Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection

Evan Bloch, Tzong Hae Lee, Peter J. Krause, Sam R. Telford, Lani Montalvo, Daniel Chafets, Sahar Usmani-Brown, Timothy J. Lepore, Michael P. Busch

Research output: Contribution to journalArticle

Abstract

Background Babesia microti, the most frequently implicated pathogen in transfusion-transmitted babesiosis, is widely endemic in the Northeast and upper Midwestern United States. High seroprevalence in endemic areas limits antibody-based donor screening. A high-performance molecular test is needed to identify donors in the preseroconversion window phase as well as to discriminate past serologic exposure with parasite clearance from continued parasitemia. Study Design and Methods Frozen Babesia-spiked whole blood was microcentrifuged, and the supernatant transferred and microcentrifuged again to concentrate the parasite. The DNA was extracted and amplified using real-time polymerase chain reaction (PCR) using Babesia-specific primers. The assay was employed in three series of experiments: 1) a validation and optimization spiking experiment, 2) a blinded serial dilution probit analysis to determine the limit of detection, and 3) evaluation of two blinded panels of clinical samples from possible babesiosis cases. Results At a decreasing inoculum of 445, 44.5, and 4.45 copies/mL, the assay had positive rates of 100, 97.5, and 81%, respectively. The blinded probit analysis demonstrated a detection rate of 95 and 50% at 12.92 and 1.52 parasites/2 mL of whole blood, respectively. Evaluation of clinical samples showed 13 of 21 samples to be positive, with a range of 85 to 4.8 million parasites/mL. There were no positives detected among 48 healthy donors Conclusion We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

Original languageEnglish (US)
Pages (from-to)2299-2306
Number of pages8
JournalTransfusion
Volume53
Issue number10
DOIs
StatePublished - Oct 2013
Externally publishedYes

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Babesia microti
Babesia
Real-Time Polymerase Chain Reaction
Parasites
Babesiosis
Infection
Midwestern United States
Donor Selection
Parasitemia
Seroepidemiologic Studies
Allergy and Immunology
Limit of Detection
Epidemiology
Antibodies
DNA

ASJC Scopus subject areas

  • Hematology
  • Immunology
  • Immunology and Allergy

Cite this

Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection. / Bloch, Evan; Lee, Tzong Hae; Krause, Peter J.; Telford, Sam R.; Montalvo, Lani; Chafets, Daniel; Usmani-Brown, Sahar; Lepore, Timothy J.; Busch, Michael P.

In: Transfusion, Vol. 53, No. 10, 10.2013, p. 2299-2306.

Research output: Contribution to journalArticle

Bloch, E, Lee, TH, Krause, PJ, Telford, SR, Montalvo, L, Chafets, D, Usmani-Brown, S, Lepore, TJ & Busch, MP 2013, 'Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection', Transfusion, vol. 53, no. 10, pp. 2299-2306. https://doi.org/10.1111/trf.12098
Bloch, Evan ; Lee, Tzong Hae ; Krause, Peter J. ; Telford, Sam R. ; Montalvo, Lani ; Chafets, Daniel ; Usmani-Brown, Sahar ; Lepore, Timothy J. ; Busch, Michael P. / Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection. In: Transfusion. 2013 ; Vol. 53, No. 10. pp. 2299-2306.
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AU - Telford, Sam R.

AU - Montalvo, Lani

AU - Chafets, Daniel

AU - Usmani-Brown, Sahar

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AB - Background Babesia microti, the most frequently implicated pathogen in transfusion-transmitted babesiosis, is widely endemic in the Northeast and upper Midwestern United States. High seroprevalence in endemic areas limits antibody-based donor screening. A high-performance molecular test is needed to identify donors in the preseroconversion window phase as well as to discriminate past serologic exposure with parasite clearance from continued parasitemia. Study Design and Methods Frozen Babesia-spiked whole blood was microcentrifuged, and the supernatant transferred and microcentrifuged again to concentrate the parasite. The DNA was extracted and amplified using real-time polymerase chain reaction (PCR) using Babesia-specific primers. The assay was employed in three series of experiments: 1) a validation and optimization spiking experiment, 2) a blinded serial dilution probit analysis to determine the limit of detection, and 3) evaluation of two blinded panels of clinical samples from possible babesiosis cases. Results At a decreasing inoculum of 445, 44.5, and 4.45 copies/mL, the assay had positive rates of 100, 97.5, and 81%, respectively. The blinded probit analysis demonstrated a detection rate of 95 and 50% at 12.92 and 1.52 parasites/2 mL of whole blood, respectively. Evaluation of clinical samples showed 13 of 21 samples to be positive, with a range of 85 to 4.8 million parasites/mL. There were no positives detected among 48 healthy donors Conclusion We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

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