Seroepidemiological survey on sporotrichosis-infection in rural areas of the south of Minas Gerais State, Brazil

Abstract

Sporotrichosis is a subcutaneous mycosis caused by traumatic inoculation into the skin by fungi species of the genus Sporothrix. The disease has different clinical manifestations (cutaneous, lymphocutaneous, and disseminated), and can also progress to a systemic infection. Despite having a worldwide distribution, sporotrichosis is most prevalent in tropical and subtropical countries. In Brazil, reports of the disease are higher frequent, where cases of the disease were found in Rio de Janeiro, Sao Paulo, Curitiba, Pernambuco, and Paraiba, among others. Certain groups of people may be more exposed to the causative agent of disease, such as residents of rural areas. Thus, this work aimed to carry out a seroepidemiological survey of the prevalence of sporotrichosis in four rural locations in the south of Minas Gerais State, Brazil. In this study, we used an indirect ELISA test in the survey on the prevalence of sporotrichosis. Data obtained in this study evaluated a population of 631 individuals and showed a prevalence of 44.69%. The distribution of seroprevalence of sporotrichosis with respect to age groups and gender showed no significant statistical difference. Thus, we found a high seroprevalence of sporotrichosis-infection in rural regions of southern Minas Gerais State, Brazil, with no difference in prevalence in relation to gender and age.

Introduction

Sporotrichosis is a subcutaneous mycosis characterized by lesions in the skin and subcutaneous tissue caused by inoculation of species of the Sporothrix genus. Infection with this fungus is related to areas of the body exposed to trauma, such as upper limbs, face, and lower limbs, usually caused by traumatic inoculation with material contaminated with Sporothrix conidia [1]. Sporothrix schenckii was considered a unique pathogenic species on the earliest reports of sporotrichosis; however, genetic sequencing studies confirmed that S. schenckii was composed of the cryptic species: S. albicans, S. brasiliensis, S. globosa, S. luriei, S. mexicana, and S. schenckii [2–4]. S. brasiliensis and S. schenckii are common in Brazil, S. mexicana is present in Mexico, and S. globosa is common in countries such as China, India, Japan, USA, Spain, and Italy [5, 6].

This disease presents the following clinical forms: localized, disseminated, lymphocutaneous, and extracutaneous [7, 8]. The lymphocutaneous form presents nodular and ulcerated infiltrations at the site of inoculation and may compromise lymphatic vessels evolving into a systemic form [1]. In disseminated cutaneous form, there may be involvement of one or more organs, usually associated with some debilitating disease [9]. The pulmonary (extracutaneous) form manifests itself through the inhalation of conidia, usually related to immunodeficiency [10]. The severity of the clinical forms of sporotrichosis depends on the site of inoculation, depth of infection, the virulence of the fungal strain, and the patient’s immune status [11, 12].

There is a difference between sporotrichosis-disease and sporotrichosis-infection. In the case of sporotrichosis-infection, the individuals present a healthy appearance and a positive reaction to Sporothrix spp. antigens in intradermal or serological tests but show no sign of the clinical disease. On the other hand, patients with sporotrichosis-disease have cutaneous and serological positive tests and present clinical signs of the disease [13, 14]. In the last years in Brazil, a significant increase in confirmed clinical cases of sporotrichosis in cats and in humans leads to an interest in studies on this disease [15–17].

The serological tests used in the routine diagnostic include agglutination, immunoprecipitation, and immunoenzymatic tests. Enzyme-linked immunosorbent assay (ELISA) has been used to diagnose different pathologies. For sporotrichosis, ELISA has been used with antigens produced from the crude extract of yeast or filamentous form of the fungus or purified antigens. Studies performed by Almeida-Paes et al. [13] found a specificity of 89% and a sensitivity of 97%. Fernandes et al. [18] using the crude exoantigen extract obtained a sensitivity of 96% and specificity of 98%.

Due to the lack of data on the prevalence of sporotrichosis-infection, the present study was carried out with the objective to access the seroprevalence of sporotrichosis in rural areas of the south of Minas Gerais State.

Materials and methods

Location, study population, and sample selection

A total of 631 serum samples were collected from residents of rural areas from south of Minas Gerais State, Brazil. The main work activity was related to coffee plantation. Most workers also cultivated vegetable gardens as a subsistence activity. The graphical representation of the cities is shown in Fig. 1. Recruitment of residents was done by spontaneous demand. The health worker from each locality announced the day of the meeting. After clarifying the objectives of the research, free and informed consent term was read and signed. Pregnant women and children under 10 were excluded from the study. Subsequently, the registration form was filled out, with personal data such as full name, date of birth, profession, marital status, gender, residence, naturalness, telephone number, general health status, and medications in use. During the interview, it was assessed the resident’s knowledge of signs and symptoms that might indicate prior contact with sporotrichosis. They also reported had not previously undergone sporotrichin skin test assessment. After authorization of the residents, blood was collected to obtain the serum. This work was approved by the Research Ethics Committee of UNIFAL-MG. Also, in the accomplishment of this study, 10 sera were used from sporotrichin-negative individuals as a negative control. The sporotrichin-negative group consisted of students reporting no previous sporotrichosis and did not use immunosuppressive drugs, in addition to the sporotrichin-negative skin test. And as positive control in the ELISA test, a group composed of sera from 3 patients with sporotrichosis. Sera from patients with paracoccidioidomycosis were used to check cross-reaction because this mycosis is also present in rural areas. Confirmation of both diseases was performed by clinical and laboratory examination (visualization of fungal elements compatible with Sporothrix sp. in culture, sporotrichin test for sporotrichosis, and histopathological and double immunodiffusion tests for paracoccidioidomycosis).

Fig. 1.

Map of the region where the data were collected showing the different cities in the south of Minas Gerais State, Brazil

Production of S. schenckii antigens

The strain (Ss118) used in this study is available in the culture collection of the Mycology bank of the Evandro Chagas Clinical Research Institute, Fiocruz, Brazil. The S. schenckii antigen was produced according to the method described by Mendoza et al. [19]. Briefly, pure yeast Sporothrix schenckii cultures were centrifuged at 5000g for 10 min at 4 °C and washed 3 times in sterile saline, where supernatants were discarded after centrifugation for removing impurities, and the pellets were frozen in liquid nitrogen. The stored pellets were macerated to a fine powder, vortexed for 30 min at 4 °C in Tris-Ca2 buffer (20 mM Tris-HCl pH 8.8, 2 mM CaCl2) containing a commercial cocktail of protease inhibitors (1:1000) (GE Healthcare), RNAse and DNAse (1:1000) (GE Healthcare), and 600 μm glass beads (1:1) (Sigma). Cell debris and glass beads were removed by centrifugation at 5000g for 10 min at 4 °C, at the end dithiothreitol (20 mM) was added. For determination of total antigen proteins, the Bradford method was used (BRADFORD; 1976), after the extracts were kept at − 80 °C until use.

Enzyme-linked immunosorbent assay

The ELISA technique was performed according to the protocol proposed by Coelho et al. [20]. Briefly, 96-well polystyrene plates were sensitized with crude extract of S. schenckii fungus at a concentration of 5 μg/mL in 0.1 M carbonate-bicarbonate buffer, pH 9.6 overnight at 4 °C. Afterward, the plate was washed with 0.9% NaCl solution and blocked with 200 μL per well with 5% skimmed milk powder solution in PBS-TWEEN at 37 °C for 1 h. The plate was washed again with 0.9% NaCl solution and dilutions of the sera (1/800, 1/1600, 1/3200, 1/6400) in PBS-TWEEN with 3% gelatin were added to respective wells. The plate was then incubated for 1 h at 37 °C and after this time and was washed with 0.9% NaCl solution. After this period, it was added 100 μL of 6 M urea solution for 5 min and the plate washed again as described above. One hundred microliters per well of the peroxidase-labeled goat anti-human IgG serum conjugate (SIGMA-ALDRICH) at 1:8000 dilution in PBS-TWEEN containing 3% gelatin was incubated for 1 h at 37 °C. The plate was washed with 0.9% NaCl solution. In each well, 100 μL of o-phenylenediamine (OPD) (SIGMA-ALDRICH) substrate was added (20 mg of OPD, 40 mL of 0.1 M citrate buffer, pH 4.5 and 40 μL of 30% H₂O₂) and allowed to stand for 8 min in the absence of light. The reaction was then stopped by the addition of 30 μL of 2 N H₂SO₄ per well. The plate reading was performed by optical density using the wavelength of 490 nm in a plate reader (ZENITH). The cut-off point was calculated between the mean optical density of sera from sporotrichin-negative individuals plus twice the standard deviation for each plate at serum dilution at 1/1600. Optical density values above the cut-off point were considered positive and below negative.

Data analysis

Frequency distributions of socio-demographic data (age and gender) were performed and these were distributed according to the results obtained in the positive or negative ELISA test. The descriptive analysis of the data was performed using the statistical program SPSS (version 19); analysis of the frequency and distribution of the main variables were performed, aiming to characterize the population served. For the continuous variables, the mean, median, and quartiles were calculated. The existence of an association between the continuous variables was evaluated using analysis of variance for comparisons between groups. Categorical variables were compared using the chi-square test.

Results

Population characterization

A total of 631 sera of individuals living in the rural areas from south of Minas Gerais State, Brazil (Alfenas, Divisa Nova, Campos Gerais, and Fama) were evaluated. Among these individuals, 344 were male (54.52%) and 287 were female (45.48%). The mean age was 42.52 ± 15.67 years. The minimum age was 10 years and the maximum age was 86 years (Table 1).

Table 1.

Distribution of gender and age average (minimum to maximum) among rural dwellers

	Male	Female	Total
Number (%)	344 (54.52%)	287 (45.48%)	631 (100%)
Age average (minimum–maximum)	43.31 ± 16.35 (10–86)	41.57 ± 14.92 (12–82)	42.52 ± 15.67 (10–86)

Seroprevalence of sporotrichosis by the ELISA test using sera from rural dwellers

The data presented in Table 2 shows that the prevalence of sporotrichosis in rural dwellers was 44.69% (153 males and 129 females, respectively), presenting a positivity of 44.48% for males and 44.95% for females gender (p = 0.428) using the sera dilution at 1/1600. The same result was found using the cut-off point for sera dilutions of 1/3200 and 1/6400 (data not shown).

Table 2.

Distribution of seroprevalence of sporotrichosis in rural dwellers by gender

	Gender			p*
ELISA	Male	Female	Total
Negative	55.52% (191)	55.05% (158)	55.31% (349)	0.428
Positive	44.48 (153)	44.95% (129)	44.69% (282)
Total	100% (344)	100% (287)	100% (631)

*p value obtained by chi-square test and significance level at 5%

Regarding the distribution by age group, the prevalence in the age group of 16 to 29 years was 44.44% and in the age group of 30 to 49 years was 44.68%. In individuals older than 50 years old, the prevalence was 44.86%. The data did not differ statistically (p = 0.997) (Table 3).

Table 3.

Distribution of seroprevalence of sporotrichosis in rural dwellers by age group

ELISA	Age range (years)			Total	p*
	16–29	30–49	> 50
Negative	55.56% (80)	55.31% (151)	55.14% (118)	55.31% (349)	0.997
Positive	44.44% (64)	44.68% (122)	44.86% (96)	44.69% (62)
Total	100% (144)	100% (273)	100% (214)	100% (631)

*p value obtained by chi-square test and significance level at 5%

Seroprevalence distributions of sporotrichosis by cities

The prevalence found in the rural areas studied ranged from 36.9 to 48.08% and did not present statistical difference (p = 0.375) (Table 4).

Table 4.

Distribution of sporotrichosis prevalence by different localities

ELISA	Cities				Total	p*
	Divisa Nova	Campos Gerais	Fama	Alfenas
Negative	51.92% (27)	54.13% (131)	63.1% (65)	53.84% (126)	55.31% (349)
Positive	48.08% (25)	45.87% (111)	36.9% (38)	46.16% (108)	44.69% (282)	0.375
Total	100% (52)	100% (242)	100% (103)	100% (234)	100% (631)

*p value obtained by chi-square test and significance level at 5%

Regarding the female gender in relation to the studied localities, the prevalence of sporotrichosis also did not present a significant statistical difference (p = 0.582) (Table 5).

Table 5.

Distribution of the prevalence of sporotrichosis by female gender in relation to different localities

	Cities
ELISA	Divisa Nova	Campos Gerais	Fama	Alfenas	Total	p*
Negative	8.23% (13)	38.61% (61)	20.89% (33)	32.28% (51)	100% (158)
Positive	6.98% (9)	39.53% (51)	13.95% (18)	39.53% (51)	100% (129)	0.582
Total	7.67% (22)	39.02% (112)	17.77% (51)	35.54% (102)	100% (287)

*p value obtained by chi-square test and significance level at 5%

The prevalence of sporotrichosis in the male gender in relation to the studied localities did not present a statistically significant difference (p = 0.371) (Table 6).

Table 6.

Distribution of prevalence of sporotrichosis by male gender in relation to different localities

	Cities
ELISA	Divisa Nova	Campos Gerais	Fama	Alfenas	Total	p*
Negative	7.33% (14)	36.65% (70)	16.75% (32)	39.27% (75)	100% (191)
Positive	10.46% (16)	39.22% (60)	13.07% (20)	37.25% (57)	100% (153)	0.371
Total	8.72% (30)	37.79% (130)	15.12% (52)	38.37% (132)	100% (344)

*p value obtained by chi-square test and significance level at 5%

Discussion

Sporotrichosis has a wide geographic distribution, presenting a variable prevalence between countries and regions [8], being more frequent in tropical zones, where the environmental conditions favor the propagation of the fungus [21]. In Brazil, the most common species are S. schenckii and S. brasiliensis [5]. Cases of the disease vary from region to region, and the State of Rio de Janeiro is considered endemic to the disease due to the high rate of zoonotic transmission by cats [16, 22]. In the State of Minas Gerais, data on sporotrichosis prevalence are scarce [23].

The soil has always been considered one of the main reservoirs of Sporothrix genus, constituting an important source of contamination. The disease is most commonly observed in individuals who are in close contact with plants and soil, reinforcing the importance of this study carried out in rural locations [24].

The gold standard diagnosis for sporotrichosis-disease is the identification of the fungus in biological samples or in culture. Although the test has a high specificity, its sensitivity is low, which can lead to false-negative diagnoses [25]. For the diagnosis of sporotrichosis and other subcutaneous diseases, serological tests are also used, among them the enzyme immunoassay (ELISA). The ELISA test for sporotrichosis was described in the literature presenting a good specificity and sensitivity [26]. In this work, an ELISA test was used as a tool to survey the seroprevalence of sporotrichosis-infection in rural locations. In order to decrease possible cross-reactions, several dilutions of serum and treatment with 6M urea solution were used. The treatment with 6M urea was able to eliminate the low-affinity Ag–Ac bonds, aiming the detection of antibodies with high affinity and decreasing false-positive results [27, 28]. The study conducted by Coelho et al. [20] showed efficient discrimination among sera from patients with sporotrichosis, patients with paracoccidioidomycosis, and from sporotrichin-negative individuals using the same ELISA technique [27]. In the present work, the seroprevalence of sporotrichosis-infection was done using the same technique described by Coelho et al. Namujju et al. [28] were able to associate high- and low-affinity antibodies in African women at risk of infection by certain human papillomavirus (HPV) strains using the ELISA test similar to that employed by Coelho et al. [20].

Barros et al. [29] described an epidemic of sporotrichosis-disease in humans in Rio de Janeiro, resulting from the zoonotic transmission. The study was based on information provided by 255 individuals, being 94 infected and 161 without symptoms of the disease. The prevalence of sporotrichosis was four times higher among individuals who lived in contact with infected cats. In this way, there was a higher prevalence in women over 40 years old, who were involved in domestic work. Data obtained from hunters in the south of Minas Gerais State showed the prevalence of sporotrichosis-infection with a higher prevalence in men over 40 years of age, being these rural workers with an exposition of a high risk of contamination [21].

The seroprevalence of sporotrichosis-infection found in this work was 44.69% with a similar distribution between male and female gender (Table 2). Data on the prevalence of sporotrichosis-infection are rare. Rodrigues and de Resende [23] found a prevalence of 13.67% for sporotrichosis-infection in 417 employees of Morro Velho mining in Minas Gerais State using intradermal sporotrichin test. In turn, Sánchez-Alemán, Araiza, and Bonifaz [30] described a prevalence of 6.25% in a sample of 144 individuals for sporotrichosis in Mexico also using intradermal test. Data reported by Ghosh et al. [31] in India report prevalence of sporotrichosis ranging from 22.9 to 40.0% also using the intradermal test. They also observed that the prevalence increased according to the increase of the age and in both genders, being higher in populations that engaged in horticulture, gardening, and carpentry (43.2%) and agriculture (33.9%), similar prevalence to the ones found in the present study.

Regarding the age groups, seroprevalence did not present a statistical difference either (p = 0.997), and the prevalence found in individuals aged between 10 and 29 years was 44.44%, in the age range of 30 to 49 years was 44.68%, and in the age group over 50 years was 44.86% (Table 3). The data presented in this work are different from the data presented by Ghosh et al. [31] who reported that the prevalence increased as the age groups increased. The data presented herein related to the age groups also differ from those presented by Alves et al. [21] evaluating sporotrichosis-disease in rural workers and Barros et al. [29] evaluating domestic workers, since both identified a higher prevalence of sporotrichosis over 40 years.

No significant difference in the prevalence among the different locations in rural areas analyzed was found (48.08%, 45.87%, 36.9%, and 46.16% in the localities of Divisa Nova, Campos Gerais, Fama, and Alfenas, respectively) (p = 0.375) (Table 4). Also, the prevalence did not vary statistically in the male and female genders relative to the rural areas studied (p = 0.371 and p = 0.582, respectively) (Tables 5 and 6).

According to Barros et al. [29], sporotrichosis affects both genders and all age groups. Regarding the prevalence between men and women and among the age groups, it is both related to the exposure to the fungus. Brazil has had sporotrichosis outbreaks where the main means of transmission occur due to contact with infected felines. This is a very particular disease transmission profile of urban localities (cities), whose residents adopt the cat as a pet [14, 16, 17, 22]. In the rural locations studied, the main domestic animal adopted by the residents was the dog raised in the peridomicile. Obviously, we cannot rule out transmission of the disease via contact with infected cats, but we believe this is less likely because of the profile of the population studied.

It is important to emphasize that the present study was carried out with residents of rural areas, where coffee culture stands out. These localities have proximity among them, characterized as a mesothermal tropical climate region (hot and humid). This might explain why no statistically significant differences were found in the prevalence of sporotrichosis in relation to gender and age groups and among rural areas studied.

The seroprevalence of sporotrichosis-infection in rural localities of the South of Minas Gerais State was reported for the first time and data here presented show no difference in prevalence between age groups or gender.

Compliance with ethical standards

After clarifying the objectives of the research, free and informed consent term was read and signed. This work was approved by the Research Ethics Committee of UNIFAL-MG.

Conflict of interest

The authors declare that they have no conflict of interest.