ABSTRACT
Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction [Multiplex PCR] is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. For detection and differentiation of the three-microscopy indistinguishable Entamoeba species in human, multiplex PCR assay using different DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction. We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys
Subject(s)
Multiplex Polymerase Chain Reaction , Entamoeba histolytica , DNAABSTRACT
Background: Surgery is one of the best choices for the treatment of hydatidosis
The use of effective scolicidal agents during surgery for hydatid cyst is essential to prevent the secondary infection. Up to now no effective and safe agent has been identified for this purpose
Berberis vulgaris called [Zereshk] in Persian has been traditionally used as herbal remedy for the treatment of complaints and it is widely cultivated in Iran. Many studies have shown that it has antibacterial, antifungal and antiparasitic effect
Methods: In this study the scolicidal effect of Berberis vulgaris aqueous and hydro-alcohol extract for different concentrations [for aqueous: 0.5, 1, 2 and 4 nig/ml and for hydro-alcohol: 0.25, 0.5, 1 and 2 mg/ml] at different exposure times [5, 15 and 30 minutes] was evaluated. For this purpose, we obtained liver hydatid-cysts from a slaughter house
Viability of protoscolices was assessed by 0.1% eosin staining. Normal saline and hypertonic saline were used as negative and positive controls respectively
Results: All the different concentrations of Berberis vulgaris aqueous and hydro-alcoholic extracts had scolicidal effect
An aqueous extract with 4mg/ml concentration acted as positive control and we observed to lead to the death of 100% of protoscolices in the first 5 minutes
The least scolicidal effect [12.3%] was observed in an aqueous extract with 0.5 mg/ml concentration.The scolicidal activity of hydro-alcoholic extract with concentration of 2 mg/ml was 100% after 5 min of application, which was the same as positive control group
We noticed a significant increase in protoscolicidal activity with an increase in concentration in the two extracts of Berberis vulgaris [P<0.001]
Conclusion: It is important to mention that all the concentration levels and exposure times applied in this experiment are relatively low, since scolitical activity in both of the extracts is at its highest in this low spectrum. For further experiments, we suggest that the stability of cyst fluid in both of the extracts should be assessed
Therefore, after In vivo examination and additional experiments, it may be used as a suitable and effective scolicidal in surgery
ABSTRACT
The aim of this study is evaluation of molecular assay and the standard staining method. Cryptosporidium is a protozoon from coccidian subclass, which is one of the most important causes of diarrhea in children and immunocompromised individuals around the world. Diagnosis and treatment are necessary for mentioned cases. Usual diagnostic method for this parasite is fecal smear preparation, modified ziehl-neelsen staining, microscopic consideration and oocyst observation. A totally of 2510 stool samples collected from children with diarrhea of 4 pediatric hospitals. Direct smears prepared from fresh fecal samples and from the sediment of formalin-ether method of the same samples. The smears stained with modified ziehl-neelsen method then considered with microscope. The 30 positive samples with staining method considered with DNA extraction and PCR method for cryptosporidiosis infection determination and sensitivity evaluation. 114 random negative samples considered with DNA extraction and PCR method for cryptosporidiosis infection diagnosis and specificity evaluation. 30 positive cases from 2510 fecal samples detected by modified ziehl-neelsen staining and PCR method. We did not have any false positive cases by staining method but 2 cases of negative samples by staining method were positive by PCR technique, which informed us of 2 false negative. The positive samples sequenced for reconfirmation. Thus, sensitivity of staining method was computed to be 94% and specificity was 100% but sensitivity and specificity of PCR method was calculated to be 100%.