Clinical Utility of PNA-FISH for Burn Wound Diagnostics: A Noninvasive, Culture-Independent Technique for Rapid Identification of Pathogenic Organisms in Burn Wounds

Abstract

Burn injury results in an immediate compromised skin state, which puts the affected patient at an immediate risk for infection, including sepsis. For burn patients that develop infections, it is critical to rapidly identify the etiology so that an appropriate treatment can be administered. Current clinical standards rely heavily on culture-based methods for local and systemic infection testing, which can often take days to complete. While more advanced methods (ie, MALDI or NAAT) have improved turnaround times, they may still suffer from either the need for pure culture or sensitivity and specificity issues. Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) offers a way to reduce this time from days to hours and provide species-specific identification. While PNA-FISH has had great utility in research, its use in clinical microbiology diagnostics has been minimal (including burn wound diagnostics). This work describes a nonculture-based identification technique using commercial available U.S. FDA-approved PNA-FISH probes for the identification of common clinical pathogens, Pseudomonas aeruginosa and Staphylococcus aureus , present in burn wound infections. Additionally, calcofluor white was included for identification of Candida albicans . All three pathogens were identified from a tri-species infected deep-partial thickness rat burn wound model. These species were clearly identifiable in swab and tissue samples that were collected, with minimal autofluorescence from any species. Although autofluorescence of the tissue was present, it did not interfere or was otherwise minimized through sample preparation and analysis. The methodology developed was done so with patient care and diagnostic laboratories in mind that it might be easily transferred to the clinical setting.

Formation of Pseudomonas aeruginosa Biofilms in Full-thickness Scald Burn Wounds in Rats

“…Images were taken with both the 20× and 63× objectives of a Leica Aperio Versa 200 slide scanner (Leica Biosystems, Inc. Buffalo Grove, IL). Additionally, peptide nucleic acid (PNA)-fluorescent in situ hybridization (FISH) was used to confirm the presence of P. aeruginosa deep within the burned tissue section using a standardized method in our laboratory 51 . The E. coli / P. aeruginosa PNA FISH ® Kit was used to visualize the penetration of P. aeruginosa into the burn eschar.…”

Rapid and Simple Approaches for Diagnosis of Staphylococcus aureus in Bloodstream Infections

“…Fluorescent in situ hybridization (FISH) is a diagnostic technique using a probe (e.g., peptide nucleic (PNA) probe) to target the 16S rRNA gene of S. aureus directly in the blood culture smears. Fluorescence in situ hybridization using the PNA probes is a useful diagnostic tool for pathogen-directed therapy ( Weaver et al 2019 ). PNA probes have unique performance characteristics that render PNA FISH to be applied widely to positive blood cultures.…”

Clinical metagenomics—challenges and future prospects

“…It provides more timely results compared to traditional culturing-based methods ( Calderaro et al, 2014 ). PNA-FISH is validated by the U.S. Food and Drug Administration (FDA) for diagnosis of blood samples, however, it is not available to use at the tissue level ( Weaver et al, 2019 ). A summary of the current technologies used in routine diagnostics in the clinical setting is provided in Table 1 .…”