Introduction

Paracoccidioidomycosis (PCM) is the most important systemic mycosis in Latin America, especially in Brazil, from where the majority of the cases are reported [1]. In this country, PCM is the eighth cause of death among chronic or recurrent infectious and parasitic diseases [2] and the most prevalent systemic mycosis, covering 35 % of the Brazilian territory [3].

Two major clinical manifestations have been observed in PCM disease: an acute or juvenile type that affects both gender of any age; and a chronic or adult type with lung, skin and mucosal involvement in male adults predominantly [1]. Despite its high prevalence and mortality, PCM is not classified as a mandatory reportable disease. Moreover, the paucity of access to diagnosis and treatment characterizes PCM as a serious neglected disease in Brazil [3].

For almost a century, PCM was thought to be caused by the single species Paracoccidioides brasiliensis [1]. However, a phylogenetic study indicated that this species consists in fact of three distinct species named P. brasiliensis S1, PS2 and PS3 [4]. Further phylogenetic studies based on the genealogical concordance method of phylogenetic species recognition (GCPSR) revealed that the best studied P. brasiliensis strain in Brazil, Pb01, and other 16 strains comprise a clade totally distinct from the previously described S1/PS2/PS3 clade [5]. This so-called Pb01-like group is now recognized as the new species Paracoccidioides lutzii [6]. Moreover, a new cryptic phylogenetic species, PS4, was also recently described [7, 8].

The species P. brasiliensis S1 and PS2 are very closely related phylogenetically [9, 10]. They share morphological characteristics of both yeast and mycelial phases [11, 12], genomic structure [13], low expression of genes related to sexual reproduction [14], in vitro susceptibilities to the combination sulfamethoxazole/trimethoprim (SMZ/TMP) and to other antifungal drugs [15–20], and also families of transposable elements in the genome [21]. On the other hand, significant differences in metabolism pathways [22], antigenic composition [23] and gp43 expression (the main exocellular antigen recognized by sera from patients with paracoccidioidomycosis) [24] are observed among them as well.

It has been described that P. brasiliensis and P. lutzii have differences in terms of conidial and yeast morphology [6, 25, 26], and also antigenic composition [27]. In fact, different serological responses are noticed in patients with PCM caused by P. brasiliensis or P. lutzii when antigens from these two species are used in serological tests [8, 28]. It is also speculated that disease caused by the cryptic species of the genus Paracoccidioides may present differences in severity, tissue involvement and response to therapy [8, 29]. In this paper, we report the first autochthonous case of chronic pulmonary PCM caused by P. brasiliensis PS2 in a patient from Rio de Janeiro, Brazil, an important endemic area of this mycosis [30].

Case Report

A 42-year-old man presented to Evandro Chagas National Institute of Infectious Diseases complaining of hoarseness, non-quantified weight loss, mucosal lesions and mild dyspnea in the preceding 6 months. The patient was referred from Casimiro de Abreu, Rio de Janeiro State, Brazil (22°28′51″S and 42°12′14″W). He reported to be a former farmer in this city and was denied recent or previous travels to other states in the country. Important medical history information to notice was as follows: seizures due to epilepsy since childhood controlled with phenobarbital and carbamazepine, tobacco smoking (8 pack-years), alcohol use disorder and chronic hepatitis B. Reticular interstitial pattern was present in chest radiology, cranial computerized tomography did not present suggestive images of fungal infection, and laboratory analyses (hematology and biochemistry) were within normal ranges, except the ACTH (cosyntropin) stimulation test: 14.48 μg/dL (reference value higher than 18–20 μg/dL after 30 min). The results of Anti-HIV ELISA serological test was negative. Pulmonary tuberculosis was ruled out by negative Ziehl–Neelsen staining and Lowenstein-Jensen culture of the sputum. The PCM diagnosis was confirmed by a positive direct KOH examination of the sputum; isolation and identification of the fungus from samples of mucosal lesions; and also positive antibodies detection for PCM in Ouchterlony immunodiffusion test (ID) (1:8). Genomic DNA was obtained from the yeast phase according to Ferrer et al. [31]. Automated partial nucleotide sequencing of two protein-encoding genes was done in the sequencing platform at Fundação Oswaldo Cruz—PDTIS/FIOCRUZ, based on amplicon products of polymerase chain reaction (PCR) using the arf (ADP-rybosilation factor) and gp43 (glucan 1,3-beta-glucosidase) primers [4, 6]. The sequences of the isolate (IPEC 20960) were deposited in GenBank (KU645890 and KU645891 for gp43 and arf loci, respectively), and a BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) comparing these sequences with those from isolates belonging to the Paracoccidioides brasiliensis complex previously deposited by Matute et al. [4] presented 100 % similarity with P. brasiliensis PS2 strain T10B1 (B7) (Fig. 1). The patient presented clinical, imaging and serological cure after receiving sulfamethoxazole/trimethoprim (SMZ/TMP 800/160 mg b.i.d) for 3  years. He was followed up for 4 years after drug suspension due to dysphonia and low adrenal reserve sequelae that lead to the need of continuous phonoaudiology and endocrinology support.

Fig. 1
figure 1

Phylogenetic relationships of arf and gp43 genes among the clinical isolate of this case report and other 11 P. brasiliensis PS2 strains deposited at GenBank. One P. brasiliensis S1 (B10 strain), one P. brasiliensis PS3 (C18 strain), one P. lutzii (Pb01 strain) and Lacazia loboi were included in the analysis. The phylogenetic tree was estimated in the SeaView version 4 software. Bootstrap values after 1000 replicates are presented in the branch nodes

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Discussion

Despite the clinical relevance that may be hidden within the cryptic species of the Paracoccidioides genus, there is a paucity of PCM case reports in the literature with molecular characterization of the strains (Table 1). Two of these reports describe P. lutzii as the agent of two severe PCM cases, one of which with a fatal outcome, and a third mild case, in which the patient presented emaciation, mucosal lesions and ganglia, but without fever or cough [32, 33]. Another report describes an acute P. brasiliensis case in Colombia, where the fungus was identified by molecular methods, but without description of its phylogenetic species [34]. More recently, Macedo et al. [35] described an acute and severe paracoccidioidomycosis case due to P. brasiliensis S1 that was misdiagnosed as hypereosinophilic syndrome because of a high eosinophil count in peripheral blood and a massive splenomegaly. To the best of our knowledge, there are no case reports of PCM due to the P. brasiliensis phylogenetic species PS2, PS3 or PS4 describing clinical and therapeutic aspects of the patients.

Table 1 PCM cases reported in the literature with molecular characterization of the causative agent and clinical aspects of the patient
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The case herein described was caused by P. brasiliensis PS2. The number of P. brasiliensis PS2 strains in the literature is small, when comparing with the phylogenetic species S1 and PS3 [9, 36]. It was found, in a literature review, 13 papers describing 20 different strains (Table 2) from four different countries (Brazil, Argentina, Venezuela and Uruguay) belonging to this phylogenetic species (Fig. 2). Besides, two PCM cases were assigned to P. brasiliensis PS2 by the sequencing of the product of a semi-nested PCR targeting the gp43 gene, but without isolation of the fungus in culture [37]. These works showed that P. brasiliensis PS2 is a paraphyletic [9], heterothallic species [38] that can be differentiated from S1 by multilocus sequence typing (MLST) [4, 39], microsatellite evaluation [9], restriction fragment length polymorphism of the α-tubulin gene using the MspI and BclI restriction enzymes [36], and single nucleotide polymorphism analysis that can be performed by quantitative real-time polymerase chain reaction (qPCR) or SNaPshot® [11]. In the present work, the strain was successfully identified as P. brasiliensis PS2 by the sequencing of gp43 and arf genes according to methodologies described by Matute et al. [4] and Teixeira et al. [6].

Table 2 Strains of P. brasiliensis PS2 described in the literature
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Fig. 2
figure 2

Geographic distribution of P. brasiliensis PS2 strains described in the literature. Cities marked in yellow or in red present one or two strains, respectively. The orange marker represents Casimiro de Abreu municipality, place of birth and residence of the patient described in this study

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Initially, virulence of P. brasiliensis PS2 was thought to be lower than P. brasiliensis S1 [9]. The supposed lower virulence of P. brasiliensis PS2 might lead to mild PCM cases. However, other studies demonstrated that P. brasiliensis PS2 strains can present high and intermediary virulence, similarly to P. brasiliensis S1 [40], which may yield PCM presenting more severe symptoms and sequelae. The case reported herein reinforces this finding, since the patient infected by P. brasiliensis PS2 had adrenal insufficiency, a serious complication of PCM [41]. Moreover, the patient presented dysphonia, another frequent sequel of this mycosis [42].

It is also important to consider that a good clinical therapeutic response to SMZ/TMP was previously supposed to be more related to P. lutzii. However, the case herein discussed also presented a satisfactory outcome after receiving these drugs.

The positive antibody detection in the ID test performed in this case with mixed exoantigens of strains Pb01 and Pb18, P. lutzii and P. brasiliensis S1, respectively [8], shows that these two species can be used for Paracoccidioides antigen preparations for serologic tests to detect antibodies in infections caused by P. brasiliensis PS2. Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains, and this molecule appeared to be more variable within P. lutzii, suggesting a different evolutionary process. However, the variation on gp43 production also occurs between isolates belonging to the same species, indicating that speciation events are important, but not enough to explain the diversity in terms of antigen production between the cryptic species and nonreactive results by immunodiffusion assays [43]. Therefore, it is recommended that different antigenic preparations from several P. brasiliensis complex species should be applied to improve PCM serodiagnosis by decreasing the number of false-negative patients seen in other studies [8, 44]. One candidate molecule to be used as antigen in the serodiagnosis of PCM is the PbP27 protein produced by P. brasiliensis S1, PS2, PS3, and P. lutzii especially in the parasitic yeast phase [45].

Although there is a paucity of case reports due to this phylogenetic species, seven P. brasiliensis PS2 strains (35 %) are described in the literature as isolated from patients with the chronic form of PCM [9, 11, 15, 36], as observed in our case. One PS2 strain was isolated from a 17-year-old patient [11] that we suppose to be related to the acute PCM form. However, the lack of clinical information of this case occurred in 1970 does not allow us to conclude that P. brasiliensis PS2 can cause both clinical forms of the disease. Further, more studies must be done in order to clarify the clinical implications and response to treatment of this phylogenetic species of P. brasiliensis.