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Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial.
PLoS Negl Trop Dis. 2016 Jan; 10(1):e0004380.PN

Abstract

BACKGROUND

Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key 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 for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy.

METHODOLOGY/PRINCIPAL FINDINGS

Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. 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 WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections.

CONCLUSIONS/SIGNIFICANCE

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.

Authors+Show Affiliations

Clinical Tropical Medicine Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.Department of Veterinary Disease Biology, Faculty of Health and Medical Science, University of Copenhagen, Copenhagen, Denmark; Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.Research School of Population Health, College of Medicine, Biology and Environment, The Australian National University, Canberra, Australian Capital Territory, Australia.Research School of Population Health, College of Medicine, Biology and Environment, The Australian National University, Canberra, Australian Capital Territory, Australia. Molecular Parasitology Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. School of Public Health, University of Queensland, Herston, Queensland, Australia.Laboratory for Medical Microbiology and Immunology, St Elisabeth Hospital, Tilburg, the Netherlands.Research School of Population Health, College of Medicine, Biology and Environment, The Australian National University, Canberra, Australian Capital Territory, Australia.Laboratorio Nacional da Saúde, Ministério da Saúde, Dili, Timor-Leste.Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria, Australia.Clinical Tropical Medicine Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. School of Medicine, University of Queensland, Queensland, Australia.

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

26820626

Citation

Llewellyn, Stacey, et al. "Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity of Intestinal Parasite Infections in a Controlled Clinical Trial." PLoS Neglected Tropical Diseases, vol. 10, no. 1, 2016, pp. e0004380.
Llewellyn S, Inpankaew T, Nery SV, et al. Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial. PLoS Negl Trop Dis. 2016;10(1):e0004380.
Llewellyn, S., Inpankaew, T., Nery, S. V., Gray, D. J., Verweij, J. J., Clements, A. C., Gomes, S. J., Traub, R., & McCarthy, J. S. (2016). Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial. PLoS Neglected Tropical Diseases, 10(1), e0004380. https://doi.org/10.1371/journal.pntd.0004380
Llewellyn S, et al. Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity of Intestinal Parasite Infections in a Controlled Clinical Trial. PLoS Negl Trop Dis. 2016;10(1):e0004380. PubMed PMID: 26820626.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial. AU - Llewellyn,Stacey, AU - Inpankaew,Tawin, AU - Nery,Susana Vaz, AU - Gray,Darren J, AU - Verweij,Jaco J, AU - Clements,Archie C A, AU - Gomes,Santina J, AU - Traub,Rebecca, AU - McCarthy,James S, Y1 - 2016/01/28/ PY - 2015/10/25/received PY - 2015/12/18/accepted PY - 2016/1/29/entrez PY - 2016/1/29/pubmed PY - 2016/6/2/medline SP - e0004380 EP - e0004380 JF - PLoS neglected tropical diseases JO - PLoS Negl Trop Dis VL - 10 IS - 1 N2 - BACKGROUND: Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key 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 for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy. METHODOLOGY/PRINCIPAL FINDINGS: Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. 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 WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections. CONCLUSIONS/SIGNIFICANCE: 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. SN - 1935-2735 UR - https://www.unboundmedicine.com/medline/citation/26820626/Application_of_a_Multiplex_Quantitative_PCR_to_Assess_Prevalence_and_Intensity_Of_Intestinal_Parasite_Infections_in_a_Controlled_Clinical_Trial_ L2 - http://dx.plos.org/10.1371/journal.pntd.0004380 DB - PRIME DP - Unbound Medicine ER -