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American International Journal of Available online at http://www.iasir.net Research in Formal, Applied & Natural Sciences ISSN (Print): 2328-3777, ISSN (Online): 2328-3785, ISSN (CD-ROM): 2328-3793 AIJRFANS is a refereed, indexed, peer-reviewed, multidisciplinary and open access journal published by International Association of Scientific Innovation and Research (IASIR), USA (An Association Unifying the Sciences, Engineering, and Applied Research)

Coccidioides immitis as a potential agent of onychomycosis from a North Indian district of Jammu and Kashmir State: a new Case Report

Sandeep Kotwal and Geeta Sumbali Department of Botany, University of Jammu, Jammu, India, 180006

Abstract: Coccidioides immitis is an endemic, most virulent, systemic, disseminated fungal pathogen of human and other animals. It is generally a soil-dwelling fungus which is present in dimorphic form and has been found to cause a number of broad spectrum diseases ranging from flu-like symptoms to severe life threatening infections. It generally causes coccidioidomycosis (valley fever), a serious fungal disease of the lungs and other tissues which is acquired by inhalation of the contaminated dust or soil which is embedded with the arthroconidia of this fungus. Survey of literature confirmed that C. immitis is not reported earlier as onychomycotic agent; hence it constitutes a new report to the list of the nail infecting fungi. The predisposing factors like conducive climatic conditions of the studied area, visiting some endemic areas by the patient during his service tenure, latent- reactivation infection, exposure of the cut or bruise in any body part of the patient to the arthrospores; may help this pathogen to establish the host-pathogen relationship and then to act as an etiological agent of onychomycosis in the studied case.

Keywords: Coccidioides, Onychomycosis; Coccidioidomycosis, Arthroconidia; DLSO.

I. Introduction Coccidioides species are generally soil-dwelling fungi, which causes a broad spectrum of diseases, ranging from an asymptomatic infection to mild respiratory illness to severe life-threatening infections [1]. Coccidioides Stiles is characterized by its dimorphism: a mycelial saprophytic phase with arthroconidia and a yeast-like parasitic phase with spherules (sporangia). In the saprophytic phase colonies are fast-growing, reaching 3.7 cm of diameter in 14 days; at first white, but later becoming yellow, pale brown or brownish. Aerial hyphae transformed into arthroconidia by a regularly spaced septation and subsequent degrading of alternating cells, so that gaps are left between the conidia. Spherical chlamydospores also formed. In parasitic phase, spherules formed, over 80 µm in diameter, mostly smooth or with fine excrescences; numerous endospores formed by repeated cleavage and released by breakage of the wall [2]. The genus Coccidioides has two genetically distinct species viz., C. immitis and C. posadassi [3]; which causes similar clinical diseases, however they are endemic to the different geographical regions of the world [4,5]. Coccidioides (meaning: resembling Coccidia- a protozoan) immitis (meaning: not mild), is the important species of this genus which generally developed coccidioidomycosis (valley fever), a serious fungal disease of the lungs and other tissues [6] and is common in the south-western United States, Mexico and South America [3]. The infection of Coccidioides species is acquired via inhalation of contaminated dust or soil which is embedded with the arthroconidia. Human to human transmission [7] and animal to human transmission [8] of coccidioidal infection was also reported. The coccidioidal infection generally occurs when a person visits an endemic area [3]; the visitor become symptomatic shortly after returning home, although latent or reactivation infection presenting months to years later was also reported in immune-compromised persons [9, 10]. Coccidioides species are generally considered as potential bioterrorism agents and C. immitis is the only fungal aetiological agent designated as Biosafety Level 3 in “Biosafety in Microbiological and Biomedical Laboratories, 3rdedn” [11] and in the USA and elsewhere, these organisms fall under stringent regulations that govern their possession, use and transfer [12]. II. Material and Methods During the onychomycotic survey in between May2017 to June 2018, different onychodystrophied samples were collected from the patients residing in district Doda of Jammu and Kashmir State (India). The infected nail samples and scrapings were collected by following the standard procedures; first by swabbing the infected nails with 70% ethanol and then collecting these samples in pre-sterilized polythene bags. These nail samples were placed on dermatophyte test medium (DTM) supplemented with chloramphenicol (0.05mg/ml) and cycloheximide (0.5mg/ml). Subculturing was done on Sabourauds dextrose agar (SDA) medium supplemented

AIJRFANS 18-313; © 2018, AIJRFANS All Rights Reserved Page 59 Kotwal and Sumbali, American International Journal of Research in Formal, Applied & Natural Sciences, 23(1), June-August, 2018, pp. 59- 62 with chloramphenicol (0.05mg/ml). The pathogen was identified by studying their growth rate, texture, colouretc and by preparing their lactophenol cotton blue mounts and observing their micro-morphological characters and by following the descriptions given by McGinnis and Hilger [13] and Domsch et al. [2]. All procedures performed during the present study are in accordance with the ethical standards of the Animal and Human Experimentation Ethics Committee (AHEEC), University of Jammu, Jammu.

III. Results Case Report: Onychomycosis was detected in 51 years old retired army personnel on both of his finger and toe nails. On examination, the finger nails of his left hand were highly involved in nail infection. The finger nails were yellowish brown in colour; rough in texture with slight onycholysed regions visualized on his nail plate (Fig 1a). The distal regions of the nails were highly involved in infection and hyperkeratosis was also observed in the hyponychinum region. The skin of the hand was moderately loose with less sensation; dry with pinkish red colouration (Fig 1 a). The infection first evolved from the distal region of the nail and then it spreads both to distal and lateral regions of the nails and these symptoms suggests a case of Distal Lateral subungual onychomycosis (DLSO). All the toe nails were also involved in infection. The toe nails were rough in texture, onychodystrophied, with crumble of nail pieces shedding off; pale yellowish in colouration (Fig 1b). The toe nails were loosely attached to the nail bed of the basal tissue of the nail and frequently blood oozes out from that region (Fig 1b). The skin of the feet was loose, dry; slightly pinkish in colour. The patient also experienced irritation and etching on his skin. The infection evolved 10 years back due to an accident which he met during his service tenure. Mycological details: On dermatophyte test medium colonies grew directly on infected nail sample and reached a diameter of 37-45 mm after 15 days of incubation at 28 ± 2oC; cottony, floccose, fluffy, initially white later changing to yellowish-brown in colour (Fig 1c); reverse pale brown. On SDA medium, colonies reached a diameter of 25-30 mm after 15 days of incubation, slimy to slightly floccose, initially white, becoming off white (Fig 1d); reverse pale. On microscopic examination, mycelium was observed smooth-walled, septate, measuring 2.1-3.2 µm in width. The hyphae formed barrel-shaped arthrospores with age which became wider than the main hypha; measuring 4.5-10.5 x 1.9-4.1 µm (Fig 1 e, f); some arthrospores dissolved with age but remain intact with the mycelium (Fig 1e), the reason of having gaps between some arthroconidia (Fig 1f). Colonies formed hyphae with embedded arthrospores only on those cultures which grew directly on infected nail samples; sub- cultured colonies on other media formed only sterile mycelium. Arthrospores cannot be converted to spherule form in in-vitro conditions. The causal pathogen recovered from the infected nails was isolated as Coccidioides immitis Stiles.

IV. Discussion C. immitis is endemic, most virulent, systemic, disseminated fungal pathogen of humans and other animals and itwas also reported earlier from an Argentinean soldier from his histopathological specimens who went on to die and leave his samples for study [11]. It is the only fungal aetiological agent designated as Biosafety Level 3 and can be used as a select agent of bioterrorism [11]. The infection can occur via inhalation of aerosolized arthroconidia from a primary environmental focus and can also occur from traumatic implantation [11]. In our present study, C. immitis was recovered as an etiological agent of onychomycosis from 51 years old retired Army personnel. There are five possible explanations for the presence of C. immitis in the nail samples of the suspected individual. First, he was an army personnel and he visited different parts of the country during his service tenure viz., UP, Bihar, Rajasthan, J&K etc, so it was possible that he visited some endemic areas of the country (still not known) and the fungal spores (arthroconidia) got transmitted in his body as also described by Litvintseva et al. [1] who confirmed that visiting different endemic areas can cause this disseminated infection and C. immitis was also reported as a threat to the Southern California military personnel’s, deployed populations and US military personnel’s as it generally causes coccidioidomycosis [14,15,11]. Second, the temperature of some parts of the district Doda is conducive for the growth and expansion of C. immitis as described by Pappagianis [16] who reported that temperature plays a pivotal role in spreading this disease. Third, it was possible that C. immitis was present in latent form in his body and now become reactivated to cause infection of the skin and nails as also described by Brown et al. [3] who documented that the infection dose of arthrospores of C. immitis is very small and even a brief exposure in an endemic area may result in symptomatic infection later on. Fourth, he was also a part time farmer and the pathogen enters his body via some cut and wound or he was exposed to arthrospores while digging as also described by Davis and Smith [7] who earlier reported that a group of students had been exposed to Coccidioides while digging a rattlesnake out of a ground squirrel hole. Fifth, the pathogen form host-pathogen interaction and then reached the nail tissue via blood vessels and then it utilizes the keratin of the nail and cause onychomycosis. More research using molecular methods of detection and better epidemiological surveillance is necessary to determine the prevalence of C. immitis in J&K and the surrounding states.

C. immitis was not earlier reported as etiological agent of onychomycosis and hence it constitutes a new addition to the list of onychomycotic fungi.

V. Conclusion Coccidioides immitis which was earlier reported as a pathogen responsible for causing coccidioidomycosis is also able to cause onychomycosis. The results and findings have significant public health implications. Doctors, medical practitioners, healthcare providers, diagnostic laboratories and the public need to be aware that this pathogen can be contracted outside the traditionally established endemic areas and can cause onychomycosis and skin irritation. Elaborate research is essential throughout the world to determine the prevalence of this pathogen as an etiological agent of onychomycosis which suggests that more infections may occur by this pathogen.

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[13]. McGinnis, M.R. and Hilger, A.E. A key to the genera of medically important fungi. Mycopathologia. 1971; 45 (3-4): 269-283. doi: 10.1007/BF02051975. [14]. Hooper, R., Curley, R., Poppell, G., Husted, S. and Shillaci, R. Coccidioidomycosis among military personnel in Southern California. Military Medicine. 1980; 145: 620-623. [15]. Rush, W.L., Dooley, D.P., Blatt, S.P. and Drehner, D.M. Coccidioidomycosis: a persistent threat to deployed populations. Aviation, Space and Environmental Medicine. 1993; 64: 653-657. [16]. Pappagianis, D. Epidemiology of coccidioidomycosis. Curr Top Med Mycol. 1988; 2:199-238.

VII. Acknowledgments The first author is thankful to the University Grant Commission, New Delhi and Higher Education Department, Jammu and Kashmir for providing an opportunity under UGC-FIP scheme. VIII. Conflict of Interest The authors have no conflict of interest to declare.

Figure 1 a: Onychomycotic finger nails and loose pinkish skin of the case report. b: Onychomycotic toe nails and dry skin of the case report. c: Colonies of Coccidioides immitis growing from direct nail sample on DTM. d: Colonies of C. immitis on SDA medium. e-f:Microphotographs showing hyphae,arthroconidia and dissolved arthroconidia of C. immitis (Bars=10 µm).