16S Ribosomal RNA PCR Versus Conventional Diagnostic Culture in the Investigation of Suspected Bacterial Keratitis

Abstract

The purpose of this study was to compare conventional diagnostic culture (CDC) to 16S ribosomal RNA polymerase chain reaction (PCR) analysis for diagnosing bacterial keratitis. Methods: Samples collected from 100 consecutive patients presenting to the Royal Liverpool University Hospital with bacterial keratitis were processed using CDC and 16S PCR analysis. Results: The overall detection rate using both methods was 36%. Of these, 72.2% (26/36) were detected by PCR and 63.9% (23/36) isolated by CDC (P = 0.62). Using a combination of both PCR and CDC increased the detection rate for pathogenic bacteria by 13% compared to using CDC alone (P = 0.04). In CDC negative samples, 16S PCR identified more pathogens than CDC in 16S PCR negative samples. Neither order of sample collection nor prior antimicrobial use affected the detection rate. Conclusions: 16S rRNA gene PCR performed in addition to CDC on corneal samples from patients with clinically suspected bacterial keratitis led to additional pathogen detection. Translational Relevance: 16S rRNA gene PCR should be developed to become an additional part of clinical service for patients with bacterial keratitis rather than used in isolation.

Risk Factors, Clinical Outcomes, and Prognostic Factors of Bacterial Keratitis: The Nottingham Infectious Keratitis Study

“…The issue with low culture yield in infectious keratitis has been consistently highlighted in many studies ( 1 , 2 ). To overcome this clinical barrier, a number of novel and emerging technologies, including MALDI-TOF mass spectrometry ( 39 , 40 ), IVCM ( 41 ), polymerase chain reaction (PCR) ( 42 ), next generation sequencing ( 43 ), and artificial intelligence-assisted platforms ( 44 ), have been developed and/or implemented in clinical practise. In addition, emerging treatment such as therapeutic corneal collagen cross-linking, ultraviolet C treatment, polymer-based treatment, and antimicrobial peptides may serve as useful adjunctive treatment in the near future, improving the management and treatment outcome of infectious keratitis ( 45 – 49 ).…”

Clinical Characteristics and Outcomes of Fungal Keratitis in the United Kingdom 2011–2020: A 10-Year Study

“…The issue with low culture yield in infectious keratitis has been a recurrent clinical theme [1,25,45]. In the future, it is envisaged that novel technologies, including polymerase chain reaction (PCR), [46,47], IVCM [48], next-generation sequencing, [49][50][51], matrix-assisted laser/desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) [14,52] and artificial intelligence-assisted platforms [53][54][55][56] would be able to enhance the diagnostic yield and accuracy of infectious keratitis. As contact lenses serve as an important risk factor for FK, future studies examining the influence of the types and brands of CL on the microbiological profiles and risk of infection would be valuable.…”

Isolation, Identification, and Antibacterial Mechanisms of Bacillus amyloliquefaciens QSB-6 and Its Effect on Plant Roots

“…Genomic DNA was extracted using the EasyPure Bacteria Genomic DNA Kit ( Del Sal et al, 1988 ). To confirm the species identity of strain QSB-6, we obtained the DNA sequences of the 16S ribosomal RNA gene (16S rDNA), DNA gyrase subunit A (gyrA), DNA gyrase subunit B (gyrB), and RNA polymerase subunit B (rpoB) ( Gardes and Bruns, 1993 ; Yamamoto and Harayama, 1995 ; Chun and Bae, 2000 ; Zalila-Kolsi et al, 2016 ; Somerville et al, 2020 ). PCR amplification was performed in a final volume of 50 μL that contained 2.0 μL of genomic DNA (20 ng/μL), 1.0 μL of each primer (10 μM), 0.5 μL of PrimeSTAR HS DNA polymerase (2.5 U/μL), 10.0 μL of 5× PrimeSTAR Buffer (Mg 2+ Plus), 4.0 μL of dNTP Mixture (2.5 mM each), and 31.5 μL of ddH 2 O ( Weisburg et al, 1991 ).…”