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A study to compare and classify etiological agents of onychomycosis
G Model INJMS 147 No. of Pages 5
Indian Journal of Medical Specialities xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Indian Journal of Medical Specialities journal homepage: www.elsevier.com/locate/injms
Original article
A study to compare and classify etiological agents of onychomycosis Prabhav Aggarwal, Bineeta Kashyap* , Jitu Mani Kalita Department of Microbiology, Maulana Azad Medical College & Associated Hospitals, New Delhi, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 August 2017 Received in revised form 5 August 2017 Accepted 5 August 2017 Available online xxx
Background: Onychomycosis, the condition that was always regarded as merely a cosmetic problem, now has become an important medical issue. Etiological prevalence of onychomycosis varies subject to the population demography, geographical location or the mycological technique used for the diagnosis. Aim: This study aims to identify and classify the fungal etiology of onychomycosis from a tertiary care hospital of north India over a duration of two years. Methods: The study included 169 clinically suspected patients of onychomycosis. The fungal etiology was determined by direct examination in 20% KOH after overnight incubation at 37 C and culture on Sabouraud’s Dextrose Agar. Results: 135 (79.9%) out of the total patients were confirmed cases of onychomycosis as per the diagnostic criteria. Hyaline hyphomycetes were the most common group of fungi isolated (50.0% of all fungal isolates) with only 15.4% isolates of the classical dermatophytes. A positivity of 18.3% and 78.7% by KOH examination and culture respectively was reported. .A sensitivity and specificity of 21.48% and 100% was found for KOH positivity; though culture was 98.52% sensitive and 82.35% specific. Conclusions: Hyaline hyphomycetes are a common cause of onychomycosis. KOH examination can not be relied upon for diagnosis of onychomycosis, for which fungal culture appears to be an appropriate choice along with KOH examination. © 2017 Published by Elsevier, a division of RELX India, Pvt. Ltd on behalf of Indian Journal of Medical Specialities.
Keywords: Onychomycosis Fungus Etiology
1. Introduction Onychomycosis remains an extremely obstinate and frequent fungal infection of the toenails or fingernails that can hugely impact the patients’ physical, social or emotional quality of life. The condition, that was always regarded as merely a cosmetic problem unworthy of any significant medical attention; now has become an important medical issue due to recurrence, chronicity, probability of transmission, therapeutic difficulties or association with immunosuppression [1]. Infected nails also are a potential reservoir of fungi which can spread to other sites of the patient [2]. Though the prevalence of onychomycosis varies subject to the population demography, geographical location or the mycological technique for diagnosis, an estimated 3–26% of the global population are affected by this significant health problem which accounts for approximately half of all onychopathies [3,4]. In a previous study done at our tertiary care centre the prevalence of
* Corresponding author at: Flat C-402, Vimal CGHS Ltd Plot 3, Sector 12, Dwarka, New Delhi-78, India . E-mail addresses: [email protected] (P. Aggarwal), [email protected] (B. Kashyap), [email protected] (J.M. Kalita).
onychomycosis was confirmed in as high as 45% of the analyzed patients [5]. Dermatophytic filamentous fungi by far remain the predominant etiology in most of the cases of onychomycosis, though yeasts and non-dermatophyte molds (NDM) are now implicated as the other common causes accounting for approximately 8–11% and 1.45–17.6% cases respectively [3,6,7,8].NDMs are increasingly reported as secondary or emerging causes of onychomycosis triggered by various factors like traumatic nails, comorbidities leading to compromised circulation or immunocompromised states [9]. Despite optimal management by the current antifungal regime, clinical as well as mycological cure is often unattainable in all onychomycosis cases [10]. The reasons for the limited success in cure rate could be many, some of which are incorrect identification of the fungal pathogen, co-existent co-morbidity, inherent characteristics of the nail which worsen the response to treatment or a plethora of host related factors [1]. Several non infectious causes of nail dystrophies mimicking onychomycosis further complicate the diagnosis [11]. Onychomycosis can be divided into several categories based upon the site involved, clinical pattern, mode of invasion or fungal etiology [12]. Such a classification aids in initiating a correct diagnosis and predicting the response to treatment and its prognosis. A laboratory confirmed clinical
http://dx.doi.org/10.1016/j.injms.2017.08.002 0976-2884/© 2017 Published by Elsevier, a division of RELX India, Pvt. Ltd on behalf of Indian Journal of Medical Specialities.
Please cite this article in press as: P. Aggarwal, et al., A study to compare and classify etiological agents of onychomycosis, Indian J Med Spec. (2017), http://dx.doi.org/10.1016/j.injms.2017.08.002
G Model INJMS 147 No. of Pages 5
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P. Aggarwal et al. / Indian Journal of Medical Specialities xxx (2017) xxx–xxx
diagnosis with the identification of the underlying fungal pathogen further helps to exclude non fungal etiology or a mixed infection and hence guide appropriate antifungal treatment especially in less responsive cases of onychomycosis. Onychomycosis continues to be a diagnostic dilemma for a dermatologist and a microbiologist as the etiology spectrum is diverse and the techniques for diagnosis are still evolving. This study aims to identify and classify the fungal etiology of onychomycosis from a tertiary care hospital of north India over a duration of two years. 2. Materials and methods This was a retrospective study spanning a period of two years (January 2014–December 2015) conducted in the Mycology Laboratory, Department of Microbiology of a tertiary care hospital in New Delhi, India. The study included 169 patients, who presented with the signs and symptoms representative of the fungal infection of either toe or finger nails (onychomycosis). The affected area was cleaned with 70% ethanol and allowed to dry. Nail clippings along with the deep sub-ungual material were collected from the most affected nails. The specimens were transported to the Mycology laboratory as soon as possible in a dry, sterile, screw capped container. Nail specimens were incubated overnight at 37 C in 20% KOH; next day wet mounts were prepared and observed under 100 x and then 400 x light microscopy magnification to look for presence of fungal hyphae and/or yeast cells. “Specimens were inoculated onto two sets of sealed tubes: (i) Sabouraud’s Dextrose Agar and (ii) Sabouraud’s Dextrose Agar with chloramphenicol (0.05 mg/ml); one tube of each set was incubated at 25 C and another at 37 C.” The culture tubes were examined twice a week for any suspected fungal growth. Tubes showing no growth were discarded after a period of 6 weeks. Isolates were identified by the morphologic characteristics of the colony growth, pigment production and microscopic examination of the Lactophenol cotton blue (LPCB) preparation. In case identification was not possible, slide cultures were put up using corn meal agar to stimulate sporulation and incubated at 25 C for 4–7 days. Subsequently, LPCB preparations were made and observed under light microscope. Onychomycosis was diagnosed when fungal elements were seen on 20% KOH preparation, and/or repeated cultures or growth in multiple tubes yielded same fungal agent. Repeat samples were collected and processed when direct KOH wet mount was negative and fungal growth was obtained in a single tube, raising suspicion of contamination. Other fungal growths were labeled as contaminants and treated as false positive cultures for statistical calculations. 2.1. Statistical analysis The data was presented as percentages and proportions. 95% confidence intervals were calculated wherever applicable. Fischer’s exact test was used for testing the statistically significant differences; p values of <0.05 were considered statistically significant. All calculations were done using SPSS version 23.0. 3. Results A total of 169 patients were involved retrospectively in the study, including 98 (58.0%; 95% CI = 50.45%–-65.17%) males and 71 (42.0%; 95% CI = 34.83%–49.55%) females. Majority of patients were adults (>18 years of age), with only 11 (6.5%; 95% CI = 3.55%– 11.40%) subjects being under 18 years of age; maximum patients belonged to 20-30 years of age group (Fig. 1). Majority of patients (60.4%) had fingernail infection, 37.8% had toenail infection, while 1.8% patients had infections of both toenail and fingernail.
Fig. 1. Age-wise distribution of male and female patients.
KOH wet mount of the nail clippings and material collected deep under nail bed were prepared; out of 169, a total of 31 (18.3%; 95% CI = 13.19%–24.90%) specimens showed presence of fungal elements (fungal hyphae and/or yeast cells). Out of these 31 specimens, 29 turned out to be positive on culture; while 2 showed no growth even after 6 weeks of incubation (Table 1). Twelve KOH positive specimens yielded growth of dermatophytes, while remaining showed non-dermatophytic growths. A total of 133 (78.7%; 95% CI = 71.89%–84.23%) specimens showed growth on SDA, some showing co-infections of two or more fungi; with a total of 156 fungal isolates. Thus, 135 (79.9%) patients were diagnosed as cases of onychomycosis. KOH mount preparation had a low sensitivity of 21.48% as compared to the culture method (98.52%). As per the criteria, six cultures were labeled as contaminants/false positives. Isolated fungi were classified as shown in Table 2. Hyaline hyphomycetes were the most common group of fungi isolated, constituting 50.0% (78/156) of all fungal isolates. Aspergillus niger and A. flavus were the most common fungi in this group as well as among all groups. Twenty-four (15.4%) isolates of the classical dermatophytes were cultured, Trichophyton spp. (T. schoenleinni, T. verrucosum, T. mentagrophytes and T. rubrum) being the most common. Members of Class Zygomycetes (now Class Glomerulomycetes) and the dematiaceous fungi were relatively less common, constituting 13.5% and 9.6% of fungal isolates, respectively. Rhizopus spp. and Mucor spp. (9 isolates of each) were common fungi of Class Zygomycetes. Dematiaceous fungi were least common of all the groups, constituting 9.6% of all fungal isolates. Among yeast-like fungi, non-albicans Candida (14 isolates) were isolated more frequently as compared to Candida albicans (3 isolates). A single isolate of Geotrichum spp. was also identified. Though, there were differences between males and females with respect to rates of isolation of these fungi (dermatophytes and yeast-like fungi being more common in males), the differences were not statistically significant (Table 2). Fig. 2 depicts the month-wise rates of isolation of various groups of fungi; it is easily discernible that these rates were highest during the months of July to September. Highest isolation of hyaline hyphomycetes, dermatophytes and zygomycetes were seen during the months of July, August and October, respectively. Twenty two specimens showing simultaneous growths of two fungi were considered as co-infections. The details are presented in Table 3. 4. Discussion Onychomycosis is increasingly being recognized as a common problem in dermatological practice. Dermatophytes, yeasts as well as molds have all been implicated as potential pathogens in
Please cite this article in press as: P. Aggarwal, et al., A study to compare and classify etiological agents of onychomycosis, Indian J Med Spec. (2017), http://dx.doi.org/10.1016/j.injms.2017.08.002
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Table 1 Comparison between KOH wet mount preparation and culture on SDA. Onychomycosis Present
Onychomycosis not present
Total
% agreement between KOH wet mount preparation and culture on SDA
KOH Positive KOH negative
29 106 135
0 34 34
29 140 169
32.3%
Culture positive Culture negative
133 2 135
6 28 34
139 30 169
Sensitivity Specificity
KOH Culture KOH Culture
21.48 98.52% 100 82.35%
onychomycosis [13]. In the present study the isolated fungi were classified under five large groups based on their morphological and/or taxonomical classification. Dermatophytes, the filamentous fungi that are able to digest and obtain nutrients from keratin (skin, hair and nails), affect the outermost layers of these tissues without invading the living tissue and comprise of three main genera: Trichophyton, Microsporon and Epidermophyton. Hyaline hyphomycetes, possessing colorless septate hyphae often producing variously colored conidia, are mostly saprophytic in nature causing opportunistic infections in predisposed individuals. Zygomycetes (now renamed as Class Glomeromycetes) differs from hyaline hyphomycetes in possessing hyphae which are almost nonseptate. The group of Dematiaceous fungi is characterized by dark colored hyphae with dark colonies (olive, brown, gray or black in color) due to presence of melanin in their cell walls. Yeast like fungi
differ from all the rest in being unicellular that reproduce by budding; these buds may remain attached to the parent cell resulting in formation of pseudohyphae. In our study 135 (79.9%) out of the total patients were confirmed cases of onychomycosis as per the diagnostic criteria. Fungi were isolated in 78.7% specimens with a total of 156 fungal isolates including co-infections of two or more fungi. Fungal isolation as low as 20% has been reported in a previous study [14]. The authors have suggested that non viable cultures or faulty sample collection or culture inoculation techniques could possibly account for such a low percentage positivity of fungal culture. Hyaline hyphomycetes were the most common group of fungi isolated (50.0% of all fungal isolates) with only 15.4% isolates of the classical dermatophytes. The relatively less common members Zygomycetes, yeast like fungi and the dematiaceous fungi
Table 2 Etiological Classification of Fungal agents isolated from cases of onychomycosis.
Dermatophytes: Trichophyton mentagrophytes Trichophyton rubrum Trichophyton schoenleinii Trichophyton verrucosum Trichophyton violaceum Trichophyton tonsurans Microsporum nanum Total Hyaline hyphomycetes Aspergillus niger Aspergillus flavus Aspergillus fumigatus Aspergillus terreus Penicillium spp. Fusarium spp. Scopulariopsis spp. Scytalidium dimidiatum Total Zygomycetes/ Glomeromycetes Rhizopus spp. Mucor spp. Cunninghamella bertholletiae Syncephalastrum racemosum Total Dematiaceous fungi Alternaria spp. Curvularia spp. Bipolaris spp. Total Yeast-like fungi Candida albicans Non albicans Candida Geotrichum spp. Total Total fungi isolates No growth
Male (n=98)
Female (n=71)
P value
Total
% (of total fungal isolates, n=156)
4 3 2 4 1 0 2 16
0 1 3 1 2 1 0 8
0.0774
4 4 5 5 3 1 2 24
2.6 2.6 3.2 3.2 1.9 0.6 1.3 15.4
16 13 2 1 5 2 0 1 40
18 8 3 0 7 1 1 0 38
0.1190
34 21 5 1 12 3 1 1 78
21.8 13.5 3.2 0.6 7.7 1.9 0.6 0.6 50.0
4 6 0 0 10
5 3 2 1 11
0.3490
9 9 2 1 21
5.8 5.8 1.3 0.6 13.5
5 2 1 8
5 2 0 7
0.7867
10 4 1 15
6.4 2.6 0.6 9.6
1 12 0 13 87 23/98
2 2 1 5 69 13/71
0.1249
3 14 1 18 156 36/169
1.9 9.0 0.6 11.5 100 21.3
0.5608 0.4523
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Fig. 2. Month-wise distribution of onychomycosis cases due to various fungal groups.
constituting 13.5%, 11.5% and 9.6% of fungal isolates, respectively. Several authors have reported dermatophytes and most studies have shown dermatophyte as the predominant cause of onychomycosis (29.5–50%) when compared with yeasts (5.6–43.3%) and non-dermatophyte moulds (8.2–13.6%) [14–17], several others report candida species as the most common cause (40.7–46%) followed by dermatophytes (38–43%) and non-dermatophyte molds (11–14%) [18,19]. Another recent report from central part of India reported culture positivity in 24 out of 44 patients contributing to 12.5% dermatophytes, 8.3% candida species and 75% Non-Dermatophytic Moulds. The same study found KOH and culture positivity in 45.5% and 38.6% of 44 patients respectively with a sensitivity, specificity, PPV and NPV of 76.5%, 74%, 65% and 83.3% which rose to 100%, 74.7%, 70.8% and 100% respectively when combined with culture [20]. Our study reported a positivity of 18.3% and 78.7% by KOH examination and culture respectively. A
sensitivity and specificity of 21.48% and 100% was found in our study for KOH positivity; though culture was 98.52% sensitive and 82.35% specific. Manjunath Shenoy et al and Das et al, however report variable rates of positivity by direct microscopy and culture ranging from 32.94% to 53% and 35% to 49.4% respectively [21,22]. Another recent Indian study done from a tertiary care hospital in Delhi reports 66.1% positivity and 68% sensitivity for KOH examination [23]. A study from Uttar Pradesh, India reported KOH- and culture-positivity in 76.76% and 37.37% cases, respectively [24]. Direct microscopic examination, when done under experienced hands, along with culture can prove an efficient tool in the epidemiology of onychomycosis and help hasten the management of such patients [22]. While all yeasts identified were Candida albicans and Aspergillus species being the commonest NDM, Trichophyton rubrum and Trichophyton mentagrophytes were reported to be the predominant dermatophytic species in a previous Indian study [20]. In our study, Trichophyton schoenleinii and Trichophyton verrucosum were slightly more common; while, non albicans Candida out-numbered Candida albicans. Our study found NDM accounting for 88.46% of all fungi isolated, though a study reports NDM prevalence between 1.45% and 17.6% [25]. As far as the population dynamics and the seasonal trend in our study is concerned only 6.5% of the total cases were less than 18 years of age and the month-wise analysis of the fungi isolated suggested highest rates during the months of July to September. A study done on onychomycosis in children reported a relative percentage of 2.3% of pediatric (under 18 years of age) to adult onychomycosis. The same study reported a higher incidence of onychomycosis in winter months [26]. Smaller available surface, increased nail growth rate or reduced external exposure were some of the factors suggested by the authors for the lower prevalence in this group. Twenty-two instances of multiple fungi were identified in a total of 135 defined cases of onychomycosis amounting to 16.3% of mixed infection along with 21.3% sterile growth among 169 cases. 0.1% and 10.9% cases of mixed growth and no mycological growth was reported in a series covering 80,396
Table 3 Details of Co-infections evidenced on culture. Fungal groups
Details of co-infections seen
Hyaline hyphomycetes + yeast like fungi (n = 5)
1. 2. 3. 4. 5.
Zygomycetes + yeast like fungi (n = 2)
1. Mucor spp., Non albicans Candida 2. Rhizopus, Candida albicans
Zygomycetes + Hyaline hyphomycetes (n = 5)
1. 2. 3. 4.
Mucor spp., Aspergillus niger (n = 2) Syncephalastrum racemosum, Aspergillus niger Mucor spp., Aspergillus fumigatus Rhizopus spp., Aspergillus niger
Dermatophytes + Hyaline hyphomycetes (n = 4)
1. 2. 3. 4.
Trichophyton Trichophyton Trichophyton Trichophyton
Dermatophytes + Dematiaceous fungi (n = 2)
1. Trichophyton verrucosum, Curvularia spp. 2. Trichophyton spp., Alternaria species
Dematiaceous fungi + Hyaline hyphomycetes (n = 1) Dematiaceous fungi + Zygomycetes (n = 1) Hyaline hyphomycetes + Hyaline hyphomycetes (n = 2)
1. Alternaria spp., Scopulariopsis spp.
Penicillium spp., Candida albicans Aspergillus niger, Candida albicans Aspergillus flavus, Non albicans Candida Aspergillus terrus, Non albicans candida Non albicans Candida, Penicillium spp.
violaceum, Aspergillus flavus schoenleinii, Aspergillus fumigatus mentagrophytes, Fusarium spp. verrucosum, Aspergillus flavus
1. Alternaria spp., Rhizopus spp. 1. Aspergillus niger, Penicillium spp. 2. Aspergillus niger, Fusarium spp.
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patients from East Asia and Europe under the Achilles foot project [26]. Onychomycosis is there to remain a disease of significance and such an epidemiological data would help to guide the management by improving the diagnostic methods that remain the priority area deserving our attention. 5. Conclusion To conclude, present study shows that direct KOH examination can not be relied upon for diagnosis of onychomycosis, for which fungal culture appears to be an appropriate choice along with KOH examination. Conflict of interest None. References [1] Elewski BE. Onychomycosis: pathogenesis, diagnosis, and management. Clin Microbiol Rev 1998;11:415–29. [2] Ghannoum M, Isham N. Fungal nail infections (onychomycosis): a neverending story? PLOS Pathog 2014;10(6):e1004105. [3] Grover C, Khurana A. Onychomycosis: newer insights in pathogenesis and diagnosis. Ind J Dermatol Venereol Leprol 2012;78(3):263–70. [4] Haghani I, Shokohi T, Hajheidari Z, Khalilian A, Aghili SR. Comparison of diagnostic methods in the evaluation of onychomycosis. Mycopathologia 2013;175(3–4):315–21. [5] Kaur R, Kashyap B, Bhalla P. A five-year survey of onychomycosis in New Delhi, India: epidemiological and laboratory aspects. Ind J Dermatol 2007;52:39–42. [6] De A, Bala NN, Taher A. Onychomycosis and its treatment. Int J Adv Pharm Biol Chem 2013;2(1):123–8. [7] Tosti A, Piraccini BM, Lorenzi S. Onychomycosis caused by nondermatophytic molds: clinical features and response to treatment of 59 cases. J Am Acad Dermatol 2000;42:217–24. [8] Moreno G, Arenas R. Other fungi causing onychomycosis. Clin Dermatol 2010;28:160–3. [9] Gunduz T, Metin DY, Sacar T, Hilmioglu S, Baydur H, Inci R, et al. Onychomycosis in primary school children: association with socioeconomic conditions. Mycoses 2006;49:431–3.
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[10] Westerberg DP, Voyack MJ. Onychomycosis: current trends in diagnosis and treatment. Ind J Clin Practice 2014;25(4):309–19. [11] Ameen M, Lear JT, Madan V, Mohd Mustapa MF, Richardson M. British Association of Dermatologists’ guidelines for the management ofonychomycosis 2014. Brit J Dermatol 2014;171:937–58. [12] Kaur R, Kashyap B, Bhalla P. Onychomycosis-epidemiology: diagnosis and management. Ind J Med Microbiol 2008;26(2):108–16. [13] Mugge C, Haustein UF, Neroff P. Causative agents of onychomycosis-a retrospective study. J Dtsch Dermatol Ges 2006;4:218–28. [14] Malik NA, Raza N. Nasiruddin: non-dermatophyte moulds and yeasts as causative agents in onychomycosis. J Pak Assoc Dermatol 2009;19:74–8. [15] Malik NA, Nasiruddin Dar NR, et al. Comparison of plain potassium hydroxide mounts, fungal cultures and nail plate biopsies in the diagnosis of onychomycosis. J Coll Phys Surg Pak 2006;16:641–4. [16] Veer P, Pathwardhan S, Damle AS. Study of onychomycosis: prevailing fungi and pattern of infection. Ind J Med Microbiol 2007;25:53–6. [17] Khosravi AR, Mansouri P. Onychomycosis in Tehran, Iran: prevailing fungi and treatment with itraconazole. Mycopathologica 2001;150:9–13. [18] Bokhari MA, Hussain I, Jahangir M, et al. Onychomycosis in Lahore, Pakistan. Int J Dermatol 1999;38:591–5. [19] Alvarez M, Gonzalez LA, Castro LA. Onychomycosis in Cali, Colombia. Mycopathologica 2004;158:181–6. [20] Attal RO, Chaudhary RA, Deotale VS, Jain SP. A clinicomycological study of onycho-mycosis in a rural hospital in Central India. Int J Res Med Sci 2015;3:1131–7. [21] Shenoy MM, Teerthnath S, Karnaker VK, Girisha BS, Krishna Prasad MS, Pinto J. Comparison of potassium hydroxide mount and mycological culture with histopathologic examination using periodic acid-Schiff staining of nail clippings in the diagnosis of onychomycosis. Ind J Dermatolol Veneorol Leprol 2008;74:226–9. [22] Das NK, Ghosh P, Das S, Bhattacharya S, Dutta RN, Sengupta SR. A study on the etiological agent and clinico-mycological correlation of fingernail onychomycosis in Eastern India. Ind J Dermatol 2008;53(2):75–9. [23] Yadav S, Saxena AK, Capoor MR, Ramesh V. Comparison of direct microscopic methods using potassium hydroxide, periodic acid Schiff, and calcofluor white with culture in the diagnosis of onychomycosis. Ind J Dermatol Venereol Leprol 2013;79(2):242–3. [24] Jaiswal A, Sharma RP, Gupta K. Onychomycosis: A clinicomycological study from western Uttar Pradesh, India. Ind J Med Special 2015;6(1):8–12. [25] Hwang SM, Kyu Suh M, Yim Ha G. Onychomycosis due to nondermatophytic molds. Ann Dermatol 2012;24(2):175–80. [26] Haneke E, Roseeuw D. The scope of onychomycosis: Epidemiology and clinical features. Int J Dermatol 1999;38:7–12.
Please cite this article in press as: P. Aggarwal, et al., A study to compare and classify etiological agents of onychomycosis, Indian J Med Spec. (2017), http://dx.doi.org/10.1016/j.injms.2017.08.002
Indian Journal of Medical Specialities xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Indian Journal of Medical Specialities journal homepage: www.elsevier.com/locate/injms
Original article
A study to compare and classify etiological agents of onychomycosis Prabhav Aggarwal, Bineeta Kashyap* , Jitu Mani Kalita Department of Microbiology, Maulana Azad Medical College & Associated Hospitals, New Delhi, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 August 2017 Received in revised form 5 August 2017 Accepted 5 August 2017 Available online xxx
Background: Onychomycosis, the condition that was always regarded as merely a cosmetic problem, now has become an important medical issue. Etiological prevalence of onychomycosis varies subject to the population demography, geographical location or the mycological technique used for the diagnosis. Aim: This study aims to identify and classify the fungal etiology of onychomycosis from a tertiary care hospital of north India over a duration of two years. Methods: The study included 169 clinically suspected patients of onychomycosis. The fungal etiology was determined by direct examination in 20% KOH after overnight incubation at 37 C and culture on Sabouraud’s Dextrose Agar. Results: 135 (79.9%) out of the total patients were confirmed cases of onychomycosis as per the diagnostic criteria. Hyaline hyphomycetes were the most common group of fungi isolated (50.0% of all fungal isolates) with only 15.4% isolates of the classical dermatophytes. A positivity of 18.3% and 78.7% by KOH examination and culture respectively was reported. .A sensitivity and specificity of 21.48% and 100% was found for KOH positivity; though culture was 98.52% sensitive and 82.35% specific. Conclusions: Hyaline hyphomycetes are a common cause of onychomycosis. KOH examination can not be relied upon for diagnosis of onychomycosis, for which fungal culture appears to be an appropriate choice along with KOH examination. © 2017 Published by Elsevier, a division of RELX India, Pvt. Ltd on behalf of Indian Journal of Medical Specialities.
Keywords: Onychomycosis Fungus Etiology
1. Introduction Onychomycosis remains an extremely obstinate and frequent fungal infection of the toenails or fingernails that can hugely impact the patients’ physical, social or emotional quality of life. The condition, that was always regarded as merely a cosmetic problem unworthy of any significant medical attention; now has become an important medical issue due to recurrence, chronicity, probability of transmission, therapeutic difficulties or association with immunosuppression [1]. Infected nails also are a potential reservoir of fungi which can spread to other sites of the patient [2]. Though the prevalence of onychomycosis varies subject to the population demography, geographical location or the mycological technique for diagnosis, an estimated 3–26% of the global population are affected by this significant health problem which accounts for approximately half of all onychopathies [3,4]. In a previous study done at our tertiary care centre the prevalence of
* Corresponding author at: Flat C-402, Vimal CGHS Ltd Plot 3, Sector 12, Dwarka, New Delhi-78, India . E-mail addresses: [email protected] (P. Aggarwal), [email protected] (B. Kashyap), [email protected] (J.M. Kalita).
onychomycosis was confirmed in as high as 45% of the analyzed patients [5]. Dermatophytic filamentous fungi by far remain the predominant etiology in most of the cases of onychomycosis, though yeasts and non-dermatophyte molds (NDM) are now implicated as the other common causes accounting for approximately 8–11% and 1.45–17.6% cases respectively [3,6,7,8].NDMs are increasingly reported as secondary or emerging causes of onychomycosis triggered by various factors like traumatic nails, comorbidities leading to compromised circulation or immunocompromised states [9]. Despite optimal management by the current antifungal regime, clinical as well as mycological cure is often unattainable in all onychomycosis cases [10]. The reasons for the limited success in cure rate could be many, some of which are incorrect identification of the fungal pathogen, co-existent co-morbidity, inherent characteristics of the nail which worsen the response to treatment or a plethora of host related factors [1]. Several non infectious causes of nail dystrophies mimicking onychomycosis further complicate the diagnosis [11]. Onychomycosis can be divided into several categories based upon the site involved, clinical pattern, mode of invasion or fungal etiology [12]. Such a classification aids in initiating a correct diagnosis and predicting the response to treatment and its prognosis. A laboratory confirmed clinical
http://dx.doi.org/10.1016/j.injms.2017.08.002 0976-2884/© 2017 Published by Elsevier, a division of RELX India, Pvt. Ltd on behalf of Indian Journal of Medical Specialities.
Please cite this article in press as: P. Aggarwal, et al., A study to compare and classify etiological agents of onychomycosis, Indian J Med Spec. (2017), http://dx.doi.org/10.1016/j.injms.2017.08.002
G Model INJMS 147 No. of Pages 5
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diagnosis with the identification of the underlying fungal pathogen further helps to exclude non fungal etiology or a mixed infection and hence guide appropriate antifungal treatment especially in less responsive cases of onychomycosis. Onychomycosis continues to be a diagnostic dilemma for a dermatologist and a microbiologist as the etiology spectrum is diverse and the techniques for diagnosis are still evolving. This study aims to identify and classify the fungal etiology of onychomycosis from a tertiary care hospital of north India over a duration of two years. 2. Materials and methods This was a retrospective study spanning a period of two years (January 2014–December 2015) conducted in the Mycology Laboratory, Department of Microbiology of a tertiary care hospital in New Delhi, India. The study included 169 patients, who presented with the signs and symptoms representative of the fungal infection of either toe or finger nails (onychomycosis). The affected area was cleaned with 70% ethanol and allowed to dry. Nail clippings along with the deep sub-ungual material were collected from the most affected nails. The specimens were transported to the Mycology laboratory as soon as possible in a dry, sterile, screw capped container. Nail specimens were incubated overnight at 37 C in 20% KOH; next day wet mounts were prepared and observed under 100 x and then 400 x light microscopy magnification to look for presence of fungal hyphae and/or yeast cells. “Specimens were inoculated onto two sets of sealed tubes: (i) Sabouraud’s Dextrose Agar and (ii) Sabouraud’s Dextrose Agar with chloramphenicol (0.05 mg/ml); one tube of each set was incubated at 25 C and another at 37 C.” The culture tubes were examined twice a week for any suspected fungal growth. Tubes showing no growth were discarded after a period of 6 weeks. Isolates were identified by the morphologic characteristics of the colony growth, pigment production and microscopic examination of the Lactophenol cotton blue (LPCB) preparation. In case identification was not possible, slide cultures were put up using corn meal agar to stimulate sporulation and incubated at 25 C for 4–7 days. Subsequently, LPCB preparations were made and observed under light microscope. Onychomycosis was diagnosed when fungal elements were seen on 20% KOH preparation, and/or repeated cultures or growth in multiple tubes yielded same fungal agent. Repeat samples were collected and processed when direct KOH wet mount was negative and fungal growth was obtained in a single tube, raising suspicion of contamination. Other fungal growths were labeled as contaminants and treated as false positive cultures for statistical calculations. 2.1. Statistical analysis The data was presented as percentages and proportions. 95% confidence intervals were calculated wherever applicable. Fischer’s exact test was used for testing the statistically significant differences; p values of <0.05 were considered statistically significant. All calculations were done using SPSS version 23.0. 3. Results A total of 169 patients were involved retrospectively in the study, including 98 (58.0%; 95% CI = 50.45%–-65.17%) males and 71 (42.0%; 95% CI = 34.83%–49.55%) females. Majority of patients were adults (>18 years of age), with only 11 (6.5%; 95% CI = 3.55%– 11.40%) subjects being under 18 years of age; maximum patients belonged to 20-30 years of age group (Fig. 1). Majority of patients (60.4%) had fingernail infection, 37.8% had toenail infection, while 1.8% patients had infections of both toenail and fingernail.
Fig. 1. Age-wise distribution of male and female patients.
KOH wet mount of the nail clippings and material collected deep under nail bed were prepared; out of 169, a total of 31 (18.3%; 95% CI = 13.19%–24.90%) specimens showed presence of fungal elements (fungal hyphae and/or yeast cells). Out of these 31 specimens, 29 turned out to be positive on culture; while 2 showed no growth even after 6 weeks of incubation (Table 1). Twelve KOH positive specimens yielded growth of dermatophytes, while remaining showed non-dermatophytic growths. A total of 133 (78.7%; 95% CI = 71.89%–84.23%) specimens showed growth on SDA, some showing co-infections of two or more fungi; with a total of 156 fungal isolates. Thus, 135 (79.9%) patients were diagnosed as cases of onychomycosis. KOH mount preparation had a low sensitivity of 21.48% as compared to the culture method (98.52%). As per the criteria, six cultures were labeled as contaminants/false positives. Isolated fungi were classified as shown in Table 2. Hyaline hyphomycetes were the most common group of fungi isolated, constituting 50.0% (78/156) of all fungal isolates. Aspergillus niger and A. flavus were the most common fungi in this group as well as among all groups. Twenty-four (15.4%) isolates of the classical dermatophytes were cultured, Trichophyton spp. (T. schoenleinni, T. verrucosum, T. mentagrophytes and T. rubrum) being the most common. Members of Class Zygomycetes (now Class Glomerulomycetes) and the dematiaceous fungi were relatively less common, constituting 13.5% and 9.6% of fungal isolates, respectively. Rhizopus spp. and Mucor spp. (9 isolates of each) were common fungi of Class Zygomycetes. Dematiaceous fungi were least common of all the groups, constituting 9.6% of all fungal isolates. Among yeast-like fungi, non-albicans Candida (14 isolates) were isolated more frequently as compared to Candida albicans (3 isolates). A single isolate of Geotrichum spp. was also identified. Though, there were differences between males and females with respect to rates of isolation of these fungi (dermatophytes and yeast-like fungi being more common in males), the differences were not statistically significant (Table 2). Fig. 2 depicts the month-wise rates of isolation of various groups of fungi; it is easily discernible that these rates were highest during the months of July to September. Highest isolation of hyaline hyphomycetes, dermatophytes and zygomycetes were seen during the months of July, August and October, respectively. Twenty two specimens showing simultaneous growths of two fungi were considered as co-infections. The details are presented in Table 3. 4. Discussion Onychomycosis is increasingly being recognized as a common problem in dermatological practice. Dermatophytes, yeasts as well as molds have all been implicated as potential pathogens in
Please cite this article in press as: P. Aggarwal, et al., A study to compare and classify etiological agents of onychomycosis, Indian J Med Spec. (2017), http://dx.doi.org/10.1016/j.injms.2017.08.002
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Table 1 Comparison between KOH wet mount preparation and culture on SDA. Onychomycosis Present
Onychomycosis not present
Total
% agreement between KOH wet mount preparation and culture on SDA
KOH Positive KOH negative
29 106 135
0 34 34
29 140 169
32.3%
Culture positive Culture negative
133 2 135
6 28 34
139 30 169
Sensitivity Specificity
KOH Culture KOH Culture
21.48 98.52% 100 82.35%
onychomycosis [13]. In the present study the isolated fungi were classified under five large groups based on their morphological and/or taxonomical classification. Dermatophytes, the filamentous fungi that are able to digest and obtain nutrients from keratin (skin, hair and nails), affect the outermost layers of these tissues without invading the living tissue and comprise of three main genera: Trichophyton, Microsporon and Epidermophyton. Hyaline hyphomycetes, possessing colorless septate hyphae often producing variously colored conidia, are mostly saprophytic in nature causing opportunistic infections in predisposed individuals. Zygomycetes (now renamed as Class Glomeromycetes) differs from hyaline hyphomycetes in possessing hyphae which are almost nonseptate. The group of Dematiaceous fungi is characterized by dark colored hyphae with dark colonies (olive, brown, gray or black in color) due to presence of melanin in their cell walls. Yeast like fungi
differ from all the rest in being unicellular that reproduce by budding; these buds may remain attached to the parent cell resulting in formation of pseudohyphae. In our study 135 (79.9%) out of the total patients were confirmed cases of onychomycosis as per the diagnostic criteria. Fungi were isolated in 78.7% specimens with a total of 156 fungal isolates including co-infections of two or more fungi. Fungal isolation as low as 20% has been reported in a previous study [14]. The authors have suggested that non viable cultures or faulty sample collection or culture inoculation techniques could possibly account for such a low percentage positivity of fungal culture. Hyaline hyphomycetes were the most common group of fungi isolated (50.0% of all fungal isolates) with only 15.4% isolates of the classical dermatophytes. The relatively less common members Zygomycetes, yeast like fungi and the dematiaceous fungi
Table 2 Etiological Classification of Fungal agents isolated from cases of onychomycosis.
Dermatophytes: Trichophyton mentagrophytes Trichophyton rubrum Trichophyton schoenleinii Trichophyton verrucosum Trichophyton violaceum Trichophyton tonsurans Microsporum nanum Total Hyaline hyphomycetes Aspergillus niger Aspergillus flavus Aspergillus fumigatus Aspergillus terreus Penicillium spp. Fusarium spp. Scopulariopsis spp. Scytalidium dimidiatum Total Zygomycetes/ Glomeromycetes Rhizopus spp. Mucor spp. Cunninghamella bertholletiae Syncephalastrum racemosum Total Dematiaceous fungi Alternaria spp. Curvularia spp. Bipolaris spp. Total Yeast-like fungi Candida albicans Non albicans Candida Geotrichum spp. Total Total fungi isolates No growth
Male (n=98)
Female (n=71)
P value
Total
% (of total fungal isolates, n=156)
4 3 2 4 1 0 2 16
0 1 3 1 2 1 0 8
0.0774
4 4 5 5 3 1 2 24
2.6 2.6 3.2 3.2 1.9 0.6 1.3 15.4
16 13 2 1 5 2 0 1 40
18 8 3 0 7 1 1 0 38
0.1190
34 21 5 1 12 3 1 1 78
21.8 13.5 3.2 0.6 7.7 1.9 0.6 0.6 50.0
4 6 0 0 10
5 3 2 1 11
0.3490
9 9 2 1 21
5.8 5.8 1.3 0.6 13.5
5 2 1 8
5 2 0 7
0.7867
10 4 1 15
6.4 2.6 0.6 9.6
1 12 0 13 87 23/98
2 2 1 5 69 13/71
0.1249
3 14 1 18 156 36/169
1.9 9.0 0.6 11.5 100 21.3
0.5608 0.4523
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Fig. 2. Month-wise distribution of onychomycosis cases due to various fungal groups.
constituting 13.5%, 11.5% and 9.6% of fungal isolates, respectively. Several authors have reported dermatophytes and most studies have shown dermatophyte as the predominant cause of onychomycosis (29.5–50%) when compared with yeasts (5.6–43.3%) and non-dermatophyte moulds (8.2–13.6%) [14–17], several others report candida species as the most common cause (40.7–46%) followed by dermatophytes (38–43%) and non-dermatophyte molds (11–14%) [18,19]. Another recent report from central part of India reported culture positivity in 24 out of 44 patients contributing to 12.5% dermatophytes, 8.3% candida species and 75% Non-Dermatophytic Moulds. The same study found KOH and culture positivity in 45.5% and 38.6% of 44 patients respectively with a sensitivity, specificity, PPV and NPV of 76.5%, 74%, 65% and 83.3% which rose to 100%, 74.7%, 70.8% and 100% respectively when combined with culture [20]. Our study reported a positivity of 18.3% and 78.7% by KOH examination and culture respectively. A
sensitivity and specificity of 21.48% and 100% was found in our study for KOH positivity; though culture was 98.52% sensitive and 82.35% specific. Manjunath Shenoy et al and Das et al, however report variable rates of positivity by direct microscopy and culture ranging from 32.94% to 53% and 35% to 49.4% respectively [21,22]. Another recent Indian study done from a tertiary care hospital in Delhi reports 66.1% positivity and 68% sensitivity for KOH examination [23]. A study from Uttar Pradesh, India reported KOH- and culture-positivity in 76.76% and 37.37% cases, respectively [24]. Direct microscopic examination, when done under experienced hands, along with culture can prove an efficient tool in the epidemiology of onychomycosis and help hasten the management of such patients [22]. While all yeasts identified were Candida albicans and Aspergillus species being the commonest NDM, Trichophyton rubrum and Trichophyton mentagrophytes were reported to be the predominant dermatophytic species in a previous Indian study [20]. In our study, Trichophyton schoenleinii and Trichophyton verrucosum were slightly more common; while, non albicans Candida out-numbered Candida albicans. Our study found NDM accounting for 88.46% of all fungi isolated, though a study reports NDM prevalence between 1.45% and 17.6% [25]. As far as the population dynamics and the seasonal trend in our study is concerned only 6.5% of the total cases were less than 18 years of age and the month-wise analysis of the fungi isolated suggested highest rates during the months of July to September. A study done on onychomycosis in children reported a relative percentage of 2.3% of pediatric (under 18 years of age) to adult onychomycosis. The same study reported a higher incidence of onychomycosis in winter months [26]. Smaller available surface, increased nail growth rate or reduced external exposure were some of the factors suggested by the authors for the lower prevalence in this group. Twenty-two instances of multiple fungi were identified in a total of 135 defined cases of onychomycosis amounting to 16.3% of mixed infection along with 21.3% sterile growth among 169 cases. 0.1% and 10.9% cases of mixed growth and no mycological growth was reported in a series covering 80,396
Table 3 Details of Co-infections evidenced on culture. Fungal groups
Details of co-infections seen
Hyaline hyphomycetes + yeast like fungi (n = 5)
1. 2. 3. 4. 5.
Zygomycetes + yeast like fungi (n = 2)
1. Mucor spp., Non albicans Candida 2. Rhizopus, Candida albicans
Zygomycetes + Hyaline hyphomycetes (n = 5)
1. 2. 3. 4.
Mucor spp., Aspergillus niger (n = 2) Syncephalastrum racemosum, Aspergillus niger Mucor spp., Aspergillus fumigatus Rhizopus spp., Aspergillus niger
Dermatophytes + Hyaline hyphomycetes (n = 4)
1. 2. 3. 4.
Trichophyton Trichophyton Trichophyton Trichophyton
Dermatophytes + Dematiaceous fungi (n = 2)
1. Trichophyton verrucosum, Curvularia spp. 2. Trichophyton spp., Alternaria species
Dematiaceous fungi + Hyaline hyphomycetes (n = 1) Dematiaceous fungi + Zygomycetes (n = 1) Hyaline hyphomycetes + Hyaline hyphomycetes (n = 2)
1. Alternaria spp., Scopulariopsis spp.
Penicillium spp., Candida albicans Aspergillus niger, Candida albicans Aspergillus flavus, Non albicans Candida Aspergillus terrus, Non albicans candida Non albicans Candida, Penicillium spp.
violaceum, Aspergillus flavus schoenleinii, Aspergillus fumigatus mentagrophytes, Fusarium spp. verrucosum, Aspergillus flavus
1. Alternaria spp., Rhizopus spp. 1. Aspergillus niger, Penicillium spp. 2. Aspergillus niger, Fusarium spp.
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