|Year : 2019 | Volume
| Issue : 4 | Page : 159-166
Histopathology of hair follicle epithelium in patients of recurrent and recalcitrant dermatophytosis: A diagnostic cross-sectional study
Payal Dilip Patil1, Sushil Pande1, Sadhana Mahore2, Milind Borkar1
1 Department of Dermatology, NKP Salve Institute of Medical Sciences and Lata Mangeshkar Hospital, Nagpur, Maharashtra, India
2 Department of Pathology, NKP Salve Institute of Medical Sciences and Lata Mangeshkar Hospital, Nagpur, Maharashtra, India
|Date of Web Publication||19-Aug-2019|
Dr Sushil Pande
Department of Dermatology, NKP Salve Institute of Medical Sciences and Lata Mangeshkar Hospital, Digdoh Hills, Hingna Road, Nagpur - 440 019, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Recurrent and recalcitrant dermatophytosis is a major evolving health problem in India. Histopathology is important in the diagnosis of recurrent and resistant cases, as it is postulated that hair follicle epithelium is affected, acting as the reservoir leading to recurrence and recalcitrance. Aims: This study aims to study histopathology of hair follicle epithelium in patients of recurrent and recalcitrant dermatophytosis. Settings and Design: A diagnostic cross-sectional study was performed at tertiary care hospital and referral center in central India. Materials and Methods: A 3 mm-sized punch skin biopsy was taken from 108 patients of recurrent or recalcitrant dermatophytosis involving hair follicles. Sections were stained by hematoxylin and eosin (H and E) stain in these patients followed by special stains such as periodic acid–Schiff (PAS) stain and Gomori methenamine silver (GMS) stain in 78 individuals where hair follicle was seen on H and E stain. Results: In H and E stain, surface epidermis fungus was observed in 57/108 patients (52.7%). In hair follicle-positive sections (78), surface fungus was seen in 52.6% with H and E stain, 84.6% with PAS stain, and 91% with GMS stain. H and E was 62.12% sensitive and 100% specific to diagnose fungus when compared with PAS stain and 57.7% sensitive and 100% specific when compared with GMS stain. PAS was 91.5% sensitive and 85.7% specific when compared with GMS stain. Ectothrix infection was the most common form of hair infection observed in 87.2% cases with GMS stain. Simultaneous presence of fungus in hair follicle and stratum corneum was observed in 87.3% of patients. Conclusion: Majority of patients showed affection of hair follicles by dermatophytes in clinically chronic, recurrent, and recalcitrant dermatophytic infections. GMS stain is the acceptable gold standard for detection of fungal elements.
Keywords: Dermatophytosis, hair follicle epithelium, histopathology
|How to cite this article:|
Patil PD, Pande S, Mahore S, Borkar M. Histopathology of hair follicle epithelium in patients of recurrent and recalcitrant dermatophytosis: A diagnostic cross-sectional study. Int J Trichol 2019;11:159-66
|How to cite this URL:|
Patil PD, Pande S, Mahore S, Borkar M. Histopathology of hair follicle epithelium in patients of recurrent and recalcitrant dermatophytosis: A diagnostic cross-sectional study. Int J Trichol [serial online] 2019 [cited 2019 Nov 20];11:159-66. Available from: http://www.ijtrichology.com/text.asp?2019/11/4/159/264730
| Introduction|| |
Superficial fungal infections (SFIs) of the skin are major evolving health problem worldwide leading to significant morbidity. SFIs affect around 20%–25% of the world's population and are associated with interference with daily activities, poor quality of life, and health-care expenditure. Dermatophytosis affects nonliving cornified layers of the skin, hair, and nail.Trichophyton rubrum is known to involve terminal hair follicle progressing to form chronic inflammatory lesions.,
Histopathology is important in the diagnosis of resistant and recurrent cases as it is postulated that hair follicle epithelium is affected in recurrent and recalcitrant dermatophytosis. The presence of fungal elements within hair follicle unit or epithelium has been postulated as the reservoir leading to recurrence and recalcitrance of dermatophytosis. Vellus hair infection in sites other than the scalp may also give rise to recurrent episodes of infection and may maintain chronicity. However, there is no published study from India or abroad where the presence of fungal elements has been studied in recalcitrant dermatophytosis of hair-bearing skin apart from the scalp. Thus, our study focuses on histopathology of recurrent and recalcitrant dermatophytosis with the aim to assess the presence of fungal hyphae in hair follicle epithelium.
| Materials and Methods|| |
A total of 108 patients of recurrent or recalcitrant dermatophytosis were enrolled in the study after approval from the Institutional Ethics committee (IEC) of the institution and after obtaining written informed consent. All the eligible patients of chronic or recurrent or recalcitrant tinea corporis of hairy area presenting to the outpatient department were included. Patients not consenting to participate in the study or not willing for skin biopsy, patients of nonrecurrent and treatment-responsive dermatophytosis, patients who were undergoing systemic treatment or topical antifungal therapy or had taken treatment in the last 1 month or 2 weeks, respectively, for fungal infections, patients of dermatophyte infection of nonhair-bearing area like palms and soles, and patients of tinea cruris alone were excluded. Recurrent dermatophytosis was defined as reoccurrence of the dermatophyte infection within few weeks (<6 weeks) of completion of treatment associated with clinical resolution., Recalcitrant dermatophytosis means patient not responding to conventional or extended therapies for a period of 2 months. Chronic dermatophytosis was described as patients who had suffered from the disease for more than 6 months to 1 year, with or without recurrence, in spite of being treated.,
A detailed history and thorough clinical examination were done. A 3 mm-sized punch skin biopsy was taken under local anesthesia under all aseptic precautions from the active margin of fungal infection patch including hair follicle. Specimen was formalin fixed and was embedded in paraffin and was then subjected to serial oblique sectioning to include upper part of hair follicle, i.e., follicular infundibulum where chances of finding the fungus were maximum due to the fact that this is keratinized zone of the hair follicle. Sections were first stained by Hematoxylin and Eosin (H and E) stain. Histomorphological changes in the skin and the presence of surface fungus, i.e., fungus in stratum corneum were recorded from all sections. Furthermore, all sections were screened to look for the presence of hair follicle. Specimens of sections showing hair follicle were further sectioned, additionally stained with special stains such as periodic acid–Schiff (PAS) stain and Gomori methenamine silver (GMS) stain, and were analyzed for sensitivity and specificity comparing positivity for surface fungus. Then, fungal elements in hair follicles were observed in all three stained sections in 78 patients, and proportions for positivity for fungus with all 3 stains were evaluated.
Evaluation of histopathology sections was done with special emphasis on hair follicle pathology. Various histomorphological changes were recorded by a dermatopathologist with reference to histologic pattern, type of cellular infiltrate, presence or absence of “Sandwich sign,” and identification of the fungus. The presence of fungal elements in stratum corneum and the hair follicle unit, in two forms, i.e., ectothrix and endothrix, was evaluated with H and E stain first followed by PAS and GMS stain. This was done separately by the dermatopathologist in different sittings to avoid observer bias. Findings were recorded to compare different stains.
All the data were compiled. Data analysis was done using appropriate statistical test. Histologic pattern or histomorphological findings were presented in proportions. Positivity for fungus by different stains was recorded in proportions or percentages. Sensitivity and specificity analysis were also done for H and E stain and PAS stain using GMS stain as the gold standard.
Data were expressed as percentage and mean ± standard deviation. Kolmogorov–Smirnov analysis was performed for checking linearity of the data. Student's t-test was used to check the significance of difference between two parameters in parametric data, and Mann–Whitney U-test was used to check the significance of difference between two parameters in nonparametric data. Fischer's exact test or Chi-square test was used to analyze the significance of difference between frequency distribution of the data. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using standard formulae. P <0.05 was considered as statistically significant. SPSS © software for Windows™ Vs 17, IBM™ Corp NY and Microsoft Excel™ 2007, Microsoft ® Inc., USA was used to perform the statistical analysis.
| Results|| |
The most common age group in our study was 26–30 years of age (23.1%) followed by 21–25 years of age group (20.3%) and 31–35 years group (17.6%).
Out of 108 patients, 25.9% were females and 74% were males. Male to female ratio was 2.9:1. Duration of disease among study participants varied from 3 months to 1 year. Maximum individuals (51.8%) presented at 4–6 months of their symptom, 34.2% individuals at 7–9 months, 10.2% patients at 3 or less months whereas only 3.7% patients were having symptoms for more than 10 months' duration. The most common site involved was found to be buttocks in 52.78%, to be followed by abdomen (48.15%), back (31.5%), and thighs (27.78%). Axilla, arms, and chest were involved in 17.6% each. Shoulder (1.8%) and knee (0.93%) were the least commonly involved sites. Among study population, maximum number of cases were recalcitrant, i.e., 37.04%, followed by chronic (33.3%) and remaining were having recurrence (29.63%). Topical steroid abuse was observed in 53.7% of individuals, 11.11% patients were immunosuppressed due to use of systemic corticosteroids (3.7%) and diabetes mellitus (7.4%). Atopy was observed in 10.2% of total individuals and 1 individual (0.93%) had ichthyosis vulgaris.
Histomorphological changes in 108 patients of dermatophytosis
The most common histomorphological change observed was superficial perivascular infiltrate, observed in 70.3%, followed by hyperkeratosis in 70.3%, acanthosis in 55.5%, spongiosis in 30.5%, and prominent papillary dermal edema in 19.4% [Figure 1] and [Figure 2].
|Figure 2: Histomorphological changes; H and E-stained oblique section of skin showing: (A) intraepidermal (intracorneal) neutrophils, (B) hyperkeratosis, (C) parakeratosis, (D) acanthosis, (E) superficial perivascular infiltrate ×10 (left), ×40 (right). (F) perifollicular infilitrate|
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Surface (epidermis) fungus detected through various investigations
Initially, the presence of stratum corneum fungus was assessed with the help of H and E stain for a total of 108 patients, of which surface fungus was seen in 52.7% individuals (57/108). Special stains were performed for 78 sections; those were positive for hair follicle, of which surface fungus was seen in 84.6% with PAS stain and 91% with GMS stain. In 78 such sections, H and E stain could detect surface fungus in only 52.6% patients (41/78) [Figure 3] and [Figure 4].
|Figure 3: Surface fungus (fungus in epidermis) observed in different stains|
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|Figure 4: Surface fungus; oblique section of skin showing intracorneal fungal elements in H and E (left), periodic acid–Schiff (middle), and Gomori methenamine silver (right) staining and inside hair follicle in H and E-stained section (black arrow), (×40)|
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Diagnostic significance of hematoxylin and eosin stain compared to periodic acid–Schiff and Gomori methenamine silver stains and of periodic acid–Schiff stain compared to Gomori methenamine silver stain
H and E was found to be 62.12% sensitive and 100% specific to diagnose fungus when compared with PAS stain (P = 0.0001) and 57.7% sensitive and 100% specific to diagnose fungus when compared with GMS stain (P = 0.004). Diagnostic significance of PAS stain compared to GMS stain to detect fungus in skin was assessed (P = 0.08). PAS was found to be 91.5% sensitive and 85.7% specific to diagnose skin fungus.
Ectothrix versus endothrix infection in recurrent and recalcitrant dermatophytosis
Ectothrix form of hair infection was observed in many study cases, with maximum positivity in GMS stain with 87.2%. PAS stain could diagnose 78.2% of ectothrix infections; whereas, H and E showed positivity for only 23.1%. Total number of patients with endothrix form of infection was lower than that of ectothrix. GMS showed endothrix in 26.9% patients. PAS had positivity for 19.2% infections, and H and E had lowest positivity of 6.4% [Figure 5], [Figure 6], [Figure 7], [Figure 8].
|Figure 5: Hair follicle fungus; bar chart showing percentages of two forms of hair follicle fungus, i.e., ectothrix and endothrix|
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|Figure 6: Hair follicle fungus; oblique sections of hair follicle showing fungal spores and hyphae; lightly pinkish in H and E stain (left), bright reddish to purplish in periodic acid–Schiff stain (middle) and Gomori methenamine silver-stained (right) oblique section showing black-stained fungal spores and hyphae in ectothrix and endothrix forms (black arrows) ×100|
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|Figure 7: Hair follicle fungus; oblique sections showing hyperkeratosis in H and E stain (left), fungal hyphae inside hair follicle in periodic acid–Schiff stain in reddish to purplish color (middle) and fungal hyphae and arthrospores in stratum corneum and hair follicle in Gomori methenamine silver stain in black color (right) (black arrows) ×100|
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|Figure 8: Hair follicle fungus; oblique sections showing fungal hyphae and spores inside hair follicle in H and E stain (left), periodic acid–Schiff stain (middle), and Gomori methenamine silver stain (right) (black arrows) ×100|
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Association of hair follicle fungus with surface fungus (Gomori methenamine silver stain as gold standard)
Association of hair follicle fungus with surface fungus was assessed using Fisher's exact test. Simultaneous presence of fungus in hair follicle and stratum corneum is observed in 87.3% of patients, which means hair follicle is affected simultaneously in majority of chronic/resistant/recalcitrant cases of dermatophytosis. However, association was not statistically significant (P = 0.633).
| Discussion|| |
Fungal infections account for 3%–5% of all cases in dermatology practice. The most frequent causative agents among fungi are dermatophytes. Histopathological analysis of specimens is required in all cases of deep and/or SFI, for example, dermatophytosis or tinea with negative direct microscopic and culture results. We conducted this diagnostic cross-sectional study at our tertiary care hospital and referral center in central India, for the period of 2 years, from October 2016 to September 2018. The present study was undertaken to find out the presence of fungal elements in the hair follicle epithelium in patients of recurrent and recalcitrant dermatophytosis and to determine sensitivity and specificity of H and E stain over special stains, i.e., PAS stain and GMS stain.
The most common associated predisposing condition was found to be topical steroid abuse (53.7%) followed by atopy in 10.19% of patients. This can be due to unchecked availability of inexpensive and irrational corticosteroid–antifungal–antibacterial combinations which are sold over the counter. These drugs are being prescribed rampantly by general physicians or even self-medicated by the patients as a treatment for virtually each kind of dermatosis. Immunosuppression due to diabetes mellitus, systemic steroids/immunosuppressants, and HIV/AIDS was seen in 7.41%, 3.7%, and 0% patients, respectively. These findings are supported by Bishnoi et al.
In our study, we adopted oblique sectioning of paraffin-embedded blocks containing skin biopsy specimens. This was in contrast to vertical sectioning as done regularly for skin biopsy specimens. The oblique sectioning was adopted after we performed a pilot study at our institute in which we found that the hair follicle was missing in majority of skin biopsy specimens with vertical sectioning. Oblique sectioning was aimed at getting a part of hair follicle above Adamson's fringe, a lowest site of zone of keratinization, where the chances of finding fungal elements are maximum as the fungus thrives on dead keratin for its growth. Thus, oblique sectioning ensured the presence of hair follicle above Adamson's fringe along with epidermal surface to look for the presence of fungus in stratum corneum.
In the present study, total number of patients was 108 in whom skin punch biopsy was performed. H and E stain was done for all 108 patients. However, special stains, i.e., PAS and GMS stains could be done only for 78 patients' specimen in which hair follicle was observed due to lack of resources and availability of kit for PAS and GMS stains. This was in accordance with the primary objective of our study, i.e., to evaluate the presence of fungus in hair follicle and its correlation with occurrence of fungi in the stratum corneum. Therefore, we decided to perform GMS and PAS stain in 78 patients.
Histomorphological characteristics in dermatophytosis
We did histomorphological analysis in all 108 patients with the help of H and E staining. The most common histomorphological finding was superficial perivascular infiltrate as observed in 77.7% cases followed by hyperkeratosis (70.3%), acanthosis (55.5%), spongiosis (30.5%), and prominent papillary dermal edema (19.4%). This can be correlated with the study by Kaliyamoorthy and Srinivasan, who observed various such histomorphological changes. Furthermore, a study by Gocev and Damevska  reported that majority cases of SFIs may present with histomorphological changes such as perivascular infiltrate, hyperkeratosis, acanthosis, and dermal edema, and also, spongiotic, psoriasiform, vasculopathic, and folliculitis/perifolliculitis histopathological reaction patterns can be observed. The observations of histomorphological changes in a study conducted by Al-Amiri et al. using PAS stain were closely correlating with our study.
Sandwich sign is the presence of fungal hyphae between two zones of stratum corneum with orthokeratotic lamellae above and parakeratotic lamellae beneath with formation of fissure in between. Fungal elements were seen within this fissure. This was also observed by Gottlieb and Ackerman. In our study, sandwich sign was observed in 11% of patients. Although this is a very specific sign, this could be seen in very few patients which is comparable to observation of Al-Amiri et al. who could observe sandwich sign in 12% patients. Thus, histomorphological changes are of great significance in diagnosing dermatophytic infection, and infection should be suspected when there are histomorphological changes in skin even when fungal elements are not evident in H and E-stained sections.
Identification of fungus in surface epidermis (stratum corneum)
In tissues of an infected host like that of a skin, dermatophytes do not form conidia. In the host tissue, they occur in two forms: hyaline, broad, and branched septate hyphae and small arthrospores as described by Weedon  and Chandler et al. Both of these forms were observed in our study sections. We attempted to identify these two above-described forms of the fungus, i.e., hyphae and arthrospores, in our study. In our study, while surface fungus was detected by H and E in only 52.6% patients, PAS was able to diagnose 84.6% patients with fungal infection and 91% patients were diagnosed with GMS. According to Gocev and Damevska, two factors make the histopathological diagnosis of dermatophytoses very difficult. First, hyphae cannot be seen easily in H and E-stained sections as in their study, only 25% of PAS-positive cases presented with visible hyphae in H and E-stained sections. Second, in most of the biopsies, dermatophytosis is suspected rarely. Thus, for reducing false-positive cases, special stains are needed to be done. Al-Almiri et al. reported that tinea was the differential diagnosis after H and E staining only in 45% of PAS-positive cases of tinea. Similarly, in a study by Mohan et al., only 57% of the PAS-positive cases of tinea showed hyphae on H and E stain. In a study by Reza Kermanshahi and Rhatigan, 4 of 30 cases were initially negative with original PAS stain, but after performing GMS stain, the fungal organisms were highlighted. Similarly, D'Hue et al. reported that GMS highlighted fungal hyphae in all PAS-positive cases and also in 5 out of 51 PAS-negative cases. In one of the PAS-negative cases, the GMS stain highlighted numerous fungal hyphae. GMS stain could detect significantly more cases of onychomycosis than PAS stains (35% vs. 28%, P = 0.0253).
Identification of fungus in hair follicle (ectothrix + endothrix)
Ectothrix form of hair infection was seen in many study cases, with maximum (87.2%) positivity in GMS stain. PAS stain could diagnose 78.2% of ectothrix infections; whereas, H and E showed positivity for only 23.1%. Total number of patients with endothrix form of infection was lower than that of ectothrix. GMS showed endothrix in 26.9% patients. PAS had positivity for 19.2% infections, and H and E had lowest positivity of 6.4%. Similarly, in a study by Farias et al. which included 17 children, ectothrix hair shaft invasion was seen in 13 children and endothrix in 4 children. Lower proportion of endothrix in this study could be due to loss of hair shaft from specimen while sectioning. However, there is no study published in the current literature, to the best of our knowledge, which describes the presence of fungal elements as ectothrix or endothrix in nonscalp dermatophytosis. In tinea capitis, it is easier to differentiate between endothrix and ectothrix because of deeply located terminal hair with intact hair shaft within the scalp biopsy. However, in superficially located vellus hair of nonscalp hairy skin, chances of finding intact hair shaft within follicular canal are lesser. In our study, hyphae and arthrospores were observed above isthmus, infundibulum, and keratinized portion of inner root sheath. Involvement of these areas suggests cyclic reinfection of the same hair during the next cycle. This can be responsible for chronicity as observed in a study by Lee and Hsu.
Sensitivity and specificity analysis
In the present study, diagnostic significance of H and E stain as compared to PAS stain was assessed. H and E was found to be 62.12% sensitive and 100% specific to diagnose fungus with PPV of 100% and NPV of 32.43%. When diagnostic significance of H and E stain compared to GMS stain was assessed, H and E was found to be 57.7% sensitive and 100% specific to diagnose fungus with PPV of 100% and NPV of 18.91%. This once again demonstrates superiority of GMS stain vis-a-vis H and E stain and PAS stain. Furthermore, when diagnostic significance of PAS stain compared to GMS stain to detect fungus in skin was assessed, PAS was found to be 91.5% sensitive and 85.7% specific to diagnose skin fungus with PPV of 98.48% and NPV of 50%.
Consistent with our findings, Heaton et al. evaluated the utility of routine staining, histochemical stains, and frozen section for fungal element identification. They found that results of GMS and PAS were 84% concordant in findings. Sensitivity for fungal detection was found to be 56% and 85% for PAS and GMS, respectively. The false-negative rate of fungal detection was 15% for GMS, and for PAS, it was 44%, suggesting that GMS was more sensitive. D'Hue et al. reported that the GMS stain is considered as the most sensitive histochemical method for the detection of fungal organisms in deep infections. According to Weinberg et al., the sensitivity was 92%, specificity was 72%, PPV was 89.7%, and NPV was 77% for PAS stain.
It has been reported by Haque  that the PAS stain performs almost as well as GMS while screening of fungi, and it actually demonstrates fungal morphology better than the GMS stain. PAS can stain degenerated fungi that may not be visible on H and E stain. However, in our study, we found GMS to be more helpful than PAS in identifying fungal hyphae. While H and E and PAS stains required higher magnifications (×100) for differentiating and correctly observing fungal elements, GMS stain highlighted fungal elements on lower and intermediate magnifications (×10 and ×40). Furthermore, GMS stain showed fungal elements in a better contrast than other two stains. PAS stain highlighted fungus in a dark pink to reddish color which was better seen than that in H and E stain, which stained fungi very light. This was correlated with the findings in a study by D'Hue et al. which showed that GMS stain is qualitatively as well as quantitatively better than PAS stain in diagnosing fungus. This suggests that GMS stain is the easiest to interpret for identifying fungus, followed by PAS stain. Whereas, identification of fungal elements is also possible with the H and E stain on the basis of tissue inflammatory reaction. This is in concordance with observations by Kaliyamoorthy and Srinivasan.
In this study, statistical association of hair follicle fungus with surface fungus was assessed using Fisher's exact test. Simultaneous presence of fungus in hair follicle and stratum corneum is observed in 87.3% of patients, which means that hair follicle is affected in majority of chronic/resistant/recalcitrant cases of dermatophytosis. However, association was not statistically significant (P = 0.633). This shows the possibility that hair follicle may get infected during longstanding dermatophytosis of hair-bearing area and may act as a reservoir. This also states the importance of using antifungal medications both topical and systemic, which has adequate penetration and potential to have sufficient concentration within hair follicle to kill this reservoir fungus. As this was cross-sectional and descriptive study, we could not evaluate the outcome of antifungal treatment and its effect on so-called “reservoir fungus” within hair follicle.
| Conclusion|| |
Affection of the hair follicle by dermatophytes was seen in majority of clinically chronic, recurrent, and recalcitrant dermatophytic infections. Thus, hair follicle is affected along with surface epithelium in majority of cases of recurrent or recalcitrant dermatophytosis. This hair follicle fungus may act as a reservoir and may be responsible for resistance, recurrence, and chronicity of the infection. Histopathology is mandatory for ensuring diagnosis and for further management in chronic, recalcitrant cases as it helps in confirming the diagnosis and extent of spread of fungal elements to other structures like hair. Histopathological clues and signs such as hyperkeratosis and sandwich sign have very important role in the diagnosis of dermatophytosis as in the Indian scenario. Special stains such as PAS and GMS stains are desirable but may not be possible in every center due to the lack of resources and skills. H and E is a basic stain for detection of fungi and histopathological changes in the skin and adnexa whereas PAS stain is better for identification of fungal elements, as it shows fungi darker. GMS stain is the acceptable gold standard for detection of fungal elements, as it provides much better contrast and fungi can be seen at lower magnification. This is especially important when facilities for fungal cultures are not universally available and positivity rates in fungal cultures are minimal.
Our study was the first study from India which evaluated the presence of fungus in hair follicle in patients of chronic recurrent and recalcitrant dermatophytosis. However, more such studies of adequate sample size are needed for the same. This will help us to formulate treatment strategies for eradication of dermatophytes from hair follicle epithelium that has been cited as the common cause of chronicity and recalcitrance of dermatophytosis in India.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]