Clinical Forms Of Paracoccidioidomycosis Research Articles

Differential production of interleukin-1 family cytokines (IL-1β, IL-18, IL-33 and IL-37) in patients with paracoccidioidomycosis: correlation with clinical form and antifungal therapy.

Besides interleukin (IL)-1β and IL-18, the newly described cytokines of IL-1 family IL-33 and IL-37 can contribute to the differentiation and maintenance of different population of T cells. IL-33 acts as an allarmin and promotes a predominant Th2 inflammatory response, whereas IL-37 plays an important role as an antagonist of inflammation. In paracoccidioidomycosis (PCM), caused by the dimorphic fungi Paracoccidioides brasiliensis and P. lutzii, it has been shown that the acquired immune responses are associated with the diverse clinical manifestations. The severe and disseminated forms (acute form [AF] and multifocal chronic form [CF-MF]) are characterized by high Th2 cytokines and antibody production, impaired cellular immune response, and eosinophilia. In contrast, in the localized form (unifocal chronic form [CF-UF]), the cellular immune response is preserved, with high production of Th1 and Th17 cytokines, and low antibody titers. This study aimed to quantify interleukin-1 family cytokines (IL-1β, IL-18, IL-37, IL-33, and the soluble IL-33 receptor sST2) in sera of patients presenting different clinical forms of PCM before, during, and after antifungal treatment, as well as to analyze the expression of these cytokines in lesions of PCM patients. We found that AF patients presented high serum levels of IL-1β, IL-18, IL-33, sST2, and IL-37, and that these cytokines are strongly expressed in lymph nodes lesions. Furthermore, antifungal therapy resulted in the diminution of circulating cytokines and sST2 levels in all groups of patients. These results indicate that, besides IL-1β and IL-18, IL-33, IL-37, and sST2 can be associated with the disease activity and severity.

Polymorphisms on IFNG, IL12B and IL12RB1 genes and paracoccidioidomycosis in the Brazilian population

Paracoccidioidomycosis (PCM) is a systemic chronic mycosis, endemic in Latin America, especially Brazil, and is the eighth leading cause of death among chronic and recurrent infectious diseases. PCM infection is characterized by the presence of Th1 immune response; the acute form, by a mixed Th2/Th9, while the chronic form is characterized by Th17/Th22 profiles. The occurrence and severity of human PCM may also be associated with genetic factors such as single nucleotide polymorphisms (SNP) on cytokines encoding genes. We investigated the association between these polymorphisms and the different clinical forms of PCM. We included 156 patients with PCM (40 with the acute form, 99 with the chronic multifocal and 17 with the chronic unifocal form) and assayed their DNA samples for IFNG +874 T/A SNP by PCR-ARMS (Amplification Refractory Mutational System), IL12B +1188 A/C SNP on 3′ UTR and IL12RB1 641 A/G SNP on exon 7 by PCR-RFLP (Restriction Fragment Length Polymorphism). We found similar genotypic and allelic frequencies of the investigated SNPs among the clinical forms of PCM. Considering male patients, the IL12RB1 641 AA genotype was more frequent in the chronic multifocal form while heterozygosis was in the chronic unifocal form of PCM (p=0.048). Although our data suggest that the AA genotype (IL12RB1) may be associated with the more disseminated chronic disease, more patients of the chronic unifocal PCM group need to be analyzed as well as the secretion patterns of IFN-γ combined with the IL-12Rβ1 expression for a better comprehension of this association.

Evaluation of polymerase chain reaction for the detection of Paracoccidioides brasiliensis DNA on serum samples from patients with paracoccidioidomycosis

The aim of this study was to demonstrate the DNA of Paracoccidioides brasiliensis in human serum samples of patients with paracoccidioidomycosis using the polymerase chain reaction (PCR). The diagnosis of paracoccidioidomycosis (PCM) was defined by microscopic observation of the fungus on direct exam or histopathology, culture, and serological positivity. DNA from serum of 33 patients with PCM was extracted and submitted to nested-PCR using primers from the gp 43 gene. Only one sample was positive on nested-PCR. We conclude that the prevalence of fungemia in patients with different clinical forms of PCM is low, limiting the use of serum DNA detection as an alternative diagnostic tool.

Anti-idiotypic antibodies in patients with different clinical forms of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Patients with PCM show a wide spectrum of clinical and pathological manifestations depending on both host and pathogen factors. Two clinical forms of the disease are recognized: the acute or juvenile form and the chronic or adult form. The major antigenic component of the parasite is a glycoprotein of 43 kDa (gp43). All patient sera present antibodies against gp43 (anti-gp43) and, as demonstrated before by our group, spontaneous anti-idiotypic (anti-Id) antibodies (Ab2) can be detected in patient sera with high titers of anti-gp43. Since it has been postulated that anti-Id antibodies may have a modulating function, we decided to purify and characterize anti-Id antibodies in this system. The possible correlation of Ab2 titers with different clinical forms of disease was also verified. Results showed that purified human anti-Id antibodies (human Ab2) recognized specifically the idiotype of some murine monoclonal anti-gp43 (17c and 3e) but not others (40.d7, 27a, and 8a). Spontaneous anti-Id antibodies were found in all clinical forms of disease. The majority of patients (88%, n = 8) with the acute form of PCM had high titers of Ab2. However, among patients with the multifocal chronic form of the disease, only 29% (n = 14) had high titers of Ab2; 70% (n = 10) of patients with the unifocal chronic form had low titers of Ab2. A correlation between Ab2 titers and anti-gp43 titers was observed before and during antimycotic treatment. Our results suggest that titers of anti-Id antibodies correlate with the severity of PCM in humans.

Paracoccidioides brasilienses isolates obtained from patients with acute and chronic disease exhibit morphological differences after animal passage.

The basis for virulence in Paracoccidioides brasiliensis is not completely understood. There is a consensus that the sequential in vitro subcultivation of P. brasiliensis leads to loss of its pathogenicity, which can be reverted by reisolation from animal passage. Attention to morphological and biochemical properties that are regained or demonstrated after animal passage may provide new insights into factors related to the pathogenicity and virulence of P. brasiliensis. We evaluated morphological characters: the percentage of budding cells, number of buds by cell and the diameter of 100 mother cells of yeast-like cells of 30 P. brasiliensis isolates, before and after animal passage. The isolates were obtained from patients with different clinical forms of paracoccidioidomycosis (PCM): acute form (group A, n=15) and chronic form (group C, n=15). The measurement of the yeast cell sizes was carried out with the aid of an Olympus CBB microscope coupled with a micrometer disc. We measured the major transverse and longitudinal axes of 100 viable cells of each preparation. The percentage of budding cells as also the number of buds by cell was not influenced by animal passage, regardless of the source of the strain (acute or chronic groups). The size values of P. brasiliensis isolates from groups A and C, measured before the animal passage exhibited the same behavior. After animal passage, there was a statistically significant difference between the cell sizes of P. brasiliensis isolates recovered from testicles inoculated with strains from groups A and C. The maximum diameter of mother cells from group A isolates exhibited a size of 42.1 microm in contrast with 32.9 microm exhibited by mother cells from group C (p<0.05). The diameter of 1500 mother cells from group A isolates exhibited a medium size of 16.0 microm (SD +/- 4.0), a value significantly higher than the 14.1 microm (SD = +/- 3.3) exhibited by 1500 mother cells from group C isolates (p<0.05). Our results reinforce the polymorphism exhibited by P. brasiliensis in biological material and the need for further investigations to elucidate the role of morphological parameters of the fungus in the natural history of the disease.

Short report: use of the polymerase chain reaction to detect Paracoccidioides brasiliensis in murine paracoccidioidomycosis.

The polymerase chain reaction (PCR) was used to detect the presence of Paracoccidioides brasiliensis in a murine model of disseminated paracoccidioidomycosis. Using a previously identified P. brasiliensis-specific DNA sequence, P. brasiliensis DNA was detected in serum of five experimentally infected mice. The PCR method was able to detect as little as 10 pg of P. brasiliensis DNA in serum, and it was more sensitive than blood culture isolation (five of five were PCR positive versus two of five blood culture positive). There were no amplified fragments in serum from three noninfected control mice. Lung colony counts were similar in all infected mice and reflected a similar degree of P. brasiliensis infection at the time the samples were drawn. The relatively short processing time for the PCR, when compared with culture, its sensitivity, and the possibility of using serum samples for analysis, are important factors favoring this method for the diagnosis of paracoccidioidomycosis. Future studies should include the detection of P. brasiliensis in patients with different clinical forms of paracoccidioidomycosis.

Isoenzyme profile ofParacoccidioides brasiliensis

Isoenzyme profiles of 10 strains of Paracoccidioides brasiliensis from different origins (nine strains from patients with different clinical forms of paracoccidioidomycosis and one from the faeces of a penguin) were determined by polyacrylamide gel electrophoresis using 37 different enzymes. Differences in carbonate dehydratase, phosphoglucomutase, phosphoglucoseisomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and beta-esterase were detected among the isolates studied allowing the characterization of nine zymodemes. Two isolates showed identical profiles. There was no correlation between the zymodeme patterns and virulence, clinical forms of the disease nor age of the cultures.

Immunological response to cell-free antigens of Paracoccidioides brasiliensis: relationship with clinical forms of paracoccidioidomycosis

Sera from patients with the acute (AF) and chronic (CF) forms of paracoccidioidomycosis (PCM) were tested against Paracoccidioides brasiliensis cell-free antigens by Western blot (immunoblot). The CFA preparation contained components ranging in molecular mass from 18 to 102 kDa. The immunoglobulin G (IgG) reactivity profiles were similar for patients with both forms of the disease, and the 43-kDa component was recognized by 100% of the sera. IgM antibodies from the AF- and the CF-PCM sera recognized 21 and 20 components, respectively, the AF-PCM sera reacting preferentially with components with molecular masses above 50 kDa. None of the AF-PCM sera (IgM) reacted with the 43-kDa component, and only 10% of the CF-PCM sera recognized this molecule. The IgA response was more significant in the CF-PCM group than in the AF-PCM group, and the 43- and 74-kDa components were the most reactive ones (about 40% each). Our results showed that the cell-free antigen preparation is very appropriate for the immunoblotting analysis of PCM sera, and they also showed that the detection of IgG anti-gp43 is the best marker for the diagnosis and the following up of patients with the acute or the chronic form of the disease.

Association of Major Histocompatibility Complex Class III Complement Components C2, BF, and C4 with Brazilian Paracoccidioidomycosis

A genetic influence of the major histocompatibility complex (MHC) on the susceptibility and the development of the different clinical forms of paracoccidioidomycosis (PCM) has been postulated. In the present investigation allotypes of MHC-coded class III gene products (complement components C2, BF, C4A, and B) were determined in 69 Brazilian PCM patients and 225 healthy control individuals matched for ethnic and geographic origin. The frequency of the non-expressed C4B allele (C4B*Q0) was significantly elevated in comparison to the controls (p less than 0.01; Fisher's exact test). Three out of 69 patients had a complete C4B deficiency as against 2 among 223 control individuals. The C4A*Q0 allele was also more frequent in the patients. Other C4 alleles were not seen to differ between the two groups. The analysis of BF allotypes showed a non-significant predominance of the rarer allele BF*S07 in the patients, whereas no difference in the distribution of C2 alleles was seen. The data on MHC class III association may support the hypothesis of immune response modulation in PCM and suggest a functional genetic role of complement action against the fungus and in the outcome of PCM infection. We conclude that MHC class III products, especially C4B*Q0, are associated with chronic uni- or multifocal PCM and may influence the course of the infection.

Mononuclear cell subsets in patients with different clinical forms of paracoccidioidomycosis.

Seventy untreated paracoccidioidomycosis patients, 15 with the acute or subacute form of the disease and 55 with the chronic form, were compared with two normal control groups of the same age range. Peripheral blood mononuclear cell subsets were defined by monoclonal antibodies directed at total T cells, helper/inducer and suppressor/cytotoxic T cell subpopulations; B cells, cortical thymocytes and monocyte/null cells. Both groups of patients showed an increased number of monocyte/null cells, a low helper/suppressor ratio and a reduced percentage of total T cells and their helper/inducer subsets. In addition patients with the acute form of the disease exhibited high levels of suppressor/cytotoxic T cells and B cells. These findings are of importance in our attempts to understand the pathogenesis of this mycosis and also to evaluate its prognosis in individual patients.

Correlation between cell-mediated immunity and clinical forms of paracoccidioidomycosis

Cellular immune response to specific and non-specific stimulants was investigated, both in vivo and in vitro, in 29 healthy controls and in 53 previously untreated patients with the chronic isolated organic form (CIOF), the chronic mixed form (CMF) and the acute progressive form (APF) of paracoccidioidomycosis. The study included skin tests to Paracoccidioides brasiliensis antigen (PbAg) and phytohaemagglutinin (PHA), DNCB sensitization, determination of T lymphocytes and complement rosette-forming cells, lymphocyte transformation and leucocyte migration inhibition tests using PbAg and PHA. Patients displayed staggered cutaneous response to PHA and to PbAg, with marked decrease in intensity in the APF group. DNCB sensitization test and proliferative response of lymphocytes to PHA and PbAg were severely depressed in most of the patients. Leucocyte migration inhibition indices to PbAg were highly positive, while response to PHA was slightly decreased regardless of the clinical form. The number of T lymphocytes was reduced in most of patients and in them the number of complement-rosette forming cells was normal. The distribution of patients according to a suppression index, based in the results of the tests employed, revealed a tendency towards an increased degree of cellular immunosuppression from the least severe (CIOF) to the most severe (APF) clinical form of the disease. On the whole, the present study demonstrated a gamut of immunological reactivity in paracoccidioidomycosis.