DETECTION OFGIARDIA LAMBLIA, CRYPTOSPORIDIUMSPP. ANDENTAMOEBA HISTOLYTICAIN CLINICAL STOOL SAMPLES BY USING MULTIPLEX REAL-TIME PCR AFTER AUTOMATED DNA ISOLATION

Abstract

Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated as an alternative approach for diagnosing Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in stool samples.Therefore, a total of 631 faecal samples were analysed both by microscopy as well as by real-time PCR following automated DNA extraction. Results showed that real-time PCR exhibited sensitivity and specificity of both 100%, whereas traditional microscopy exhibited sensitivity and specificity of 37.5% and 99.8% respectively. As real-time PCR provides simple, sensitive and specific detection of these three important pathogenic protozoan parasites, this technique, rather than microscopy, has become our diagnostic method of choice for the detection of enteric protozoan parasites for the majority of patients.

Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis

“…The traditional microscopic ova and parasite exam of stool is insensitive and labor-intensive, and laboratories may have difficulties maintaining technologist proficiency, further reducing the efficacy of the test (16). Antigen detection tests are more sensitive and less laborious than microscopy (31) but have reduced sensitivity compared to that of molecular methods (32). Consequently, molecular tests were utilized in this study as the comparator method for detecting protozoa, with no FN detections but six FP Cryptosporidium and seven FP G. lamblia detections.…”

Molecular Detection of the Carriage Rate of Four Intestinal Protozoa with Real-Time Polymerase Chain Reaction: Possible Overdiagnosis of Entamoeba histolytica in Nigeria

“…The observation of fewer Giardia-positive samples and no Cryptosporiodium-positive samples by DIF microscopy compared with real-time PCR was not unexpected; a previous study showed PCR to be the more sensitive method for detecting these organisms. 43 The high CT values in DIF-microscopy-negative cases indicate that PCR testing for Giardia may be too sensitive to discriminate between clinically relevant infection and asymptomatic carriage. Further studies should address this question and also clarify the potential importance of an asymptomatic carrier state.…”

Cryptosporidium in humans and animals—a one health approach to prophylaxis

“…60 Multiplex qPCR assays have also been developed for the detection of Cryptosporidium and other common causes of diarrhoea such as Giardia duodenalis and Entamoeba histolytica, which have the advantage of identifying mixed infections. [64][65][66] The most widely used molecular markers for identification and typing of Cryptosporidium species are the 18S ribosomal RNA (18S rRNA) gene and the 60-kDa glycoprotein (gp60) gene, respectively. 4,10 Miniaturized fluidic devices, which can detect to species level, have also been developed, mainly for the water industry (reviewed by Bridle et al 67 ), but as with antibody-based biosensor chips, have yet to be fully validated and are costly.…”