Authors (including presenting author) :
Tsang YM (1), Luk KS (2), To WK (1,2)
Affiliation :
(1)Department of Pathology, North Lantau Hospital
(2)Department of Pathology, Princess Margaret Hospital
Introduction :
Culture-negative infection remains a diagnostic challenge in clinical microbiology necessitate the incorporation of molecular diagnostic tools into the routine diagnostic workflow. Sanger sequencing can serve as supplementary diagnostic method, but has low throughput and poor resolution for polymicrobial infections. Nanopore-based next-generation 16S rRNA gene sequencing likely has tremendous potential given its higher sensitivity, simple sample preparation, quick turnaround time and being relatively inexpensive.
Objectives :
We aimed to evaluate the performance of Nanopore-based 16s rRNA gene sequencing to identify bacterial species from culture-negative specimens from a wide spectrum of infections including meningitis, septic arthritis, prosthetic joint infection, pleural infection, and peritoneal dialysis-related peritonitis. Clinical applicability of incorporating this technique into routine diagnostic workflow of Princess Margaret Hospital (PMH) Microbiology laboratory was also assessed.
Methodology :
We retrospectively retrieved culture-negative sterile specimens processed in PMH Microbiology laboratory for Nanopore sequencing upon clinical suspicion of bacterial infection. We compared the sequencing results with Microseq-based Sanger sequencing.
Result & Outcome :
Among the 20 clinical samples tested, Nanopore identified significant bacterial species in 11 (55%) of these samples, compared to 2 (10%) by Microseq-based Sanger sequencing. Five of these samples with positive pathogens tested by Nanopore were confirmed by culture, nucleic acid amplification test or Sanger sequencing, showing good result correlation. The real-time sequencing offered by Nanopore could shorten the result turnaround time from 2 to 3 days to same day when compared to conventional culture. By multiplexing specimens, the sequencing cost per sample can be lowered to comparable price as the Sanger platform which is more affordable for clinical diagnostic use.
With the promising result from pilot study, PMH Microbiology Laboratory has routinely incorporated Nanopore sequencing in assisting the diagnosis of culture-negative bacterial infection starting from July 2022. Culture-negative sterile specimens fulfilling diagnostic criteria for infection would prospectively undergo Nanopore sequencing on a bi-weekly regular basis. Specimens with positive bacterial targets sequenced would be issued in the laboratory reports upon Microbiologists’ interpretation and clinical correlation. Case-clinicians, Microbiologists, and Infectious Disease physicians could then adjust antibiotics or proceed to further investigations accordingly.
