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A Novel Real Time PCR Assay for the Detection of Babesia microti in a Highly Endemic Area Using Luminex Aries System

BACKGROUND: Babesiosis is a tick-borne infection of erythrocytes caused by parasites of the genus Babesia. In United States most of the reported cases occur in the Northeast and upper Midwestern states, New York being an endemic area. Majority of the cases in the United States is due to infection wi...

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Autores principales: Chandrasekaran, Alamelu, Zhang, Fan, Joseph, Ansamma, Bythrow, Maureen, Khare, Reeti, Juretschko, Stefan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5631332/
http://dx.doi.org/10.1093/ofid/ofx163.1617
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author Chandrasekaran, Alamelu
Zhang, Fan
Joseph, Ansamma
Bythrow, Maureen
Khare, Reeti
Juretschko, Stefan
author_facet Chandrasekaran, Alamelu
Zhang, Fan
Joseph, Ansamma
Bythrow, Maureen
Khare, Reeti
Juretschko, Stefan
author_sort Chandrasekaran, Alamelu
collection PubMed
description BACKGROUND: Babesiosis is a tick-borne infection of erythrocytes caused by parasites of the genus Babesia. In United States most of the reported cases occur in the Northeast and upper Midwestern states, New York being an endemic area. Majority of the cases in the United States is due to infection with Babesia microti. Most patients with babesiosis are asymptomatic or have a mild illness, but some may develop fatal illness. Current laboratory diagnosis of Babesiosis including parasitemia is by microscopic demonstration of intraerythrocytic Babesia parasites in blood films which requires specially trained personnel. We have developed a simple and rapid PCR assay for the detection of B. microti 18S rRNA gene (BMPCR) which can be used as an alternative screening assay. METHODS: BMPCR assay was developed based on Luminex Multicode technology using Luminex Aries instrument and in-house developed primers. Babesia positive clinical samples confirmed by NY State Department of Health and clinical samples negative for Babesia were used to determine accuracy. Specificity was evaluated using malaria positive samples, several bacterial isolates, selected B. microti strains and other Babesia species. Limit of Detection was determined using known copies of B. microti DNA. Reproducibility of the assay was assessed by testing samples on different days and runs by different analysts. RESULTS: Accuracy of the BMPCR assay was 100% for the 30 Babesia positive and 30 negative samples. All B. microti ATCC strains were positive by BMPCR. 95% LOD of the assay as determined by probit analysis was 1177 copies/50µl of blood. Sensitivity of the assay was higher than that of microscopic detection and specificity was 100%. No cross reactivity was observed with any of the malarial species tested. Babesia MO1, Babesia duncani and all bacterial isolates tested were negative by the BMPCR. Intra-run, inter-run and day to day reproducibility of the assay was 100%. CONCLUSION: The B. microti real time PCR assay developed by Northwell Health Laboratories is rapid, sensitive, specific and reproducible. With the sample to result turnaround time of 2.5 hours and hands on time of only 5 minutes per sample, BMPCR can be used as screening assay for B. microti in clinical laboratories. DISCLOSURES: All authors: No reported disclosures.
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spelling pubmed-56313322017-11-07 A Novel Real Time PCR Assay for the Detection of Babesia microti in a Highly Endemic Area Using Luminex Aries System Chandrasekaran, Alamelu Zhang, Fan Joseph, Ansamma Bythrow, Maureen Khare, Reeti Juretschko, Stefan Open Forum Infect Dis Abstracts BACKGROUND: Babesiosis is a tick-borne infection of erythrocytes caused by parasites of the genus Babesia. In United States most of the reported cases occur in the Northeast and upper Midwestern states, New York being an endemic area. Majority of the cases in the United States is due to infection with Babesia microti. Most patients with babesiosis are asymptomatic or have a mild illness, but some may develop fatal illness. Current laboratory diagnosis of Babesiosis including parasitemia is by microscopic demonstration of intraerythrocytic Babesia parasites in blood films which requires specially trained personnel. We have developed a simple and rapid PCR assay for the detection of B. microti 18S rRNA gene (BMPCR) which can be used as an alternative screening assay. METHODS: BMPCR assay was developed based on Luminex Multicode technology using Luminex Aries instrument and in-house developed primers. Babesia positive clinical samples confirmed by NY State Department of Health and clinical samples negative for Babesia were used to determine accuracy. Specificity was evaluated using malaria positive samples, several bacterial isolates, selected B. microti strains and other Babesia species. Limit of Detection was determined using known copies of B. microti DNA. Reproducibility of the assay was assessed by testing samples on different days and runs by different analysts. RESULTS: Accuracy of the BMPCR assay was 100% for the 30 Babesia positive and 30 negative samples. All B. microti ATCC strains were positive by BMPCR. 95% LOD of the assay as determined by probit analysis was 1177 copies/50µl of blood. Sensitivity of the assay was higher than that of microscopic detection and specificity was 100%. No cross reactivity was observed with any of the malarial species tested. Babesia MO1, Babesia duncani and all bacterial isolates tested were negative by the BMPCR. Intra-run, inter-run and day to day reproducibility of the assay was 100%. CONCLUSION: The B. microti real time PCR assay developed by Northwell Health Laboratories is rapid, sensitive, specific and reproducible. With the sample to result turnaround time of 2.5 hours and hands on time of only 5 minutes per sample, BMPCR can be used as screening assay for B. microti in clinical laboratories. DISCLOSURES: All authors: No reported disclosures. Oxford University Press 2017-10-04 /pmc/articles/PMC5631332/ http://dx.doi.org/10.1093/ofid/ofx163.1617 Text en © The Author 2017. Published by Oxford University Press on behalf of Infectious Diseases Society of America. http://creativecommons.org/licenses/by-nc-nd/4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Abstracts
Chandrasekaran, Alamelu
Zhang, Fan
Joseph, Ansamma
Bythrow, Maureen
Khare, Reeti
Juretschko, Stefan
A Novel Real Time PCR Assay for the Detection of Babesia microti in a Highly Endemic Area Using Luminex Aries System
title A Novel Real Time PCR Assay for the Detection of Babesia microti in a Highly Endemic Area Using Luminex Aries System
title_full A Novel Real Time PCR Assay for the Detection of Babesia microti in a Highly Endemic Area Using Luminex Aries System
title_fullStr A Novel Real Time PCR Assay for the Detection of Babesia microti in a Highly Endemic Area Using Luminex Aries System
title_full_unstemmed A Novel Real Time PCR Assay for the Detection of Babesia microti in a Highly Endemic Area Using Luminex Aries System
title_short A Novel Real Time PCR Assay for the Detection of Babesia microti in a Highly Endemic Area Using Luminex Aries System
title_sort a novel real time pcr assay for the detection of babesia microti in a highly endemic area using luminex aries system
topic Abstracts
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5631332/
http://dx.doi.org/10.1093/ofid/ofx163.1617
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