Etiologic significance of Aspergillus terreus in primary cutaneous mycosis of an agricultural worker  

Pratibha Dave1 , Raj Mahendra2 , Mahendra Pal3
1. Department of Dermatology, Welfare Hospital and Research Center, Bharauch-392001, Bharauch, Gujarat, India
2. Shashwat Skin Clinic, Bharauch-392001,Gujarat, India
3. Department of Microbiology, Immunology and Public Health, College of Veterinary Medicine and Agriculture ,Addis Ababa University,P.B.No.34,Debre Zeit, Ethiopia
Author    Correspondence author
Molecular Microbiology Research, 2015, Vol. 5, No. 2   doi: 10.5376/mmr.2015.05.0002
Received: 13 Nov., 2014    Accepted: 18 Jan., 2015    Published: 17 Feb., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Dave et al., 2015, Etiologic significance of Aspergillus terreus in primary cutaneous mycosis of an agricultural worker, Molecular Microbiology Research, Vol.5, No.2 1-4 (doi: 10.5376/mmr.2015.05.0002)

Abstract

This pilot study was aimed to elucidate the growing role of non-dermatophytic filamentous moulds in the cutaneous lesions of laborers who worked in agriculture field in the villages of Bharauch, Gujarat ,India. Eighteen patients (13 males and 5 females, aged 21 to 46 years) with various skin problems presented to the outpatient department (OPD) of Welfare Hospital and Research Center, and Shashwat Skin Clinic, Bharauch, India were examined for the non-dermatophytic filamentous fungi by using standard mycological techniques. All the specimens were treated in 10 % potassium hydroxide solution of direct microscopy; and cultural isolation was done onto Sabouraud medium with chloramphenicol. The detailed identification of fungal isolates was carried out in “ Narayan” stain. The treatment of patient was attempted with oral administration of itraconazole. Aspergillus terreus was identified in the cutaneous lesion of 1 of the 18 patients both by direct microscopy as well as by cultural isolation. The cinnamon-brown coloured colonies of A. terreus grew in pure culture from the biopsied tissue on Sabouraud dextrose agar with chloramphenicol. In “Narayan” stain, conidial heads of A. terreus were found compact, biseriate, and densely columnar. The patient had received trauma on the skin of right lower leg by wooden splinter while working in the field. The oral administration of itraconazole for 12 weeks showed good clinical response. The demonstration of A. terreus in the skin lesion by direct microscopy and cultural isolation, and good response to antifungal drugs clearly indicated that our patient was suffering with cutaneous mycosis. The patient with chronic cutaneous lesion, history of the skin trauma, and occupational exposure to the soil should be investigated for cutaneous mycosis. The immediate attention to traumatic injury to the skin is highly imperative to prevent the further complications. This seems to be first report of primary cutaneous mycosis due to A. terreus in an immunocompetent patient from this part of India.

Keywords
Aspergillus terreus; Cutaneous lesion; Itraconazole; Narayan stain, Soil; Trauma

Cutaneous diseases of multiple etiologies are commonly encountered in human and animal clinical practice. In recent years, mycotic infections due to opportunistic fungi are gaining significance both in developed and developing countries of the world (Pal, 2007). Among such group of fungi, Aspergillus infections are being reported with increasing frequency in humans and animals from many regions of the world including India (Pal and Torres-Rodrigues, 1990; Baddley et al., 2003; Judson, 2004; Nucci and Marr, 2005; Pal, 2007). There are about 600 species of Aspergillus prevalent in our environment, of which 27 species of Aspergillus are implicated in various clinical disorders of humans and animals (Pal, 2007; Pal et al., 2012) . Aspergillosis is primarily caused by A.fumigatus, however, other species such as A. amstelodami, A. candidus, A.chevallieri, A.clvatus, A.deflectus, A.flavus, A.glaucus, A.nidulans, A.niger, A.ochraceous, A.restrictus, A.syowii, A.tamari, A. terreus, A.udagawae, A ustus, and A. versicolor are also incriminated in the etiology of disease (Levitz, 1989; Naidu et al., 1992; Pal and Dave, 2006; Pal, 2007; Pal et al., 2012; Venugopal and Venugopal, 2012). Disseminated aspergillosis is associated with a high mortality rate of about 90 %. Maximum cases of aspergillosis are encountered in immunocompromised patients (Pal, 2007).The prolonged neutropenia predisposes the humans and animals to Aspergillus infection. Therefore, invasive aspergillosis has become a leading cause of death in neutropenic patients. The cutaneous form of aspergillosis is rarely encountered in immnunocompetent hosts. It may be either primary in origin following traumatic implantation of fungi with contaminated objects, or occurs due to haematogenous dissemination of the infection from the lungs to other sites (Pal and Dave, 2007). Occasional outbreaks of cutaneous aspergillosis are traced to the fungal contaminated biomedical devices (Pal and Dave, 2006). Certain occupational groups such as gardeners, agricultural workers, brick manufacturers, etc., who remain in direct contact with the soil are at a greater risk of acquiring fungal infections (Pal and Dave, 2006; Pal, 2007). A plethora of drugs such as amphotericin B, caspofungin, itraconazole, posaconazole, terbinafine, and voriconazole have been tried with variable success in the management of aspergillosis Nucci and Marr, 2005; Pal and Dave, 2006; Pal, 2007).The paucity of information on cutaneous aspergillosis from this region of India prompted us investigate the causative role of non-dermatophytic filamentous fungi in primary cutaneous mycosis in persons who were engaged in agricultural occupation.

1 Materials and Methods
In all, 18 patients of both sexes (13 males and 5 females) and different age groups (21 to 46 years) with various dermatological disorders were presented at the Skin OPD of Welfare Hospital and Research Center, and Shashwat Skin Clinic, Bharauch, Gujarat, India for diagnosis and treatment. All the patients belonged to nearby villages of Bharauch, India. The suitable clinical materials such as swab, scrapings from the border of the lesion, pus, aspirate, and biopsy (punch method) etc., were collected aseptically from all the patients for mycological diagnosis. Each specimen was subjected for detailed mycological investigation using standard techniques. Direct microscopy was done in 10 % solution of potassium hydroxide (KOH) (Pal, 2007), India ink, Gram stain; and the cultural isolation was attempted on nutrient agar, Sabouraud dextrose agar, Sabouraud dextrose agar with chloramphenicol, and Pal sunflower seed medium (Pal, 1997).The examination of the fungal isolates under light microscope was carried out by preparing mount in Narayan stain which contained 6.0 ml of dimethyl sulfoxide (DMSO), 0.5 ml of 3 % aqueous solution of methylene blue, and 4.0 ml of glycerin (Pal, 2004).
2 Results
There were 13 males and 5 females, and their age varied from 21 to 46 years. All the patients did not use any protective wears while working in the agriculture field. The lesions were observed on different parts of the body especially on the legs and hands. Clinical findings in these patients included erythema, vesicles, cellulitis, papules, plaques, nodules, and ulcers. Aspergillus terreus was demonstrated in the cutaneous lesion of 1 of the 18 patients. The patient who yielded A. terreus was 27- years- old male; and he had one ulcerated lesion on the lower part of the right leg. As narrated by the patient, he had received injury with wooden splinter when planting in the field. The direct microscopy of the punch biopsy sample (taken from the ulcer edge) in 10% KOH mounts showed the presence of thin ,hyaline, septate, and dichotomously branched hyphae morphologically resembling Aspergillus. However, India ink preparation failed to detect any capsule of Cryptococcus neoformans. Similarly, Nocardia was absent in the impression smear when stained by Gram’s technique. There was no growth of bacteria, Nocardia, and Cryptococcus neoformans on nutrient agar, Sabouraud dextrose agar, and Pal’s sunflower seed medium, respectively. However, velvety cinnamon-brown coloured colonies were isolated in pure and luxuriant from the ulcer biopsy material on Sabouraud dextrose agar with chloramphenicol medium after 3 days of incubation at 37 C. As A.terrerus is sensitive to cycloheximide, it should not be incorporated in the medium. The growth of fungal isolate in Narayan stain revealed small, smooth walled, globose- shaped conidia, thin walled smooth condiophores, hemispherical vesicles, and biseriate sterigmata (Pal, 2007).Based on the gross cultural,and microscopic morphology, the fungal isolate was identified as A. terreus. The laboratory examination of our patient for HIV, diabetes mellitus, and tuberculosis was non-committal indicating that he was not immunocompromised, and his immune status was normal. Moreover, the negative culture of blood, and urine on mycological media ruled out the possibility of dissemination of A. terreus infection. The patient was put on itraconazole (200 mg ,12 hourly, orally daily for 4 weeks, followed by 100 mg, 12 hourly orally daily for 8 weeks) therapy for the management of cutaneous mycosis. In addition, supportive drugs such as multivitamin, multimineral, liver tonic, and unienzyme were also prescribed. The oral therapy with itraconazole showed good clinical response in our patient.
3 Discussion
Cutaneous mycosis (dermatomycosis, fungal dermatitis) is an infectious, sporadic, global fungal disease caused by a large number of non-dermatophytic fungi which are widely prevalent in our environment (Pal, 2007). Most of the fungi responsible for cutaneous mycosis are opportunistic pathogens; and are recovered from a wide variety of natural substrates including the soil (Pal, 2007). In majority of cases, transmission of the infection occurs by the introduction of fungi into the skin through traumatic injury from saprobic environment (Pal and Dave, 2006; Pal, 2007). Clinical history and laboratory investigations indicated that our patient was immunocompetent who developed primary cutaneous aspergillosis due to A. terreus following traumatic injury in the agriculture field. Cases of primary cutaneous aspergillosis in immunocompetent patients have been reported by several investigators (Romano and Miracco, 2003; Zhang et al., 2005; Mohapatra et al., 2009; Sharma et al., 2011). Our finding is in accordance with Ozer and co-workers (2009) who isolated A. terreus from the cutaneous lesions of an immunocompetent patient. As A. terreus resembles to other filamentous fungi in direct microscopy, hence cultural isolation of the fungus is very essential to confirm the specific diagnosis. Our experience had indicated that the skin biopsy by punch method is a very good specimen to establish an unequivocal diagnosis of cutaneous mycosis due to non-dermatophytic filamentous fungi. Our patient ignored the skin injury, and did not seek medical advice due to financial constraints .However, when the lesion became very apparent on the lower part of the right leg after about five months, the patient visited the hospital for treatment. The duration of lesion in primary cutaneous aspergillosis in an immunocompetent patient was recorded 10 years by some workers (Mohapatra et al., 2009). As A. terreus is widely prevalent in Indian environment, we believe that our patient probably acquired the infection from the fungal contaminated soil after receiving traumatic injury on the skin. The role of trauma in the development of cutaneous aspergillosis is described by earlier investigators (Romano and Miracco, 2003; Ozar et al., 2009). Since the lesion was chronic in nature, long duration of therapy was recommended. As A. terreus is refractory to treatment with amphotericin B, we tried itraconazole in our patient, and the clinical response was encouraging. The drug was well tolerated as our patient did not exhibit any side effects. The drug itraconazole has been found effective to treat cutaneous aspergillosis in an immunocompetent patient (Sharma et al., 2011). Moreover, it was observed in our clinical practice of over two decades that the patients who were given vitamins, minerals, liver and unienzyme preparations along with antibacterial antibiotics or antifungal antibiotics showed better results. The clinical efficacy of newer drugs such as caspofungin, posaconazole, and voriconazole should be further studied in immunocompromised as well as immunocompetent patients. It is, therefore, advised that person with a history of traumatic injury to the skin from the environment must immediately visit the physician for medical treatment to avert the further complications of disease. Moreover, the high risk groups should be provided protective wears; and they should be educated about the skin hygiene. It is emphasized that antifungal therapy is warranted in all patients with localized lesions in order to prevent the risk of dissemination. As A. terreus is an emerging human and animal pathogen, its etiologic role in various clinical disorders should be further studied.
Acknowledgements
The authors are thankful to the technical assistance rendered by the staff of the Welfare Hospital and Research Center, and also Shashwat Skin Clinic, Bharauch, Gujarat, India. Thanks are also due to the patients for their cooperation.
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