An update on the occurrence of Paracoccidioides species in the Midwest region, Brazil: Molecular epidemiology, clinical aspects and serological profile of patients from Mato Grosso do Sul State

Background Paracoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii. Methodology and main findings This study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome. Conclusions This study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented.

Introduction Paracoccidioidomycosis (PCM) is an endemic systemic fungal infection exclusive to Latin American countries such as Brazil, Argentina, Colombia and Venezuela, where approximately 10 million people have already been infected [1]. Infection in humans with Paracoccidiodes spp. occurs through the development of activities that involve the management of soil contaminated by conidia, such as agriculture, gardening, soil preparation and earthworks [2]. The chronic form of PCM affects mainly adults aged 30 years or older, usually male patients. The acute/sub-acute form of PCM occurs mainly in children and young adults, who represent approximately 10% to 25% of PCM cases [3].
Etiological agents of PCM are thermodimorphic fungi belonging to the Paracoccidioides genus, Ajellomycetaceae family, Onygenales order and Eurotiomycetes class [4]. Currently Paracoccidioides spp. complex is composed of five phylogenetic species: Paracoccidiodes brasiliensis sensu stricto (S1a and S1b), belonging to the paraphyletic group distributed in Brazil, Argentina, Paraguay, Peru and Venezuela; Paracoccidiodes americana (PS2), belonging to the monophyletic group distributed in Brazil and Venezuela; Paracoccidiodes restrepiensis (PS3), belonging to the monophyletic group found mainly in Colombia; and Paracoccidiodes venezuelensis (PS4), belonging to the monophyletic group found exclusively in Venezuela [5][6][7][8]. Meanwhile, the Paracoccidiodes lutzii genotype includes just one species, which is the etiologic agent found in the area endemic to the states of Mato Grosso and Goiás, located in the Midwest region of Brazil, as well as in the Amazon [7,[9][10][11][12].
PCM diagnosis is performed by visualization or isolation and culture of the fungus and indirectly by detection of antibodies in serological tests, where the reactivity and specificity of the tests are directly related to the preparation of exoantigens produced in house [13]. In addition to its low sensitivity in serological analyses, Paracoccidioides spp. may also show cross reactions with other microorganisms, such as Histoplasma spp., Candida spp. and Aspergillus spp [14]. Therefore, the observation of Paracoccidioides spp. suggestive structures using the microbiological (fresh examination or culture) and histopathological techniques is considered as the gold standard diagnosis [15].
Molecular biology tools have been a great ally in the identification of phylogenetic species of the genus Paracoccidioides spp. for genotypic studies and clinical diagnosis performed directly from samples of patients with PCM [5,11,12,16,17]. Some methodologies such as PCR-Nested [18], conventional PCR [16], qualitative PCR real time [19,20], microsatellites [8,21], mitochondrial markers [22], Multilocus Sequence Typing (MLST) [5] and whole genomic sequencing [23] have been used for the purpose of phylogenetic understanding and for answering important questions that relate genotypic data to epidemiological and serological data. A technique that identifies the species (not variety) of the Paracoccidioides spp. complex and P. lutzii, both environmental and human samples, is the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the alpha tubulin (tub1) gene [24].
The species P. brasiliensis sensu stricto (S1a and S1b) has a geographic distribution in the South and Southeast regions of Brazil and its genotypic frequency is found in the states of São Paulo, Rio de Janeiro, Minas Gerais and Paraná, both in clinical and environmental samples [2,13,18,[24][25][26][27][28]. P. americana (PS2) has its genotype frequency in several regions of Brazil, mainly in the South (States of Paraná and Rio Grande do Sul) and Southeast (States of São Paulo and Rio de Janeiro) [12,24,[26][27][28]. The phylogenetic species P. restrepiensis (PS3) has been described as geographically restricted to Colombia and neighboring territories [5,8]. However, the occurrence of P. restrepiensis (PS3) in Brazil was first shown by  in a PCM endemic area from the Southeastern region (Ribeirão Preto, São Paulo, Brazil) [29]. In addition, this species also has been identified in Botucatu, São Paulo, Brazil [30]. In both municipalities, located in the central-west and northwest of the state of São Paulo respectively, P. brasiliensis sensu stricto (S1) is the prevalent species [26,28,30].
In short, the territorial distribution of Paracoccidioides species is not yet completely known [12,25,31]. In the state of Mato Grosso do Sul, there are no genetic data about the occurrence of Paracoccidioides spp. species. In this sense, our aim was to describe, for the first time, the occurrence of Paracoccidioides species using genotyping of clinical isolates, instead of serology. Furthermore, the study aim was also to determine the reactivity profile of exoantigens produced from genotyped strains of the Paracoccidioides spp. species found circulating in the state.

Ethics statement
This study was approved by the Human Research Ethics Committee (CAAE: 69793917.0.0000.0021) from the Universidade Federal de Mato Grosso do Sul (UFMS). The informed consent document was signed by all participants for research agreement. The parental consent for the participation of the child was obtained in writing, as well the child assent.

Patients
Location, period and design. This study was conducted with PCM cases diagnosed between May 2016 and October 2019, from the reference center for Infectious and Parasitic Diseases at the UFMS hospital, located in the state of Mato Grosso do Sul, Brazil. Thirteen patients, from whom fungus of the Paracoccidioides genus was isolated, participated in the study.
Clinical and sociodemographic analysis. All patients with Paracoccidioides species isolated from their clinical sample cultures were included in this study. Clinical and sociodemographic data collected from the medical records database of PCM patients comprise: age, gender, municipality where they live, occupation, agricultural activity, HIV infection, symptoms, clinical form of PCM, severity, affected organs, therapeutic regimen, outcome and sequelae.
The affected organs were identified by clinical examination (skin, peripheral lymph nodes and pharynx), computed tomography (CT) (lungs, deep-chain lymph nodes, spleen, liver, central nervous system and adrenal glands) or by videoscopy (for larynx and intestine). The PCM clinical form of PCM was classified into acute/subacute or chronic, according to Mendes et al., 2017 [3].
Migratory history. Information about patients' migratory history was obtained by questionnaire applied to each patient and/or the responsible family member. These collected data were: hometown, municipalities they have worked or lived in and previous occupations.
Treatment. PCM cases were treated with Amphotericin B, Itraconazole or Sulfamethoxazole-Trimethoprim combination (also known as Cotrimoxazole), according to the recommendations of the Brazilian guidelines on PCM [13].

Fungal strains
Sixteen clinical strains from 13 PCM cases, collected from different sources, including 5 skin lesion fragments, 4 lymph node aspirates, 4 oral lesion scrapings, 2 sputum and 1 tracheal aspiration were identified by classical methods of fungal identification and identified as belonging to the genus Paracoccidioides spp. These clinical isolates were maintained at 36˚C, in the form of yeast cells, in Fava-Netto´s medium for further genotyping and/or exoantigen production.

Genomic DNA extraction from Paracoccidioides strains
The genomic DNAs isolated from Paracoccidioides yeast cells (reference and clinical strains) were obtained using the ZR Fungal/Bacterial extraction kit (Zymo Research, Irvine, California, United States), according to the manufacturer's protocol, and quantified using the NanoDrop 2000 (Thermo Fisher Scientifc Inc, Massachusetts, United States).

Identification of Paracoccidioides species, phylogenetic analisys and geographic localization for this PCM case series
Clinical isolates from PCM patients were genotyped (tub1-PCR-RFLP) to determine their phylogenetic species, following previously described methodology by Roberto et al. (2016) [24],

Production of exoantigens and accomplishment of serological tests
The exoantigens were produced in house, according to the methodology used by Camargo et al. [14], from three genotyped species o Paracoccidioides genus, circulating in the state of Mato Grosso do Sul: P. brasiliensis sensu stricto-S1b (Pb18) [8], which was designated as Pb_Ag; P. restrepiensis-PS3 (EPM01-B_339) [14] named as Pr_Ag; and P. lutzii (clinical isolate from this study-MS2451) defined as Pl_Ag. To determine the reactivity profile of produced exoantigens, the double agar gel immunodiffusion technique (DID) was performed as described previously [33], against 13 PCM patients' sera, from the same cases from which the clinical strains were isolated.

Statistical analysis
Statistical analysis was performed by Fisher's exact test for comparison of two frequencies from independent samples, and the Cochran Q test for comparison of occurrence of more than two dependent variables. Then, comparison of organs involved occurrence between P. brasiliensis and P. restrepiensis was analyzed by Fisher's exact test. While, multiple comparisons of its DID reactivity were performed using Cochran Q test. The P. lutzii data were not included because only one strain was described. Significance was set up at p�0.05. The statistical software used was SAS-Statistical Analysis System, version 9.2.

Sociodemographic and clinical aspects of this PCM case series
The sociodemographic characteristics of the PCM patients included in the study are presented in Table 1. All PCM cases, with isolated fungus from their culture exams, included in the study were male, with a mean age (SD) of 46 years (11.77), with most of them (84.6%, 11/13) over the age of 40. Regarding occupation, 46.1% (6/13) carry out activities with risk for infection by Paracoccidioides spp. (4 bricklayers and 2 rural workers), while 69.2% (9/13) are currently participating or have already participated in agricultural activities.

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Lymph node aspirate MS2791 P. brasiliensis sensu stricto (S1)  Fig 1 presents the phylogenetic analysis of the thirteen clinical isolates submitted to this study, using the NJ method to assess the genetic distance between them. The result shows that, thirteen samples evaluated phylogenetically, nine have evolutionary proximity with Pb18, thus belonging to the P. brasiliensis sensu stricto (S1). Three clinical isolates had genetic similarity with the reference strain EPM54-T2-P. restrepiensis (PS3), thus confirming their inclusion in this clade. Only one isolate (MS2451) had similarity with the reference strain Pb01, characterizing as P. lutzii. Pb01, MS2541 and EPM194-Pbdog were considered an external group into Paracoccidioides spp. species, showing genetic divergences in comparison to clinical isolates and reference strains of P. brasiliensis sensu stricto (S1) and P. restrepiensis (PS3), which that have genetic similarity between them.

Serological profile for this PCM case series
The serum reactivity of the 13 patients varied according to the exoantigen used in the DID test (S1 Table). The positivity of the test is higher with Pb_Ag and Pr_Ag than with Pl_Ag. In addition, the serum positivity with homologous antigen was 77.8% for Pb_Ag and 66.7% for Pr_Ag (p = 0.87), and the homologous negativity was not different with these two antigens (p = 0.87). The evolutionary method used to analyze the genetic distance between them was Neighbor Joining (NJ). Pb01 (P. Lutzii), MS2451 (P. Lutzii) and EPM194 (P. americana (PS2)) were considered as species of close genetic variety and external group due to the phenomenon long branch attraction (LBA). Pb18 (P. brasiliensis sensu stricto (S1)) and EPM54 (P. restrepiensis (PS3)) had similarity with the clinical isolates classified in their respective species and showing a common similarity between them. On the contrary, a few cross-reactions were observed with Pl_Ag-one with a patient infected with P. brasiliensis sensu stricto (S1) and one with P. restrepiensis (PS3) (S1 Table).

Update on occurrence of Paracoccidioides species in the Midwest region
We performed a review of the literature looking for previously reports of PCM cases in the Midwest region, that have been diagnosed by molecular techniques (S3 Table). The geographic distribution of Paracoccidioides species previously reported [6,7,9,12,24,25,31,[34][35][36] was grouped with our data to present an update on occurrence of phylogenetic species in the Midwest region, Brazil (Fig 2).  [6,7,9,12,24,25,31,[33][34][35] for this region, additionally with found phylogenetic species from this study. The black triangles (▲) represent the geographic distribution of P. brasiliensis sensu stricto (S1) in Goiás

Discussion
Molecular epidemiology is revealing the geographical distribution of Paracoccidioides species into endemic areas [9,24,27,29,31,37]. PCM cases currently are described in almost all regions of Brazil, except the interior of the Northeast [2]. This descriptive study presents a thirteen case PCM series in the state of Mato Grosso do Sul, Brazil, from which clinical Paracoccidioides strains were isolated and genotyped. Thus, for the first time in this state, the clinical Paracoccidioides species were molecularly identified, instead of identification being based on serological data. We found nine clinical isolates genotyped as P. brasiliensis sensu stricto (S1), three isolates as P. restrepiensis (PS3), and only one as P. lutzii.
The best choice to identify the Paracoccidioides species is the molecular approach using clinical isolates from culture exam; however, it grows slowly in culture media. For example, despite this study having enrolled only 13 PCM cases, this series came from the 56 identified new cases admitted to our reference center between 2016 and 2019, and the continuous character of this procedure should be considered, in order to define the present picture and future directions.
Genotype studies evaluating clinical and environmental isolates from Brazilian South and Southeast regions, bordering the state of Mato Grosso do Sul, have shown an occurrence of P. brasiliensis sensu stricto (S1), confirming the predominance of this species in these areas [2,13,[24][25][26][27][28]. In addition, P. restrepiensis (PS3), a phylogenetic species characterized by Matute et al. (2006), previously considered to be restricted to Colombia, was already found (two isolates-human and soil) in Venezuela in 2016, and recently in Southeastern Brazil [5,6,24,29]. Now, we present the first report of P. restrepiensis (PS3) occurrence in the Brazilian Midwest region. Our findings could suggest that endemic areas for PCM would be expanding from the South and Southeast regions to the Midwest and North in Brazil, maybe due to migration for the establishment of agriculture and animal husbandry [2]. However, further studies are needed to explore this potential spread.
Studies have suggested that the Brazilian Midwest region is the area in which P. lutzii is identified with higher frequency than in other regions, based mainly on reports from the state of Mato Grosso [7,9,12,18]. This finding was frequently misinterpreted as meaning that P. lutzii predominates other species in this region. In a recent publication, Hahn et al. (2019) reported that during the period 2011 to 2017 only 34 PCM patients at the reference center were treated due to P. lutzii in the state of Mato Grosso [9]. In addition, studies on PCM caused by P. lutzii from the state of Mato Grosso do Sul were performed based on serological identification [25,30]. The genotyping identification of a great number of fungi of the Paracoccidioides genus should be performed to identify the species distribution in different Brazilian regions.
Our findings partially differed from previous expectations, with only one P. lutzii isolate. The incidence rate of this disease in adults, who frequently migrate, and the long period of latency after infection, ranging from years to decades, means that it becomes difficult, if not impossible, to determine the geographic origin of these phylogenetic species. Thus, it is possible that the patients had acquired the infection in other locales. Five of 13 (38.5%) patients presented a past history of having lived in other Brazilian states. Four were born in different regions of Brazil (Northeast, South and Southeast), and currently are living in the state of Mato Grosso do Sul. For instance, one patient was born in the state of Ceará, where PCM is rare [38], and another one in Minas Gerais, an important endemic area [39,40]; both patients were infected by P. brasiliensis sensu stricto (S1). In addition, two patients were born in the state of Paraná-one infected by P. restrepiensis (PS3) and another by P. brasiliensis sensu stricto (S1), respectively rare and common genotypes in this Brazilian region. On the other hand, the patient infected by P. lutzii was born in the state of Mato Grosso do Sul, but he lived for some time in the state of Rondônia (Northern region), where this phylogenetic species had already been reported in clinical and environmental samples [11,18].
The evolutionary method used in our study, NJ classified P. lutzii and P. americana (PS2) as genetically close species, but it was a mistake because in the proposed nomenclature classification for species of the genus Paracoccidioides spp. these are considered to genetic diverge from each other [6,7]. Thus, P. lutzii and P. americana (PS2) were considered species that evolved quickly in the Paracoccidoides spp, phenomenon known as LBA. Due to the techniques used to identify species and the phylogenetic analysis used in this study, we are limited in terms of the availability of evolutionary methods to assess similarity between strains, but the tub1-PCR RFLP method and the phylogenetic tree built by the NJ method were able to answer the aspects of occurrence of Paracoccidioides species in the state of Mato Grosso do Sul.
Knowledge of the geographical distribution of species regarding genotype is important to define the antigen used in the serological tests, for diagnosis and control of cure, and in specific clinical, radiological and therapeutic aspects if differences are detected. Our serological results showed a high positivity for heterologous antigens between P. brasiliensis sensu stricto (S1) and P. restrepiensis (PS3), both from the Paracoccidoides spp. complex. Similar results were recently observed between P. brasiliensis sensu stricto (S1) and P. americana (PS2), once again between species from the Paracoccidoides spp. complex [27]. On the other hand, serum from only two PCM patients-one infected by P. brasiliensis sensu stricto (S1) and one by P. restrepiensis (PS3) reacted with the P. lutzii antigen, and serum from the patient infected by P. lutzii did not react with the P. brasiliensis sensu stricto (S1) antigen nor with the P. restrepiensis (PS3) antigen. This explains the previous results of false negative reactions in the serum of patients infected by P. lutzii tested with the antigen from P. restrepiensis (PS3)-B339, which belongs to the Paracoccidioides spp. complex. Species from the Paracoccidioides spp. complex present similar protein profiles, explaining the cross reactions among its cryptic species [42], while P. lutzii isolates present different protein profiles, which will demand future studies to define a better antigen for a more specific serological diagnosis [35,42].
The genotypic identification of clinical and environmental isolates of Paracoccidioides spp in countries in Latin America such as Brazil, Argentina, Paraguay and Colombia has been important to understand the geographic distribution of this species in these endemic regions of the PCM [2]. Some countries in South America have the genotypic classification determined according to the relationship between the phylogenetic frequency of each species and its geographic region. P. restrepiensis (PS3) was determined to be a species found exclusively in Colombia, but has been identified in countries such as Venezuela, Brazil, Argentina, Peru and Bolivia [5,23,24,29]. These reports of the presence of P. restrepiensis (PS3) in countries outside of Colombia suggest a possible evidence of geographic expansion of the species in countries that the presence of this clade is unexpected in other regions of South America, but genotypic studies must be carried out to respond to the genotypic frequency of species of Paracoccidioides spp. genus so far exclusive in certain regions and countries of Latin America. Another species of the genus Paracoccidioides spp. that has been observed in a phylogenetic and genotypic study in South American countries is P. brasiliensis sensu stricto (S1) where, its genotypic frequency found in Argentina and Paraguay, corroborating with data from the South and Southeast of Brazil, as well as observed in the Mato Grosso do Sul, MS shown in this study [23]. P. lutzii was found in Ecuador [10], showing unexpected evidence of his clade outside its endemic region in the Midwest region of Brazil. With that, we observe the geographic distribution of species until then exclusive in certain countries of South America.
Our findings reinforce the importance of the identification at molecular level of the fungal species occurring in every endemic area. Which could be the key target for the best development of the diagnosis and follow-up of PCM patients.  Table 2). M: 50 bp DNA ladder molecular weight marker (Sinapse Inc., USA). (TIF) S1