Validation of a serodiagnostic test for sporotrichosis: a follow-up study of patients related to the Rio de Janeiro zoonotic outbreak

Medical Mycology, 2015, 53, 28 33 doi: 10.1093/mmy/myu058 Advance Access Publication Date: 4 December 2014 Original Article Original Article Validation of a serodiagnostic test for sporotrichosis: a follow-up study of patients related to the Rio de Janeiro zoonotic outbreak Andréa Reis Bernardes-Engemann 1,,Mônica de Lima Barros 2, Tamara Zeitune 1, Daniela Cotrim Russi 1, Rosane Orofino-Costa 1 and Leila M. Lopes-Bezerra 3 1 Departamento de Dermatologia, Hospital Universitário Pedro Ernesto, Universidade do Estado do Rio e Janeiro, Rio de Janeiro, Brazil, 2 Fundação Oswaldo Cruz, Rio de Janeiro, Brazil and 3 Laboratório de Micologia Celular e Proteômica, Universidade do Estado do Rio e Janeiro, Rio de Janeiro, Brazil *To whom correspondence should be addressed. Andréa Reis Bernardes Engemann, Departamento de Dermatologia/HUPE/UERJ, Av. 28 de setembro 77, 2 o andar CEP: 20551-030, Vila Isabel, Rio de Janeiro, Brazil. Tel: 55 21 2868-8203/2868-8478; Fax: 55-21-2868-8326; E-mail: andreaengemann@yahoo.com.br Received 6 May 2014; Revised 10 July 2014; Accepted 7 August 2014 Abstract The gold standard for the diagnosis of sporotrichosis consists of the isolation and identification of the fungus from clinical exudates and/or biopsy specimens. However, new technologies offer several advantages over the traditional methods because they are noninvasive and more sensitive in the differential diagnosis of infectious diseases. In the present study, we performed a validation, impact evaluation, and analysis of the applicability of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of sporotrichosis in different groups of patients in comparison with the reference protocols for the evaluation of diagnostic tests for infectious diseases. We used 177 serum samples that were collected between 1998 and 2008 from patients in a geographic area related to the Rio de Janeiro outbreak of sporotrichosis. The ELISA had a low rate of cross-reactivity based on the reference values for diagnostic tests, and the analysis of the receiver operating characteristic curve revealed an area under the curve of 0.9154. Furthermore, higher accuracy was observed in the serodiagnosis of infections in special groups of patients such as pediatric and human immunodeficiency virus positive patients. We concluded that this ELISA had a good clinical serological correlation and, therefore, can be considered a new diagnostic tool applicable to all clinical presentations of sporotrichosis. Key words: sporotrichosis, serodiagnosis, ELISA, SsCBF antigen, ROC curve. Introduction Sporotrichosis is a subacute or chronic mycosis caused by thermodimorphic pathogenic species belonging to the genus Sporothrix [1,2]. It is an infection that usually affects the skin and subcutaneous tissue. However, extracutaneous and atypical manifestations have been observed 28 C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Bernardes-Engemann et al. 29 frequently in zoonotic sporotrichosis outbreaks in the state of Rio de Janeiro, Brazil [3 5]. The main pathogenic species known to exist within the complex Sporothrix schenckii are S. schenckii sensu strictu, S. brasiliensis, S. globosa, ands. luriei [6]. The diagnosis of sporotrichosis is confirmed by the isolation and identification of the etiologic agent (known as the gold standard). Although this is a simple and inexpensive method, isolation of the fungus is difficult in some cases due to the location of the lesions. Despite the need to develop more specific molecular methods for the diagnosis of different Sporothrix species, this type of test has only been used in clinical research and has not yet been validated for use in routine diagnoses [7]. The development of a serological test such as an enzyme-linked immunosorbent assay (ELISA) with the antigen SsCBF [8] is important as it can be used as a complementary tool for molecular studies, including therapeutic monitoring [7,9]. In addition, it could be used for the laboratory diagnosis of sporotrichosis in medical practice, particularly in cases where there is no need to identify the species or when there is difficulty in isolating the agent. The identification of species-specific antigens increases the possibility that rapid, noninvasive, and highly sensitive serological methods will be used for diagnosis, diagnostic screening, and monitoring treatment [10]. According to the reference protocols for evaluation of the diagnosis of infectious diseases, the first steps are to identify the diagnostic target and optimize the test components and reagents. Subsequently, the performance of the serological test and the operating characteristics are evaluated [11,12]. Previously, we showed that an ELISA that uses the antigenic SsCBF fraction reached 90% sensitivity and 80% specificity with samples from patients who had different clinical manifestations of sporotrichosis that had been diagnosed by isolation of the etiologic agent (gold standard) [8]. Our aim in this study was to validate this ELISA using the purified SsCBF antigenic fraction in the serodiagnosis of sporotrichosis according to the reference protocols for the evaluation of diagnostic tests for infectious diseases. Methods Study design We performed a cross-sectional study that included 307 serum samples in order to determine the sensitivity, specificity, positive and negative predictive values, and efficiency of the test. A total of 177 serum samples from patients with sporotrichosis confirmed via mycological examination and culture were analyzed. The samples were collected from September 1998 through October 2008 at the Mycology Laboratory of the Pedro Ernesto University Hospital, Universidade do Estado do Rio de Janeiro, and the Evandro Chagas Clinical Research Institute, Fundação Oswaldo Cruz. As controls, 77 samples of sera from patients with heterologous infections (aspergillosis, n = 6; paracoccidioidomycosis, n = 35; histoplasmosis, n = 8; cryptococcosis, n = 18; and chromoblastomycosis, n = 10), 15 serum samples from patients with other nonfungal diseases, and 38 serum samples from healthy individuals were included. Specimens from human immunodeficiency virus (HIV) positive and pediatric patients were analyzed independently and denominated as special groups. The ethics committees of both institutions approved this study. ELISA performance characteristics To calculate the performance characteristics of the test, the following four types of results were analyzed: truepositive (TP), false-positive (FP), false-negative (FN), and true-negative (TN) [12]. S. schenckii strain and isolation of SsCBF fraction The S. schenckii strain 1099 18 (American Type Culture Collection MYA 4821), originally obtained from the Mycology Section, Department of Dermatology, Columbia University, New York, USA, was used. The SsCBF fraction was isolated according to the method of Penha and Lopes-Bezerra [13]. Briefly, SsCBF was purified from the cell wall peptide-ramnomanan fraction isolated from yeast cells through affinity chromatography on sepharose 4B- ConA (Sigma-Aldrich, St. Louis, MO, USA) followed by fractionation on a Bio-Gel P4 column (Bio-Rad, Hercules, CA, USA). ELISA The serum samples from patients with confirmed sporotrichosis (using the gold standard) were evaluated using ELISA to determine the titer of the anti-sscbf immunoglobulin G (IgG) antibody fraction as previously described [8]. Briefly, the wells of the ELISA plates (Maxisorp; Nunc, Roskilde, Denmark) were coated with 50 µl of SsCBF antigen solution followed by an overnight incubation at 4 C. After blocking with fetal calf serum as previously described [13], the plates were incubated with either human serum samples at a dilution of 1:6400 or, when appropriate, the test was performed with 1:2 serial dilutions of the serum samples (starting at a 1:6400 dilution). The plates were incubated with an anti-human IgG peroxidase-conjugate (Gibco BRL, St. Louis, MO, USA) and developed with a substrate solution (2.5 mg/ml O-phenylenediamine and 0.006% H 2 O 2 in citrate-phosphate buffer, ph 5.6). The reaction was carried

30 Medical Mycology, 2015, Vol. 53, No. 1 out over a 20-min incubation period and stopped with 3 M H 2 SO 4 ; the absorbance was then determined at 490 nm. Analysis of the receiver operating characteristic curves To determine the discriminatory power of the test, receiver operating characteristic (ROC) curves were computed, and the test performance characteristics were calculated as functions of the different cutoffs to determine the best cutoff point. The area under the ROC curve (AUC) was used to determine the accuracy of the test, with data analyses performed via Stata software, version 8.2 (StataCorp LP, USA). Statistical analysis The cutoff (A 490 = 0.230) was established by calculating the mean plus two times the standard deviation of the absorbance values of the serum samples from the healthy individuals to build the table for comparison with the gold standard test results. The comparison of the means between different groups and a reproducibility analysis of the test were performed using Student unpaired t test and the Pearson correlation coefficient, respectively. Figure 1. Receiver operating characteristic (ROC) curve obtained in standardizing the serological enzyme-linked immunosorbent assay (ELISA) with 307 serum samples (sporotrichosis, other mycosis, other nonfungal diseases, and health individuals) using the purified fraction SsCBF. The area under the ROC curve represents the accuracy of the ELISA; the value is 0.9154. Results Evaluation of the performance characteristics of the ELISA The analysis of 177 serum samples from confirmed sporotrichosis patients had an AUC of 0.9154 ± 0.0163 (95% confidence interval, 0.88346 0.9431; Fig. 1). The reproducibility of the ELISA data (Pearson correlation coefficient, r = 0.98) suggests that there is a match among tests performed during the test standardization and in the computation of r = 0.89 when fresh and archived samples were compared, indicating that archived samples (a retrospective analysis) can be used without impairing the outcome. The test performance characteristics calculation showed that of the 177 evaluated samples, 158 were correctly classified as true-positive samples and only 19 were false negatives. Of the 130 serum samples used as negative gold standards, 106 were correctly classified as true negatives and only 24 samples were classified as false-positive samples (Table 1). The ELISA had a sensitivity of 89%, a specificity of 82%, a positive predictive value of 87%, a negative predictive value of 85%, and an overall efficiency of 86%. Sporotrichosis seroepidemiology The seroepidemiology data from the 177 patients were analyzed as described in the following paragraphs. Regarding gender, 65.5% of the patients were female (116/177) and 34.5% were male (61/177). While the serological analysis indicated that the seropositivity was similar for both sexes, male serum samples had a lower number of false-negative results (Fig. 2). Regarding the clinical classification, the lymphcutaneous form was predominant (68.4%), followed by Table 1. A2 2 table for performance evaluation of the diagnostic test. Test under evaluation Reference standard test a Total Positive Negative Positive True positive = 158 False positive = 24 182 Negative False negative = 19 True negative = 106 125 Total 177 130 307 a Reference standard was based on positive or negative mycological exam.

Bernardes-Engemann et al. 31 89 years (mean = 38.1 years), with the highest number of cases found in patients aged 40 and <60 years (38%) and the highest number of cases with false-negative results concentrated in those aged 19 and <40 years. This same group had the highest serological titers (Fig. 3). The geographical area with the highest number of sporotrichosis cases was underserved places in the state of Rio de Janeiro (31.5%). Figure 2. Reactivity of serum samples at a dilution of 1:6400 from men and women in the 177-patient sporotrichosis group determined via an enzyme-linked immunosorbent assay specific for the SsCBF antigen. The dotted line represents the set cutoff point (A 490, 0.23). Figure 3. Reactivity determined by the enzyme-linked immunosorbent assay in 109 sporotrichosis serum samples at a dilution of 1:6400 from patients of different ages. The different age groups are represented by the following numbers on the x-axis: (1) pediatric patients, aged 19 years; (2) adult patients, aged 20 and <40 years; (3) adult patients, aged 40 and <60 years; and (4) adult patients, aged 60 years. The dotted line represents the set cutoff point (A 490, 0.23). the fixed cutaneous (15.8%) and disseminated cutaneous (9.4%) forms. Extracutaneous forms and special manifestations represented 6.5% of the total cases. The serological analysis revealed that only 5.4% of the samples from the patients with a lymph-cutaneous form had a false-negative result and confirmed a high sensitivity for other cutaneous manifestations. For the extracutaneous and atypical manifestations, the test had a sensitivity of 100%, which demonstrates the relevance of the test in cases of difficult clinical and mycological diagnoses. Most lesions were on the upper limbs (63%), followed by the lower limbs (13%) and face (5%). The progression of the disease varied from 2 weeks to 24 months, with a mean of 12.3 months. The most common route of infection was contact with sick and/or healthy felines (75%). The serological analysis showed that only 7.5% of the samples were false negatives for these patients. The age parameter varied from 3 to Applicability of ELISA analysis for serum samples from special groups The serum samples from five HIV-positive male patients, mean age 30.8 years, were all positive in the ELISA test. Of the five patients, four had the disseminated cutaneous form of the disease and were unaware of the contagious nature of the disease, while the remaining patient had the extracutaneous form (meningitis). In all of the cases, the suspicion and subsequent diagnosis of an HIV infection was due to the severe clinical manifestations of sporotrichosis, which suggested an impaired immune system. The serological monitoring for sporotrichosis was performed before and after treatment and the majority presented high titers of IgG antibodies. In addition, a strong clinical serological correlation could be observed in all of the cases (Fig. 4A). Serum samples from the group of patients aged <19 years were all seriologically positive in the ELISA. In this group, the titers were the highest and, in most cases, increased approximately 1 month after the initiation of treatment, similar to what occurs with syphilis (Jarisch- Herxheimer phenomenon). In most of these patients, the post-treatment serum antibody titer was significantly lower than the initial diagnosis titer, decreasing to the cutoff level or even reaching undetectable antibody levels (Fig. 4B). Discussion Recently, sporotrichosis has become an important zoonosis in the state of Rio de Janeiro, and also in the south and southeast regions of Brazil [14]. The number of patients who acquire the infection from felines (Felix catus) is progressively increasing [3,4,15,16]. According to the literature, this outbreak of the zoonotic transmission of sporotrichosis is unique, as outbreaks in other regions and continents result from the contamination of damaged skin by natural sources [17]. Currently, contact with a cat is the most common route of transmission of sporotrichosis infection in the state of Rio de Janeiro, mainly in areas with low socioeconomic levels and bad sanitation conditions, which is consistent with the data observed by other researchers [3].

32 Medical Mycology, 2015, Vol. 53, No. 1 Figure 4. (A) Serological analysis of five human immunodeficiency virus positive patients with disseminated cutaneous and extracutaneous forms of sporotrichosis. The light gray bar represents the control serum. (B) Serological follow-up of seven serum samples from one pediatric patient. Pretreatment sample ( ), sample after 1 month of treatment with a saturated solution of potassium iodide ( ), one month posttreatment sample ( ), 3 months post-treatment sample ( ), 6 months post-treatment sample ( ), 9 months post-treatment sample ( ), 12 months post-treatment sample ( ), and control serum sample ( ). Sporotrichosis affects men and women indiscriminately, as well as patients of all ages. In the group of patients that we studied, we observed a prevalence of females, aged 40 60 years, and a ratio of women to men of 2:1. Lima Barros et al. [3] found the highest prevalence of the disease in women with an average age of 41 years; according to the authors, the prevalence was twice as high in women as in men. The data suggest a correlation with the household activities performed by women, which frequently include contact with and care of domestic animals. The serological analyses of the patients of both sexes showed that the average reactivity (antibody titer) in the patients sera was similar between men and women but that there was a lower proportion of false-negative results in the serum samples from males. Even though sporotrichosis is considered an uncommon mycosis in childhood, we observed a frequency of 9.7% in individuals aged <19 years. All of the patients had positive serology for sporotrichosis (100% true-positive results). Cutaneous manifestations were the most common in this age group, with the upper limbs and face being the most affected sites. Lima Barros et al. [3] assessed a group of 81 sporotrichosis patients aged <15 years and found similar epidemiologic data. In the present study, the lymphcutaneous form was the most common clinical manifestation of the infection (68%). However, the extracutaneous and atypical forms represented approximately 7% of the cases analyzed, suggesting a trend toward an increasing incidence of these clinical forms that were once considered rare. These values are consistent with the distribution of cases reported by Lopes-Bezerra et al. [18]. The serologic testing had 100% efficiency in diagnosing these two clinical forms of the disease, wherein the isolation of the agent is more difficult, and a late diagnosis can lead to the worsening of clinical symptoms including death [7,19]. Currently, the definitive diagnosis of sporotrichosis is based on the isolation and identification of the etiologic agent, which are still regarded as gold standards. This type of diagnosis is considered low cost, but it is not very effective in diagnosis of the extracutaneous and atypical forms of the disease. In contrast, serological tests can be performed quickly, have a high accuracy, and do not require an invasive intervention in most cases. New methods for the more accurate diagnosis of infectious diseases are currently under intense investigation. Most diagnostic methods have significant shortcomings due to the large number of false-positive or false-negative results [20]. The ELISA evaluated in this study had high sensitivity and specificity, in line with the highest international standards set for accuracy in testing [21]. The ROC curve analysis revealed an AUC of 0.9154 for a sample of 177 patients, and the test had 98% reproducibility, making use in multicenter studies possible. The largest percentage of patients with sporotrichosis independent of the clinical manifestation was correctly classified as either a true positive or true negative, according to the serological data. However, false-negative results may occur for several reasons, either individually or in combination, such as a specific anergy within a patient [22]. Another aspect is the fact that serology with the SsCBF fraction is effective and relevant to the diagnosis of severe clinical cases caused by emergent species such as S. brasiliensis [5]. Our data reinforce the importance of the SsCBF-fraction ELISA as a new highly accurate tool for the differential diagnosis of sporotrichosis that could be made available for routine use in clinics and health centers, particularly in Brazil s Unified Health System.

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