Real-time PCR and DNA sequencing for detection and identification of Trichophyton rubrum as a cause of culture negative chronic

Medical Mycology August 2009, 47, 508-514

JEAN-FRANC ̧ OIS DURANT*, PIERRE-ALAIN FONTEYNE*, PAULINE RICHEZ**, LILIANE MAROT**, LEILA BELKHIR***, DOMINIQUE TENNSTEDT** & JEAN-LUC GALA*§

*Center for Applied Molecular Technologies/DLD-Bio, Universite ́ catholique de Louvain, Brussels, **Department Dermatology, and ***Department of Infectious Disease, Saint-Luc University Hospital, Brussels, and §Defense Laboratories Department, ACOS Ops & Trg, Belgian Armed Forces

Materials and methods Case report Patient (male, 31 years old) presented with a 5-year long history of dermatophytosis which was not responding to antifungal therapy. At presentation, the diagnosis of dermatophytosis was first suggested by the rare presence of hyphae in squamae on direct mycologic examination but culture remained consistently negative. Accordingly, the patient was initially treated on the basis of a presumed fungal infection with fluconazole 200 mg once a week for five months. However, since the patient did not respond, a second protocol was applied consisting of griseofulvine 500 mg a day which was replaced after five months with terbinafine 250 mg a day for 20 months. These antifungals did not prevent extension of the lesions. Therefore, at this time a pulse therapy involving itraconazole 200 mg twice a day for one week per month resulted in a significant improvement. However, after five months there was a reoccurrence of the lesions and treatment with itraconazole pulse and terbinafine 250 mg was begun. This combined therapy did not result in significant improvement and was replaced after 12 months by voriconazole 600 mg a day. While the cutaneous lesions regressed initially, they were still present 19 months after the initiation of this therapy. At this time, a periodic control of the biological parameters revealed that the patient was developing toxic hepatitis. Voriconazole was then replaced by pozaconazole 400 mg twice a day, but without major impact on cutaneous lesions.

Despite continuous antifungal therapy, the dermatophytosis progressed in the following years, slowly invading both feet and one leg and leading with progressive increase in the number and size of the nodules (Fig. 1).

The hypothesis of primary immunodeficiency disorder as a cause for the progression of the disease was not supported by CD4 counts and the different types and subtypes of immunoglobulins. Swabs of the wound were repeatedly collected over a 5-year period to ascertain the initial diagnosis of dermatophytosis. While bacteria were occasionally recovered in cultures inoculated with these swabs and hyphae rarely noted on direct examination of skin scrapping, cultures for fungi remained consistently negative. In 2004, a biopsy was performed which provided histologic evidence compatible with the initial diagnosis of dermatophytic granuloma. In August 2007, the lack of favourable therapeutic outcome prompted investigations with alternative methods to confirm the initial diagnosis of dermatophytic granuloma. Molecular and histological examinations were carried out on successive cutaneous deep biopsies and swabs from a nodule. Samples were collected over seven months.

Compared to the reported DNA extraction procedure [8], this semi-automated DNA extraction method appeared easy and extremely fast (turn around time about 90 min vs. about 3 h) while having the potential for high throughput analysis without the risk of molecular carryover. It appeared to be highly effective, allowing successful fungal DNA recovery from the three successive skin biopsies and one of two successive swabs. To the best of our knowledge, this is the first report describing the use of combined rapid semi-automated methods for direct fungal DNA extraction from unfixed clinical samples.

A second issue was to select the fungal genetic target. In order to screen our sample for a wide spectrum of dermatophytic and non dermatophytic species, it was decided to amplify the internal transcribed spacer region 1 rather than other specific fungal molecular targets. Indeed, sequencing ITS1 appears to be a reliable method to discriminate among most clinically relevant yeasts and moulds species [10,11]. In the current study, identification of T. rubrum relied on ITS1 sequence homology with T. rubrum sequences accessible in GenBank/EMBL database. In contrast, consistent ITS1 mismatches differenciated the T. rubrum cluster from another cluster which included T. soudanense and T. violaceum species. The resulting tree view was also in agreement with the molecular taxonomy of the T. rubrum complex, as previously published [12].

Thirdly, the SYBR® Green dye was preferred to a fluorescent probe for the real-time PCR assay. The former allowed comparing the amplicon melting curve profiles of successively processed clinical samples. Moreover, the value of amplicon melting curves as a screening test for identifying fungal DNA in clinical specimens was recently suggested [13].

However, SYBR® Green real-time amplification of ITS1 requires DNA sequencing for identifying fungi at the species level. In the current case, a species belonging to the T. rubrum group was consistently identified in clinical samples collected over more than half a year. It is of note that the species with ITS DNA sequences common to T. rubrum have been reduced to synonomy or reclassified within this species on the basis of phenotypic and molecular methods [12]. Results of the sequence analysis were in line with the diagnostic hypothesis of the dermatologist, the clinical features of the lesions, the histological evidence of dermal granuloma with hyphae in the same clinical samples, and the fact that T. rubrum is the most frequent cause of superficial as well as deep dermatophytosis [14].

A last issue was related to the specificity and sensitivity of our molecular approach. This was addressed at two different levels, i.e., the real-time PCR Cp results and interpretation of DNA sequence alignement. The panfungal primers used in our real-time PCR assay were published in 1990 and have been used ever since for amplifying DNA from fungal cultures, especially in the framework of taxonomical studies on clinically relevant fungi [9]. We are also routinely using them for genotyping fungal colony. The mean Cp value obtained from a panel of 18 control DNA samples confirmed the fungal specificity of our real-time PCR assay. However, these primers are not specific for the T. rubrum group. Samples contamination with another ITS-1 positive species would have led to co-amplifica- tion of both genetic targets and mixed amplicons. However, this was not seen on sequence analysis. In contrast, the presence of ITS1-negative contaminating fungus cannot be ruled out. It could also be argued that identification of T. rubrum in the biopsies resulted from skin colonization with this fungal species and not from an invasive infection. The consistent positive and negative results from patient and control samples respectively, as well as the weak PCR signal with only one of the two skin swabs make this hypothesis very unlikely.

Despite the advantages of DNA-based identification methods, the place of fungal genotyping in routine microbiological diagnosis is yet poorly defined. The costs of processing specimens as well as the need for skilled personal, sophisticated equipment, and well adapted infrastructure are clear limitations to a widescale use. While current technological constraints do not yet allow for widespread implementation of fungi genotyping in routine clinical mycology laboratories, those methods should be available within reference facilities. Indeed, as highlighted here, fungal genotyping has proved its ability to elucidate difficult cases (i.e., presumed fungal infection based on a suggestive histology but unconfirmed by culture) by bringing rapid and valuable DNA-based identification. In most cases of presumed dermatophytosis, the initial clinical diagnosis relying on the rare presence of fungal hyphae in skin samples would have been satisfactory. In the current case, the unusually long and bad evolution of the disease and its therapeutic antifungal resistance requested an unambiguous identification of the causative agent.

In conclusion, we report the case of a patient with a long-standing history of granulomatous dermatophytosis which was not responding to most antifungal agents. This case pinpoints the benefit of combining semiautomated DNA preparation methods, analysis of ITS1 amplicon melting curve profiles and sequence analysis on repeated skin biopsy samples for unambiguous identification of the causative fungal species, especially when microbiologic confirmation is lacking.