Seroprevalences Against Paracoccidioides cetii: A Causative Agent for Paracoccidiomycosis Ceti (PCM-C) and Coccidioides posadasii; for Coccidioidomycosis (CCM) in Dall's Porpoise (Phocoenoides dalli) and Harbor Porpoise (Phocoena phocoena) Stranded at Hokkaido, Japan.

Paracoccidiodomycosis ceti (PCM-C) is a zoonotic mycosis characterized by chronic granulomatous cutaneous lesions in cetaceans. It is distributed worldwide and is caused by an unculturable fungus; Paracoccidioides cetii. On the other hand, coccidioidomycosis (CCM), caused by Coccidioides spp., is also a zoonotic and highly pathogenic fungal infection endemic in both American continents. Even though the Far East is not an endemic area of CCM, an autochthonous case has been reported in China. Although the seroprevalence against P. cetii in captive dolphins was 61.0%, there is no information on wild dolphins living in cold waters. The present study aimed to investigate the seroprevalence against P. cetii and C. posadasii in 15 Dall's porpoises (Phocoenoides dalli) and 11 harbor porpoises (Phocoena phocoena) stranded in Hokkaido, Japan. The seroprevalence against P. cetii in the above dolphins was 26.9% (7/26), which was recorded only in Dall's porpoises (7/15), and that against C. posadasii was 15.4% (4/26), three in Dall's porpoises and one in harbor porpoise. The present study demonstrated positive seroprevalence against P. cetii and C. posadasii in wild cetaceans living in the subarctic areas of the Far East as the first records, and would issue the warning those who live in the area were exposed to the causative agent of CCM from seawater.

First report of Paracoccidioides brasiliensis infection in fish.

The thermodimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), a deep mycosis endemic in Latin American countries that affects mainly male rural workers. Infection by P. brasiliensis has also been reported in several species of terrestrial animals; however, the capacity of the fungus to infect aquatic organisms is poorly known. The aim of this study was to detect P. brasiliensis in a fish species, Nile tilapia (Oreochromis niloticus), the most farmed and widely distributed fish in endemic areas for human PCM in Brazil. As a first step, the humoral immune response against the fungus was evaluated in an experimental group of three fish immunized with inactivated P. brasiliensis yeast cells. For the seroepidemiological study, serum samples of Nile tilapia raised in cages (n = 109) and in ponds (n = 105), collected from a fish slaughterhouse, were analyzed for P. brasiliensis antibodies by ELISA using gp43 as antigen. All the inoculated fish produced antibodies against the fungus. The seropositivity observed in fish raised in cages and ponds was 17.4 and 5.7%, respectively. Due to the higher seropositivity observed in caged fish, 100 tissue samples (encephalon, liver, and kidney), from another group of tilapia raised in cages, were analyzed by polymerase chain reaction (PCR; Pb-ITSR and Pb-ITSE). Three tissue samples (liver n = 1, kidney n = 1, and enchepahlon n = 1) from three different fish resulted positive to PCR. This is the first report to show serological and molecular evidence of P. brasiliensis infection in a fish species.

Parengyodontium album Isolated from Cutaneous Lesions of a Pacific White-Sided Dolphin (Lagenorhynchus obliquidens) During Treatment for Paracoccidioidomycosis Ceti.

The prominence of seafood in Japan motivates close monitoring of its seas and marine lives for potentially pathogenic fungi. During the treatments of the male Pacific white-sided dolphin (Lagenorhynchus obliquidens) for paracoccidioidomycosis ceti (PCM-C), 5 white and floccose colonies showing identical genotype and morphological characteristics were isolated from two skin biopsy samples of cutaneous granulomatous lesions in 2018. The isolates were identified as Parengyodontium album known as one of fungal species having abilities to produce industrially important proteases, and to become a causative agent for emerging mycosis based on morphological and molecular biological characteristics. These lesions consisted of non-malignant pearl-like structures of hyperplastic keratinocytes. Interestingly, although the isolates could grow at 35 °C, their DNA sequences were phylogenetically located in a cluster consisting of environmental and clinical isolates lacking the ability to grow at 35 °C, based on previous reports. The opportunistic infection we observed in the dolphin might be caused by immune disorder due to PCM-C. Notably, although P. album is recognized as non-harmful, and has significant industrial importance and antitumor activity, it has potential to cause not only superficial but also systemic infection, and presents difficulties in treatment because of its high resistance to antifungal compounds.

Seroprevalence of Antibodies Against Paracoccidioides Spp. in Captive Dolphins from Three Aquaria in Japan.

The skin disease paracoccidioidomycosis ceti occurs in several dolphin species globally. Infection by the unculturable fungi Paracoccidioides brasilensis or other Paracoccidioides spp. results in chronic cutaneous and granulomatous lesions. In this study we used immunohistochemistry to investigate the seroprevalence of antibodies to Paracoccidioides spp. in captive dolphins from three aquaria in Japan. We had previously reported that there were serological cross-reactions for Paracoccidioides spp. with related species in the order Onygenales. We hypothesized that the degree of serological cross-reactions for Paracoccidioides spp. might be lower in areas, such as Japan, where the fungal diseases coccidiodomycosis and paracoccidiodomycosis are not endemic. Sera from 41 apparently healthy dolphins, including 20 Atlantic bottlenose dolphins (BD: Tursiops truncatus), 6 Indo-Pacific bottlenose dolphins (IPBD: Tursiops aduncus), 2 F1 generation of a cross between BD and IPBD (F1), 3 Pacific white-sided dolphins (PWD: Lagenorhynchus obliquidens), 2 pantropical spotted dolphins (PSD: Stenella attenuata), 6 false killer whales (FKW: Pseudorca crassidens), and 2 rough-toothed dolphins (RTD: Steno bredanensis) were investigated. Sera from three dolphins with paracoccidioidomycosis ceti were used as a positive control. The yeast-form cells of Paracoccidioides spp. in the cutaneous tissue sample derived from the first Japanese paracoccidioidomycosis ceti case were used as the antigen for the immunohistochemistry. Of the 41 dolphins tested, 61.0% had antibodies against Paracoccidioides spp. This indicates that dolphins of several species in Japanese aquaria have likely been exposed to the pathogen Paracoccidioides spp.

Immunohistochemical Cross-Reactivity Between Arthrographis kalrae and Highly Pathogenic Coccidioides posadasii, Histoplasma capsulatum, and Paracoccidioides Fungal Species.

Recently, we have reported serological cross-reactivity between paracoccidioidomycosis ceti and paracoccidioidomycosis, histoplasmosis, and coccidioidomycosis. However, data on the interaction of Arthrographis kalrae with the above pathogenic fungal infections are lacking. A. kalrae is a widely occurring ascomycetous fungus; causes superficial and deep mycoses; shows thermally dependent dimorphism; and has a genomic profile related to the above-mentioned fungal species. Our study aims to investigate cross-reactivity using eight murine sera, obtained from experimental infection with two A. kalrae isolates. The murine sera were incubated with fungal cells of A. kalrae, Coccidioides posadasii, Histoplasma capsulatum, Paracoccidioides sp., and P. brasiliensis. Thirty murine sera, obtained from experimental infection with six isolates of H. capsulatum, sera from three cases of dolphin paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum sample from a healthy person with a history of coccidioidomycosis, were also incubated with A. kalrae fungal cells and the respective fungal cells that caused the infection as positive controls. Sera derived from the mice infected with A. kalrae reacted strongly when incubated with the Paracoccidioides sp., P. brasiliensis, and C. posadasii, but no positive reaction was observed against the fungal cells of H. capsulatum. The murine sera infected with three out of six isolates of H. capsulatum, and all cetacean and human serum samples reacted positively with the fungal cells of A. kalrae. The present study demonstrated serological cross-reactions among A. kalrae infection, coccidioidomycosis, paracoccidioidomycosis, paracoccidioidomycosis ceti, and histoplasmosis.

Paracoccidioides restrepiensis B339 (PS3) and P. lutzii LDR2 yeast cells and soluble components display in vitro hemolytic and hemagglutinating activities on human erythrocytes.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermodimorfic fungi of Paracoccidioides species complex. Several pathogenic fungi produce hemagglutinins and hemolysins, which are virulence factors involved in adhesion of pathogens to host tissues or cells and in destruction of erythrocytes. The present research investigated hemolytic and hemagglutinating activities of yeast cells and soluble components from P. restrepiensis (PS3; former P. brasiliensis B339) and P. lutzii (LDR2). Different concentrations of live and heat-killed yeast cells and soluble components from cell free antigen preparation (CFA) (native or heated - 56 and 100 °C, 30 min) were mixed with 1% human erythrocyte suspension. Yeast cells from both species caused hemolysis, but P. lutzii LDR2 was more hemolytic than P. restrepiensis B339, while the opposite phenomena occurred with soluble components in most conditions. Live or heat-killed yeast cells of both fungi agglutinated erythrocytes, but only heated soluble components from P. restrepiensis B339 showed hemagglutinating activity. In conclusion, yeast cells of P. restrepiensis B339 and P. lutzii LDR2 produce hemolysins and hemagglutinins, which most likely are more restricted to yeast cells in P. lutzii LDR2 and are more released in soluble form byP. restrepiensis B339, requiring further study.

Study of differential expression of miRNAs in lung tissue of mice submitted to experimental infection by Paracoccidioides brasiliensis.

MicroRNAs (miRNAs) are small single stranded RNA sequences involved in post-transcriptional regulation of different biological and physiological processes. Paracoccidioidomycosis (PCM) is an infection caused by Paracoccidioides brasiliensis, and it is a major cause of mortality due to systemic mycoses in Brazil. To date, there have been few reports on the role of miRNAs in the immune response against fungi, especially PCM. The objective of this study was to evaluate the differential expression of miRNAs related to the inflammatory response associated with pulmonary infection by P. brasiliensis. For this purpose, lungs from BALB/c mice, intravenously infected with P. brasiliensis (2.7×107 yeast cells/ml, n = 12) and noninfected BALB/c mice (n = 8), were collected at the 28 and 56 day after infection. The lung parenchyma presented a great number of yeast cells, granulomas, and edema at 28 days and a framework of resolution of the inflammatory process after 56 days. The mRNAs gata-3, ror-γt, foxp3, and IL-6 were positively regulated at the moment at the 56 day, while the TGF-ß1 mRNA was positively regulated at both moments. The miRNAs 126a-5p, 340-5p, 30b-5p, 19b-3p, 221-3p, 20a-5p, 130a-3p, and 301a-3p, 466k presented the greatest increase in expression levels 28 days after infection, and the miRNAs let-7f-5p, let-7a-5p, 5p-26b, let-7e-5p and 369-3p, 466k presented a greater increase in levels of expression 56 days after infection. This study shows a set of differentially expressed miRNAs possibly involved in the immune response in mice during pulmonary infection by P. brasiliensis.

Trichosporon asteroides Isolated from Cutaneous Lesions of a Suspected Case of "paracoccidioidomycosis ceti" in a Bottlenose Dolphin (Tursiops truncatus).

"Paracoccidioidomycosis ceti" is a rare zoonotic fungal infection affecting dolphins and is endemic worldwide. The causative agents are Paracoccidioides species; however, it is impossible to isolate the fungal species. We isolated Trichosporon asteroides from multifocal, irregularly raised skin lesions on a female bottlenose dolphin (Tursiops truncatus) captured off coast of Japan, which was suspected to have "paracoccidioidomycosis ceti." An abundance of round, yeast-like cells was detected in a potassium hydroxide direct-mount specimen of the skin samples; however, nested PCR targeting the partial sequence of 43-kDa glycoprotein-coding gene correspondent to Paracoccidioides sp. was negative. Biopsied tissue samples were cultured on brain heart infusion agar plates supplemented with chloramphenicol, 1% yeast extract, and 4% sodium chloride (4% NaCl-BHI), on Mycosel agar with 4% sodium chloride (4% NaCl-Mycosel), and on potato dextrose agar supplemented with chloramphenicol (CPDA) at 35 °C for 4 weeks. Cream-colored and wrinkled colonies consisting of hyphae and arthroconidia grew on 4% NaCl-BHI and CPDA, while film-like colonies composed of arthroconidia and round yeast-like cells developed on 4% NaCl-Mycosel. Although these primary cultures resembled fresh isolates of P. brasiliensis, they were identified as Trichosporon asteroides based on routine mycological studies and the internal transcribed spacer regions of ribosomal RNA sequences. The results suggested that trichosporonosis caused by T. asteroides might remain latent among cases of "paracoccidioidomycosis ceti" diagnosed without cultures and molecular biological analysis.

Serological Evidence of Infection by Paracoccidioides brasiliensis in Dogs with Leishmaniasis.

Paracoccidioidomycosis is a systemic mycosis prevalent in Latin American countries, caused by the dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. The habitat of these fungi in nature remains undefined, although it is believed that infection occurs by inhalation of infective propagules present in soil. Sentinel animals, such as dogs, can be valuable epidemiological markers of paracoccidioidomycosis. Taking into account that paracoccidioidomycosis and visceral leishmaniasis may occur in the same area, the objective of this study was to evaluate the occurrence of P. brasiliensis infection in dogs positive for Leishmania sp. Serum samples of dogs positive (n = 199) and negative (n = 101) for Leishmania sp. were analyzed by the immunodiffusion test using P. brasiliensis exoantigen, and 22 samples (7.3%) were positive. The serum samples positive in the immunodiffusion test were also analyzed by Western blotting using the P. brasiliensis gp43 recombinant protein, and 86% of the samples were positive. A high positive correlation (r = 0.96) between positivity for Leishmania sp. and P. brasiliensis was observed. These data suggest an association between leishmaniasis and paracoccidioidomycosis in dogs.

Paracoccidioides brasiliensis-associated dermatitis and lymphadenitis in a dog.

Paracoccidioidomycosis (PCM) is an endemic disease of humans from Latin America that is caused by Paracoccidioides brasiliensis and P. lutzii, with most cases of PCM in domestic animals being associated with P. brasiliensis. This study presents the clinical, cytological, mycological, serological, and molecular findings associated with P. brasiliensis in a dog from Southern Brazil. Fine needle biopsies were collected from the skin and several lymph nodes of a 5-year-old female Labrador dog that had enlargement of most superficial lymph nodes. Cytology of the skin and lymph nodes revealed pyogranulomatous dermatitis and lymphadenitis associated with fine-necked, budding fungal structures consistent with the Paracoccidioides genus of organisms; mycological culture derived from the lymph node aspirate demonstrated similar budding structures. Serological assays using exoantigens obtained from the fungal culture demonstrated that the fungal organisms derived from the lymph node were antigenically similar to P. brasiliensis by immunodiffusion and Western blot. A PCR assay, using the fungal culture as input, amplified a partial segment of the internal transcribed spacer 1 and 2 regions of P. brasiliensis; direct sequencing and phylogenetic analyses confirmed the PCR product as P. brasiliensis. The combined cytological, mycological, serological, and molecular findings confirmed a diagnosis of fungal dermatitis and lymphadenitis due to P. brasiliensis in this dog. This case represents the third description of clinical PCM in dogs and the first confirmation of mycotic dermatitis associated with P. brasiliensis in this species. The participation of dogs in the possible dissemination of PCM is reviewed, and it is proposed that dogs are probable accidental hosts in the epidemiological cycle associated with P. brasiliensis.

Circulating dentinal antigen-antibody immune complexes during orthodontic tooth movement in rats.

BACKGROUND: The immunogenic potential of dentin has been reported through dentin-reactive autoantibodies detection in human and animal model. This study aimed to investigate the formation and diagnostic value of immune complexes formation after autoantibodies production, and soluble dentin antigens levels associated to root resorption, in the course of orthodontic tooth movement, in rat experimental model. METHODS: Forty Wistar rats (n = 8 for each group) were submitted to orthodontic tooth movement, in which the maxillary right first molar was mesially moved by applying of 55 g of force for 3, 7, 14, or 21 days. Untreated group was used as control. Circulating autoantibodies to rat dentinal extract, immune complexes, and soluble dentinal antigen levels were determined by immunoenzyme assays. Additionally, dentinal antigens were analyzed by immunoblot. RESULTS: Higher serum dentin-reactive IgG and immune complex levels were detected in the 14- and 21-day groups (p < 0.05 and p < 0.001 respectively) but not in circulating dentinal antigen levels (p > 0.05), as compared to the control group. Reactivity was found to dentinal components with molecular mass (MM) ~120 and ~150 kDa, by immunoblot. CONCLUSION: This work represents the first evidence of immune complexes formation and circulating soluble dentin antigens associated to root resorption in orthodontic tooth movement. Immune complexes formation could be used to early diagnosis of external root resorption.

Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

Immunoblotting of soluble antigens in Paracoccidioides brasiliensis culture.

This study investigated the major soluble antigens produced by Paracoccidioides brasiliensis (Pb339) cultured in solid Sabouraud (pH 5.6 and 8.5), Sabouraud plus brain heart infusion and liquid tomato juice-enriched complex medium media at intervals of 3 days over 30 days by immunoblotting and concluded that, to optimize the source of each antigen, both time and growth conditions should be considered.

Detection of antibodies against Paracoccidioides brasiliensis in free-range domestic pigs (Sus scrofa).

Paracoccidioidomycosis, caused by the thermodimorphic fungus Paracoccidioides brasiliensis, is a human systemic mycosis prevalent in Latin America. Paracoccidioidomycosis affects mainly male rural workers, causing granulomatous lesions in several organs such as the lungs, liver and spleen. The participation of other animal species in the fungus epidemiology is not well understood. The objective of this study was to evaluate the infection of free-range domestic pigs by P. brasiliensis. Serum samples from 106 pigs were analyzed by ELISA and the immunodiffusion test, using P. brasiliensis gp43 and exoantigen as antigens, respectively. The overall positivity to gp43 in ELISA was 37.7 %, although no reactivity was observed in the immunodiffusion test and nor was P. brasiliensis detected in tissue samples (spleen, lung, liver and lymph nodes) from slaughtered animals submitted to culture, histopathological examination and PCR analysis. Five pigs seronegative to gp43 were exposed to natural infection by P. brasiliensis, and all animals seroconverted 3 months after exposure. The results suggest that free-range pigs are frequently infected with P. brasiliensis but are resistant to disease development. This is the first report of paracoccidioidomycosis in pigs.

An atypical case of chronic paracoccidioidomycosis in a dog caused by a fungus from the Paracoccidioides brasiliensis complex.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin American countries and one of the most important fungal diseases regarding incidence and mortality in humans. PCM has also been described in some animal species such as dogs. In this study we describe a new case of PCM disease in a dog that differed from previous records in the literature which includes a progressive evolution of fungal dermatitis causing a deforming lesion in the nose, like those found in human patients, and humoral response against gp70 instead of gp43, the major diagnostic antigen for human PCM. The clinical isolate through the ITS and partial gp43 gene phylogenetic analysis was grouped in the Paracoccidioides brasiliensis complex. This case describes several features which may contribute to improving diagnosis and understanding of canine paracoccidioidomycosis.

In vitro Arthrographis kalrae biofilm formation: Scanning electron microscopy and cytotoxic analysis.

To our knowledge, this study represents the first demonstration of Arthrographis kalrae biofilm formation in vitro by scanning electron microscopy and the distinct cytotoxic activity between planktonic and biofilm extracts on RAW 264.7 cell line. Higher activity was observed with biofilm. It could impact host immune response, that require furthers study.

Experimental chemotherapy in paracoccidioidomycosis using ruthenium NO donor.

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb). To determine the influence of nitric oxide (NO) on this disease, we tested cis-[Ru(bpy)2(NO)SO3](PF6), ruthenium nitrosyl, which releases NO when activated by biological reducing agents, in BALB/c mice infected intravenously with Pb 18 isolate. In a previous study by our group, the fungicidal activity of ruthenium nitrosyl was evaluated in a mouse model of acute PCM, by measuring the immune cellular response (DTH), histopathological characteristics of the granulomatous lesions (and numbers), cytokines, and NO production. We found that cis-[Ru(bpy)2(NO)SO3](PF6)-treated mice were more resistant to infection, since they exhibited higher survival when compared with the control group. Furthermore, we observed a decreased influx of inflammatory cells in the lung and liver tissue of treated mice, possibly because of a minor reduction in fungal cell numbers. Moreover, an increased production of IL-10 and a decrease in TNF-α levels were detected in lung tissues of infected mice treated with cis-[Ru(bpy)2(NO)SO3](PF6). Immunohistochemistry showed that there was no difference in the number of VEGF- expressing cells. The animals treated with cis-[Ru(bpy)2(NO)SO3](PF6) showed high NO levels at 40 days after infection. These results show that NO is effectively involved in the mechanism that regulates the immune response in lung of Pb-infected mice. These data suggest that NO is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, influencing cytokine production, and consequently moderating the development of a strong inflammatory response.

Anti-dentine antibodies with root resorption during orthodontic treatment.

The aim of this study was to analyse serum IgG levels and salivary secretory IgA (sIgA) levels in human dentine extract (HDE) before (T0) and 6 months after (T6) orthodontic treatment and to correlate anti-HDE autoantibodies to root resorption. Fifty orthodontic patients were selected, 19 males (15.6 ± 8.5 years) and 31 females (21.4 ± 11.2 years), 19 in the mixed dentition (10.3 ± 1.9 years) and 31 in the permanent dentition (24.6 ± 9.9 years). Fifty individuals not undergoing orthodontic treatment matched by gender and age were selected as the controls. Periapical radiographs of the upper central incisors and saliva sampling were obtained of all patients at T0 and T6. Serum samples were collected from the permanent dentition patients (n = 31). Antibody levels were determined by means of immunoenzyme assay. At T6, root resorption was classified as grade 0 (no resorption), grade 1 (slight resorption), and grade 2 (moderate to severe resorption). Differences between antibody levels at T0 and T6 and among different grades of resorption were determined by paired t- and Kruskal-Wallis tests, respectively. Spearman's rank correlation coefficient was applied to detect correlation between sIgA and IgG levels, and logistic regression to determine the association of root resorption grade and the studied variables. Differences were considered significant at P < 0.05. Serum anti-HDE IgG levels decreased (P < 0.01) in grade 2 root resorption patients during treatment and was not correlated to salivary sIgA levels or other variables. Patients who had grade 2 root resorption at T6 showed higher levels of anti-HDE sIgA (P < 0.001). Anti-HDE sIgA levels at T0 and root shape were the main factors associated with the degree of root resorption. The results suggest that variations to systemic and local humoural immune response to dentine antigens may occur during orthodontic treatment. High levels of salivary sIgA before treatment were associated with more advanced lesions after 6 months of treatment.

IgG reactivity profile to Paracoccidioides spp. antigens in people with asymptomatic Paracoccidioidomycosis.

Introduction. Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. As the disease is known to affect mostly men over 40 years old who previously worked handling soil, some cities of agricultural economy in endemic regions may have more cases of paracoccidioidal infection.Gap statement. The true frequency of PCM cannot be established in Brazil because it is not a disease of mandatory reporting. The detection of paracoccidioidal infection may assist in the planning of health services, in order to provide early detection of the disease and to prevent its worsening or even progression to death. In addition, little is described about sera reactivity with antigens from different species of Paracoccidiodes, especially P. lutzii.Aim. Current research was conducted in an inland municipality of southern Brazil, in order to assess infection rate within this endemic region of PCM disease.Methodology. ELISA was employed to evaluate 359 sera from random volunteers from Guarapuava, Paraná, Brazil, to detect IgG against cell-free antigens (CFA) from P. restrepiensis B339, P. americana LDR3 and P. lutzii LDR2. Confirmatory ELISA employed gp43 from B339. Reduction of cross-reactions was sought by treatment with sodium metaperiodate (SMP-CFA, SMP-gp43). Immunoblot was performed with 37 selected sera among those reactive in ELISA. Epidemiological profile was assessed by questionnaire.Results. ELISA reactivity was: CFA/SMP-CFA in general 37.3/17.8 %, B339 25.3/14.5 %, LDR3 24.5/1.4 %, LDR2 8.3/5.8 %; gp43/SMP-gp43 7.2/4.7 %. There were sera reactive with multiple CFAs. In immunoblot, five sera showed the same reaction profile with P. lutzii's antigens as PCM disease sera. Rural residence and soil-related professions were risk factors for paracoccidioidal infection.Conclusion. The low prevalence is in accordance with previous reports of lower PCM disease endemicity in Guarapuava than in other areas of Paraná. Although P. brasiliensis seems to be the prevalent strain of the region, 21 sera from people who only lived in Guarapuava reacted with P. lutzii LDR2. CFA-ELISA with whole antigens seems a good option for serological screening in epidemiological surveys.