Evaluation of two molecular techniques for rapid detection of the main dermatophytic agents of tinea capitis.
BACKGROUND
Tinea capitis is very common in Western China, with the most widespread aetiological agent being Trichophyton violaceum, while Microsporum canis is prevalent in the remainder of China. Conventional diagnostics and internal transcribed spacer (ITS) sequencing analyses have proven relatively limited due to the close phylogenetic relationship of anthropophilic dermatophytes. Therefore, alternative molecular tools with sufficient specificity, reproducibility and sensitivity are necessary.


OBJECTIVE
To evaluate two molecular techniques [multiplex ligation-dependent probe amplification (MLPA) and rolling circle amplification (RCA)] for rapid detection of the aetiological agents of tinea capitis, T. violaceum and M. canis.


METHODS
Probes of RCA and MLPA were designed with target sequences in the rDNA ITS gene region. Strains tested consist of 31 T. violaceum, 22 M. canis and 24 reference strains of species that are taxonomically close to the target species.


RESULTS
The specificity and reproducibility of RCA and MLPA in detection of T. violaceum and M. canis were both 100% in both species. Sensitivity testing showed that RCA was positive at concentrations down to 1·68 × 10(6) copies of DNA in the TvioRCA probe, and 2·7 × 10(8) copies of DNA in McRCA. MLPA yielded positive results at concentrations of DNA down to 1·68 × 10(1) copies in the TvioMLPA probe and 2·7 × 10(2) in McMLPA.


CONCLUSIONS
The two techniques were sufficiently specific and sensitive for discriminating the target DNA of T. violaceum and M. canis from that of closely related dermatophytes. RCA and MLPA are advantageous in their reliability and ease of operation compared with standard polymerase chain reaction and conventional methods.
Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.|Department of Dermatology, First Hospital of Xinjiang Medical University, Urumqi, Xinjiang, China.
S
S
Deng
Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.|Department of Dermatology, Puyang Oilfield General Hospital, Puyang, Henan, China.
Z
Z
Zhou
Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.|CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands.|Basic Pathology Department, Federal University of Paraná State, Curitiba, Paraná, Brazil.|King Abdulaziz University, Jeddah, Saudi Arabia.
G S
GS
de Hoog
CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands.
X
X
Wang
CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands.
P
P
Abliz
Guangdong Provincial Institute of Public Health, Guangdong Provincial Center for Disease Control and Prevention, China.
J
J
Sun
Department of Parasitology and Mycology & Cancer Molecular Pathology Research Center, School of Medicine, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran.
M J
MJ
Najafzadeh
Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
W
W
Pan
Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
W
W
Lei
Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
S
S
Zhu
CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands.
H
H
Hasimu
Department of Dermatology, The Second Affiliated Hospital of Kunming Medical University, Kunming, China.
P
P
Zhang
Department of Dermatology, The Second Affiliated Hospital of Kunming Medical University, Kunming, China.
Y
Y
Guo
Department of Dermatology, The Second Affiliated Hospital of Kunming Medical University, Kunming, China.
D
D
Deng
Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
W
W
Liao
D015342:DNA Probes; D042241:Early Diagnosis; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008864:Microsporum / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction; D021141:Nucleic Acid Amplification Techniques; D012016:Reference Values; D012680:Sensitivity and Specificity; D014006:Tinea Capitis / Q000175:diagnosis*; D014249:Trichophyton / Q000302:isolation & purification*
D015342:DNA Probes