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DERMATO-MICROBIOLOGY ROUND
Year : 2007  |  Volume : 52  |  Issue : 1  |  Page : 61-63
Diagnosis of onychomycosis by trypsin treatment method


1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India

Correspondence Address:
Immaculata Xess
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-5154.31930

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   Abstract 

Background: Fungal infection of the nails is a common, difficult to treat problem, prevalent worldwide. A discrepancy in the microscopic examination and culture findings can create problems in the diagnosis of this common infection.
Aim:
This study was designed to evaluate a new method for accurate diagnosis of onychomycosis. Materials and Methods: Nail samples from 25 patients of suspected onychomycosis were taken. A portion of the samples was treated with 2% trypsin before culturing and the rest was processed by the standard mycological technique. Results: A higher number of culture positive samples were obtained by the trypsin treatment method as compared to the standard technique. Conclusion: Trypsin treatment prior to culture increases the isolation of fungi from nail samples.


Keywords: Candida spp., onychomycosis, trypsin treatment


How to cite this article:
Xess I, Dubey D, Purva M. Diagnosis of onychomycosis by trypsin treatment method. Indian J Dermatol 2007;52:61-3

How to cite this URL:
Xess I, Dubey D, Purva M. Diagnosis of onychomycosis by trypsin treatment method. Indian J Dermatol [serial online] 2007 [cited 2019 Dec 6];52:61-3. Available from: http://www.e-ijd.org/text.asp?2007/52/1/61/31930


Onychomycosis, defined as fungal infection of nail affects approximately 5% of the population worldwide and represents around 30% of all superficial mycotic infection and 50% of nail disorders.[1] The infection has profound social consequences for the affected patients, who often have diminished confidence or self esteem and experience embarrassment in social and work situations. Onychomycosis in immunocompromised patients, such as those infected with human immunodeficiency virus (HIV), can pose a more serious health problem.[2] Over the recent years, an upsurge in cases of onychomycosis due to nondermatophytes has been documented.[2],[3] Of the nondermatophytic filamentous fungi, agents implicated in onychomycosis include members of Scopulariopsis and Scytalidium (the two most common genera), which are both thought to digest keratin in vivo , as well as members of the genera Alternaria , Aspergillus , Acremonium and Fusarium .[3] The most common yeast that is involved in onychomycosis is C. albicans. The fact that the infection is difficult to treat and treatment consists of prolonged courses of potentially toxic drugs makes it imperative to make an early and accurate diagnosis. Diagnosis of onychomycosis depends on direct microscopy, supplemented by culture results. Direct microscopy is often time-consuming, because nail debris is thick and coarse and hyphae are usually only sparsely present.[2] Although direct microscopy can provide clues about the identity of the microorganism, careful matching of microscopic and culture results is necessary for the clinician to be confident of the diagnosis.[2] Almost half of all specimens taken from onychomycotic nails fail to yield a pathogen in culture. We have previously reported a modified method for diagnosis of dermatophytes, which is highly sensitive and specific as compared to the conventional culture methods.[4] In this paper, we have used the same method for diagnosing onychomycosis and document its superiority for onychomycosis caused by Candida Spp.


   Materials and Methods Top


The study population consisted of 25 patients, whose clinical diagnosis was distal or lateral subungual onychomycosis. The nail scraping from these patients were collected from the dorsal surface and nail bed, on sterile brown paper. All samples were processed on the day of collection. For processing of samples, the method of Naka et al was followed.[5]

A part of the scrapings were dipped in 1 ml of 2% trypsin solution and kept at 37C for two hours. The scrapings were then washed with phosphate buffered saline (PBS) by centrifugation three times (1500 g X 10 min). The deposit was suspended in 300 l PBS. 200 l of this was cultured on Saboraud's dextrose agar (SDA) supplemented with cyclohexamide and gentamycin (0.026 mg/ml) and cyclohexamide (0.05 mg/ml) only. 100 l was mixed with equal amount of 1% neutral red (Merck) and kept at room temperature for one hour. This was then microscopically examined as a wet mount under high dry objective (40x).[5]

Another part of sample was not treated with trypsin and processed by standard mycological techniques: A direct microscopic examination was done after treating the scraping with 20% KOH and gentle heating.[6],[7] The scrapings were also cultured on SDA supplemented with gentamycin and cyclohexamide and on SDA with cyclohexamide.

All the culture tubes were incubated at 25C and 37C for up to one month before declaring a negative result. The growth obtained was identified microscopically. For Candida speciation was done by growth characteristics on corn meal agar and pigmentation on TTZ (2, 3, 5 triphenyle tetrazolium chloride) agar.


   Results Top


Nail scrapings from a total of 25 patients whose clinical diagnosis was distal or lateral subungual onychomycosis were studied. The study population consisted of 18 males and seven females. The age of the patients ranged from 10-67 years. Of these patients, three had a history of noninsulin dependent diabetes and two had insulin dependent diabetes. None of them were HIV positive. Two patients had a history of receiving treatment for onychomycosis previously. All the samples were examined by direct microscopy and cultured both before and after trypsin treatment. The results of microscopy and culture both before and after trypsin treatment is shown in [Table - 1].

The etiological agent most frequently found in cases of onychomycosis was Candida parapsilosis. Candida albicans and geotrichum Spp. w ere isolated in two cases, whereas T. rubrum was isolated in one case. It was found that in nine cases (36%), yeast was isolated by the trypsin treatment method, whereas the standard method of culture did not yield any growth. In only one case, yeast was isolated by the standard method, while the cultures were sterile by trypsin method.


   Discussion Top


Fungal infection of the nail is a chronic, extremely difficult to treat condition, with profound social implications. Since the treatment consists of prolonged courses of antifungals, an accurate diagnosis is of utmost importance.

In this study, all the samples were examined microscopically and were cultured. Part of the sample, treated with trypsin and part without trypsin, was processed by the standard mycological protocol. After clearing of the specimen by 10-20% KOH, it was directly cultured on supplemented SDA.

We found that of the nail samples from 25 cases of suspected onychomycosis, culture by standard methods yielded no growth in nine samples, all of which were positive for Candida Spp. In our previous study, we had reported that trypsin treatment also increased the sensitivity of diagnosis of dermatophytes. [4] Further, the results of standard culture and trypsin method were concordant in all cases except one, where the trypsin method failed to yield any growth as compared to the standard culture, which yielded C. albicans .

Over the years, many modifications have been attempted to increase the sensitivity of direct microscopy for diagnosis of onychomycosis. The specimen can be mounted in a solution of 20 to 25% KOH or NaOH mixed with 5% glycerol and heated to emulsify lipids before examination. Alternatively, 20% KOH and 36% dimethyl sulfoxide can be used for clearing the specimen. However, culture is the only method by which the causative microorganism can be identified and the diagnosis truly confirmed.[2],[5],[6] The suboptimal sensitivity of standard method of culture makes it difficult for the clinicians to treat the patients appropriately.

The trypsin treatment method for culture-based diagnosis of onychomycosis is simple, economic and user-friendly, which can be performed in a busy mycology laboratory. The present study reiterates the fact that treatment of specimens with trypsin prior to culture increases the sensitivity of the culture technique and ultimately will help in proper treatment of the patients.

 
   References Top

1.Brilhante RS, Cordeiro RA, Medrano DJ, Rocha MF, Monteiro AJ, Cavalcante CS, et al . Onychomycosis in Ceara (Northeast Brazil): Epidemiological and laboratory aspects. Mem Inst Oswaldo Cruz 2005;100:131-5.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]  
2.Elewski BE. Onychomycosis: Pathogenesis, diagnosis and management. Clin Microbiol Rev 1998;11:415-29.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]  
3.Migdley G, Moore MK, Cookk JC, Phan QG. Mycology of nail disorders. J Am Acad Derm 1994;31:S68-74.  Back to cited text no. 3      
4.Xess I, Mathur P, Sirka CS, Banerjee U. Comparison of trypsin treatment method and standard laboratory technique for diagnosis of dermatomycosis. Southeast Asian J Trop Med Public Health 2004;35:396-8.  Back to cited text no. 4  [PUBMED]    
5.Naka W, Hanyaku H, Tajima S, Harda T, Nishikara T. Application of neutral red staining for evaluation of the viability of dermatophytes and Candida in human skin scales. J Med Vet Mycol 1994;32:31-5.  Back to cited text no. 5      
6.Rippon JW, editor. The pathogenic fungi and pathogenic actinomycetes. Medical Mycology. 3rd ed. Saunders: Philadelphia; 1998. p. 169-275.  Back to cited text no. 6      
7.Campbell CK, Johnson EM. The dermatophytes. In : Collier L, Balows A, Sussman, editors. Topley and Wilson's Microbiology and Microbial infections, Vol 4. 9th ed. Arnold: London; 1998. p. 215-36.  Back to cited text no. 7      



 
 
    Tables

  [Table - 1]

This article has been cited by
1 Comparison of potassium hydroxide mount and mycological culture with histopathologic examination using periodic acid-Schiff staining of the nail clippings in the diagnosis of onychomycosis
Shenoy, M., Teerthanath, S., Karnaker, V., Girisha, B., Krishna Prasad, M., Pinto, J.
Indian Journal of Dermatology, Venereology and Leprology. 2008; 74(3): 226-229
[Pubmed]



 

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