Histopathology of Nail Clippings III Onychomycosis

Objective: To demonstr3le histopathologic I1ndings of nail dipping specimens In patients with onychomycosis. Material and Methods: Pmients with clinically suspected onychomycosis were enrolled in this study. Afler cleansing affected nails with 70% ethyl alcohol, the most proximal pan of abnormal nail and subungual keratinous debris were collected by sterile nail clippers and scalpel blade scraping. Each specimen was fixed in 10')6 formalin solution and processed for routine histologic examination. Hematoxylin-eosin (H&E), and special stainings for fungus by periodic acid -Schiff (PAS) and Gomeri methenamine silver (GMS) were applied. Slides were examined for hyphae using light microscopy. Concomitant fungal culture was performed in each case. Results: 75 nail specimens were evaluated. Histologically, 30 specimens (40%) were positive for fungus demonstraled by PAS and GMS stainings showing fungal elements lying between laminae of nail plales. Conclusion: Histopathologic examination of nail clippings can be used to diagnose onychomycosis. Procedure is painless, simple to perform and can be used as screening test of early diagnosis in cases of KOH-negative, clinically suspected onychomycosis before culture results are acheived. Introduction Onychomycosis is the fungal infection of the nails. It is very common and is up to 50% of all nail diseases I The causative organisms in­ clude dermatophytes, yeasls, or nonderm alophyte molds. Onychomycosis may affect the patients' emotion, social, and occupational funclions, and can cost considerable health care expen,e. " Onychomycosis especially due to demlato­ phytes needs long-term duration of systemic an­ tifungal treatment. It is not only expensive but also carries potential side effec ts and it is necessary to diagnose the infection correctly. Patients with onychomycosis may present as onycholysis, sub­ ungual hyperkeratosis and/or change in nail color. However, it should he kept in mind that many nail diseases may clinically mimic onychomycosis, including psoriasis, lichen planus, and contact dermalitis' which needed to be excluded. For these reasons, laboratory investigations for confirmation of mycosis are necessary. ConvenlionalJy, direct microscopic exami ­ nation using potassium hydroxide method (KOH) and fungal cuhure are used to diagnose onycho­ . Department of Pathology *'" Internal Medicine, Faculty of Medicine, Thammasat University ______ _ ______________ 44 mycosI s. These methods are technically difficult even when performed by experienced handS"', and may yield false -negative results. ' Culture requires at least 4 weeks to confinn the causative agents, especially in nondermatophyte mold infection. In cases of which the common mycological exami­ nations are negative but the clinical features are highly suggestive of onychomycosis, nail unit biopsy is recommended by SOme authors. 13 However, this procedure is painful and can cause permanent damage to the nail plate The nail clipping of detaching distal portions of the nails including the subungual keratinous debris can be performed without any surgical traumatization, and has now been used for histological diagnosis of onychomycosis in many laboratories. Several studies confirmed its hi gh sensitivity and specificity, in comparison to those of the direct microscopy and the fungal culture. 1:; 1:J Aim of this study is to demonstrate the histopathologic findings of nail clippings in patients with onychomycosis. Materials & Methods Patients of e ither sex from the inpatient and outpatient departments of Thammasat University Hospital with nail dystrophy clinically suggestive of onychomycosi s, evaluated by a dermatologist (M.A.), were enrolled in this study. Normal nail samples were includes as the nega tive controls. After cleansing of the affected nail s wi th 70% ethyl alcohol, the full-thickness nail clippings were obtained with steri le standard dual-action nail clipper. Each specimen was clipped at the most proximal point or the nail's separation from the nail bed. Subungual keratinous dehri" if any, was collected hy scalpel blade scraping. Histologic examination and fungal culture were pclionned in each sample. For his tologic exa mination, nail clippings were placed in 10% formalin solution and processed _ ___ ___ ___ Thammasal Medical Journal for routine histology. After em bedded in paraffin blocks and sectioned at 4 11m, the sections were stained with hematoxylin-eosin (H&E), periodic acid-Schiff (PAS) and Gomeri methenamine s ilve r (GMS ), following the s tandard staining protocols'"o Each histologic slide was examined for fungal e l­ ements under light microscope by a pathologist (O.s.) who was blinded to the clinical findings. For microbiologic studies ) nail dipping spec imens were plated on Sabouraud's dextrose agar, Sabouraud's dextrose agar plus cycloheximide, and Sabouraud's dextrose agar plus chloramphenicol and incubated using standard mycological technique. Culture specimens were checked periodically for growth for at least 4 weeks by a microbiologist who was unaware of the results of the other tests. Cultures were considered positive jf a dermatophyte grew. Cultures yielding non-dermatophyte molds or yeasts were considered positive jf the same organ­ isms growing on a second cu lture were obtained from the portion of O,e original specimens reserved in the sterile container. Species were identified for each positive culture by means of light mI­ croscopy and subculture, if required. Results 75 nail clippings obtained from 68 patients were enrolled in the study. Among these, 15 specimens were from the clinically normal nails, submitted as the negative controls. Histologically, 30 specimens ( 40% of all , 52% of clinically abnormal nails) were positive for fungus. The fungal hyphae or yeasts were visible by H&E stain in few cases. In all positive cases, PAS and GMS stainings dem­ onstrated fungal elements lying between laminae of the nail plates ( Fig. 1-3). These fungal filaments stain bright pink by PAS staining and stain black in sharp contrast to the green background by GMS staining. In some cases, the included subungual keratinous debris contained the same-appearing orgaOisms as in the nail plate tissue (Fig. 38). Volume 6 NO.1 September-December 2005 _ ___ _____________ _______ _ 45 Fig. 1 Presence of brightly pink fungal filaments invading the nail plate. The culture in this case was positive for Aspergillus niger. (PAS x 200) Fig. 2 The hyphae stain black in contrast to the greenish nail lamina. (OMS x 400) Additionally, coccal and short rod bacteria were found in the nail anfractuousities in 3 cases (Fig. 4). However, their cultures for bacteria were negative. There were 14 cases yielding bacteria on cultures but histology-negative. Among these) concomitant growth of molds was found in 5 cases) and 4 with concomitant yeasts. 22 specimens were positive for fungus by both histology and culture. The identified organisms were summarized in Table 1. Our results showed a significantly larger proportion of nondermatophyte fungi (21 cases, 96*). Among these, 16 cases (73*) were nondermatophyte molds and 5 (23*) A)