Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples

Abstract

Although PCR offers the potential for sensitive detection of parasites there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

High occurrence of Blastocystis sp. subtypes 1–3 and Giardia intestinalis assemblage B among patients in Zanzibar, Tanzania

“…For a number of years in the early 2010s clinical stool samples from patients seeking care at an out-patient health clinic in Jambiani, were analyzed for parasites at the Clinical Microbiology laboratory, Umeå, Sweden due to a personal contract between the local health clinic authorities and one of the researchers (BE). Two samples per patient, one stored in L2-buffer containing guanidine thiocyanate [ 23 ] and one stored in sodium acetate-acetic acid-formalin fixative (SAF), were sent numbered from anonymised patients for molecular and microscopic parasite detection. In consultation with the medical staff at the health clinic, treatment was given to patients that were diagnosed with what was interpreted as clinically relevant intestinal parasites.…”

“…DNA was extracted from samples stored in L2 buffer (guanidine-thiocyanate 0.96 g/ml dissolved in 0.1 M Tris, pH 6.4) using a method previously developed in our laboratory [ 23 ]. Briefly, 150–200 mg faeces was collected with a flocked nylon swab (ESwab, Copan) and transferred to 1 ml of Amies medium, 400 μl of this suspension was transferred to a tube with Lysing matrix E that contains beads (MP Biomedicals, Nordic Biolabs AB, Täby, Sweden).…”

Intestinal Protozoan Parasitic Infections in Immunocompromised Child Patients with Diarrhea

“…PCR (28,29). In the study, three reactions were inhibited PCR and the one sample changed to positive after resolving the inhibitions.…”

Optimization of routine microscopic and molecular detection of parasitic protozoa in SAF-fixed faecal samples in Sweden

“…The most common gastrointestinal parasites included in NAbased diagnostics in Sweden are E. histolytica, G. intestinalis and Cryptosporidium spp. Different cell lysis methods have been used prior to protozoan DNA extraction to minimize inhibitors of the PCR process [32,33] and to disrupt robust cell walls [34][35][36][37][38][39][40]. A reduced yield of parasite-DNA has been reported from fixed samples [41,42].…”