Polymerase chain reaction in the diagnosis of onychomycosis

ARTICLE

Auteur(s) : Ercan ARCA, M. Ali SARACLI*, Ahmet AKAR, S. Taner YILDIRAN*, Zafer KURUMLU, Ali Riza GUR

Department of Dermatology Glhane Military Medical Faculty, School of Medicine Etlik, 06018 Ankara, Turkey
*Department of Microbiology, Glhane Military Medical Faculty, School of Medicine, Etlik, 06018 Ankara, Turkey

Article accepted on 30/10/2003

Onychomycosis, a mycotic infection of the nail unit, is caused by members of the fungal triad, namely dermatophytes, yeasts and other molds [1]. It is estimated that fungi cause 50% of all nail disorders, and onychomycosis accounts for 30% of all superficial fungal infections [2, 3]. Although onychomycosis is regarded as causing only aesthetic problems/cosmetic nuisance and often it is trivialized, it has become a significant medical disorder that poses physical, psychosocial, and occupational problems [4]. The incidence of onychomycosis has been steadily increasing in parallel with an expanding number of elderly persons, immunocompromised patients, increased awareness/vigilance, changing life styles with tight clothing/footwear, and increasing participation in fitness-related activities [4, 5]. Numerous other conditions, such as psoriasis and lichen planus, can mimic onychomycosis, especially clinically, and the use of traditional antifungal agents, which have potential side effects, requires many months of therapy. For these reasons, an accurate diagnosis with laboratory confirmation is essential before treatment of onychomycosis [6].
Unfortunately, a predictably successful diagnostic laboratory approach to onychomycosis does not yet exist. Microscopy and culture are generally accepted as gold standards' for confirmation, but culture, in particular, requires time to produce adequate results, and may yield false-negative results [7, 8]. Nail histopathology may be undertaken to differentiate fungal infection of the nail from nonmycotic psoriasis and lichen planus onychodystrophy, but the procedure is time consuming, may be painful and is potentially disfiguring [8]. A method using KOH-treated nail clippings that were taken crushed and finally stained with periodic acid-Schiff (PAS) stain (KONCPA) might be used for diagnosis of onychomycosis as suggested by some authors [9, 10]. Recently, immunochemistry and flow cytometry have emerged as valuable techniques for diagnosis. But these methods cannot be applied routinely because of their complexity, their cost and the fact that access is limited to the rare centers equipped for them [4].
Application of polymerase chain reaction (PCR) technology to molecular diagnosis allows early and accurate identification of important pathogens such as viruses and microorganisms. PCR analysis of clinical specimens, including blood, sputum, urine, and cerebrospinal fluid, collected from patients systemically infected with fungi is a sensitive diagnostic method [6]. But there are not many studies about its use in superficial fungal diseases.
In the present study, we aimed to evaluate the feasibility of PCR in the diagnosis of onychomycosis and compare the results with those of microscopy and culture.

Materials and methods

Fifty-two patients with clinically suspected onychomycosis of the toenails were included in this study, excluding the patients with changes suggestive of proximal subungual onychomycosis, psoriasis and lichen planus. The diagnosis of onychomycosis was established if one of the three methods, e.g. microscopy, culture or PCR, was positive. Sampling was performed at the first dermatological examination and no treatment had been administered previously to the patients. Nails were cleaned by swabbing liberally with alcohol (70%) to exclude the possibility of the presence of contaminants attached only to the scarification from the active edges of the lesion. A sufficient amount of nail samples was taken from each patient for examination by three methods. Then, the nail scrapings were separated into three parts.
The first part of the specimen was examined by direct microscopy with 20% KOH. The second part was cultured onto Sabourauds dextrose agar slants containing chloramphenicol and cycloheximide.
The third part of the specimen was taken to the sterile centrifuge tubes for DNA extraction. In the case that very small amounts of nail scraping were available; lesser amounts were used for molecular diagnosis. This process was performed according to the method described by Turin et al. [11]. Briefly, nail scrapings were suspended in 400 µL of Tris-based buffer [Tris-HCl 50 mM, pH8.0, sodium-ethylenediamine tetra acetic acid (EDTA) 25 mM, NaCl 75 mM] in 1.5 mL sterile Eppendorf tubes, and lysed by incubation with 3 µg (15 U/sample lyticase (30 min. at 30 °C), followed by incubation with 50 µg (4 U/sample RNAse A for 20 min at 37 °C and then with 100 µL of [10%] sodium do decyl sulphate (SDS) and 200 µg (2 U/sample proteinase K for 60 min at 55 °C. After phenol-chloroform extraction, DNA precipitate was dissolved in 100 µL of sterile distilled water. Amplification was carried out in a reaction mixture of 100 µL containing 10 µL of 10x-concentrated buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl), 4 µL 25 mM MgCl2, 4 µL of a deoxynucleotide triphosphate (dNTPs) mixture (10 mM for each dNTP), 2.5 units of Ampli Taq DNA polymerase, 0.5 µL of each primer (0.5 µg/ul). Thermal cycler (MJ Research, Watertown, USA) according to the following cycle profile: hot start denaturation at 95 °C for 5 min, followed by 30 cycles consisting of template denaturation at 95 °C for 1 min, primer annealing at 55 °C for 1 min, extension reaction at 72 °C for 1 min and final extension at 72 °C for 7 min. Internal transcribed spacer (ITS) 1 and 4 primers were used in PCR amplication. The sequences of the ITS1 primer were “5'-TCC GTA GGT GAA CCT GC-3'” and of the ITS4 primer “5'-TCC TCC GCT TAT TGA TAT G-3'”. We preferred these primer pairs since their target region is sufficiently variable to differentiate fungal species. Positive and negative controls were also included in the study. A clinical isolate of dermatophyte, Trichophyton rubrum, was used as positive control and distilled water as negative control. [11, 12]. In practice, Turin et al. tested three different primer pairs against six dermatophytes, seven yeasts, eight moulds, the imperfect fungus Acremonium sp., Trichoderma sp., six mammals and two prokaryotes and found that primers ITS 1 and ITS 4 were more successful in the early detection of medically relevant fungal DNA [11]. In order to detect PCR amplified fragments, ranging from 400 to 900 base pairs (bp) in length; 10 µL of each reaction was run on 1.2% agarose gel with 0.5x TBE buffer for 60 min at 75 V constant in the presence of ethidium bromide (0.5% w/v).

Results

A total of 52 patients (26 men and 26 women; ages ranged from 22 to 72 years) were enrolled in the study. Of them, forty-four (84.6%) were diagnosed as having onychomycosis according to the criteria that any positivity resulted from any of the three tests. The results of KOH examination, culture and PCR analysis are shown in Table I. Of all 52 specimens, 40 (77%) were found to be positive with KOH examination, 12 (23%) with culture and 20 (38%) with PCR. Eight (15.4%) specimens were negative by all three methods. In all of the 40 culture-negative cases, 14 were positive with PCR. Also, in all 12 microscopy-negative cases, four were positive with PCR. The four cases that were negative both with culture and microscopy were positive with PCR. In three of 20 PCR-positive cases, two products were amplified. These secondary amplifications were due to Malassezia pachydermatis in one case, and the other two cases were due to Candida albicans. Conventional identification of the growths of PCR-positive cases was the same as their molecular identifications. None of the inoculations yielded contaminant molds by culture and all PCR products amplified from culture or microscopy or both-negative cases had a size of ca. 680 bps, suggesting the presence of M. gypseum, T. mentographytes or T. rubrum (Fig. 1).

Discussion

The mycological diagnosis of onychomycosis cannot be made on the basis of clinical observation alone and requires an assessment of both clinical and laboratory features. The two most important methods used to diagnose a fungal infection are direct microscopy (a KOH preparation) and fungal culture. Nevertheless, 50-70% of samples clinically diagnosed as positive for fungi were in fact negative on both microscopy and culture. Therefore these approaches are compared by false-negative and false-positive results which has confused treatment outcomes [7, 13]. In an analysis of over 2000 nails with suspected fungal disease examined in a diagnostic mycological laboratory, 47% of the toenails and 62% of fingernails were negative on microscopy and on culture [7]. And also the false negative rate of cultures and KOH technique has been reported as approximately 30% [9].
Although the KOH examination is fast and easy to perform, it may not provide a definite diagnosis because of artifacts, heterologous fibers, and environmental contaminants and possibly requires a lot of experience to be read. However, a microscopic examination by an expert gives important clues for further steps of definitive identification of the causative agent.
Cultures suffer from an exceptionally high level of false positive and false negative results. The false positives constitute cases of contamination of the nail by a fungus that does not play a pathogenic role in the situation concerned. All types of fungi -dermatophytes, yeasts and moulds- may be the protagonists in this type of false positive results. False negatives are not rare either because they represent at least 20% of cases. This is due to the fact that the fungi contained in the distal part of the nail have sometimes died at this point [4]. The fungal culture takes at least one to two weeks to obtain visible growth and some additional time for identification. Cultures should be read after 2 and 4 weeks and only be discarded after 6 weeks if negative. The negative report sometimes may suggest the presence of nonvital hyphae in nails.
In some cases, a nail biopsy can be performed, or the nail clippings can be sent for histological analysis. Histology of nail clippings is very useful with the same limitations as KOH. Nail histopathology can be used to confirm the presence of fungi, but cannot identify the specific pathogen [8].
Lawry et al. [14] compared the available diagnostic tests for onychomycosis, such as culture on Sabouraud agar with chloramphenicol and cycloheximide, culture on Litmann-oxgall agar, routine histopathologic examination with PAS (PATHPAS), KOH dissolution of nail and centrifugation of nail (KONC) with PAS (KONCPA), KONC with fluorescent stain (KONCFLU), and KONC with chlorazol black E stain (KONCBE). They obtained samples with the use of standard nail clippers. The distal free edge of the nail plate, along with any attached subungual debris, was clipped just distal to its attachment to the nail bed, resulting in no discomfort to the patient. They stated that PATHPAS was significantly more sensitive in detecting onychomycosis than any of the other single tests. Although their sample collection technique is probably not optimal for fungal cultures, it has been shown that the frequency of positive culture results obtained individually from distal nail plate clipping, subungual curettage, and nail plate shaving is not statistically different and they concluded that this might result from the fact that onychomycosis begins as a disease of the distal nail bed. A portion of the distal nail bed is adherent to the ventral nail plate and is therefore included in nail plate clippings. However Daniel III and Elewski [15] disagreed with Lawry et al. and said that they felt that the article had well substantiated the use of PATHPAS for the diagnosis of onychomycosis in some patients, and PATHPAS may well be the most sensitive test on nail clippings. They stated that in the article by Lawry et al., the specimens were all obtained from nail plate, which would result in false-negative results in those patients with early stages of the disease, involving only the nail bed. However, distal subungual onychomycosis is primarily a disease of the nail bed. And they also stated that the article substantiated the poor effectiveness of direct microscopy and fungal cultures in the diagnosis of onychomycosis using nail clippings, which is to be expected since the nail plate is only secondarily involved, and subungual nail debris is the best material to use for direct microscopy and fungal cultures [15].Recently, the new diagnostic techniques of immunohistochemistry and flow cytometry have provided a more accurate and specific diagnosis. The nail plate must be removed for histopathologic examination, immunohistochemistry and flow cytometry, and this may be an unreasonable demand. The preparation of fungal-specific monoclonal antibodies and the availability of standard and reference data for immunohistochemistry and flow cytometry are limitations [6].
Because, at present, there is no reliable, reproducible, and fast assay for onychomycosis, we investigated the applicability of a PCR-based diagnostic method.
PCR is a very powerful tool for parasitology and clinical mycology. It allows selective amplication of very small amounts of nucleic acids. These can be directly extracted from clinical specimens [11, 16]. Several highly specific primer pairs have already been described, showing how PCR amplification methods can bypass false-positive results due to contaminant nucleic acids from most saprophyte fungi, bacteria, parasites, plants, animals or humans. Also, random amplification polymorphic DNA assay has been shown to work effectively on fungal DNA, in both diagnostic and phylogenetic studies [11, 17, 18].
Back et al. questioned whether PCR analysis is also applicable to onychomycosis. They purified fungal DNA from fungus-infected nails, and positive PCR results were obtained for all species of fungus-infected nails, but not for normal non-infected nails and the causative pathogen was proven by fungus culture. They examined 69 routinely collected skins and nails specimens by PCR and culture. They found that PCR detected dermatophytes in 35 and culture in 28 of 38 specimens that were classified as positive, and the sensitivity of PCR (92%) was higher than of culture (73%) [14]. Their method also involved restriction enzyme analysis of PCR products obtained with primers common to all fungi. They stated that combined PCR-restriction enzyme analysis is not only sensitive but also has the potential to decrease the time taken for the laboratory identification of pathogens that grow slowly or are difficult to culture [6]. We did not perform restriction enzyme analysis in our study, and we preferred the ITS primer pairs (ITS1 and ITS4), because ITS sequences in rDNA genes have been shown to be highly polymorphic in different species [16]. Turin et al., tested the different primer pair systems, e.g. TR1-TR2, B2F-B4F and ITS1-ITS4, described previously for broad detection of fungi directly in dermatological clinical specimens in both human and veterinary fields; and aimed to develop an efficient small-scale, highly sensitive and fast procedure by which to prepare PCR-fit DNA samples; and to establish a method to detect important fungal pathogens directly from clinical specimens. They found that PCR detected dermatophytes in 10 and culture in 12 of 20 human clinical specimens that were classified as positive [11].
In our study, out of all the 40 microscopy-positive cases, 24 were PCR-negative. This might be explained by the small amount of samples and lower sensitivity of the assay which should be increased by additional optimization studies and by obtaining sufficient amounts of clinical material. In 12 cases of both KOH examination and culture negative cases, four were PCR positive. It might be relevant that the method can detect the genome of the dead fungal cells which could not be grown by culture.
Contamination might be a problem in the clinical application of PCR as a cause of false positiveness which may occur during sampling or sample processing. In order to minimize the risk of contamination, a series of well-known tricks and devices have to be applied, such as laboratory organization, inclusion of sufficient positive and negative controls.
Major disadvantages of PCR methodology are that it requires training, equipment and standardization. On the other hand, it is not only sensitive and specific, but also has the potential to decrease the time taken for the laboratory identification of pathogens that grow slowly or are difficult to culture. This method can detect the existence of fungal species such as Trichophyton species, Candida and Malassezia, but without doing sequence analysis as the species have close amplification ranges. However the PCR method could be useful in early diagnostic methods before acquiring the results of the culture.
In conclusion, in clinical mycology laboratories, the simple standard use of wet preparations, cultures on plates and a trained mycologist to interpret those results is still the best method for diagnosis of onychomycosis and PCR is beyond the scope of routine practice, but if enough material can be taken, PCR method appears to be a valuable diagnostic method especially in the patients that cannot be detected by conventional methods. The subject needs wide and well organized studies for confirmation. n

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