Capture Sequencing Enables Sensitive Detection of Tick-Borne Agents in Human Blood

Abstract

Assay sensitivity can be a limiting factor in the use of PCR as a tool for the detection of tick-borne pathogens in blood. We evaluated the performance of Tick-borne disease Capture Sequencing Assay (TBDCapSeq), a capture sequencing assay targeting tick-borne agents, to test 158 whole blood specimens obtained from the Lyme Disease Biobank. These included samples from 98 individuals with signs and symptoms of acute Lyme disease, 25 healthy individuals residing in Lyme disease endemic areas, and 35 samples collected from patients admitted to the Massachusetts General Hospital or referred to the infectious disease clinic. Compared to PCR, TBDCapSeq had better sensitivity and could identify infections with a wider range of tick-borne agents. TBDCapSeq identified a higher rate of samples positive for Borrelia burgdorferi (8 vs. 1 by PCR) and Babesia microti (26 vs. 15 by PCR). TBDCapSeq also identified previously unknown infections with Borrelia miyamotoi, Ehrlichia, and Rickettsia species. Overall, TBDCapSeq identified a pathogen in 43 samples vs. 23 using PCR, with four co-infections detected versus zero by PCR. We conclude that capture sequencing enables superior detection of tick-borne agents relative to PCR.

BosR and PlzA reciprocally regulate RpoS function to sustain Borrelia burgdorferi in ticks and mammals

“…In a pilot RNA-Seq study using ribodepleted RNA from engorged nymphs infected with WT strain B31, only approximately 6,700 reads mapped to protein coding genes (0.034% of approximately 20 million total raw reads) ( Supplemental Table 1 ; supplemental material available online with this article; https://doi.org/10.1172/JCI166710DS1 ), a value too low to obtain comprehensive transcriptomic data. To overcome this bottleneck, we took advantage of an enrichment strategy, designated TBDCapSeq, developed by Tokarz and colleagues ( 26 , 27 ), which uses hybridization probes to ‘capture’ pathogen-specific amplicons prior to sequencing ( Figure 1 ). Using TBDCapSeq, we compared the transcriptomes of WT and Δ rpoS Bb in fed nymphs and DMCs.…”

“…A total of 1 μg of library pool was mixed with 5 μg of COT human DNA (Thermo Fisher Scientific) and 2 nmol of blocking oligo pool (Roche) and then dehydrated. To enrich for Bb -specific transcripts, the dried pool was resuspended in 7.5 μL Hybridization Buffer and 3 μL Hybridization Component A (Roche) and heated at 95°C for 5 minutes before the addition of 4.5 μL of custom biotinylated TBD SeqCap EZ Probes ( 26 , 27 ). The mixture was heated at 95°C for 5 min and incubated at 47°C for 16–20 h. After incubation, the probes were pulled down using magnetic streptavidin SeqCap Capture beads (Roche) and washed with buffers of decreasing stringency (SeqCap EZ Hybridization and Wash Kit; Roche).…”

Hybrid-Capture Target Enrichment in Human Pathogens: Identification, Evolution, Biosurveillance, and Genomic Epidemiology

“…Crucially, POWV is a re-emerging TBD that causes meningoencephalitis, making it a model target for HC and biosurveillance [ 204 ]. Subsequently, TBDCapSeq was updated by adding one bacterium ( Francisella tularensis ) and two viruses (Colorado tick fever virus (CTFV) and Bourbon virus (BRBV)) [ 205 ]. Currently, TBD is typically not routinely included in the standard CSF PCR, but HC can be used to complement the identification of diseases.…”

Tick-Borne Co-Infections: Challenges in Molecular and Serologic Diagnoses

“…The utility of this approach has been documented for viral and bacterial agents, and the development of the TBD capture sequencing assay (TBDCapSeq) demonstrated its superior capacity for the detection of tick-borne pathogens in ticks, rodents and clinical specimens with a sensitivity equal to, or occasionally, superior to PCR [187,188]. Capture sequencing can provide a substantial improvement over PCR for the detection of B. burgdorferi in blood, and it can simultaneously detect any tick-borne infection including concur- The utility of this approach has been documented for viral and bacterial agents, and the development of the TBD capture sequencing assay (TBDCapSeq) demonstrated its superior capacity for the detection of tick-borne pathogens in ticks, rodents and clinical specimens with a sensitivity equal to, or occasionally, superior to PCR [187,188]. Capture sequencing can provide a substantial improvement over PCR for the detection of B. burgdorferi in blood, and it can simultaneously detect any tick-borne infection including concurrent infections [188] (Figure 2).…”

“…Capture sequencing can provide a substantial improvement over PCR for the detection of B. burgdorferi in blood, and it can simultaneously detect any tick-borne infection including concur- The utility of this approach has been documented for viral and bacterial agents, and the development of the TBD capture sequencing assay (TBDCapSeq) demonstrated its superior capacity for the detection of tick-borne pathogens in ticks, rodents and clinical specimens with a sensitivity equal to, or occasionally, superior to PCR [187,188]. Capture sequencing can provide a substantial improvement over PCR for the detection of B. burgdorferi in blood, and it can simultaneously detect any tick-borne infection including concurrent infections [188] (Figure 2). The high costs and size of NGS sequencers, coupled with the current assay time of >12 h present another prohibitive roadblock to NGS-based diagnostics.…”