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Specific detection of fungal pathogens by 18S rRNA gene PCR in microbial keratitis.

ABSTRACT: BACKGROUND: The sensitivity and specificity of 18S rRNA polymerase chain reaction (PCR) in the detection of fungal aetiology of microbial keratitis was determined in thirty patients with clinical diagnosis of microbial keratitis. METHODS: Corneal scrapings from patients were used for Gram stain, culture and PCR analysis. PCR was performed with primer pairs targeted to the 18S rRNA gene. The result of the PCR was compared with conventional culture and Gram staining method. The PCR positive samples were identified by DNA sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. Main outcome measures were sensitivity and specificity of PCR in the detection of fungus in corneal keratitis. RESULTS: Combination of microscopy and culture gave a positive result in 11 of 30 samples of microbial keratitis. PCR detected 10 of 11 samples that were positive by conventional method. One of the 19 samples that was negative by conventional method was positive by PCR. Statistical analysis revealed that the PCR to have a sensitivity of 90.9% and specificity of 94.7% in the detection of a fungal aetiology in microbial keratitis. CONCLUSION: PCR is a rapid, sensitive and useful method to detect fungal aetiology in microbial keratitis.

SUBMITTER: Embong Z

PROVIDER: S-EPMC2413208 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Specific detection of fungal pathogens by 18S rRNA gene PCR in microbial keratitis.

Embong Zunaina Z Wan Hitam Wan Hazabbah WH Yean Chan Yean CY Rashid Nur Haslindawaty Abdul NH Kamarudin Balqis B Abidin Siti Khaironi Zainal SK Osman Sabariah S Zainuddin Zainul F ZF Ravichandran Manickam M

BMC ophthalmology 20080429

<h4>Background</h4>The sensitivity and specificity of 18S rRNA polymerase chain reaction (PCR) in the detection of fungal aetiology of microbial keratitis was determined in thirty patients with clinical diagnosis of microbial keratitis.<h4>Methods</h4>Corneal scrapings from patients were used for Gram stain, culture and PCR analysis. PCR was performed with primer pairs targeted to the 18S rRNA gene. The result of the PCR was compared with conventional culture and Gram staining method. The PCR po ...[more]

PMID: 18445283

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Project description:BackgroundMicrobial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis.MethodsUsing full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods.ResultsWe found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results.ConclusionWe have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.

Dataset Information

Specific detection of fungal pathogens by 18S rRNA gene PCR in microbial keratitis.

Publications

Specific detection of fungal pathogens by 18S rRNA gene PCR in microbial keratitis.

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