KOH-mounts were performed with 20% KOH and were microscopically checked for fungal elements after at least 20 min of exposure. Fungal cultures were prepared on Sabouraud glucose agars (bioMérieux, Marcy-l’Etoile, France) containing antibiotics (chloramphenicol 0.05 g l−1 selleck kinase inhibitor and gentamycin 0.01 g l−1) with and without addition of cycloheximide (0.4 g l−1; AppliChem, Darmstadt, Germany). The cultures were
incubated at 26 °C for at least 3 weeks before assessed as negative. Positive cultures were identified by established criteria based on morphology and physiology.12 If necessary, subcultures were prepared on special media according to the specific requirements. In particular, the urease test, cultures on potato-dextrose agar and the hair perforation test were used
if a strain could not be identified on morphological basis. The sample shares allocated to genetic analysis were submitted to DNA-extraction using a QiAmp DNA Mini Kit (Qiagen, Hilden, Germany). A PCR was then performed using a HotStarTaq Plus Master Mix Kit (Qiagen) according to the manufacturer’s instructions. The following primers were used that amplify a 280 base pair fragment containing a GT-microsatellite repeat specific for strains of the closely selleck chemical related T. rubrum/Trichophyton violaceum-clade6: forward 5′TGG TCT GGC CTT GAC TGA CC3′, reversed 5′GTA AGG ATG GCT AGT TAG GGG G3′. The fungal DNA was amplified by 30 cycles in a thermocycler (Thermomixer Comfort; Eppendorf, Hamburg, Germany): 30 s at 95 °C denaturation, 30 s mafosfamide at 60 °C annealing and 45 s at 42 °C extension. The amplification product was then checked for bands with 280 bp by gel electrophoresis in a 2% agarose gel in comparison with a negative control and a positive control with DNA extracted from Trichophyton mentagrophytes (Fig. 1). For a total of 464 samples of scales, a corresponding detection of T. rubrum by PCR and culture was seen in 75
cases. A positive T. rubrum PCR, but negative T. rubrum culture, was found in 40 scale samples, whereas a positive T. rubrum culture, but negative T. rubrum PCR, was found in 13 cases. Trichophyton rubrum culture and T. rubrum PCR were both negative in 336 cases. In Fig. 2, these results are shown in rounded percentage of all scale samples. In 66 samples of scales, other fungi than T. rubrum were detected by cultures; all of these samples had a negative PCR for T. rubrum. Twenty-one scale samples were positive for fungal elements in the KOH-mounts only and were negative in cultures and T. rubrum PCR. For a total of 230 nail samples, a corresponding detection of T. rubrum by PCR and culture was seen in 40 cases. A positive T. rubrum PCR, but negative T. rubrum culture, was found in 47 nail samples, whereas a positive T. rubrum culture, but negative T. rubrum PCR, was found in 8 samples. In Fig. 3, these results are shown in rounded percentage of all nail samples. In 25 samples of nails, other fungi than T.