Direct 16S/18S rRNA Gene PCR Followed by Sanger Sequencing as a Clinical Diagnostic Tool for Detection of Bacterial and Fungal Infections: a Systematic Review and Meta-Analysis

Dřevı́nek, Pavel; Hollweck, Regina; Lorenz, Michael G.; Lustig, Michael; Bjarnsholt, Thomas

doi:10.1128/jcm.00338-23

Cited by 13 publications

(2 citation statements)

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“…varied clinical severity and types of infectious keratitis). 16,19,26,37,39 Although our study was set out to include fungal keratitis cases, none of the culture or PCR yielded any fungal pathogen, limiting the interpretation of 18S rRNA PCR for diagnosing fungal keratitis. That said, the lack of false positive results and the perfect concordance between culture and 18S rRNA PCR suggests a high specificity of this test for ruling out fungal keratitis (though the sensitivity remains to be elucidated in our patient cohort).…”

Section: Discussionmentioning

confidence: 99%

“…Among various assays and techniques, broad-range 16S/18S ribosomal RNA (rRNA)-based PCR has emerged as one of the most popular methods. 3,[14][15][16] 16S rRNA is a major component of the 30S small subunit present in all prokaryotes, including bacteria. It contains multiple highly conserved regions (useful for universal PCR priming) and nine variable regions (V1-V9; pertinent for phylogenetic analysis and taxonomic resolution).…”

Section: Introductionmentioning

confidence: 99%

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Microbiological Culture Versus 16S/18S Ribosomal RNA PCR-Sanger Sequencing for Infectious Keratitis: A Three-Arm, Diagnostic Cross-Sectional Study

Hammoudeh,

Suresh,

Ong

et al. 2023

Preprint

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Purpose: To compare the diagnostic performance of microbiological culture and 16S/18S polymerase chain reaction (PCR) for infectious keratitis (IK). Methods: This was a monocentric, three-arm, diagnostic comparative study. We included 81 patients (86 episodes of IK) who presented with presumed bacterial/fungal keratitis to the Queens Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous microbiological culture (either direct culture, indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. Main outcome measures included diagnostic yield, performance, and inter-test agreement. Results: All organisms identified were of bacterial origin. Diagnostic yields were similar among direct culture (52.3%), indirect culture (50.8%), and PCR (43.1%; p=0.13, Cochran Q test). The addition of PCR enabled a positive diagnostic yield in 3 (9.7%) direct culture-negative cases. Based on composite reference standard, direct culture had the highest sensitivity (87.5%; 95% CI, 72.4-95.3%), followed by indirect culture (85.4%; 95% CI, 71.6-93.5%) and PCR (73.5%; 95% CI, 59.0-84.6%), with 100% specificity noted in all three tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement=81.8-86.2%, Cohen k=0.67-0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with worse presenting visual acuity and greater severity of infection. Conclusion: This study highlights a similar diagnostic performance of direct culture, indirect culture and 16S rRNA PCR for bacterial keratitis, with substantial inter-test concordance. PCR serves as a useful diagnostic adjuvant to culture, particularly in culture-negative cases or those with lesser disease severity (where culture-PCR concordance is lower).

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