Multiplex Polymerase Chain Reaction Tests for Detection of Pathogens Associated with Gastroenteritis

Zhang, Hongwei; Morrison, Scott; Tang, Yi‐Wei

doi:10.1016/j.cll.2015.02.006

Cited by 152 publications

(94 citation statements)

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“…Indeed, the pore sizes of the SPD filters are large enough to collect bacteria and parasites. Recently, several multiplex molecular assays have been developed to detect several pathogens, including viruses, bacteria, and parasites, in a single assay (25). They demonstrate higher sensitivities than those of conventional methods, with reduced turnaround and hands-on times.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a New Device for Simplifying and Standardizing Stool Sample Preparation for Viral Molecular Testing with Limited Hands-On Time

et al. 2016

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Sensitive molecular assays have greatly improved the diagnosis of viral gastroenteritis. However, the proper preparation of stool samples for clinical testing remains an issue. bioMérieux has developed a stool preprocessing device (SPD) that includes a spoon for calibrated sampling and a vial containing buffer, glass beads, and two filters. The resulting stool filtrate is used for nucleic acid extraction. The purpose of this study was to evaluate the performance of the SPD for the quantification of human adenovirus (HAdV) DNA in stool samples collected from hematopoietic stem cell transplant (HSCT) recipients. HAdV DNA was quantified with the Adenovirus R-gene kit. The suitability of the device to reproducibly quantify HAdV DNA in stools using different extraction platforms (easyMAG and QIAsymphony) was determined using archived HAdV-positive stool samples. Coefficients of variation of HAdV DNA quantifications ranged from 1.79% to 1.83%, and no difference in quantification was observed between the two extraction systems. The HAdV DNA limit of quantification using the SPD was 3.75 log 10 copies/g of stool. HAdV DNA quantification using the SPD was then compared to that of the routine preprocessing technique on 75 fresh stool samples collected prospectively from pediatric HSCT recipients at risk for HAdV infections. Thirty-eight samples were HAdV DNA positive with both the SPD and routine preprocessing methods. HAdV DNA loads were on average 1.14-log 10 copies/g of stool higher with the SPD (P < 0.0001) than with routine methods. This new device enabled a standardized preparation of stool samples in <5 min and a reproducible and sensitive quantification of HAdV DNA. The use of the SPD for the detection of other gastrointestinal infections warrants further evaluation. PCR is a very rapid and reliable tool for molecular biologybased diagnosis of a variety of infectious diseases, using a wide range of clinical sample types (1).However, stool samples are among the most difficult clinical samples to process for molecular pathogen detection because of the presence of very potent inhibitors of nucleic acid (NA) amplification that are often coextracted along with pathogen NAs (2-4). Bile salts, hemoglobin, polysaccharides, heme, and bilirubin have been identified as factors that inhibit amplification assays (3, 5-8). Inhibitory effects can be reduced by adding amplification facilitators, such as bovine serum albumin, to the PCR mixture (9) by using thermostable polymerases that are more resistant to PCR inhibition (10), or by using more efficient processes for extracting NAs from stool samples (11). The efficiency of NA extraction and purification has been shown to influence the sensitivity, reproducibility, and accuracy of NA amplification-based target detection (12).Due to the potential presence of amplification inhibitors, sample preprocessing before NA extraction is an essential step to improve amplification-based pathogen detection from stool samples. Currently, stool preparation is not standardized and can be labor-...

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Section: Discussionmentioning

confidence: 99%

Evaluation of a New Device for Simplifying and Standardizing Stool Sample Preparation for Viral Molecular Testing with Limited Hands-On Time

et al. 2016

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“…El cultivo microbiológico "clásico" se ha enfocado principalmente en el cultivo de patógenos como Salmonella spp., Shigella spp., Campylobacter spp. ; y pruebas rápidas [4][5] .…”

Section: Introductionunclassified

Panel Filmarray GI® en la detección de patógenos entéricos en deposiciones: experiencia preliminar

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Labra

et al. 2016

Rev. chil. infectol.

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Introduction: Gastrointestinal infections remain a major public health problem worldwide and its etiologic diagnosis is one of the main challenges. In molecular diagnostic techniques recently developed, the Filmarray GI ® panel allows detection of 23 pathogens (14 bacteria, virus 5 and 4 parasites) within an hour. Objective: To describe the experience of Filmarray GI ® panel in the Molecular Biology Laboratory of Clinica Las Condes. Method: A cross-sectional observational study that includes the results of 305 stool samples tested by Filmarray GI ® panel was performed. Results: Of the 305 tests requested 99 (32.5%) were negative and 206 were positive (67.5%). Of the positive samples, in 107 samples (51.9%) one pathogen was detected and in 99 samples (48.1%) more than one pathogen was found. Conclusions: Describe the experience of using FilmArray GI ® panel in stool specimens, which highlights the large number of positive samples for a microorganism and co-detection of enteric pathogens.

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“…Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3)(4)(5)(6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays.…”

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Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

et al. 2016

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Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P < 0.05) and improved the sensitivity of pathogen detection (P < 0.01), with a 20-to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples. Clinical microbiology is one of the most rapidly changing areas of laboratory medicine today due to the introduction of new technologies and automation (1). Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3-6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays. These less common organisms, for which tests are not readily available, are likely responsible for many cases of undiagnosed illness, particularly in critically ill patients and those with compromised immunity (7,8). Therefore, there is increasing interest in the application of novel technologies, such as next-generation sequencing (NGS), for unbiased detection of pathogens in clinical samples.Among the various challenges with the implementation of NGS for routine pathogen detection using metagenomics, the presence of an overwhelming amount of host DNA is one of the most important problems to be addressed. A previous metagenomic study on nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections revealed that up to ϳ95% of raw NGS reads were of human DNA (9). The su...

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Multiplex Polymerase Chain Reaction Tests for Detection of Pathogens Associated with Gastroenteritis

Cited by 152 publications

References 64 publications

Evaluation of a New Device for Simplifying and Standardizing Stool Sample Preparation for Viral Molecular Testing with Limited Hands-On Time

Evaluation of a New Device for Simplifying and Standardizing Stool Sample Preparation for Viral Molecular Testing with Limited Hands-On Time

Panel Filmarray GI® en la detección de patógenos entéricos en deposiciones: experiencia preliminar

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

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