Exploring genetic diversity, population structure, and phylogeography in Paracoccidioides species using AFLP markers.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii. DNA-sequencing of protein-coding loci (e.g., GP43, ARF, and TUB1) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides. We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67-87 fragments), the values of polymorphism information content (PIC = 0.3345-0.3456), marker index (MI = 0.0018), effective multiplex ratio (E = 44.6788-60.3818), resolving power (Rp = 22.3152-34.3152), discriminating power (D = 0.5183-0.5553), expected heterozygosity (H = 0.4247-0.4443), and mean heterozygosity (H avp  = 0.00002-0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651-0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ2 = 1.025; P = 0.3113), P. venezuelensis (χ2 = 0.692; P = 0.4054), and P. lutzii (χ2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found for P. americana (χ2 = 8.909; P = 0.0028) and P. restrepiensis (χ2 = 4.571; P = 0.0325) with a preponderance of MAT1-1. Geographical distributions confirmed that P. americana, P. restrepiensis, and P. lutzii are more widespread than previously thought. P. brasiliensis s. str. is by far the most widely occurring lineage in Latin America countries, occurring in all regions of Brazil. Our new DNA fingerprint assay proved to be rapid, reproducible, and highly discriminatory, to give insights into the taxonomy, ecology, and epidemiology of Paracoccidioides species, guiding disease-control strategies to mitigate PCM.

Trends in the molecular epidemiology and population genetics of emerging Sporothrix species.

Sporothrix (Ophiostomatales) comprises species that are pathogenic to humans and other mammals as well as environmental fungi. Developments in molecular phylogeny have changed our perceptions about the epidemiology, host-association, and virulence of Sporothrix. The classical agent of sporotrichosis, Sporothrix schenckii, now comprises several species nested in a clinical clade with S. brasiliensis, S. globosa, and S. luriei. To gain a more precise view of outbreaks dynamics, structure, and origin of genetic variation within and among populations of Sporothrix, we applied three sets of discriminatory AFLP markers (#3 EcoRI-GA/MseI-TT, #5 EcoRI-GA/MseI-AG, and #6 EcoRI-TA/MseI-AA) and mating-type analysis to a large collection of human, animal and environmental isolates spanning the major endemic areas. A total of 451 polymorphic loci were amplified in vitro from 188 samples, and revealed high polymorphism information content (PIC = 0.1765-0.2253), marker index (MI = 0.0001-0.0002), effective multiplex ratio (E = 15.1720-23.5591), resolving power (Rp = 26.1075-40.2795), discriminating power (D = 0.9766-0.9879), expected heterozygosity (H = 0.1957-0.2588), and mean heterozygosity (Havp  = 0.000007-0.000009), demonstrating the effectiveness of AFLP markers to speciate Sporothrix. Analysis using the program structure indicated three genetic clusters matching S. brasiliensis (population 1), S. schenckii (population 2), and S. globosa (population 3), with the presence of patterns of admixture amongst all populations. AMOVA revealed highly structured clusters (PhiPT = 0.458-0.484, P < 0.0001), with roughly equivalent genetic variability within (46-48 %) and between (52-54 %) populations. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for S. schenckii (χ2 = 2.522; P = 0.1122), supporting random mating. In contrast, skewed distributions were found for S. globosa (χ2 = 9.529; P = 0.0020) with a predominance of MAT1-1 isolates, and regional differences were highlighted for S. brasiliensis with the overwhelming occurrence of MAT1-2 in Rio de Janeiro (χ2 = 14.222; P = 0.0002) and Pernambuco (χ2 = 7.364; P = 0.0067), in comparison to a higher prevalence of MAT1-1 in the Rio Grande do Sul (χ2 = 7.364; P = 0.0067). Epidemiological trends reveal the geographic expansion of cat-transmitted sporotrichosis due to S. brasiliensis via founder effect. These data support Rio de Janeiro as the centre of origin that has led to the spread of this disease to other regions in Brazil. Our ability to reconstruct the source, spread, and evolution of the ongoing outbreaks from molecular data provides high-quality information for decision-making aimed at mitigating the progression of the disease. Other uses include surveillance, rapid diagnosis, case connectivity, and guiding access to appropriate antifungal treatment.

The global epidemiology of emerging Histoplasma species in recent years.

Histoplasmosis is a serious infectious disease in humans caused by Histoplasma spp. (Onygenales), whose natural reservoirs are thought to be soil enriched with bird and bat guano. The true global burden of histoplasmosis is underestimated and frequently the pulmonary manifestations are misdiagnosed as tuberculosis. Molecular data on epidemiology of Histoplasma are still scarce, even though there is increasing recognition of histoplasmosis in recent years in areas distant from the traditional endemic regions in the Americas. We used multi-locus sequence data from protein coding loci (ADP-ribosylation factor, H antigen precursor, and delta-9 fatty acid desaturase), DNA barcoding (ITS1/2+5.8s), AFLP markers and mating type analysis to determine the genetic diversity, population structure and recognise the existence of different phylogenetic species among 436 isolates of Histoplasma obtained globally. Our study describes new phylogenetic species and the molecular characteristics of Histoplasma lineages causing outbreaks with a high number of severe outcomes in Northeast Brazil between 2011 and 2015. Genetic diversity levels provide evidence for recombination, common ancestry and clustering of Brazilian isolates at different geographic scales with the emergence of LAm C, a new genotype assigned to a separate population cluster in Northeast Brazil that exhibited low diversity indicative of isolation. The global survey revealed that the high genetic variability among Brazilian isolates along with the presence of divergent cryptic species and/or genotypes may support the hypothesis of Brazil being the center of dispersion of Histoplasma in South America, possibly with the contribution of migratory hosts such as birds and bats. Outside Brazil, the predominant species depends on the region. We confirm that histoplasmosis has significantly broadened its area of occurrence, an important feature of emerging pathogens. From a practical point of view, our data point to the emergence of histoplasmosis caused by a plethora of genotypes, and will enable epidemiological analysis focused on understanding the processes that lead to histoplasmosis. Further, the description of this diversity opens avenues for comparative genomic studies, which will allow progress toward a consensus taxonomy, improve understanding of the presence of hybrids in natural populations of medically relevant fungi, test reproductive barriers and to explore the significance of this variation.

Subtractive phage display selection for screening and identification of peptide sequences with potential use in serodiagnosis of paracoccidioidomycosis caused by Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease endemic in Latin America whose aetiologic agents are the thermodimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. Despite technological advances, some problems have been reported for the fungal antigens used for serological diagnosis, and inconsistencies among laboratories have been reported. The use of synthetic peptides in the serological diagnosis of infectious diseases has proved to be a valuable strategy because in some cases, the reactions are more specific and sensitive. In this study, we used a subtractive selection with a phage display library against purified polyclonal antibodies for negative and positive PCM sera caused by P. brasiliensis. The binding phages were sequenced and tested in a binding assay to evaluate its interaction with sera from normal individuals and PCM patients. Synthetic peptides derived from these phage clones were tested in a serological assay, and we observed a significant recognition of LP15 by sera from PCM patients infected with P. brasiliensis. Our results demonstrated that subtractive phage display selection may be useful for identifying new epitopes that can be applied to the serodiagnosis of PCM caused by P. brasiliensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, there is no standardized method for the preparation of paracoccidioidomycosis (PCM) antigens, which has resulted in differences in the antigens used for serological diagnosis. Here, we report a procedure that uses subtractive phage display selection to select and identify new epitopes for the serodiagnosis of PCM caused by Paracoccidioides brasiliensis. A synthetic peptide obtained using this methodology was successfully recognized by sera from PCM patients, thus demonstrating its potential use for improving the serodiagnosis of this mycosis. The development of synthetic peptides for the serodiagnosis of PCM could be a promising alternative for the better standardization of diagnoses among laboratories.

Reactivity of antibodies from patients with acute and chronic paracoccidioidomycosis to a high molecular mass antigen from Paracoccidioides brasiliensis.

Yeast forms of Paracoccidioides brasiliensis produce polydispersed high molecular mass (h-MM) antigens. We investigated the antibodies to an h-MM antigen from P. brasiliensis by immunoblotting and ELISA in sera from paracoccidioidomycosis (PCM) patients. IgG from the sera of chronic PCM patients was able to recognize the h-MM antigen at a higher frequency in the cell-free antigen (CFA) (8/13) than in the somatic antigen (SA) (2/13), as assessed by immunoblotting. The CFA was fractionated by Sephadex G-200 chromatography, and fraction 17 (F17) with the h-MM antigen of approximately 366 kDa was used in ELISA to analyze specific levels of IgG and IgE. Patients with the chronic form showed significantly higher levels of IgG (P<0.05) but not IgE (P>0.05) to F17 by ELISA, compared to patients with the acute form or to healthy donors. In conclusion, CFA is better than SA as a source of the P. brasiliensis h-MM antigen. This study reveals a new characteristic to differentiate between the acute and chronic forms of PCM, by demonstrating a higher level of seric IgG to h-MM antigen in chronic compared to acute PCM patients.

PCR for diagnosis of paracoccidioidomycosis.

A PCR assay based on oligonucleotide primers derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis DNA in sputa. In the standardized conditions, it could detect 10 cells/ml of sputum, providing sufficient accuracy to be useful for diagnosis of paracoccidioidomycosis.

Conventional versus molecular diagnostic tests.

In this session, emphasis was placed on the diagnosis of various mycoses through the identification of antibodies and antigens in sera, as well as on new techniques to properly identify medically important fungi through molecular biological procedures. The use of restriction fragment length polymorphism (RFLP) on fungal mitochondrial DNA (mtDNA) has enabled the identification of different strains of Sporothrix schenkii, several dermatophytes, Candida spp. and black fungi according to their species-specific mtDNA-RFLP patterns. In some species, distinct specific types where found in relation to the geographic origin of the patients. These particular molecular diagnostic tests are useful in the identification of strains and in epidemiological studies. An account of the applications of serological methods in the diagnosis of paracoccidioidomycosis was presented. Serology has been used in the identification of paracoccidioidomycosis using a specific, sensitive and rapid antibody-based immunodiagnosis method. Using the gp43 antigen, the diagnostic coverage of inmunodifussion has been improved from the 93-95% achieved with crude antigens, to 100% in an enzyme-linked immunodiffusion assay capture test. Cross-reactions were eliminated by treatment of the antigen with sodium metaperiodate. Antibody detection is useful, especially in paracoccidioidomycosis and histoplasmosis.

Current knowledge on pathogenesis and immunodiagnosis of paracoccidioidomycosis.

Host-parasite relationship and immunodiagnostic testing in paracoccidioidomycosis have been extensively investigated in recent years. We review the major advances in the understanding of pathogenesis of the disease with emphasis on the sequential steps in granuloma formation and the envolvement of immunological mechanisms in host defenses against the parasite. In addition, the several immunodiagnostic tests used for diagnosis and in the follow-up of patients are commented upon and critically analysed.

[Paracoccidioidomycosis in Brazilian Indians of the Suruí tribe: clinical-laboratory study of 2 cases]. / Paracoccidioidomicose em índios brasileiros da tribo Suruí: estudo clínico-laboratorial de 2 casos.

Paracoccidioidomycosis has been considered the most frequent endemic systemic mycosis in Latin America. Although most cases of paracoccidioidomycosis involve rural workers, this systemic fungal disease has been scarcely reported among Amerindian populations from Brazil. We report two cases of paracoccidioidomycosis in Tupi-Mondé Amerindians from Cacoal, state of Rondônia, Brazil. Both cases exhibited positive serological results by a specific immunodiffusion test only when the assay was performed with antigens obtained from the mycelial form of P. brasiliensis. The authors present a literature review of paracoccidioidomycosis in Brazilian Amerindians and discuss the need for further investigations about the impact of the antigenic diversity of P. brasiliensis from different geographic areas on the serological diagnosis of PCM.

Pattern of immune response to GP43 from Paracoccidioides brasiliensis in susceptible and resistant mice is influenced by antigen-presenting cells.

Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis. The infection can evolve to different clinical forms that are associated with various degrees of suppressed cell-mediated immunity. In the murine model, A/Sn and B10.A isogenic strains of mice are known to be resistant and susceptible, respectively, to this fungal infection. Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated the importance of two antigen-presenting cells (APCs), macrophages and B cells, in the development of the immune response to P. brasiliensis. In resistant mice, purified gp43, the main antigenic component of P. brasiliensis, seems to have been preferentially presented by macrophages and stimulated Th1 lymphokine production. On the other hand, in susceptible animals gp43 was distinguishably presented by B cells, which led to stronger activation of Th2 subsets. Moreover, T cells from resistant mice responded as those from susceptible animals when stimulated by gp43 presented by APCs from susceptible mice and vice versa, indicating that there are no significant differences in the T cell repertoires from A/Sn and B10.A mice. When T cells from F1 (A/Sn x B10.A) mice were stimulated by gp43 presented by APCs from A/Sn or B10.A, impaired behavior of B10.A macrophages in activating Th1 cells and a B10.A B cell tendency to stimulate T cells that secrete higher levels of IL-10 were observed. Taken together, our results suggest that APCs may be implicated in the outcome of P. brasiliensis infection.

Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus noveminctus) captured in an endemic area of paracoccidioidomycosis.

Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from armadillos from the Amazonian region where the mycosis is uncommon. In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease. Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, Sao Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver, and mesenteric lymph nodes were processed for histology, cultured on Mycosel agar at 37 degrees C, and homogenized for inoculation into the testis and peritoneum of hamsters. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37 degrees C. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph nodes of hamsters. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three armadillos isolates (Pb-A1, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticularly inoculated hamsters. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos are a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus.

Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu, SP, Brazil.

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp 43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

Humoral immune response to Malassezia furfur in patients with pityriasis versicolor and seborrheic dermatitis.

Humoral immune responses against exoantigen components of oval, elliptic and round yeast forms of Malassezia furfur were analysed by ELISA and Western blotting assays, using sera from patients with pityriasis versicolor (PV), seborrheic dermatitis (SD) and healthy adults (HA), as control. Sera from patients with SD showed IgG anti-oval M. furfur titers ranging from 1/400 to 1/6400 showing geometric mean (GM) of 1/1472, higher than those obtained with sera from patients with PV (1/200 to 1/6400, GM = 1/1239). Both patient groups showed mean titres statistically superior (P < 0.05) than those obtained form HA (GM = 1/229). Similar data were also obtained with the elliptic and round antigens. However, the anti-oval IgG mean titers from patients' sera were much higher than those obtained with elliptic or round antigenic components (p < 0.05) Anti-M furfur IgM titers obtained from patient's sera with PV against all three exoantigens were statistically superior (p < 0.05) than HA group. Patients with SD showed IgM titers statistically superior (p < 0.05) only to oval yeasts of M. furfur. The IgA mean titers from patients' groups against the different morphological antigens were shown be slightly higher than those HA group. By Western blot, using rabbit anti-sera, the different antigenic components of M.furfur showed a close relationship mainly between oval and elliptic yeast cells antigens. The 70 kDa component of the M. furfur exoantigen of oval morphology was recognized by 84% of the PV patients' sera. On the other hand, SD patients' sera recognized 3 principal components of 70 kDa (100%), 65 kDa (67%) and 84 kDa (53%). These components may be considered immunological markers for PV and SD. Twenty-five percent of HA sera recognized the components of 65, 70 and 94 kDa. This investigation shows that M. furfur antigens can sensitize the host, mainly the oval yeast form of M. furfur with a very important specific IgG response in patients with SD and PV.

Enzyme-linked immunosorbent assay (ELISA) in the paracoccidioidomycosis. Comparison with counterimmunoelectrophoresis and erythro-immunoassay.

An enzyme-linked immunosorbent assay (ELISA) for detection and quantification of antibodies anti-Paracoccidioides brasiliensis is described. Polystyrene plates have been used as solid phase to absorb P. brasiliensis metabolic yeast phase antigen. Twenty sera of proven paracoccidioidomycosis, 11 of histoplasmosis due Histoplasma capsulatum, 20 of aspergillosis and 20 human normal sera were tested. Ninety-five percent of the paracoccidioidomycosis sera had O.D. superior to 0.150 (from 0.163 to 2.650) at 1/400 serum dilution. ELISA assay was compared with counterimmunoelectrophoresis and erythro-immunoassay tests; a correlation was observed only with erythro-immunoassay. ELISA test should give new perspectives for the serodiagnosis of paracoccidioidomycosis.

Titration of antibodies to Paracoccidioides brasiliensis by erythro-immunoassay (EIA).

The erythro-immunoassay, a new serological procedure in which a hybrid antibody conjugate is able to bind erythrocytes, was used for the titration of antibodies against P. brasiliensis in sera from patients with paracoccidioidomycosis. A peptide-polysaccharide and a lyophilized yeast culture filtrate of P. brasiliensis were used as antigens. Absorption with dead Candida albicans whole cells was necessary to decrease cross reactions observed with heterologous sera. Erythro-immunoassay provides a sensitive system for titration of antibodies in paracoccidioidomycosis with serum dilutions up to 1:102000.

Magnetic enzyme-linked immunosorbent assay (MELISA) for determination of specific IgG in paracoccidioidomycosis.

A magnetic solid phase enzyme-linked immunosorbent assay (MELISA) for quantification of IgG antibodies to somatic and metabolic antigens of Paracoccidioides brasiliensis was developed. Activation of magnetic polyacrylamide agarose beads with concanavalin A was superior to glutaraldehyde activation, and test sensitivity was higher for somatic than for metabolic antigens. Comparative MELISA, counterimmunoelectrophoresis and erythroimmunoassay tests with sera from 33 proven cases of paracoccidioidomycosis, 14 cases of histoplasmosis and 20 normal human sera showed the MELISA could distinguish antibody levels in paracoccidioidomycosis from those in normal sera; however two sera from histoplasmosis cases cross-reacted in the MELISA. MELISA is a rapid test (5-6 h) and the results suggest it has considerable potential value for assay of anti-P. brasiliensis antibodies.

Experimental protothecosis in labortory animals.

Attemps were made to produce protothecosis in laboratory animals by intratesticular, intraperitoneal, intravenous and subcutaneous inoculation of Prototheca zopfii, P. wickerhamii and P stagnorta. Only intratesticular injection successfully caused infection, and of the inoculated protothecae, only P. zopfii and P. wickerhamii were recovered in culture from testicular material.

Paracoccidioidomycosis with palpebral and conjunctival involvement.

A case of pulmonary and lymphatic paracoccidioidomycosis followed by conjunctivo-palpebral involvement is studied in Sao Paulo (Brazil). The Brazilian literature is reviewed. The most frequent clinic forms of ocular lesions in natural infection are described. The small number of human cases observed is discussed. The severity and frequency of ocular lesions in experimental animals, intracardiacally inoculated with P. brasilensis cultures suggest haematogenic ocular dissemination of the fungus, which may occur in the septicemic stage of the human paracoccidioidomycosis.