Multiplex Quantitative Assays Detect Polyparasitism
By LabMedica International staff writers Posted on 09 Feb 2016 |
Image: The Rotor-Gene 6000 for quantifying DNA (Photo courtesy of Corbett Life Science)
Image: The Powersoil DNA Isolation Kit (Photo courtesy of Mo Bio).
Accurate quantitative assessment of infection with soil transmitted helminths and protozoa are essential to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies.
As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques, such as polymerase chain reaction (PCR) methodology, for assessment of infection intensity for both purposes.
An international team of scientists led by those at the QIMR Berghofer Medical Research Institute (Herston, Australia) collected fecal samples from a total of 680 people, originating from rural communities: 467 from samples Timor-Leste and 213 samples from Cambodia. DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions, the first targeting both helminths and protozoa. Samples were also subject to sodium nitrate flotation for identification and quantification of soil transmitted helminth (STH) eggs, and zinc sulfate centrifugal flotation for detection of protozoan parasites.
DNA extraction was performed using the Powersoil DNA Isolation Kit (Mo Bio, Carlsbad, CA USA). Extracted DNA was run in two pentaplex real-time PCR reactions. The first was a quantitative assay for Necator americanus, Ancylostoma spp. (A. duodenale, A. ceylanicum), Ascaris spp., Trichuris trichiura; the second was a semi-quantitative assay for Entamoeba histolytica, Cryptosporidium spp., Giardia duodenalis, and Strongyloides stercoralis. The Rotor-Gene 6000 (Qiagen, Melbourne, Australia) was used for all PCR assays, with reactions set up for both PCR reactions.
Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by official guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections.
The authors concluded that multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and Giardia compared to microscopy, especially in samples exhibiting polyparasitism. The superior performance of multiplex PCR to detect polyparasitism and more accurately determine infection intensity suggests that it is a more appropriate technique for use in epidemiologic studies and for monitoring large-scale intervention trials. The study was published on January 28, 2016, in the journal Public Library of Science Neglected Tropical Diseases.
Related Links:
QIMR Berghofer Medical Research Institute
Mo Bio
Qiagen
As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques, such as polymerase chain reaction (PCR) methodology, for assessment of infection intensity for both purposes.
An international team of scientists led by those at the QIMR Berghofer Medical Research Institute (Herston, Australia) collected fecal samples from a total of 680 people, originating from rural communities: 467 from samples Timor-Leste and 213 samples from Cambodia. DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions, the first targeting both helminths and protozoa. Samples were also subject to sodium nitrate flotation for identification and quantification of soil transmitted helminth (STH) eggs, and zinc sulfate centrifugal flotation for detection of protozoan parasites.
DNA extraction was performed using the Powersoil DNA Isolation Kit (Mo Bio, Carlsbad, CA USA). Extracted DNA was run in two pentaplex real-time PCR reactions. The first was a quantitative assay for Necator americanus, Ancylostoma spp. (A. duodenale, A. ceylanicum), Ascaris spp., Trichuris trichiura; the second was a semi-quantitative assay for Entamoeba histolytica, Cryptosporidium spp., Giardia duodenalis, and Strongyloides stercoralis. The Rotor-Gene 6000 (Qiagen, Melbourne, Australia) was used for all PCR assays, with reactions set up for both PCR reactions.
Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by official guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections.
The authors concluded that multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and Giardia compared to microscopy, especially in samples exhibiting polyparasitism. The superior performance of multiplex PCR to detect polyparasitism and more accurately determine infection intensity suggests that it is a more appropriate technique for use in epidemiologic studies and for monitoring large-scale intervention trials. The study was published on January 28, 2016, in the journal Public Library of Science Neglected Tropical Diseases.
Related Links:
QIMR Berghofer Medical Research Institute
Mo Bio
Qiagen
Latest Molecular Diagnostics News
- Blood Proteins Could Warn of Cancer Seven Years before Diagnosis
- New DNA Origami Technique to Advance Disease Diagnosis
- Ultrasound-Aided Blood Testing Detects Cancer Biomarkers from Cells
- New Respiratory Syndromic Testing Panel Provides Fast and Accurate Results
- New Synthetic Biomarker Technology Differentiates Between Prior Zika and Dengue Infections
- Novel Biomarkers to Improve Diagnosis of Renal Cell Carcinoma Subtypes
- RNA-Powered Molecular Test to Help Combat Early-Age Onset Colorectal Cancer
- Advanced Blood Test to Spot Alzheimer's Before Progression to Dementia
- Multi-Omic Noninvasive Urine-Based DNA Test to Improve Bladder Cancer Detection
- First of Its Kind NGS Assay for Precise Detection of BCR::ABL1 Fusion Gene to Enable Personalized Leukemia Treatment
- Urine Test to Revolutionize Lyme Disease Testing
- Simple Blood Test Could Enable First Quantitative Assessments for Future Cerebrovascular Disease
- New Genetic Testing Procedure Combined With Ultrasound Detects High Cardiovascular Risk
- Blood Samples Enhance B-Cell Lymphoma Diagnostics and Prognosis
- Blood Test Predicts Knee Osteoarthritis Eight Years Before Signs Appears On X-Rays
- Blood Test Accurately Predicts Lung Cancer Risk and Reduces Need for Scans