J Cutan Pathol 2008: 35: 1097–1099doi: 10.1111/j.1600-0560.2007.00978.xBlackwell Munksgaard. Printed in Singapore

Copyright # Blackwell Munksgaard 2008

Journal of

Cutaneous Pathology

Evaluation of skin biopsies for fungalinfections: role of routine fungal staining

Background: The diagnosis of cutaneous fungal infection is usuallymade on clinical examination of the lesion and direct microscopicexamination of skin scrapings with potassium hydroxide.Histopathological examination is rarely required to confirmdermatophytic infections. There is often a discord between the clinicaland histopathological findings and many times clinical appearance canhave a poor histopathological correlation.Methods: This retrospective study was carried out on all skinbiopsies received in a period of 1 year. The clinical details of all caseswere retrieved and slides were reviewed. Routine fungal stainingperiodic acid-Schiff (PAS) stain was performed in all cases.Results: Of 403 skin biopsies received in 1 year, material for furtherstaining was available in 338. On re-examining the slides stained withPAS stain, fungi were identified in 34 cases (10%), of which fungalinfection had been suspected clinically in five cases.Conclusions: Use of routine stains like PAS can help to reacha correct diagnosis and initiate appropriate treatment. Fungal stainingshould be done in skin biopsies with non-specific clinical details andmicroscopic findings for best patient management.

Mohan H, Bal A, Aulakh R. Evaluation of skin biopsies for fungalinfections: role of routine fungal staining.J Cutan Pathol 2008; 35: 1097–1099. # Blackwell Munksgaard 2008.

Harsh Mohan, Amanjit Bal andRakhi Aulakh

Department of Pathology, Government MedicalCollege, Chandigarh, India

Prof. Harsh Mohan, Department of Pathology,Government Medical College, Sarai Building,Sector-32A, Chandigarh 160 030, IndiaTel/Fax: 1 91 0172 2665375e-mail: drharshmohan@gmail.com

Accepted for publication December 10, 2007

The primary cutaneous fungal pathogens fall into twogroups: those that tend to cause superficial infectionsand those that cause deep infections.1 Fungi thatusually cause systemic disease and only secondarilycolonize the skin form the third group of cutaneousfungal pathogens. The immune status of the hostalong with other factors may modify the cutaneoushistologic reaction pattern. Superficial fungal infec-tions are generally characterized by hyphae orpseudohyphae and sometimes yeast forms in thekeratin layer of the epidermis and in follicles. Thetissue reaction in superficial fungal infections variesfrom an almost undetectable response to mild focalspongiosis to chronic spongiotic-psoriasiformpattern.The dermis may show a mixed inflammatoryinfiltrate in some cases and fungal hyphae or sporesare only present in cases of follicular rupture. Deep

cutaneous fungal infections are associated withpseudoepitheliomatous hyperplasia and a mixeddermal infiltrate and occasionally dermal fibrosis.Incidental infections with fungi that usually primarilyinvolve other organs show a pattern similar to deepprimary cutaneous fungi.1

Several �clues’ for the histologic diagnosis ofdermatophytoses have been described in the litera-ture; they include neutrophils in the cornified layer ofthe skin, presence of hyphae away from the serum inthe horn, compact orthokeratosis and presence offungal hyphae between two zones of cornified cells,the so called �sandwich sign’.2,3 Meymandi et al.4 re-ported findings of intraepidermal neutrophils in 63%(five of eight) cases of Tinea infection and in 41% ofcases of psoriasis or eczema, a difference, that washowever, not deemed statistically significant. Also,

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there were no specific findings or clues that weremoreprominent in the Tinea cases when compared withthe non-Tinea cases.5

The diagnosis of cutaneous fungal infection isusuallymade on clinical examination of the lesion anddirect microscopic examination of skin scrapings withpotassium hydroxide. Histopathological examinationis rarely required to confirm dermatophytic infec-tions.6 There is often a disparity between the clinicaland histopathological findings.7 Meticulous micro-scopic examination and special stains can help toreach a correct diagnosis. Confirmative evidence andspecies identification comes from fungal culture.However, histopathology provides a relatively rapidand reliable diagnosis along with information regard-ing the tissue reaction and localization of the fungus.

Materials and methods

A retrospective analysis of all skin biopsies received ina year (2004) was done. Clinical information regard-ing each case was retrieved. In all cases the hema-toxylin and eosin (H&E)-stained slides were reviewed.Periodic acid-Schiff (PAS) stain with diastase wasperformed in each case irrespective of whether fungalinfection formed one of the differential diagnoses.Grocott methenamine silver stain was done only in 19cases where clinical/histological suspicion of funguswas strong but PAS stain was negative.8

Results

Four hundred and three skin biopsies were receivedover a period of 1 year. In nine cases, fungal infectionwas one of the clinical differential diagnoses. Becauseof tiny biopsies, no tissue was available for staining in65 cases and PAS stain was performed in 338 cases.On re-examining slides stained with PAS stain,presence of fungi was identified in 34 cases (10%) ofwhich fungal infection had been suspected clinicallyin five cases. In 33 cases, yeast forms with or withoutbuddingwere noted in the keratin layer. In these cases,tissue reaction ranged from mild to dense lympho-mononuclear inflammatory infiltrate.One case showedpresence of septate hyphae in the dermis with sur-rounding giant cell reaction. In 29 cases, fungalinfection was an unexpected finding and in most ofthese it was secondary to some other pathology (23cases; Table 1 and Figs. 1 and 2), while six cases(1.7%) had been concluded as descriptive and thepresence of fungi resulted in a new diagnosis (Figs. 3and 4). Grocott’s staining done in the PAS-negativecases did not reveal any additional findings.

Discussion

In this study, 10% of the cases were positive for fungiafter re-examining PAS-stained slides. A new diagno-

sis was reached in 1.7% cases in which the initialfindings had been non-specific and fungal infectionhad not been clinically suspected. As fungal infectioncan be treated, confirmation or exclusion of fungi ina skin biopsy with non-specific findings will preventfurther unnecessary referrals and help in propermanagement of the patient.In a similar study by Murphy and Donahue,

a retrospective analysis of skin biopsies submitted over3 years was done. A total of 99 cases were studied forfungi with the PAS stain with diastase. Fungi werepresent in seven cases; fungi had been suggested in thedifferential diagnosis of three of these cases but werean unexpected finding in four cases.9 Another studyshowed that only 45%of cases of Tineawere diagnosed

Table 1. Lesions in which fungal infection was an unexpected additionalfinding

Number Diagnosis Number of cases

1 Achrochordon 22 Dermatitis herpetiformis 23 Verruca 14 Pseudoepitheliomatous hyperplasia 15 Nevus sebaceous 16 Neurotized nevus 17 Nodular hidradenoma 18 Pemphigus vegetans 19 Pemphigus vulgaris 110 Lymphocytoma cutis 111 Becker’s melanosis 112 Squamous cell carcinoma 213 Chronic dermatitis 114 Basal cell carcinoma 215 Non-Hodgkin’s lymphoma 116 Actinic lentigo 117 Pigmented solar keratosis 118 Erythema 119 Seborrheic keratosis 1

Fig. 1. Photomicrograph showing the typical changes of pemphigus

vegetans in the form of verrucous vegetative epidermal hyperplasia

with broad papillae and intraepidermal abscesses composed chiefly

of eosinophils (hematoxylin and eosin, 3200).

Mohan et al.

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clinically and only 57% of the PAS-positive casesshowed hyphae onH&E stain. In addition, there wereno significant histologic differences between Tineaand non-Tinea cases.9

For the histologic identification of cutaneousmycosis, a well-fixed biopsy is essential. Artefacts thatmay be mistaken for fungi in tissue sections includeRussel bodies, calcific bodies, karryorhectic debrisand elastic and reticulin fibres. Usually one or more

special stains will be required to identify the fungus.It is recommended that positive controls should bestained with every batch. PAS is a relatively cheap,rapid and easily performed stain for fungi. Grocott’smethenamine silver stain also shows the fungi but theprocedure is longer and more complicated and maygive variable results.1,8

References

1. Hinshaw M, Longley BJ. Fungal diseases. In Elder DE, Elenitsas

R, Johnson BL, Murphy GF, eds. Lever’s histopathology of the

skin. Philadelphia: Lippincott Williams and Wilkins, 2004; 603.

2. Ackerman AB. Histologic diagnosis of inflammatory skin

diseases: an algorithmic method based on pattern analysis, 2nd

ed. Baltimore: Williams & Wilkins, 1997; 295.

3. Gottlieb GJ, Ackerman AB. The �sandwich sign’ of dermato-

phytosis. Am J Dermatopathol 1986; 8: 347.

4. Meymandi S, Silver SG, Crawford RI. Intraepidermal neutro-

phils – a clue to dermatophytosis? J Cutan Pathol 2003; 30: 253.

5. Al Amiri A, Chatrath V, Bhawan J, Stefano CM. The periodic

acid-Schiff stain in diagnosing tinea: should it be used routinely

in inflammatory skin diseases? J Cutan Pathol 2003; 30: 611.

6. Rebell G, Taplin D. Dermatophytes: their recogonition and

identification, 2nd ed. Coral Gables: University of Miami Press,

1970.

7. Requena L, Sanchez Yus E. Invisible dermatoses additional

findings. Int J Dermatol 1991; 30: 552.

8. Swisher BL. Microorganisms. In Bancroft JD, Gamble M, eds.

Theory and practice of histological techniques. London:

Churchill Livingstone, 2002; 325.

9. Murphy JK, Donohue LO. The diagnostic value and cost

effectiveness of routine fungal stains in a dermatopathology

service of a district general hospital. J Clin Pathol 2004; 57: 139.

Fig. 2. Photomicrograph of the same case showing fungal hyphae

highlighted by the periodic acid-Schiff (PAS) stain (arrow) (PAS,

3200). The inset shows PAS-stained fungal hyphae at high power

(PAS, 3400).

Fig. 3. Photomicrograph showing no significant changes in a penile

skin biopsy in a patient who had presented with a whitish patch on

the penis (hematoxylin and eosin, 3200).

Fig. 4. Photomicrograph revealing the presence of fungal hyphae

and spores on periodic acid-Schiff (PAS) stain (PAS, 3200).

Evaluation of skin biopsies for fungal infections

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