Rapid detection of dermatophytes and Candida albicans in onychomycosis specimens by an oligonucleotide array.
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.
Institute of Biomedical Engineering, College of Engineering, National Cheng Kung University, Tainan, Taiwan. p8896116@mail.ncku.edu.tw.
Huan Wen
HW
Han
ESkin Clinic, Tainan, Taiwan. a441224@mail.ncku.edu.tw.
Mark Ming-Long
MM
Hsu
Department of Dermatology, Yeungnam University College of Medicine, Daegu, Korea. jschoi@med.yu.ac.kr.
Jong Soo
JS
Choi
Department of Dermatology, National Cheng Kung University Hospital, Tainan, Taiwan. kylehsu@mail.ncku.edu.tw.
Chao-Kai
CK
Hsu
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. toheart0824@yahoo.com.tw.
Hsin Yi
HY
Hsieh
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. b8803126@stmail.cgu.edu.tw.
Hsin Chieh
HC
Li
Institute of Biomedical Engineering, College of Engineering, National Cheng Kung University, Tainan, Taiwan. hcchang@mail.ncku.edu.tw.
Hsien Chang
HC
Chang
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. tsungcha@mail.ncku.edu.tw.
Tsung Chain
TC
Chang
D003883:Arthrodermataceae / Q000235:genetics / Q000302:isolation & purification*; D002176:Candida albicans / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis; D006801:Humans; D009262:Nails / Q000382:microbiology; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D015345:Oligonucleotide Probes; D014009:Onychomycosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004271:DNA, Fungal; D015345:Oligonucleotide Probes