Clinical evaluation of β‐tubulin real‐time PCR for rapid diagnosis of dermatophytosis, a comparison with mycological methods

Abstract

Following our previous report on evaluation of the beta tubulin real-time PCR for detection of dermatophytosis, this study aimed to compare the real-time PCR assay with conventional methods for the clinical assessment of its diagnostic performance. Samples from a total of 853 patients with suspected dermatophyte lesions were subjected to direct examination (all samples), culture (499 samples) and real-time PCR (all samples). Fungal DNA was extracted directly from clinical samples using a conical steel bullet, followed by purification with a commercial kit and subjected to the Taq-Man probe-based real-time PCR. The study showed that among the 499 specimens for which all three methods were used, 156 (31.2%), 128 (25.6%) and 205 (41.0%) were found to be positive by direct microscopy, culture and real-time PCR respectively. Real-time PCR significantly increased the detection rate of dermatophytes compared with microscopy (288 vs 229) with 87% concordance between the two methods. The sensitivity, specificity, positive predictive value, and negative predictive value of the real-time PCR was 87.5%, 85%, 66.5% and 95.2% respectively. Although real-time PCR performed better on skin than on nail samples, it should not yet fully replace conventional diagnosis.

Non-culture based assays for the detection of fungal pathogens

“…On the other hand, some multiplex PCR methods for dermathophytes or dermatophytes/Candida species in clinical samples were also developed and clinical evaluation is still in progress [38]. Recently, published results [39] showed that real-time PCR with specific pan-dermatophyte primer for detection of most agents from this group of fungi in clinical samples detected more infected patients compared with ME. However, with unsatisfactory sensitivity of 87.5% and positive predicative value of 66.5%, this method cannot be suggested yet as sufficient replacement of conventional diagnosis.…”

Assessment of a pan‐dermatophyte nested‐PCR compared with conventional methods for direct detection and identification of dermatophytosis agents in animals

“…Current standard approaches for diagnosis of human and animal dermatophytosis rely on conventional microscopic detection of fungal elements (arthroconidia and/or hyphae) in KOH preparation of clinical specimens as well as further morphological and biochemical analyses of in vitro cultures . With direct microscopy, the infection can be confirmed but its drawbacks are as follows: failure to distinguish between dermatophytic and nondermatophytic elements, inability to identify the causative agent at genus or species level, and numerous false‐negative results due to lack of training . Culture in selective media is frequently associated with a poor sensitivity, mainly due to the development of a large variety of fungal or bacterial contaminants and also the presence of nonviable arthroconidia or mycelia in infected materials .…”

“…Since late 1990s and with the advent of molecular biology, many attempts have been concentrated on the development of early and reliable PCR‐based alternatives to direct microscopy and culture for the diagnosis of dermatophytosis. Some PCR‐based methods targeting the internal transcribed spacer (ITS) regions, Chitin synthase (CHS), Topoisomerase II, and beta tubulin genes are examples of these efforts for the direct detection of dermatophytes, mainly in human dermatophytosis. However, to date, there has been no molecular study devoted to evaluate fast and reliable procedures for the diagnosis of dermatophytosis in clinical animal materials.…”

“…[7][8][9][10] With direct microscopy, the infection can be confirmed but its drawbacks are as follows: failure to distinguish between dermatophytic and nondermatophytic elements, inability to identify the causative agent at genus or species level, and numerous false-negative results due to lack of training. 6,10,11 Culture in selective media is frequently associated with a poor sensitivity, mainly due to the development of a large variety of fungal or bacterial contaminants and also the presence of nonviable arthroconidia or mycelia in infected materials. 4,5,12 Thus, a highly sensitive and specific method for fast and precise detection of dermatophytes would meet the practitioners' requirements and could significantly increase the management of cases suspected to dermatophytosis.…”

“…Some PCR-based methods targeting the internal transcribed spacer (ITS) regions, 8,9 Chitin synthase (CHS), 6,10 Topoisomerase II, 13 and beta tubulin 11 genes are examples of these efforts for the direct detection of dermatophytes, mainly in human dermatophytosis. However, to date, there has been no molecular study devoted to evaluate fast and reliable procedures for the diagnosis of dermatophytosis in clinical animal materials.…”