Other fungal species were identified using amplification of the internal transcribed
spacer (ITS) region of the rRNA gene, followed by restriction fragment length polymorphism analysis or
sequencing. In addition, polyacrylamide gel separation of the T1-PCR product allowed subtyping of T. rubrum
strains. We studied 195 nail specimens (the “nail sample”) and 66 previously collected etiologic strains (the
“strain sample”) from 261 onychomycosis patients from Bulgaria and Greece. Of the etiologic agents obtained
from both samples, T. rubrum was the most common organism, confirmed to be present in 76% of all cases and
serving as the sole or (rarely) mixed etiologic agent in 199 of 218 cases (91%) where the identity of the causal
organism(s) was confirmed. Other agents seen included molds (6% of cases with identified etiologic agents;
mainly S. brevicaulis) and other dermatophyte species (4%; most frequently Trichophyton interdigitale). Simultaneous infections with two fungal species were confirmed in a small percentage of cases (below 1%). The
proportion of morphologically identified cultures revealed by molecular study to have been misidentified was
6%. Subtyping revealed that all but five T. rubrum isolates were of the common type B that is prevalent in
Europe. In comparison to microscopy and culture, the molecular approach was superior. The PCR was more
sensitive (84%) than culture (22%) in the nail sample and was more frequently correct in specifically identifying etiologic agents (100%) than microscopy plus routine culture in either the nail or the strain samples
(correct culture identifications in 96% and 94% of cases, respectively). Using the molecular approach, the time
for diagnosing the identity of fungi causing onychomycosis could be reduced to 48 h, whereas culture techniques generally require 2 to 4 weeks. The early detection and identification of the infecting species in nails will
facilitate prompt and appropriate treatment and may be an aid for the development of new antifungal agents