DETECTION AND DIFFERENTIATION OF HUMAN PROTOZOAN PARASITES IN STOOL SPECIMENS BY USING MULTIPLEX ALLELE SPECIFIC POLYMERASE CHAIN REACTION (MAS-PCR) In New Damietta City, Egypt

Abstract

Enteric protozoa continue to be the most commonly encountered parasitic diseases causing significant morbidity and mortality in developing regions of the world affecting millions of people. This study assessed the use of Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) assay and microscopy for detection and identification of common pathogenic protozoan parasites in New Damietta city of North Delta region, Egypt. Between Jun 2013 until Sept 2013, fresh stool samples were obtained from 249 patients up to 65 years of age attending the internal clinic of the Damietta University Hospital and those visiting their general practitioner (GP) of outpatient clinics because of gastrointestinal symptoms. Stool samples collected was preserved at -200C for DNA extraction whilst the remaining was preserved in sodium acetate-acetic acid formalin and concentrated using the formol-ether technique for microscopic examination. DNA extracts were analyzed with the multiplex allele specific Polymerase Chain Reaction (MAS-PCR) for pathogenic protozoan parasites. The diagnostic results obtained using a multiplex allele specific PCR for the detection of E. histolytica/dispar, G. lamblia and C. parvum/C. hominis were compared with these obtained by routine microscopy of faecal samples from patients. 69 samples were positive by MAS-PCR assays , 9 cases of G. intestinalis infection, 34 cases of D. fragilis infection, 3 cases of E. histolytica infection, 17 cases E.dispar and 6 cases of Cryptosporidium infection in the clinical samples. By microscopy, only 32 samples were positive for one or more of the enteric protozoa, 5cases of G. intestinalis infection, 9cases of D. fragilis infection, 13 cases of E. histolytica infection, and 1 cases of Cryptosporidium infection in the clinical samples. However, there are no cases of E.dispar observed. Mixed infections were detected in 4 samples. The sensitivities varied from 58% for D. fragilis to 47% for E. histolytica, 35% for Giardia, and 30% for Cryptosporidium, while the specificities also varied from 97% for E. histolytica to 99% for D. fragilis and 100% for E.dispar . No cross-reactivity was detected in stool samples containing various other bacterial, viral, and protozoan species. This present study showed relatively high rates of protozoa infections in the study patients. The study has also demonstrated that the multiplex real time PCR assay was more sensitive compared to microscopy in the diagnosis of the intestinal protozoa parasites and thus, molecular methods must be considered the diagnostic methods of choice for enteric protozoan parasites.
Keywords: Human intestinal protozoa, Stool specimens, Microscopy, Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR), Diagnosis.