The Current Status of Feline Sporotrichosis in Malaysia

Transcription

1 Med. Mycol. J. Vol. Med. 58E, Mycol. E 107 J. Vol. E 113, 58(No ), 2017 E 107 ISSN Review The Current Status of Feline Sporotrichosis in Malaysia Han Hock Siew Former Han Veterinary Surgery ABSTRACT Feline sporotrichosis has been reported in Malaysia since the 1990ʼs. Since then, studies have revealed that clinical clade D, Sporothrix schenckii sensu stricto, of a single clonal strain is the most common cause of this disease in Malaysia. The prevalence of a single clonal strain from a clinical clade was never before reported in Asia in a specific geographical niche. This raises the possibility of a process of purifying selection and subsequent clonal proliferation. While agricultural practices may serve as the selective pressure, direct causality has yet to be established. Studies into the thermo-tolerability of the Malaysian clonal strain of S. schenckii sensu stricto revealed that a small minority of clinical isolates have the capacity to grow at 37, while the majority displayed low susceptibility to commonly used antifungals in clinical practice, such as itraconazole(itz)and terbinafine(trb). Despite unestablished breakpoints, suspected resistance(mic > 4 mg/ml)towards amphotericin B(AMB)and fluconazole(flc)was recorded in the isolates. This explains the often lack of clinical response in feline patients treated with recommended doses of antifungals, including ITZ. Coupled with the potential zoonotic transmission to clients and veterinarians, protracted treatment period, and subsequent cost of treatment, prognosis of feline sporotrichosis is often regarded to be poor. The use of a higher dose of ITZ has been reported, and an adoption of this high-dose treatment regime is reported in this manuscript, with complete cure achieved in cases of recalcitrant and/or unresponsive feline sporotrichosis, which would otherwise be euthanized. Key words:feline, Malaysia, sporotrichosis Introduction Ever since the description of the disease, the isolation, and the identification of the infectious agent by Benjamin Schenck and Erwin Smith in 1896, Sporothrix schenckii, a soil-associated fungus, has gained worldwide notoriety as an important zoonotic agent of subcutaneous mycoses in humans, dogs, cats, and many other mammals 1, 2). Recent studies have revealed a high genetic heterogeneity of this cryptic fungus, of which five species, namely Sporothrix brasiliensis, S. schenckii sensu stricto, Sporothrix globosa, Sporothrix mexicana, and Sporothrix luriei (formerly S. schenckii var. luriei)are currently considered pathogenic 3, 4). Molecular phylogenetic analyses of the internal transcribed spacer(its)region of ribosomal DNA have been utilized to study the genetic diversity of the Sporothrix complex 5). Zhou et al. suggested that isolates of S. schenckii complex can be divided into four different clades based on molecular typing and geographic distribution 5). The authors divided the isolates into: Clade B(for Sporothrix brasiliensis, a species currently restricted to Brazil), Clade C(the first group of S. schencki sensu stricto, seen predominantly in the Americas and Asia), Clade D(the second group of S. schenckii sensu stricto, seen predominantly in the Americas and Africa), and finally, Clade E(S. globosa, isolated predominantly in Asia) 5). These results not only demonstrate the evolution of isolates in different geographical areas, but also Address for correspondence : Han Hock Siew Han Veterinary Surgery, 42 JLN Ponderosa 1/9, TMN Ponderosa, Johor Bahru, Johor, Malaysia. hanhocksiew.vet@gmail.com

2 E 108 Medical Mycology Journal Volume 58, Number 3, 2017 that S. schenckii sensu stricto can be further grouped phylogenetically into two distinct clades (C and D), underlining the genetic diversity within this species. This would partially explain why different strains of S. schenckii sensu stricto, isolated from different countries like Spain, Brazil, and Malaysia display varying degrees of susceptibility profiles to commonly used antifungals and differ in their pathogenicity in the specific geographical context where they are described 6 9). The clinical manifestations of the disease in the human or animal host are dependent on the virulence factors of the particular strain, such as thermo-tolerability(ability to grow at 37 ) 2). Isolates that are able to grow at 35 but not at 37 remain as a fixed cutaneous lesion, whereas isolates that grow at 37 (close to the human body temperature)display lymphocutaneous lesions in human patients. Other reported virulence factors are melanin production, expression of adhesion molecules, high production of ergosterol peroxide, and production of other specific proteins related to virulence, such as the 70-kDa glycoprotein 10 11). In this review, we report the molecular epidemiology, thermo-tolerability, and antifungal susceptibility profile of S. schenckii sensu stricto isolates from Malaysia. We also describe the treatment of three cases of feline sporotrichosis, refractory to antifungal treatment at standard dosages, using higher oral itraconazole(itz)dosages. Sporothrichosis in the Malaysian veterinary context The first report of feline sporotrichosis as an emerging zoonotic disease in Malaysia was made by Zamri et al. in ). Since then, multiple reports of sporotrichosis in human patients have been published in Malaysia. Among these, a retrospective study reported 13/19 cases that were associated with preceding trauma, half of which were directly attributable to cat scratches or bites 13). This highlights the role of cats not only as propagators but also as a significant risk factor for acquiring this disease in Malaysia, as similarly reported in other countries like Brazil and India 14 17). Molecular epidemiology for Malaysian isolates Characterization of clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rdna ITS region, has been described by Kano et al. 18). The results suggested that a single clonal strain of S. schenckii sensu stricto, from the clinical clade D, was the prevailing causative agent of feline sporotrichosis in Malaysia. This result contrasted with those of Zhou et al., which suggested that S. globosa and S. schenckii sensu stricto clinical clade C were the most common human sporotrichosis pathogens in Asia 5). Moreover, this report by Kano et al. was the first report of the clinical clade D S. schenckii sensu stricto as the prevailing cause of sporotrichosis in feline patients in Asia. Phylogenetic analysis of Sporothrix complex has also recently been conducted 19). It was concluded that there is scarce genetic flow with low migration index and high ancestry coefficient between S. brasiliensis and S. schenckii sensu stricto, while S. globosa, S. mexicana, and S. pallida displayed highly differentiated species with no genetic exchange. The identification of a single clonal strain of S. schenckii sensu stricto of the clinical clade D from Malaysia consisting of an independent genetic population supports the hypothesis of a process of purifying selection followed by population expansion. This is postulated to be caused by divergent evolutionary processes within the species, due to environmental or host selection pressure. The possibilities of a purifying selection process on a nationwide scale are few for an agricultural country like Malaysia. Palm tree(elaeis guineensis)as the biggest cash crop is planted on a mass scale in every Malaysian state, with rice(oryza sativa), rubber(hevea brasiliensis), and others on a lesser scale. Incorporation of farm fungicide has been the cornerstone of crop management in Malaysia since the advent of modern agricultural practices to control crop-associated fungi 20).Of the many fungal diseases that affect Elaeis guineensis, the basal stem rot caused by the fungus Ganoderma boninense is one of the most destructive, directly attributed to significant economic losses to farmers, and its presence in Malaysian palm has been recorded for the past 50 years 21). While this disease has been well described in literature, and root contact with inoculum sources in the soil has been identified as the primary route of infection, its treatment is complex 22). One of its more common treatment modality is the use of hexaconazole, either

3 Med. Mycol. J. Vol. 58(No. 3), 2017 E 109 administered into the palm as trunk injections, soil drenching, or the combination of both methods 23, 24). Recent publications to ascertain the dissipation and persistence of hexaconazole from Malaysian soil samples confirmed that hexaconazole remains moderately persistent in soil samples with an average half-life of days, and its residue in soil sample ranges from to 2.74 mg/kg 23, 24). Thus, the possibility of prolonged exposure to low doses of azoles over decades could have served as an environmental selective pressure to induce selection of a lowly susceptible clonal strain of S. schenckii and its subsequent clonal expansion. Thermo-tolerance of clinical isolates from Malaysia Evaluation of thermo-tolerance was reported in a recent publication in feline isolates from Malaysia 8). In this study, 8/44 isolated strains exhibited the ability to grow at 37 (thermotolerant), which suggests that upon establishment of the infection in the human host, about one-fifth of the patients would contract the lymphocutaneous form instead of the fixed cutaneous disease. This is in contrast to another retrospective study that reported that all human cases of sporotrichosis in Malaysia attributable to cat bites or scratches were presented with the lymphocutaneous form 13). Antifungal susceptibility study for the clinical isolates from Malaysia In the above-mentioned study, estimation of minimum inhibitory antifungal concentration(mic) was also assessed 8). Despite unestablished breakpoints for moulds including S. schenckii, any MIC < 1 mg/l is usually considered susceptible, 1-4 mg/l as intermediately susceptible, and > 4 mg/ L as putatively resistant 2). When geometric means (GM)MIC of S. schenckii sensu stricto from Malaysia, Brazil, and Spain were compared, similarities and differences were found 6 8). All strains were resistant to fluconazole(> 128 mg/l), which could be attributed to its inherent water solubility. In the Brazilian and Spanish reports, terbinafine(trb)displayed the lowest MIC, between 0.05 mg/l and 0.22 mg/l, while MIC towards ITZ was mg/l and 4 mg/l, and towards amphotericin B(AMB), mg/l and mg/l, for Brazil and Spain respectively 6, 7). In contrast, the Malaysian strains displayed low MIC values towards ketoconazole(ktz)(0. 92 mg/l), were intermediately susceptible to ITZ(1. 3 mg/l)and TRB(2.85 mg/l)and resistant to AMB(9.3 mg/l) 8). This may suggest that despite being of the same species, geographical differences should be taken into consideration for the empirical systemic antifungal choices by medical and veterinary practitioners. Furthermore, based on current understanding of the pharmacokinetics and bioavailability of ITZ (10 mg/kg) and TRB (30 mg/kg)upon oral dosing in the cat, it can be extrapolated that standard doses of ITZ would not be able to breach the Malaysian S. schenckii sensu stricto MIC values, and that it would require multiple doses of TRB at 30 mg/kg per day to sufficiently maintain MIC values high enough to inhibit the growth of S. schenckii, increasing the possibility of toxicity 25 27). Nevertheless, it still remains unknown if in vitro results are concordant with in vivo results. Treatment of feline sporotrichosis in the Malaysian context It is a common impression among Malaysian veterinarians that the usage of potassium iodide (KI)capsules, TRB, KTZ, and ITZ, or their combinations at recommended dosages results in very little clinical improvement in most Malaysian sporotrichoid feline patients, despite evidence suggesting that KI, either used alone or in combination with other antifungals, is effective to manage this disease 28 30). Thus, finding an effective therapy has become a real challenge for the Malaysian veterinary clinician. A recent publication that highlighted the use of higher-dose ITZ(35 mg/kg)in cats showed promise for a more successful treatment of challenging cases of feline sporotrichosis 30). This author has been presented with multiple cases of recalcitrant, feline sporotrichosis unresponsive to regular doses of ITZ(10 mg/kg). These patients, otherwise suggested to be euthanized, were successfully treated with higher doses of ITZ(up to mg/kg)as the last resort. Three of such cases are described below with all patients receiving the same protocol. Patients were started on an initial dose of 10 mg/kg, then gradually increased by mg/kg, depending on clinical examination and biochemical tests results every 1-2 weeks, until signs of clinical improvement were observed. The maintenance dose was then continued for 1 month beyond cure, with fortnightly complete

4 E 110 Fig. 1. Case 1: Presented on 10th January 2015 with generalised sporotrichosis confirmed with culture and PCR. Nasal sinuses and ear pinnae were mainly affected with multiple granulomas forming at the ear pinnae tip. Body weight = 3.8 kg Fig. 3. Case 1: 6th January Feline patient tolerated gradual dose increment of itraconazole ITZ without detectable toxicity on biochemistry nor adverse effects on each complete physical examination during follow ups. Body weight = 6 kg physical examination, body weight measurement, and complete biochemistry tests. Other than an occasional increase in aspartate transaminase AST suggesting myopathy, seen as a stiff gait, all cats were able to tolerate the gradual dose increment of ITZ, up to mg/kg/day without liver toxicity. Body weight increased with improved appetite as amelioration of clinical signs took hold. Nevertheless, due to the risk of hepatoxicity associated with ITZ, further controlled studies need to be conducted before this treatment method can be recommended to pa- Medical Mycology Journal Volume 58, Number 3, 2017 Fig. 2. Case 1:1st June 2015, patient after six monthsʼ treatment with ketoconazole at 10 mg/kg per day. No significant clinical improvement. Body weight = 4.8 kg tients suffering from intractable, non-responsive, and life-threatening sporotrichosis. Case 1 An adult intact male, stray, domestic, shorthaired cat from an animal shelter weighing 3.8 kg was presented with the complaint of unresponsive sporotrichosis to systemic ITZ treatment 510 mg/kg administered for the past 3 months. Main lesions were located at the facial region, with marked ulceration of nasal planum and multiple granulomas at the ear pinnae Fig. 1. Culture and genetic analyses confirmed a nonthermo-tolerant S. schenckii sensu stricto. The isolateʼ s MIC for both ITZ and KTZ was 1 mg/l. The patient was then administered KTZ at 10 mg/ kg for 6 months with no significant improvement Fig. 2. With the ownerʼ s informed consent, the patient was then started on the high-dose protocol, after which significant clinical improvement was noted very quickly. The patient made complete recovery Fig. 3 within the next 6 months and remains free of disease to date over 12 months follow up. This patient was treated with high-dose protocol for approximately 7 months with no abnormal biochemistry results during the treatment period. Case 2 A 5-year-old intact male, black, domestic, shorthaired cat weighing kg was presented with the complaint of sporotrichosis unresponsive to ITZ at regular doses. The patient had multiple chronic exudative wounds located principally

5 Med. Mycol. J. Vol. 58(No. 3), 2017 E 111 Fig. 4. Case 2: Generalized sporotrichosis affecting the nasal sinuses and tip of ear pinnae. Multiple granulomas were seen forming at the ear pinnae tip, suggesting an anatomic preference of the fungi to cooler regions of the host body. Diagnosis was confirmed with culture and PCR. Body weight = 5.65 kg Fig. 5. Case 2: Final examination to determine cure in May 2016, after 8 months of continuous treatment. Body weight = 4.7 kg around the face and trunk, with several granulomas on the ear pinnae(fig. 4). For this patient, previous systemic administration of ITZ at 5-10 mg/kg failed to induce any significant improvement after 2 months. Culture and genetic analyses confirmed a thermo-tolerant S. schenckii sensu stricto with MIC of the clinical isolate at 2 mg/l for ITZ and 4 mg/l for KTZ. Patient was started on the high-dose protocol with written ownerʼs informed consent and was declared cured after 8 months of therapy(fig. 5). No abnormal biochemistry results were noted throughout the whole treatment period. Case 3 A spayed 3-year-old female cat weighing 5.35 kg was presented with the complaint of a localized chronic, ulcerative, non-healing wound at its perineal region, previously diagnosed as sporotrichosis.(fig. 6). The patientʼ s lesions were unresponsive to systemic ITZ, administered at 10 mg/kg for the previous 2 months. With written informed consent of the owner, the patient was started on the high-dose ITZ protocol plus topical application of chlorhexidine 3%, climbazole 0.5%, and phytoshingosine(douxo Pyo Mousse, Ceva Santé Animale, Libourne, France)3 times a week. During the next monthly scheduled follow up, biochemistry tests revealed increased aspartate transaminase(ast)(fig. 7). Systemic treatment was stopped and topical medication continued. Despite only being on the high-dose ITZ protocol for 1 month, complete cure was achieved in this patient on subsequent follow up. The patient remains free of disease to date, 5 months after drug withdrawal. Conclusion Malaysian sporothrix isolates have been identified as S. schenckii sensu stricto. Its infrequent thermo-tolerability may partially explain the preponderance of lesions in cooler body regions, such as ear tips and nasal planum, while its antifungal susceptibility characteristics may explain why commonly used antifungals at recommended dosages seem to be unable to inhibit fungal growth in Malaysian feline patients. The added risk of zoonosis to veterinarians and owners, protracted treatment periods, and subsequent high treatment costs are factors that often represent additional challenges while attempting the treatment of sporotrichosis. While the author has managed to reduce treatment period using higher-dose ITZ, this treatment modality has risks of hepatotoxicity in cats. In the future, controlled studies are needed to evaluate the safety and efficacy of the high-dose treatment modality in treating patients suffering from intractable, nonresponsive, and life-threatening sporotrichosis.

6 E 112 Medical Mycology Journal Volume 58, Number 3, 2017 Fig. 6. Case 3: Initial presentation on 8th September 2016 with localized chronic non-healing wound at the perineal region. Body weight = 5.35 kg th This article was presented at the 60 Annual Meeting of the Japanese Society for Medical Mycology, Asakusa, Acknowledgement The author would like to thank Dr. Chiara Noli and Dr. Rui Kano for their support, critique and comments in the preparation of the manuscript. Source of funding The study is self-funded. Conflicts of interest The author reports no conflict of interests. The author alone is responsible for the content and writing of this paper. References 1 Schenck BR: On refractory subcutaneous abscesses caused by a fungus possibly related to the sporotricha. Bull Johns Hopkins Hosp 9: , Barros MB, de Almeida Paes R, Schubach AO: Sporothrix schenckii and Sporotrichosis. Clin Microbiol Rev 24: , Marimon R, Cano J, Gené J, Sutton DA, Kawasaki M, Guarro J: Sporothrix brasiliensis, S. globose, Fig. 7. Case 3 on 10th October Patient developed increased AST on routine biochemistry tests. Patient was asymptomatic during physical examination and owner noticed slight stiff gait at home. All systemic medications were stopped with no recrudescence of clinical signs during subsequent phone call follow up. Body weight = 5.6 kg and S. mexicana, three new Sporothrix species of clinical interest. J Clin Microbiol 45: , Bickford D, Lohman DJ, Sodhi NS, Ng PK, Meier R, Winker K, Ingram KK, Das I: Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22: , Zhou X, Rodrigues AM, Feng P, de Hoog GS: Global ITS diversity in the Sporothrix schenckii complex. Fungal Divers 66: , Ottonelli Stopiglia CD, Magagnin CM, Castrillón MR, Mendes SD, Heidrich D, Valente P, Scroferneker ML: Antifungal susceptibilities and identification of species of the Sporothrix schenckii complex isolated in Brazil. Med Mycol 52: 56-64, Marimon R, Serena C, Gené J, Cano J, Guarro J: In vitro antifungal susceptibilities of five species of sporothrix. Antimicrob Agents Chemother 52: , Han HS, Kano R, Chen C, Noli C: Comparison of two in vitro antifungal sensitivity tests and monitoring during therapy of Sporothrix schenckii sensu stricto in Malaysian cats. Vet Derm 28: 156-e32, Fernandes FG, dos Santos PO, Rodrigues AM, Sasaki AA, Burger E, de Camargo ZP: Characterization of virulence profile, protein secretion and immunogenicity of different Sporothrix schenckii sensu stricto isolates compared with S. globosa and S. brasiliensis. Virulence 4: , Nascimento RC, Espindola NM, Castro RA, Teixeira PA, Loureiro y Penha CV, Lopes-Bezerra LM, Almeida SR: Passive immunization with monoclonal antibody against a 70-kDa putative adhesin of Sporothrix schenckii induces protection in murine

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