Utilization of a real-time PCR assay for diagnosis of Babesia microti infection in clinical practice

Persistence of babesiosis for >2 years in a patient on rituximab for rheumatoid arthritis

“…3 PCR testing began to be performed by an academic medical center starting on 11/1/13 utilizing a real time quantitative PCR (qPCR) assay targeting the gene encoding 18S rRNA of B. microti, as described elsewhere [3,4]. On 11/1/13, 4.44 log10 copies/mL of whole blood of the target babesia DNA were detected by the qPCR on a blood sample, despite uninterrupted treatment with azithromycin 500 mg orally per day along with 750 mg twice daily of atovaquone since 5/13/13.…”

Comparison of a quantitative PCR assay with peripheral blood smear examination for detection and quantitation of Babesia microti infection in humans

“…This qualitative PCR has a limit of detection of 1-3 parasites/μL of blood, and its analytical specificity and reproducibility were well documented (Wang et al, 2015). In the present study, we converted this qualitative PCR assay to a qPCR with the inclusion of an external standard set in each run.…”

“…We recently developed a multiplex real-time qualitative PCR assay, targeting a B. microti-specific 18S rRNA gene and a human housekeeping gene (gapdh), and validated its utility for diagnosis and follow-up of patients with B. microti infection in clinical practice (Wang et al, 2015). This qualitative PCR has a limit of detection of 1-3 parasites/μL of blood, and its analytical specificity and reproducibility were well documented (Wang et al, 2015).…”

“…Two hundred microliters of whole blood was used for DNA extraction. Extracted DNA was eluted to 50 μL of elution buffer by using a commercial DNA extraction kit (QIAamp DNA Blood Mini; Qiagen, Valencia, CA, USA) (Wang et al, 2015). qPCR was performed on the 7500 Fast Dx Real-Time PCR instrument (Applied Diagnostic Microbiology and Infectious Disease 82 (2015) 109-113 Biosystems, Foster City, CA, USA), as described elsewhere (Wang et al, 2015).…”

“…Extracted DNA was eluted to 50 μL of elution buffer by using a commercial DNA extraction kit (QIAamp DNA Blood Mini; Qiagen, Valencia, CA, USA) (Wang et al, 2015). qPCR was performed on the 7500 Fast Dx Real-Time PCR instrument (Applied Diagnostic Microbiology and Infectious Disease 82 (2015) 109-113 Biosystems, Foster City, CA, USA), as described elsewhere (Wang et al, 2015). Briefly, the PCR reaction consisted of 1x TaqMan® Fast Universal PCR Master Mix (without AmpErase; Applied Biosystems), 0.9 μmol/L forward and reverse primers and a 0.5 μmol/L probe that are specific for B. microti, and 0.025 μmol/L forward and reverse primers and a 0.02 μmol/L probe that are specific for the human gapdh gene.…”