samples of infected patients. The antigen was extracted and analyzed using SDS-PAGE. Sixty nine serum samples belonging to 3 groups of patients were analyzed and included in the study as: Group I [shedding S. stercoralis larvae in feces], group II [infected with other parasites], and group III [with negative parasitological results]. Reactivity of the resulting bands of S. stercoralis L3 antigen was analyzed with the serum samples using WB technique. Thirty four immunoreactive bands were detected in the WB analysis representing recognition of proteins with molecular weight [MW] varying from 19 to 214 kDa. Immunodominant proteins of 43, 41, 36, and 23/33 kDa were recognized respectively in 39%, 35%, 70% and 60% of sera from patients with confirmed strongyloidiasis; and in 29%, 21%, 21% and 30% of sera from those infected with other parasitic infections. One band [41 kDa] gave reaction with one serum sample from group III. It was concluded that the 43, 41, 36 and 32 kDa bands could be considered important tools for the development of diagnostic techniques for strongyloidiasis

Prevalence and clinical features of Dientamoeba Fragilis infections in patients suspected to have intestinal parasitic infection

The present study was conducted to determine the prevalence and clinical features of dientamoebiasis in patients presumed to be infected with intestinal parasites. A total of 168 patients were examined for D. fragilis using microscopy [after Wheatley's trichrome staining] and culture [using modified Boeck and Drbohlav's medium] D. fragilis trophozoites were detected in 15 samples [8.9%] examined using trichrome staining and in 50 samples [29.8%] by culture method. Other enteric parasites were common in the study population as 48.8% of patients [82/168] were found harboring intestinal parasites. Blastocystis hominis was the most common, identified in 33.3% [56/168] of the samples. Giardia lamblia was detected in 17.9% [30/168] and E. histolytica/E. dispar in 11.9% [20/168]. The symptoms most frequently encountered were diarrhea, abdominal pain and weight loss and fatigue. Diarrhea and abdominal pain were significantly more frequent in patients with dientamoebiasis compared to non pathogenic cases [P<0.05]. Diarrhea was 38.5% of patients infected with D. fragilis compared to 50% of patients infected with G. lamblia, while abdominal pain was encountered with D. fragilis in 41% compared to 33.3% with G. lamblia. These differences were insignificant [P>0.05]