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

Abstract

Co-infections are a poorly understood aspect of tick-borne diseases. In the United States alone, nineteen different tick-borne pathogens have been identified. The majority of these agents are transmitted by only two tick species, Ixodes scapularis and Amblyomma americanum. Surveillance studies have demonstrated the presence of multiple pathogens in individual ticks suggesting a risk of polymicrobial transmission to humans. However, relatively few studies have explored this relationship and its impact on human disease. One of the key factors for this deficiency are the intrinsic limitations associated with molecular and serologic assays employed for the diagnosis of tick-borne diseases. Limitations in the sensitivity, specificity and most importantly, the capacity for inclusion of multiple agents within a single assay represent the primary challenges for the accurate detection of polymicrobial tick-borne infections. This review will focus on outlining these limitations and discuss potential solutions for the enhanced diagnosis of tick-borne co-infections.

Keywords: Lyme disease; anaplasmosis; babesiosis; capture sequencing; next-generation sequencing; serology; tick-borne co-infections.

Figure 1

TBDCapSeq probe design. The probe mix targets 14 tick-borne pathogens found in the US. For each pathogen, the capture probes are designed along the entire length of each genomic segment. The probes, shown in red, are approximately 100 nucleotides (nt) in length and bind to a region within 50 to 100 nt from its next nearest genomic neighbor, resulting in thousands of probes per agent.

Figure 2

Schematic of TBDCapSeq. The workflow consists of DNA library preparation, capture of target DNA libraries and NGS sequencing. (A) For the library preparation, nucleic acid is extracted from a clinical sample and enzymatically sheared into short fragments (<200 bp). Fragmented DNA is ligated at both ends with universal adaptors to generate dual-indexed libraries. (B) Amplified libraries are pooled for hybridization with custom biotinylated TBDCapSeq enrichment probes. Subsequent probe-bound fragments are then captured with streptavidin beads. The probe-bound DNA is eluted and amplified using universal primers, followed by quantification and NGS.