Superiority of Next-Generation Sequencing (NGS) over Quantitative Polymerase Chain Reaction (qPCR) Testing in Detecting Primary Pathogens of Polymicrobial Communities Found in Infected Chronic Foot Wounds

Chronic foot wounds, whether diabetic or nondiabetic, yield a multilayered, polymicrobial community of fast and slow growing aerobic and anaerobic bacteria, as well as fungus, yeast, and viruses. The challenge in developing treatment plans for chronic wounds is sifting through the layers to identify the predominant cause of an infection within the wound microbiota, while also addressing the bacterial bioburden that may contribute to the chronicity of inflammation.  

Advances in molecular diagnostics, especially qPCR and NGS, have enabled clinicians to parse the microbiome data to direct treatment more efficiently and effectively.  However, not all molecular testing is equal. This study seeks to retrospectively review if NGS would correctly identify the primary hostile pathogen(s) more frequently and accurately than qPCR testing.  

A retrospective review was performed of 65 healed chronic forefoot and rearfoot wounds treated in one wound clinic, where both qPCR and NGS testing was performed on each sample by a single molecular lab. Results of each test on the same sample were compared. Wound chronicity was defined as being present >6 months without significant size reduction. Each wound was complicated by one or more chronic conditions that negatively impacted an appropriate immune response (diabetes, ischemia, neuropathy, or systemic inflammatory disorders, with malignancies excluded from the study). Healing was defined as being resolved >3 months.

The conclusion found in this review was NGS is superior to qPCR when identifying the primary causative microbe, thereby allowing a more targeted antimicrobial regimen. In 70.77% of wounds, the dominant pathogen was detected by NGS only. In 6.15% of wounds, PCR also missed the mutated species. Quantitative PCR testing is rapid and relatively inexpensive, but relies on comparison to known microbes. NGS identifies characteristics of multiple microbial communities, simultaneously allowing for discovery of numerous and novel variants including gene mutations, leading to optimized targeted treatments.