Development and validation of a droplet digital PCR assay for the detection and quantification of Bartonella species within human clinical samples.

Abstract

This report describes the development, optimization, and validation of a ddPCR assay for the detection of Bartonella spp. DNA within several sample matrices, including clinical blood samples from patients with or without documented Bartonella spp. bacteremia. The Bartonella spp. ddPCR assay was developed based upon previously published TaqMan-based qPCR assays that can amplify DNA of over 25 Bartonella spp. Host DNA (housekeeping gene) amplification serves as a reference target to facilitate quantification. The efficiency, sensitivity, and specificity of the Bartonella spp. ddPCR assay was assessed by direct comparison with the current qPCR methods used by the Intracellular Pathogens Research Laboratory (North Carolina State University, North Carolina, USA), and Galaxy Diagnostics (Research Triangle Park, North Carolina, USA). Bartonella spp. ddPCR assay parameters were successfully optimized to detect Bartonella concentrations equivalent to 0.5 bacterial genome copies per microliter of blood (0.001 pg/ul of bacterial DNA). The number of droplets detected (resolution) for each concentration was consistent across each of four assessed time points. The Bartonella spp. ddPCR assay detected 16 species/strains including B. henselae; B. quintana; B. vinsonii subsp. berkhoffii (genotypes I, II, III and IV); B. vinsonii subsp. vinsonii; B. melophagi; B. volans; B. monaki; B. alsatica; B. bovis; B. elizabethae; B. clarridgeiae; and B. koehlerae. Bartonella DNA was detected in only one previously negative patient sample (119/120 negative; 99% specificity). The ddPCR sensitivity (53/112) was significantly better than qPCR (6/112) when testing patient blood and enrichment blood culture samples. The development of commercial ddPCR systems with integrated technologies has significantly streamlined the DNA detection process, making it more efficient and standardized for clinical diagnostic testing. The assay described in this work is the first step toward the development of a multiplex ddPCR assay (i.e., using the QX One from Bio-Rad) for the simultaneous detection and absolute quantification of multiple vector-borne pathogens (such as Babesia, Bartonella and Borrelia) within clinical samples.