Microbiological culture versus 16S/18S rRNA gene PCR-sanger sequencing for infectious keratitis: a three-arm, diagnostic cross-sectional study
Hammoudeh, Yasmeen Suresh, Lakshmi Ong, Zun Zheng Lister, Michelle M Mohammed, Imran Thomas, D John I Cottell, Jennifer L Holden, Jennifer M Said, Dalia G Dua, Harminder S
Hammoudeh, Yasmeen
Suresh, Lakshmi
Ong, Zun Zheng
Lister, Michelle M
Mohammed, Imran
Thomas, D John I
Cottell, Jennifer L
Holden, Jennifer M
Said, Dalia G
Dua, Harminder S
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Affiliation
Queen's Medical Centre; New Cross Hospital Eye Infirmary; Cardiff University; Sandwell and West Birmingham NHS Trust; et al.
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Publication date
2024-08-12
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Abstract
Background: To compare the diagnostic performance of microbiological culture and 16S/18S rRNA gene polymerase chain reaction (PCR)-Sanger sequencing for infectious keratitis (IK) and to analyse the effect of clinical disease severity on test performance and inter-test concordance. Methods: This was a three-arm, diagnostic cross-sectional study. We included all eligible patients who presented with presumed bacterial/fungal keratitis to the Queen's Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous culture (either direct or indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. The bacterial/fungal genus and species identified on culture were confirmed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Relevant clinical data were also collected to analyze for any potential clinico-microbiological correlation. Main outcome measures included the diagnostic yield, test accuracy (including sensitivity and specificity), and inter-test agreement [including percent agreement and Cohen's kappa (k)]. Results: A total of 81 patients (86 episodes of IK) were included in this study. 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). 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 tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement = 81.8-86.2%, Cohen's k = 0.67-0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with greater infection severity. Conclusions: 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).
Citation
Hammoudeh Y, Suresh L, Ong ZZ, Lister MM, Mohammed I, Thomas DJI, Cottell JL, Holden JM, Said DG, Dua HS, Ting DSJ. Microbiological culture versus 16S/18S rRNA gene PCR-sanger sequencing for infectious keratitis: a three-arm, diagnostic cross-sectional study. Front Med (Lausanne). 2024 Aug 12;11:1393832. doi: 10.3389/fmed.2024.1393832
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Article