Sporothrix Brasiliensis Outbreaks and the Rapid Emergence of Feline Sporotrichosis

mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Original article

Sporothrix brasiliensis outbreaks and the rapid emergence of feline sporotrichosis

Karine Ortiz Sanchotene,1,2 Isabel Martins Madrid,3 Gabriel Baracy Klafke,2 Mariana Bergamashi,2 Paula Portella Della Terra,4 Anderson Messias Rodrigues,4 Zoilo Pires de Camargo4 and Melissa Orzechowski Xavier1,2,5 1Post-Graduation program in Health Science, Federal University of Rio Grande (FURG), Rio Grande, Brazil, 2Mycology Lab of Medicine Faculty (FAMED)- FURG, Rio Grande, Brazil, 3Zoonosis control, Municipal Health secretary of Pelotas, Pelotas, Brazil, 4Cell Biology Division, Department of Microbiology, Immunology and Parasitology, Federal University of Sao~ Paulo (UNIFESP), Sao~ Paulo, Brazil and 5Interdisciplinary Area of Biomedical Science (FAMED- FURG), Rio Grande, Brazil

Summary Sporotrichosis is the main subcutaneous mycosis in Brazil, and is caused by Sporo- thrix schenckii and allied species. Sporothrix propagules present on soil and plant debris may be traumatically inoculated into the cutaneous/ subcutaneous tissues of the warm-blooded host. An alternative route involves direct animal–animal and animal– human transmissions through deep scratches and bites of diseased cats. Sporotri- chosis is much more common than previously appreciated with several cases emerging over the years especially in South and Southeast Brazil. We conducted an epidemiological surveillance in endemic areas of feline sporotrichosis in the southern region of Rio Grande do Sul state, Brazil. Over the last 5-year period the number of feline sporotrichosis in Rio Grande increased from 0.75 new cases per month in 2010 to 3.33 cases per month in 2014. The wide geographic distribution of diagnosed cases highlights the dynamics of Sporothrix transmission across urban areas with high population density. Molecular identiﬁcation down to species level by PCR- RFLP of cat-transmitted Sporothrix revealed the emergence of the clonal offshoot S. brasiliensis during feline outbreaks; this scenario is similar to the epidemics taking place in the metropolitan areas of Rio de Janeiro and Sao~ Paulo. Controlling and pre- venting sporotrichosis outbreaks are essential steps to managing the disease among humans and animals.

Key words: Sporothrix brasiliensis, cats, outbreak, sporotrichosis, epidemiology.

complex differ towards ecology, virulence and mor- Introduction phology, which reﬂect in contrasting epidemics strate- Sporotrichosis is the main subcutaneous mycosis in gies and pathogenicity levels to mammals. The high Brazil and is caused by dimorphic fungus from the virulent S. brasiliensis is frequently associated with ani- Sporothrix schenckii complex. Species embedded in this mal horizontal transmission1,2 whereas S. schenckii sensu stricto (s. str.) and S. globosa may be involved Correspondence: Melissa Orzechowski Xavier, Prof. Carlos Henrique with the classic sapronotic route of transmission to – Nogueira, 268. Pelotas, Rio Grande do Sul, CEP: 96020-560, Brazil. humans.3 5 Remarkably, on the other extreme of Tel.: +55 53 3237 4636/4634. pathogenicity, strictly environmental Sporothrix, i.e. S. E-mail: [email protected] or [email protected] mexicana, S. pallida and S. chilensis evolved in associa- Integral part of the doctoral thesis of the ﬁrst author. tion with soil, plants debris and bark-beetles. True environmental species usually lacks pathogenicity to Submitted for publication 17 June 2015 Revised 4 August 2015 mammals and scarce cases of human infection were 6–8 Accepted for publication 2 September 2015 described in the literature.

The classic route of transmission, involves contami- lies at a very low altitude, at its highest point only nation of the warm-blooded host via traumatic inocu- 10 m (33 feet) above sea level. The population esti- lation of plant matter and soil containing Sporothrix mated by the Brazilian Institute of Geography and propagules. Such route reflects what is commonly Statistics (IBGE) is in 207.036 inhabitants (data from observed around the warm areas of the world, espe- 2014) and it harbours the longest beach in the world cially in the Americas and South Africa involving S. (Cassino beach). Cassino beach is approximately schenckii s. str.3,4,9,10 and in Northeast China and 250 km of uninterrupted Atlantic coastline. The biome India, involving S. globosa.4,9,10 It is suggested that is pampa, which is restricted in Brazil to the state of specific conditions in the environment should be met Rio Grande do Sul. The climate of Rio Grande is to promote Sporothrix expansion in nature to cause humid subtropical and mild, with a strong oceanic sapronotic epidemics.4,9 However, in the alternative influence and relatively cold winters, warm summers route of infection, comprising horizontal animal trans- and regular precipitation all year. The average temper- mission (e.g., cat-cat) in addition to zoonotic transmis- ature in the city is 18.3 °C and the average annual sion (e.g., cat-human), S. brasiliensis propagules may precipitation is 1.207 mm. be efficiently transferred via deep scratch and bites of The criteria for feline sporotrichotic infection to infected animals in endemic areas.1,2 qualify as an outbreak case included observing clinical Zoonotic sporotrichosis outbreaks have been symptoms consistent with Sporothrix infection accom- described since the 1990’s and are emerging as an panied with isolation of Sporothrix from ulcerative important infectious disease in South and Southeast lesions. Clinical samples from suspected cases were col- Brazil.3,11–18 These cases are mainly associated with S. lected from January 2010 to December 2014 and brasiliensis,1,3 and domestic cats play key roles in dis- referred to the Mycology Laboratory of the Faculty of ease epidemiology. Cats are highly susceptible to Sporo- Medicine at Universidade Federal do Rio Grande thrix infection, developing multiple skin lesions with a (FAMED-FURG), which drains cases of human and great load of yeast-like cells.19 Consequently, cats pre- animal sporotrichosis and other tropical diseases sent a great potential to spread the disease to other throughout the southern region of Rio Grande do Sul. animals and humans in a short period of time due to Samples were processed by direct mycological exam- their mobility and fighting behaviour.17,19,20 ination with Gram stain and culture in duplicate in The long-lasting outbreak of cat-transmitted sporotri- Sabouraud dextrose agar with and without cyclohex- chosis that originated in Rio de Janeiro presents with imide with incubation of 30 days at 30 °C. The vari- epidemic proportions.14,20 More recently, the beginning ables studied comprised the year of the diagnosis, the of a similar outbreak was described in Sao~ Paulo, and S. animal origin neighbourhood, gender, location of the brasiliensis was involved during animal–animal trans- lesions (fixed form – only one anatomical site; or dis- mission.2 Sporothrix epizootics in cats also occurs seminated – generalised lesions by skin tissue and /or through the Rio Grande do Sul state.18,21–23 However, respiratory signs), animal access to the street (free, the southern region of Rio Grande do Sul is highlighted limited or none) and period between progression of due to the expressive number of cases described com- lesions and diagnosis. pared to adjacent areas,18 with the epicentre around Molecular identification was carried out by PCR- the city of Rio Grande.17,18,24 Sporothrix infections may RFLP (Restriction Fragment Length Polymorphism) as persist in Rio Grande for a long period among symp- described elsewhere.25 Briefly, DNA was obtained and tomatic and asymptomatic animals – a challenge to purified directly from 10-day-old colonies on slants by disease control. Given the importance of feline sporotri- following the Fast DNA kit protocol (MP Biomedicals, chosis, we conducted an epidemiological surveillance Vista, CA, USA).6 Calmodulin-encoding gene (CAL) study in endemic areas of Rio Grande to investigate the was amplified by PCR using the primers CL1 and emergence of cat-transmitted disease using classical CL2A26 and thereafter digested with the enzyme and molecular tools. HhaI.25 The digested products were electrophoresed on 2.5% (w/v) agarose gels for 90 min at 100 V in the presence of GelRedTM (Biotium, Hayward, CA, USA). Materials and methods The bands were visualised using the L-Pix Touch (Loc- The city of Rio Grande is located on southern Rio cus Biotecnologia, Sao~ Paulo, Brazil) imaging system Grande do Sul, Brazil, and is surrounded by waters of under UV illumination. The reference strain CBS the Lagoa dos Patos (the largest lagoon in Brazil), Lagoa 120339 of S. brasiliensis was used as a quality control Mirim and the Atlantic Ocean. The entire municipality for CAL-RFLP.

Data were compiled and the descriptive analysis and The cases of feline sporotrichosis were scattered â chi-square test were performed with SPSS 20.0 (IBM, among 30 different areas of Rio Grande, especially Chicago, USA) considering P < 0.05 statistically signif- from Cassino beach, accounting for 25% of cases icant. Kappa index was calculated to assess the degree (n = 28), followed by Parque Marinha (n = 13/10.9%) of agreement between the diagnostic methods used and Cidade Nova (n = 12/10.1%) (Fig. 2a and (direct examination and culture), and sensitivity rates, Fig. 2b). Approximately half of the animals with specificity, positive predictive value and negative sporotrichosis developed single lesion (56.9%), and the predictive value of the direct examination using the majority were located at the cephalic region. Strik- mycological culture as the gold standard were also ingly, the disease was significantly more diagnosed in calculated. Kappa values were interpreted as follows: male animals (P = 0.007) which accounted for 74% 0.00–0.20, poor agreement; 0.21–0.40, fair agree- of cases, and with free access to street (P < 0.001) as ment; 0.41–0.60, moderate agreement; 0.61–0.80, described in 86.2% of sporotrichotic patients. In addi- good agreement; 0.81–1.00, very good agreement.27 tion, the period between lesions appearing and diagnosis ranged from 3 days to 3 years, but only 21.2% of the animals were early diagnosed in less than 30 days Results to the onset of lesions. Over the last 5-year period, 29 veterinarians joined Of the 129 animals that the culture techniques this study and referred to the Mycology Laboratory at allowed the isolation of Sporothrix, 61 (47.3%) were FAMED-FURG a total of 230 clinical samples (swabs positive in the direct mycological examination, demon- from ulcerated lesions) of cats suspected of sporotri- strating the presence of elongated and oval yeast-like chosis. Of them, 129 (56.1%) were positive in culture cells. Moderate agreement was found between these for Sporothrix spp. supporting clinical findings. The two diagnostic methods (P < 0.001; Kappa = 0.441). positivity percentile of clinical samples did not differ Judging from the mycological culture as the gold stan- between the years (P = 0.387) and ranged from dard, sensitivity, specificity, positive predictive value 66.7% (24/36) in 2012 to 48.2% (40/83) in 2014 and negative predictive value of the direct mycological (Fig. 1). examination were 47.2%, 100%, 100% and 59.8%, Considering the whole study period (60 months), an respectively. average of about two new cases were diagnosed per We employed PCR-RFLP to identify 35 Sporothrix month. However, we found a remarkable increase over isolates down to species level. Cat-transmitted isolates the years of study regarding the number of samples were submitted to CAL-RFLP and after electrophoresis received and the number of confirmed cases. In 2010, fragments were 251, 232, 198, 96 and 85 bp in only 15 samples were received, confirming nine cases length, which are compatible with the restriction pat- of sporotrichosis. Subsequently, the number of positive tern of S. brasiliensis (CBS 120339). samples increased in 2013–2014 to 85 animals (65.9%) with confirmed sporotrichosis (Fig. 1). Thus, Discussion if we consider the number of positive diagnosis for the years of the study, it increased from 0.75 new cases The Rio Grande do Sul State is an endemic area of per month in 2010 to 3.33 cases per month in 2014. sporotrichosis with several studies highlighting the

Figure 1 Number of clinical samples received from felines suspected of sporotrichosis and number of conﬁrmed cases by the isolation of Sporothrix spp. in culture during the 5 years of the study.

(a)

(b)

Figure 2 (a) Geographic localization of Rio Grande do Sul state from Brazil, highlighting Rio Grande city. (b) Aerial view of Rio Grande city (32°04028″S52°08032″); each yellow point represents conﬁrmed cases of feline sporotrichosis included in the study, highlighting the dynamics of Sporothrix transmission across urban areas with high population density. (Accessed in: October 28, 2014). threat of animal–animal and animal–human transmis- to 129 cases in only 5 years and in a single munici- sions.17,18,21–24 Eco-epidemiological characteristics of pality, of which 85 cases were diagnosed during the the Sporothrix-associated outbreaks in the Southern last 2 years of our study (2013–2014). Sporothrix is region of the state are of interesting, due to the emerg- unique in the fungal kingdom due to its frequent ing number of infected animals. Judging from previous occurrence as outbreaks. Indeed, the high number of records of feline sporotrichosis, we observed a signiﬁ- cases in a short period is compatible with the ongoing cant increase in disease incidence rising from 92 cases situation reported earlier in other endemic areas of among seven municipalities over a 10-year period,18 Brazil.1–3,6,12,14,15,19,20,28,29

The real prevalence of feline sporotrichosis is likely which is a major contributing factor to epizootics. In to be underestimated in Southern Brazil, since it is not addition, all these regions described an early outbreak an obligatorily reported disease. We considered only episode2,17,31 triggering the continued transmission, the cases referred to FAMED-FURG and with confirmed which points to negligence of the epidemic. laboratorial diagnosis of sporotrichosis, i.e., by isola- The gold standard to diagnosis sporotrichosis is tion of Sporothrix in culture. Cases diagnosed in other mycological culture; however, it can take up to laboratories, or cases with clinical suspect, were not 30 days to conclusive results. In this sense, it is rec- included. Moreover, feline sporotrichosis is a disease ommended to perform the direct mycological examina- that classically occurs in urban areas characterised by tion concomitantly to culture.34 In cats, whose poor sanitation, substandard housing and little or no injuries often develop a large number of fungal access to health services, what leads us to believe, that propagules, direct examination has a great value, most of the cases may be neglected by pet owners that allowing the presumptive diagnosis in a few minutes do not have access to veterinary care. and can help to guide specific treatment and conse- The wide geographic distribution of diagnosed cases quently improve clinical outcome.20,34 Although the highlights the dynamics of Sporothrix transmission yeast cells of Sporothrix spp. observed in the direct (e.g, cat–cat transmission) among the feline population examination are not pathognomonic and can be con- across the urban areas of Rio Grande as well as the fused with other microorganisms, such as Candida glab- Cassino beach. The scenario is aggravated by the fact rata and Histoplasma capsulatum,34 in our study the that most cases occurs in male animals, with free specificity of this test was 100%, specially taking into access to the street and that developed ulcerated consideration the association with clinical and epi- lesions within more than 30 days before diagnosis. demiological data of the patient. Thus, a positive result Indeed, male cats tend to engage in disputes over terri- in the direct examination of a clinical sample taken tory or females, and once infected they can easily from a feline with suggestive lesions of sporotrichosis transmit the disease to other animals or humans by and in areas of high endemicity of the disease, can deep scratch and bites. Thus, cats remain the main safely be used to aid diagnosis. source of infection of S. brasiliensis in South and Molecular characterisation of Sporothrix isolates is Southeast Brazil.1,3,12,18,30 It is remarkable that close now an essential tool in our attempts to understand related Sporothrix, i.e. S. schenckii s. str. and S. globosa the epidemiology of feline sporotrichosis. Sporothrix follows different strategies during outbreaks.3 There- brasiliensis is the etiological agent of all cases in our fore, dissimilar routes of transmission requires different study identified down to the species level, corroborat- approach to tackle epidemic due to Sporothrix spp. ing previous studies.1–3,20 Sporothrix brasiliensis is a Judging from the animal–animal transmission as in S. clonal offshoot of the classical species S. schenckii s. str. brasiliensis, it is unlikely that the epidemic will end and is considered the most virulent pathogen in the S. spontaneously, what is in accordance with our data schenckii complex.35 Moreover, S. brasiliensis is associ- reporting the increasing number of feline sporotri- ated with zoonotic transmission and atypical clinical chosis in Southern Brazil. presentations in humans.36–38 However, the isolates of The first outbreak of feline sporotrichosis in Rio S. brasiliensis from Rio Grande do Sul differ molecu- Grande occurred in 2000, affecting 30 animals.17,18 larly of those from Rio de Janeiro, Sao~ Paulo and Afterwards, a considerable increasing in the number Minas Gerais,1,12 which deserves more investigation. of confirmed cases in Rio Grande and Cassino beach In conclusion, this study demonstrated the presence was observed between 2008 and 2010. Montenegro of Sporothrix infection among cats in the Southeast et al. [2] also describes the emerging character of Brazil, indicating a high prevalence of the clonal off- feline sporotrichosis in the metropolitan region of Sao~ shoot S. brasiliensis. The emerging nature of feline Paulo, with 163 cases diagnosed over the last 3-year sporotrichosis alerts to the need for constant surveil- period. In addition, the long-lasting outbreak of cat- lance programs and to develop measures to prevention transmitted sporotrichosis in Rio de Janeiro started in and control sporotrichosis in endemic areas. 1990’s decade31–33 and now is accounting for over than 4000 human cases and 3800 in felines.20 Rio Acknowledgments Grande do Sul, Sao~ Paulo and Rio de Janeiro states present different prevalence of cat infections, and eco- This study was supported by grants from the Brazilian logical biomes, although they have in common the funding agencies Sao~ Paulo Research Foundation presence of urban areas with high population density, (FAPESP 2009/54024-2), National Council for

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