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Published on: July 18, 2012
Rapid detection of dermatophytes and Candida albicans in onychomycosis specimens by an oligonucleotide array
Huan Wen Han1, Mark Ming-Long Hsu2, Jong Soo Choi3
1Institute of Biomedical Engineering, College of Engineering, National Cheng Kung University, Tainan, Taiwan. p8896116@mail.ncku.edu.tw.
Background:
Onychomycosis is a fungal infection of nails, leading to the gradual destruction of the nail plate. Treatment of onychomycosis may need long-time oral antifungal therapy that can have potential side effects, thus accurate diagnosis of the disease before treatment is important. Culture for diagnosis of onychomycosis is time-consuming and has high false-negative rates. To expedite the diagnosis, an oligonucleotide array, based on hybridization between immobilized oligonucleotide probes and PCR products, for direct detection of dermatophytes and Candida albicans in clinical specimens was evaluated.
Methods:
Species-specific oligonucleotide probes designed from the internal transcribed spacer (ITS) regions of the rRNA gene were immobilized on a nylon membrane. The assay procedures consisted of PCR amplification of the ITS using universal primers, followed by hybridization of the digoxigenin-labeled amplicons to probes on the array. Thirty two nail samples (29 patients) were analyzed by the array, and the results were compared with those obtained by culture. Array-positive but culture-negative samples were confirmed by cloning and re-sequencing of the amplified ITS and by reviewing patient's clinical data. The total recovery of culture and confirmed array-positive but culture-negative results was considered 100% and was used for performance evaluation of both methods.
Results:
Concordant results were obtained in 21 samples (10 positives and 11 negatives) by both methods. Eleven samples were array-positive but culture-negative; among them, 9 samples were considered true positives after discrepant analysis. Comparing with culture, the array had significantly higher sensitivity [100% (95% CI 82.2% -100%) vs 52.6% (28.9% -75.5%), p <0.001] and negative predictive value [100% (71.3% -100%) vs 59.1% (36.4% -79.3%), p <0.05), while no significant differences were observed in specificity (84.6% vs 100%, p =0.48) and positive predictive value (90.5% vs 100%, p =1.0). The whole procedures of the array were about 24 h, whilst results from culture take 1 to 3 weeks.
Conclusions:
The array offers an accurate and rapid alternative to culture. Rapid diagnosis can expedite appropriate antifungal treatment of onychomycosis. However, the single site nature of this study conducted at a referral hospital invites caution.
Insights
This study introduces a rapid oligonucleotide array for diagnosing onychomycosis (nail fungal infection). The array offers a faster and more accurate alternative to traditional culture methods, improving timely antifungal treatment.
Area of Science:
- Medical Diagnostics
- Mycology
- Molecular Biology
Background:
- Onychomycosis is a common nail fungal infection causing nail destruction.
- Current treatments often require prolonged oral antifungal therapy with potential side effects.
- Accurate and timely diagnosis is crucial for effective treatment, but traditional culture methods are slow and have high false-negative rates.
Purpose of the Study:
- To evaluate an oligonucleotide array for the direct and rapid detection of dermatophytes and Candida albicans in clinical nail specimens.
- To compare the diagnostic performance of the oligonucleotide array with conventional culture methods.
Main Methods:
- An oligonucleotide array was designed using species-specific probes targeting the internal transcribed spacer (ITS) regions of rRNA genes.
- Clinical nail samples underwent PCR amplification of the ITS regions, followed by hybridization with labeled amplicons on the immobilized probes.
- Results from the array were compared to those from fungal culture, with discrepancies resolved through cloning, re-sequencing, and clinical data review.
Main Results:
- The oligonucleotide array demonstrated significantly higher sensitivity (100%) and negative predictive value compared to culture (52.6% sensitivity, 59.1% NPV).
- The array provided results within 24 hours, drastically reducing the diagnostic time compared to culture (1-3 weeks).
- High concordance was observed, with the array identifying true positives missed by culture.
Conclusions:
- The oligonucleotide array is an accurate and rapid diagnostic tool for onychomycosis, offering a significant improvement over traditional culture methods.
- Faster diagnosis via the array can expedite appropriate antifungal treatment, potentially improving patient outcomes.
- Further validation at multiple sites is recommended due to the single-center nature of this study.
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