Evaluation of the EasyScreen™ Enteric Parasite Detection Kit for the detection of Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis from clinical stool samples

doi:10.1016/j.diagmicrobio.2013.10.013

Diagnostic Microbiology and Infectious Disease

Volume 78, Issue 2, February 2014, Pages 149-152

https://doi.org/10.1016/j.diagmicrobio.2013.10.013 Get rights and content

Abstract

The aim of this study was to evaluate the EasyScreen™ Enteric Parasite Detection Kit (Genetic Signatures, Sydney, Australia) for the detection and identification of 5 common enteric parasites: Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis in human clinical samples. A total of 358 faecal samples were included in the study. When compared to real-time PCR and microscopy, the EasyScreen™ Enteric Parasite Detection Kit exhibited 92–100% sensitivity and 100% specificity and detected all commonly found genotypes and subtypes of clinically important human parasites. No cross reactivity was detected in stool samples containing various other bacterial, viral, and/or protozoan species. The EasyScreen™ PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the 5 most important diarrhoea-causing enteric parasites that infect humans. It should be noted, however, that the EasyScreen™ Kit does not substitute for microscopy or for additional PCRs as it does not detect the pathogenic Coccidia spp. Cystoisospora belli or Cyclospora cayetanensis and it does not differentiate between pathogenic and nonpathogenic Entamoeba spp. This study also highlights the lack of sensitivity demonstrated by microscopy; as such, molecular methods should be considered the diagnostic method of choice for enteric parasites.

Introduction

Enteric protozoa continue to be the most commonly encountered parasitic diseases affecting millions of people each year and causing significant morbidity and mortality worldwide (Stark et al., 2009). Pathogenic enteric parasites include Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis. While Blastocystis is the most common enteric parasite detected in humans, it remains a controversial cause of gastrointestinal disease (Roberts et al., 2013).

Traditional diagnostic methods for the detection of enteric protozoa rely on either microscopic examination of faecal material or enzyme immunoassays (Garcia et al., 2000). More recently numerous PCR assays have been described in the literature (Liang et al., 2010, Stark et al., 2006, Stark et al., 2011). These PCR assays have the advantage of greater sensitivity and the capacity for multiple parasite detection in the form of multiplexed reactions (Liu et al., 2013, Nazeer et al., 2013).

The EasyScreen™ Enteric Parasite Detection Kit (Genetic Signatures, Sydney, Australia) is a simple and rapid molecular method that utilises 3base™ technology that modifies the 4 usual DNA base pairs (A, C, T, G) into only 3 base pairs (A, T, G) via a novel, patented bisulphite conversion step. The EasyScreen™ kit comprises of an extraction kit/protocol and an real time PCR (RT-PCR) amplification kit containing all the primers and probes to detect the following targets: Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis. It also contains an extraction and separate amplification control for the detection of PCR inhibition. The kit can be run on various platforms and has the option to be semi-automated.

This is the first study to evaluate the EasyScreen™ Enteric Parasite Detection Kit for the simultaneous detection and identification of Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis in human clinical samples.

Section snippets

Faecal specimens

Faecal specimens submitted to the Department of Microbiology at St. Vincent's Hospital, Sydney, for investigation of diarrhoea from September 2011 to December 2012 were included in the study. A total of 358 samples were included in the study; 108 samples were positive by microscopy for the following parasites: Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis samples. Along with 200 faecal samples tested previously by microscopy using the

Results

The results are summarised in Table 1. A true test-positive result was defined as a sample that was test-positive using at least 2 of the 3 methods employed; any discordant results were repeated. A total of 53 Blastocystis, 43 D. fragilis, 26 G. intestinalis, 24 Entamoeba complex (comprising of the morphologically identical E. histolytica, E. dispar, and E. moshkovskii) and 9 Cryptosporidium spp. were detected by various methods. When compared with microscopy, the molecular methods demonstrated

Discussion

E. histolytica, G. intestinalis, Cryptosporidium, Blastocystis, and D. fragilis are 5 important and commonly occurring parasitic protozoa that infect humans. The EasyScreen™ assay provides an additional diagnostic tool for the rapid, sensitive, and specific detection of these enteric protozoa.

The EasyScreen™ assay uses a novel chemistry that universally modifies the nucleic acid genomes of pathogens by converting all cytosine bases to thymine bases (via a uracil intermediary). This modification

Acknowledgments

This work was supported by a grant from the Institute of Laboratory Science at St. Vincent's Hospital, Sydney, Australia.

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Cryptosporidium is a critical waterborne protozoan pathogen found in water resources that have been a major cause of death and serious illnesses worldwide, costing millions of dollars annually for its detection and treatment. Over the past several decades, substantial efforts have been made towards developing techniques for the detection of Cryptosporidium. Early diagnostic techniques were established based on the existing tools in laboratories, such as microscopes. Advancements in fluorescence microscopy, immunological, and molecular techniques have led to the development of several kits for the detection of Cryptosporidium spp. However, these methods have several limitations, such as long processing times, large sample volumes, the requirement for bulky and expensive laboratory tools, and the high cost of reagents. There is an urgent need to improve these existing techniques and develop low-cost, portable and rapid detection tools for applications in the water quality industry. In this review, we compare recent advances in nanotechnology, biosensing and microfluidics that have facilitated the development of sophisticated tools for the detection of Cryptosporidium spp.Finally, we highlight the advantages and disadvantages, of these state-of-the-art detection methods compared to current analytical methodologies and discuss the need for future developments to improve such methods for detecting Cryptosporidium in the water supply chain to enable real-time and on-site monitoring in water resources and remote areas.
Cryptosporidium spp. diagnosis and research in the 21<sup>st</sup> century
2021, Food and Waterborne Parasitology
The protozoan parasite Cryptosporidium has emerged as a leading cause of diarrhoeal illness worldwide, posing a significant threat to young children and immunocompromised patients. While endemic in the vast majority of developing countries, Cryptosporidium also has the potential to cause waterborne epidemics and large scale outbreaks in both developing and developed nations. Anthroponontic and zoonotic transmission routes are well defined, with the ingestion of faecally contaminated food and water supplies a common source of infection. Microscopy, the current diagnostic mainstay, is considered by many to be suboptimal. This has prompted a shift towards alternative diagnostic techniques in the advent of the molecular era. Molecular methods, particularly PCR, are gaining traction in a diagnostic capacity over microscopy in the diagnosis of cryptosporidiosis, given the laborious and often tedious nature of the latter. Until now, developments in the field of Cryptosporidium detection and research have been somewhat hampered by the intractable nature of this parasite. However, recent advances in the field have taken the tentative first steps towards bringing Cryptosporidium research into the 21^st century. Herein, we provide a review of these advances.
Diagnóstico molecular de parasitosis intestinales
2020, Enfermedades Infecciosas y Microbiologia Clinica
Las infecciones causadas por parásitos del tracto digestivo humano representan un problema de salud pública global. En países industrializados, sus particulares características epidemiológicas (baja prevalencia general de enteroparásitos), económicas (elevados costes laborales) y clínicas (incremento constante del número de muestras y determinaciones diagnósticas a realizar) han causado que las técnicas moleculares estén progresivamente reemplazando a la microscopía convencional como método de diagnóstico de primera línea de estos patógenos en el laboratorio clínico moderno. Los métodos basados en PCR, particularmente los desarrollados para la detección simultánea de varios agentes que causan la misma etiología (diagnóstico sindrómico), representan ya una opción coste-efectiva que permite automatizar procesos, optimizar flujos de trabajo, comparar resultados entre diferentes laboratorios y facilitar la acreditación de procedimientos diagnósticos. En esta revisión se detalla de forma clara y concisa el estado actual del diagnóstico molecular de las principales especies de parásitos intestinales humanos, particularmente de los protozoos entéricos causantes de diarrea (Cryptosporidium spp., Giardia duodenalis, Entamoeba histolytica), de los miembros más destacados de los filos Microsporidia (Enterocytozoon bieneusi) y Stramenopiles (Blastocystis sp.), así como de los helmintos transmitidos por suelo (Ancylostoma spp., Ascaris lumbricoides, Necator americanus, Strongyloides stercoralis y Trichuris trichiura) y alimentos (Anisakis spp., Clonorchis sinensis, Fasciola spp., Taenia solium, y Trichinella spiralis). Especial atención se ha prestado a la descripción de técnicas y formatos disponibles, a sus prestaciones diagnósticas y a los marcadores genéticos más usados, tanto para la detección en laboratorios clínicos como para el genotipado en centros de referencia e investigación.
Infections causes by parasites of the gastrointestinal tract are a global public health problem. In industrialised countries, their particular epidemiological (low general prevalence of enteroparasites), economic (high labour costs) and clinical characteristics (constant increase in the number of samples and diagnostic determinations to be performed) have led molecular techniques to progressively replace conventional microscopy as the first-line diagnostic method of these pathogens in modern clinical laboratories. PCR-based techniques, particularly those developed for the simultaneous detection of the various agents that can cause the same infectious disease (syndromic diagnosis), already represent a cost-effective option that allow process automisation, workflow optimisation, and comparison of results among different laboratories, and facilitate accreditation of diagnostic procedures. This review clearly and concisely discusses the current situation of the molecular diagnosis of the main species of intestinal parasites in humans, particularly the enteric protozoans causing diarrhoea (Cryptosporidium spp., Giardia duodenalis, Entamoeba histolytica), the most important members the Microsporidia phyla (Enterocytozoon bieneusi) and Stramenopiles phyla (Blastocystis sp.), as well as the helminths transmitted by soil (Ancylostoma spp., Ascaris lumbricoides, Necator americanus, Strongyloides stercoralis and Trichuris trichiura) and food (Anisakis spp., Clonorchis sinensis, Fasciola spp., Taenia solium, and Trichinella spiralis). Special attention is paid to the description of available techniques and formats, to their diagnostic benefits and the most widely used genetic markers for their detection, both in clinical laboratories and genotyping in referral and research centres.
Evaluation of the EasyScreen Protozoan Detection Kit for the diagnosis of Entamoeba histolytica
2019, Pathology
Rapid diagnosis of parasitic diseases: current scenario and future needs
2019, Clinical Microbiology and Infection
Parasitic diseases are one of the world's most devastating and prevalent infections, causing millions of morbidities and mortalities annually. In the past, many of these infections have been linked predominantly to tropical or subtropical areas. Nowadays, however, climatic and vector ecology changes, a significant increase in international travel, armed conflicts, and migration of humans and animals have influenced the transmission of some parasitic diseases from ‘book pages’ to reality in developed countries. It has also been noted that many patients who have never travelled to endemic areas suffer from blood-borne infections caused by protozoa. In the light of existing knowledge, this new trend can be explained by the fact that in the process of migration a large number of asymptomatic carriers become a part of the blood bank donor and transplant donor populations. Accurate and rapid diagnosis represents the crucial weapon in the fight against parasitic infections.
To review old and new approaches for rapid diagnosis of parasitic infections.
Data for this review were obtained through searches of PubMed using combinations of the following terms: parasitological diagnostics, microscopy, lateral flow assays, immunochromatographic assays, multiplex-PCR, and transplantation.
In this review, we provide a brief account of the advantages and limitations of rapid methods for diagnosis of parasitic diseases and focus our attention on current and future research in this area. The approximate costs associated with the use of different techniques and their applicability in endemic and non-endemic areas are also discussed.
Microscopy remains the cornerstone of parasitological diagnostics, especially in the field and low-resource settings, and provides epidemiological assessment of parasite burden. However, increased use and availability of point-of-care tests and molecular assays in modern era allow more rapid and accurate diagnoses and increased sensitivity in the identification of parasitic infections.
Comparison of multiplexed-tandem real-time PCR panel with reference real-time PCR molecular diagnostic assays for detection of Giardia intestinalis and Tritrichomonas foetus in cats
2019, Veterinary Parasitology
Giardia intestinalis and Tritrichomonas foetus are frequent enteric protozoan parasites of the gastrointestinal track of domestic cats. Because of different treatment options for the parasites, confirmation of presence of one or both pathogens is necessary. The PCR based assays are suitable for differential diagnosis. We evaluated the performance of Small Animal Diarrhoea panel, a multiplexed-tandem real-time PCR (MT-PCR) assay, that detects DNA of both G. intestinalis and T. foetus. The sensitivity and specificity were compared to reference real-time PCR assays using 105 faecal samples, 39.05% (n = 41) positive for G. intestinalis and 30.48% (n = 32) were positive for T. foetus. The faecal samples positive for T. foetus had a high proportion of late amplifiers, determined by an arbitrary threshold of C_t-values > 35. On the other hand, only one G. intestinalis positive sample was considered a late amplifier. For G. intestinalis DNA, the MT-PCR assay had 95.1% sensitivity and 92.1% specificity. For T. foetus DNA, the MT-PCR assay had 41.9% sensitivity and 100.0% specificity. To evaluate the interlaboratory reproducibility of the MT-PCR assay, results were compared in two different laboratories and found to be in a very good agreement (Kappa = 0.9). Further analysis of the DNA using conventional PCR determined presence of G. intestinalis Assemblage F and T. foetus genotype ‘feline’. In conclusion, the MT-PCR Small Animal Diarrhoea panel had a good and poor performance against reference assays for G. intestinalis and T. foetus, respectively. The assay is suitable for detection and differential diagnosis of G. intestinalis and moderate to high burdens of T. foetus in small animal clinical practice.

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Diagnostic Microbiology and Infectious Disease

Abstract

Introduction

Section snippets

Faecal specimens

Results

Discussion

Acknowledgments

References (23)

Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism

Parasitology

Comparison of a novel HPV test with the Hybrid Capture II (hcII) and a reference PCR method shows high specificity and positive predictive value for 13 high-risk human papillomavirus infections

J Clin Virol

PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia

J Clin Microbiol

Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum antigens in human fecal specimens using the triage parasite panel enzyme immunoassay

J Clin Microbiol

Evaluation of a new single-tube multiprobe real-time PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar

J Parasitol

A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens

J Clin Microbiol

Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples

J Infect

Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt

Parasitol Res

Identification of zoonotic Cryptosporidium and Giardia genotypes infecting animals in Sydney's water catchments

Exp Parasitol

Comparison of microscopy, culture, and conventional polymerase chain reaction for detection of Blastocystis sp. in clinical stool samples

Am J Trop Med Hyg

Subtype distribution of Blastocystis isolates identified in a Sydney population and pathogenic potential of Blastocystis

Eur J Clin Microbiol Infect Dis

Cited by (44)

Comprehensive review of conventional and state-of-the-art detection methods of Cryptosporidium

Cryptosporidium spp. diagnosis and research in the 21<sup>st</sup> century

Diagnóstico molecular de parasitosis intestinales

Evaluation of the EasyScreen Protozoan Detection Kit for the diagnosis of Entamoeba histolytica

Rapid diagnosis of parasitic diseases: current scenario and future needs

Comparison of multiplexed-tandem real-time PCR panel with reference real-time PCR molecular diagnostic assays for detection of Giardia intestinalis and Tritrichomonas foetus in cats

ParasitologyEvaluation of the EasyScreen™ Enteric Parasite Detection Kit for the detection of Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis from clinical stool samples

Abstract

Introduction

Section snippets

Faecal specimens

Results

Discussion

Acknowledgments

Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism

Parasitology

Comparison of a novel HPV test with the Hybrid Capture II (hcII) and a reference PCR method shows high specificity and positive predictive value for 13 high-risk human papillomavirus infections

J Clin Virol

PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia

J Clin Microbiol

Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum antigens in human fecal specimens using the triage parasite panel enzyme immunoassay

J Clin Microbiol

Evaluation of a new single-tube multiprobe real-time PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar

J Parasitol

A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens

J Clin Microbiol

Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples

J Infect

Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt

Parasitol Res

Identification of zoonotic Cryptosporidium and Giardia genotypes infecting animals in Sydney's water catchments

Exp Parasitol

Comparison of microscopy, culture, and conventional polymerase chain reaction for detection of Blastocystis sp. in clinical stool samples

Am J Trop Med Hyg

Subtype distribution of Blastocystis isolates identified in a Sydney population and pathogenic potential of Blastocystis

Eur J Clin Microbiol Infect Dis

Parasitology
Evaluation of the EasyScreen™ Enteric Parasite Detection Kit for the detection of Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis from clinical stool samples