Authors (including presenting author) :
Cheng LSK (1), Chau SKY (1), Wong BKC (1), Lam ILY (1), Fung KSC (1)
Affiliation :
(1) Department of Pathology, United Christian Hospital
Introduction :
Early diagnosis of infectious diseases and identification of offending pathogens is crucial in patient care. It improves disease outcomes by allowing timely administration of appropriate antimicrobials and intervention. However, not all clinical specimens properly obtained from sites of infection are culture positive, even with the advent of well-established conventional bacterial culture system.
The 16S ribosomal RNA gene codes for the RNA component of 30S subunit of the ribosome. The 16S rRNA gene in bacterial species is highly conserved and specific, and comprises usually nine variable regions of sufficient phylogenetic diversity for species identification. Through the detection of 16S rRNA gene and analysis of the rRNA gene sequence, the presence of bacteria in the clinical samples can be detected and identified at species level. This is particularly useful in situations where cultures are obtained before antibiotic administration, slow-growing or non-cultivable bacteria, in which conventional culture procedures may give initial misleading false negative results.
Objectives :
To evaluate the contributions of 16S rRNA gene sequencing directly from clinical specimens in the detection and identification of bacterial pathogens in patients with high clinical suspicion of infections yet initial culture-negative results
Methodology :
Data related to 16s rRNA gene sequencing requested by microbiologists after clinical consultations between January 2008 and December 2022 were retrospectively retrieved from the laboratory records of testing. The 16S rRNA PCR analysis was performed in the Microbiology Laboratory of United Christian Hospital using validated protocol for amplification and sequencing. Only the 16s rRNA gene sequencing directly performed on clinical samples from normally sterile sites except serum with initial negative results following routine culture were included. A normally sterile site is defined as cerebrospinal fluid, peritoneal fluid, pleural fluid, bone, joint fluid, or internal body site. The results of sequence analysis on 16S rRNA databases were reviewed and compared with the results on final reports issued by the attending microbiologists after clinical correlations.
Result & Outcome :
280 samples from 194 patients with a high index of suspicion for bacterial infections investigated by direct 16S rRNA gene sequencing were included in the analysis. Fifty-three samples (18.9%) from 44 patients (22.6%) were positive in 16S rRNA PCR, among which 45 (84.9%) out of the 53 samples identified bacteria species meeting the identify threshold for 16S ribosomal RNA and were regarded as the disease-causing pathogen after interpretation by microbiologists. Direct 16s rRNA sequencing on heart valve tissue (100%), pus from liver (50%) and brain abscesses (50%) and pleural fluid (30%) showed satisfactory performance in detecting bacterial pathogens.
The availability of sequence-based analysis on clinical samples positively impacts patient management. Yet, wide use of 16s rRNA gene PCR analysis on a routine basis is limited by certain drawbacks, including high cost, demand for technical expertise and poor performance in polymicrobial specimens and anaerobic pathogens. In selected cases with high clinical suspicion and initial negative findings in conventional bacteriological culture, direct 16s rRNA gene sequencing from samples can be a useful complementary tool for the diagnosis of bacterial infections.
