The First Isolation of Hortaea Zverneckii from a Household Guinea Pig

Jpn. J. Med. Mycol. Vol. 43, 175-180, 2002 ISSN 0916-4804

Original Article The First Isolation of Hortaea zverneckii from a Household Guinea Pig

Shahana Sharmin1, Kumiko Haritani2, Reiko Tanaka1, Paride Abliz1, Kayoko Takizawa1, Ayako Sano1, Kazutaka Fukushima1, Kazuko Nishimura1, Makoto Miyaji1

1 Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan. 2Namiki Animal Hospital , 7-2-4 Namiki, Abiko, Chiba 270-0011, Japan. Received: 14, March 2002. Accepted: 26, April 2002]

Abstract Hortaea werneckii, a black yeast and the causative agent of tinea nigra (a superficial type of dermatomycosis), is a human pathogen and is also found in the environment. It is not highly pathogenic but in the last fifteen to twenty years has been isolated from various human and environmental sources in Japan. As far as we know, there has been no report on the isolation of H. werneckii from animals. Recently, we found a case of a guinea pig with dark superficial lesions on the palm and dorsal areas. Cultural and morphological studies of scrapings from the lesion showed that the causative agent was a black yeast, which was identified as H. werneckii by morphological study and molecular biological screening. Dl/D2 region of the 26S large subunit rDNA gene of this isolate was identical to those of 11 other H. werneckii isolates used as reference strains in this study. This is the first case recorded of tinea nigra caused by H, werneckii in a guinea pig. Key words: guinea pig, Hortaea werneckii, LSU rDNA gene, tinea nigra

zverneckii. Introduction Tinea nigra is often found in the tropical and Tinea nigra, a black localized discoloration of subtropical zones and is also found in temperate the palms was first observed in a human in 1891 areas of the Western Hemisphere; cases have in Brazil. In 1921 a case was reported in Rio de been reported from the USA5~, Brazil6}, Uruguay7~, Janeiro from which Horta isolated a fungus he Canadas~, Costa Rica9 , Venezuela'0~, Panama" called Cladosporium zverneckii'. In 1970 von Arx India12~, Thailand'3~, Israel'4~, Congo'5~ and changed C. zverneckii to Exophiala zverneckii based Australia'6'8~. Reports from Great Britain'3~, on its annellidic budding2}. Until 1984, there was Germany'47, Canada8} and France's} attributed to some confusion regarding the nomenclature of tinea nigra as an imported fungal infection at the this black yeast, the causative agent of tinea time, because the patients presented with travel nigra on the palms and soles3~. Nishimura and history to subtropical or tropical areas. Miyaji4~ clarified its conidiogenesis by scanning Endophthalmitis due to H. zverneckiihas also been electron microscopy indicating that the conidia of reported in an immunocompetent host after cata- E. zverneckiiwere produced by both annellidic and ract surgery in USA1°~. Its ecological distribution sympodial conidiogenesis. Since the conidia of as halophilic saprobes in tropical and subtropical other Exophiala species are produced only by areas20~ and in natural saltpans21~ has also been annellidic conidiogenesis, a new genus, Hortaea was reported. The first human case in Japan was re- proposed to accommodate E. zverneckii as Hortaea ported from Okinawa prefecture located in the Corresponding author: Makoto Miyaji southern subtropical part in 1984 (Nakama et al.: Research Center for Pathogenic Fungi and Microbial Jpn J Med Mycol 25: 15, 1984, article in Toxicoses, Chiba University, Japanese) . More recently, other cases were re- 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan ported from Okinawa and then later from 176 真上菌誌第43巻禽八∫3号平成14年

Fig. 1. A skin lesion on the dorsal region of the guinea pig Fig. 2. The undersurface of the right palm of the guinea (arrow). pig (arrow).

Kyushu, Shikoku and Kanto areas indicating a purulent small lesion that recovered after treat- gradual migration of the disease northwards in ment with chloramphenicol for 6 days. Japan in the last 15 to 20 years4^22'.H. werneckii On October 22, 2001, his right palm had a was also isolated from the seashore, house-dust black pigmented lesion from which pus oozed out and an indoor swimming-pool 22,2si in this country. (Fig. 2). The scrapings from his palm and back To date there has been no available informa- showed no fungal elements in a direct examina- tion regarding animal infection by this organism. tion under light microscope after KOH mounting. This paper reports the first case of cutaneous in- The culture of scrapings on Mycosel Agar plates fection of H. werneckii in a household guinea pig was also negative. The guinea pig was given with mycological and molecular biological identi- griseofulvin for one week followed by ketoconazole fication of this pathogen. by mouth and bathing with an anti-fungal medi- cated shampoo was advised. Materials and Methods By November 2, 2001, both lesions had become Case history better and the animal is currently under follow- The animal in this study was a male guinea up observation. pig (English strain), born in Japan on February, Isolates 2001 and weighing 850 g. There was no history The clinical isolate collected from the guinea of its having been taken abroad. pig was evaluated in relation to the following On June 21, 2001, the male animal was taken eleven H. werneckii isolates stored at this research to an animal clinic and was said to have been center. Isolates IFM 4885, IFM 4993, IFM 41538, screaming in a strange voice, though he was vig- IFM 41540, IFM 41541, IFM 41542, IFM 46077, orous and had a good appetite. He was infested IFM 46078, IFM 46442, IFM 46445 and IFM with lice and was administered anti-lice treat- 46446 were used as reference strains in this study. ment and vitamin C. Fungal culture was negative Mycological study at that time. All the control isolates and the clinical isolate On September 16, 2001 he was found to be from the animal were subjected to the following continuously scratching his body and had an ul- mycological studies. The growth was observed on cerous lesion with loss of hair on his back (Fig. PDA slants at room temperature for two weeks, 1). Scrapings from the lesion were cultured on and slide cultures on PDA stained with lactophenol Mycosel Agar (BBL`S, Becton Dickinson and Co., cotton blue were viewed under a light micro- Cockeysville, MD) plates at room temperature scope. for two weeks. Shiny, black yeast-like fungal colo- Extraction of DNA nies developed on the plates together with other DNA was extracted from yeast cells according environmental fungal sprouts. After subculturing to the method of Makimura et al."'. Briefly, sev- on potato dextrose agar (PDA, Difco, Detroit, eral loops of yeast cells were suspended in a USA) slants for two months at room tempera- lysing solution (100 mM Tris-HC1, pH 8.0, ture, the colonies were sticky, paste-like, metallic 30 mM EDTA and 0.5% dodecyl sulphate) black in color and had a shiny yeast-like texture. and then heated at 65C for 15 minutes. The On October 7, 2001, the animal's right forearm solution was extracted with phenol-chloroform- was swollen and the skin on his right palm had isoamylalcohol (25:24:1 [v/vl). DNA was precipi- Jpn. J. Med. Mycol. Vol. 43 (No. 3 ), 2002 177

Fig. 3. Microscopic observation of yeast-form cells of the Fig. 4. Microscopic observation of a mycelial culture of isolate IFM 51425 stained with lactophenol cotton the isolate IFM 51425 stained with lactophenol cotton blue, (X 400). blue, (X 400). Annellidic conidia (arrows).

Fig. 5. Alignment of nucleotide sequences of D1/D2 region of the large subunit 26S rDNA genes of H. werneckii isolates. Phylogenic type A: IFM 41540, IFM 41541, IFM 46077, IFM 46078 and IFM 46445. Phylogenic type B: IFM 46446, IFM 46442 and IFM 4993. Phylogenic type C: IFM 41542, IFM 41538 and IFM 4885 *; indicates the bases substituted in types B and C from type A. tated with chilled isopropanol. electrophoresis in 1.5% agarose gel in Tris/boric PCR amplification of the large subunit ribo- acid/EDTA buffer (89 mM Tris, 89 mM boric somal DNA gene (D1/D2 region) acid and 2 mM EDTA [pH 8.3]) and staining The divergent domain of Dl/D2 at the 5' end with ethidium bromide. of the LSU rDNA gene was amplified by the DNA sequencing method of Kurtzman and Robnett"l using the set The PCR amplified Dl/D2 region of the ribo- of primers NL-1 (5'-GCA TAT CAA TAA GCG somal gene was directly sequenced at both GAG GAA AAG-3') and NL-4 (5'-GGT CCG strands using an ABI PRISM 3100 sequencer TGT TTC AAG ACG G-3'). Amplification was after labeling with an ABI PRISM BigDye"' performed for 30 PCR cycles after denaturation Terminator Cycle Sequencing kit (Applied at 95C for 4 min., annealing at 551C for 2.5 min., Biosystems, Foster City, Ca., USA). The primers extension at 72C for 2.5 min. followed by a final NL-1 and NL-4 were also used in labeling. The extension at 72C for 10 min. and denaturing at nucleotide sequence data was analyzed with 94'C for 1 min. The products amplified were the genetic information processing software, purified with SUPREC''"-02 (TaKaRa, Shiga, GENETYX-MAC version 11.2.0 (Software Japan) according to the manufacturer's instruc- Development, Tokyo, Japan) and was compared tion and were visualized (not shown here) by to sequences of other H. werneckii isolates used in 178 真菌誌第43巻第3号平成14年

Table 1 GenBank accession no. and IFM no. of Hortaea to GenBank. This isolate was registered as IFM werneckii isolates 51425 (Table 1) and maintained in the fungal culture collection at the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University. Discussion Tinea nigra had been assumed to be a tropical disease of humans in the past, but with the suc- cession of various case reports from different parts of the world it can no longer be considered as such. Its occurrence has been reported from Africa, Asia and both South and North America, although the condition almost always appears in a sub-clinical form. In nature, H. werneckiiusually *; the clinical isolate obtained from the guinea pig identified as is isolated in soil, humus or sewage26~.Generally, H. werneckii was submitted to GenBank with accession no. AB079595 and registered at the center as IFM 51425. it affects a younger age group with a superficial type of dermatomycosis and is limited to the out- this study. ermost horny layer of the epidermis. Children may be infected through accidental injury with Results contaminated thorny or sharp objects while play- The clinical isolate and control isolates of H. ing outdoors, but human-to-human transmission werneckii showed annellidic and sympodial forma- of the disease has not been known26>. The young- tion of conidia typical to this genus and the yeast est case was reported in a female child, 21 form cells showed a one- or two-celled arrange- months of age27~. An environmental H. werneckii ment with thick black walls. All of the isolates isolate showing pathogenicity in guinea pigs was developed to form black, shiny yeast-like colonies evaluated by Fujita's animal model experiment2s~; on PDA slants. The yeast cells in slide cultures fungal cells were seen to invade the skin of the were elongated, peanut-shaped and stained with sole at the site of inoculation, but the invasion lactophenol cotton blue, about 4 X 8 ~Cm in di- was limited to the superficial cornified ce11s2A'. ameter and had thick, black cell walls (Fig. 3). According to Kwon-Chung and Bennett, infection Annellidic and sympodial formation of conidia by H. werneckii was experimentally induced by were confirmed under light microscope (Fig. 4). rubbing some of the culture material on scarified The sequence of the D1/D2 region consisted of skin and then occluding the area with a bandage; 604 base pairs (Fig. 5) . The guinea pig and the a lesion appeared at the same place within 10 to control isolates were deposited in GenBank with 15 days26>. The present guinea pig was the first the following accession numbers: IFM 4885 case of natural infection with H. werneckii identi- (AB079584), IFM 4993 (AB079585), IFM 41538 fied by mycological and molecular biological (AB079586), IFM 41540 (AB079587), IFM methods, and it was manifested similarly to the 41541 (AB079588), IFM 41542 (AB079589), experimental models. It may be possible that the IFM 46077 (AB079590) , IFM 46078 (AB079591) , animal contracted the infection from its environ- IFM 46442 (AB079592), IFM 46445 (AB079593) ment because the cage was humid and dirty with and IFM 46446 (AB079594) and are shown in feces and urine that could create a very favorable Table 1. All these sequences were almost identi- condition for fungal growth. We can not deny the cal. The DNA sequence patterns revealed three possibility that the sample isolated was thus an different types with very high similarity among accidental contamination from the surroundings all the H, werneckii isolates screened in this study. of the animal, because direct and cultural exami- The fungal isolate from the guinea pig showed nation of skin samples from the dark, blackish 100% identity with isolates IFM 41540, IFM lesion in the palm were negative. 41541, IFM 46077, IFM 46078 and IFM 46445 For the treatment of tinea nigra, a personal (type A) . It differed at only one base pair from communication suggested an easy and simple way the isolates IFM 46446, IFM 46442 and IFM of applying scotch tape-occlusion over the black 4993 (type B) and at two base pairs from the lesion once or twice a day until it disappeared. isolates IFM 41542, IFM 41538 and IFM 4885 The drug treatment for this condition when natu- (type C) . The newly isolated fungal sequence rally acquired includes topical undecylenic acid301 was submitted with the accession no. AB079595 or an imidazole agent31~. Rarely, it may appear Jpn. J. Med. Mycol. Vol. 43 (No. 3 ), 2002 179 like a malignant melanoma and a junctional pure culture. Cramer, Lehre: p.260, 1970. nevus32, which are often slightly indurated, ele- 3) Nishimura K: Fonsecaea, Exophiala, Phialophora, vated or both and biopsy is necessary to diagnose Cladosporium, Alternaria and Hortaea. Rinsho to these conditions. Tinea nigra is characterized by Biseibutsu 21: 538-544, 1994 (in Japanese). 4) Nishimura K, Miyaji M: Hortae, a new genus to a black discoloration of the epidermis; a KOH- accommodate Cladosporium werneckii. Jpn J Med smear of skin scrapings and a culture taken from Mycol 25: 135-146, 1984. the lesion help to confirm the diagnosis33) 5) Khan A, McIver FA: A case of tinea nigra A few cases of tinea nigra from the Kanto area palmaris from Charleston, South Carolina. J S C of Japan have been reported to date22,23). In 1999, Med Assoc 76: 464-465, 1980. an isolate of H. werneckii was isolated from an 6) Severo LC, Bassanesi MC, Londero AT: Tinea indoor swimming pool in Chiba City located at nigra: report of four cases observed in Rio 3535'O9" north latitude and at 140 06'04" east Grande do Sul (Brazil) and a review of Brazilian literature. Mycopathol 126: 157- 162, longitude. The present case of the guinea pig was 1994. recorded from Abiko City, located at 3551'4O" 7) Conti-Diaz IA, Burgoa F, Civila E, Bonasse J, north latitude and at 140 01'58" east longitude, Miller A: Tinea nigra. 1st clinical case in in the northern part of Chiba prefecture, indicat- Uruguay. Mycopathol 87: 81- 83, 1984 (in ing a northward migration of this disease in our Spanish). country, possibly caused by recent environmental 8) Kane J, Birkett B, Fischer JB: Tinea nigra infec- changes34~. This phenomenon of expansion of tion in Canada. Sabouraudia 14: 327-330, 1976. 9) Marin-Rojas R: First cases of tinea nigra habitat of the fungus towards the north could be palmaris in Costa Rica (original report of another indicator of global warming and provide cases) . Rev Latinoam Microbiol 14: 25-27, 1972 a motivation to initiate a revision of certain in- (in Spanish). fections caused by organisms known to have par- 10) Kerdel-Vegas F, Bastardo de Albornoz MC: ticular zonal distribution. Tinea Nigra. Report of five cases seen in Phylogenic analysis by evaluation at the D1/D2 Venezuela. Dermatologia 132: 320-330, 1966. region of the 26S large subunit rDNA gene re- 11) Tapia A: Tinea nigra in the Republic of Panama. Arch Argent Dermatol 15: 67-74, 1965 vealed that H. werneckiihad 3 different genotypes. (in Spanish). The maximal rate of divergence was only O.330o 12) Kamalam A, Thambiah AS: Tinea nigra: first among all the H. werneckii isolates tested so far. case report from Madras. Mykosen 25: 626-628, These sequences were more divergent from se- 1982. quences of the other black fungi such as Exophiala 13) Hughes JR, Moore MK, Pembroke AC: Tinea spp., Foncecaea spp., Cladophialophora spp., nigra palmaris. Clin Exp Dermatol 18: 481-482, Aureobasidium pullulans and Alternaria alternata 1993. 14) Dorn M, Krempl-Lamprecht L: Tinea nigra (data not shown) . Using the D1/D2 region of the plantaris. Hautarzt 28: 412 - 415, 1977 (in LSU rDNA gene for identification of H. werneckii German). can be a very useful method. 15) Vittori F: Tinea nigra palmaris imported from In conclusion, H, werneckii infection occurs both Congo. Nouv Presse Med 6: 3229, 1977 (in in humans and in animals and its geographical French). distribution in Japan is gradually migrating 16) Isaacs F, Reiss-Levy E: Tinea nigra plantaris: a north. case report. Australias J Dermatol 21: 13- 15, 1980. Acknowledgement 17) Wallman LS, Ferguson RC: A case of tinea nigra. Med J Aust 2: 270, 1968. This study was performed as the program "Frontier Studies and International N 18) O'Donnell JM, McAleer R: Tinea nigra palmaris. etworking of Med J Aust 2: 270, 1968. Genetic Resources in Pathogenic Fungi and 19) Huber CE, LaBerge T, Schwiesow T, Carroll K, Actinomycetes (FN-GRPF) " through the Special Bernstein PS, Mamalis N: Exophiala werneckii Coordination Funds for Promoting Science and endophthalmitis following cataract surgery in an Technology from the Ministry of Education, immunocompetent individual. Ophthalmic Sur Lasers 31: 417-422, 2000. Culture, Sports, Science and Technology, the 20) Uijthof JM, Cock AW, de Hoog GS, Quint Japanese Government in 2001. WG, van Belkum A: Polymerase chain reaction mediated genotyping of Hortaea werneckii, causa- References tive agent of tinea nigra. Mycoses 37: 307-312, 1) Horta P: Sobre um caso de tinha preta e um 1994. novo coquemelo (Cladosporium werneckii). Rev 21) Zalar P, de Hoog GS, Gunde-Cimerman N: Med Cir Bras 29: 269-274, 1921. Ecology of halotolerant dothideaceous black 2) Arx JA von: The genera of fungi sporulating in yeasts. Stud Mycol 43: 38-48, 1999. 180 真菌誌第43巻第3号平成14年

22) Kihara A, Tanaka R, Nishimura K, Ogasawara 28) Fujita S, Matsuyama T: Experimental tinea Y, Ishikawa M, Hasegawa S, Omichi M: Tinea pedis induced by non-abrasive inoculation of nigra-causing fungus isolated from an indoor Trichophyton mentagrophytes arthrospores on the pool. Bokin Bobai 29: 147 - 151, 2001 (in plantar part of a guinea pig foot. J Med Vet Japanese). Mycol 25: 203-213, 1987. 23) Tanaka R, Nishimura K, Miyaji M: Hortaea 29) Tanaka R, Sano A, Nishimura K: Pathogenicity werneckii isolated from house dust. Jpn J Med of Hortaea werneckii isolated from house dust. Jpn Mycol 31: 23-28, 1990 (in Japanese). J Med Mycol: Proceedings of 33rd Annual 24) Makimura K, Murayama YS, Yamaguchi H: Meeting of the Japanese Society for Medical Detection of a wide range of medically impor- Mycology, Okinawa, September 22-23, p.90 (C- tant fungal species by polymerase chain reaction 20), 1989. (PCR) . J Med Microbiol 40: 358-364, 1994. 30) Merwin CF: Tinea nigra palmaris: review of the 25) Kurtzman CP, Robnett CJ: Identification of literature and case report. Pediatrics 36: 537- clinically important ascomycetous yeasts based 541, 1965. on nucleotide divergence in the 5'-end of the 31) Marks JG Jr. King RD, Davis BM: Treatment large-subunit (26S) ribosomal DNA gene. J Clin of tinea nigra palmaris with miconazole. Arch Microbiol 35: 1216-1223, 1997. Dermatol 116: 321-322, 1980. 26) Kwon-Chung KJ, Bennett JE: Tine nigra. In: 32) Babel DE, Pelachyk JM, Hurley JP: Tinea nigra Medical Mycology (Kwon-Chung KJ, Bennett masquerading as acral lentiginous melanoma. J JE, eds.) , 1st edn. p.191-197, Lea & Febiger, Dermatol Surg Oncol 12: 502-504, 1986. Philadelphia, 1992. 33) Itani Z: Tinea nigra palmaris. Mykosen 15: 27- 27) Palmer SR, Bass JW, Mandojana R, Wittler 34, 1972. RR: Tinea nigra palmaris and plantaris: a black 34) Misawa M: Secular change of temperature with fungus producing black spots on the palms and urbanization in Tokyo. Geog Rep of Chiba Univ soles. Pediatr Infect Dis J 8: 48, 1989. 11: 19-25, 2000 (in Japanese).