Negative immunodiffusion test results obtained with sera of paracoccidioidomycosis patients may be related to low-avidity immunoglobulin G2 antibodies directed against carbohydrate epitopes

Immunodiffusion (ID) is the serologic test most frequently used for the diagnosis and posttherapy follow-up of patients with paracoccidioidomycosis (PCM). the ID test is highly specific (100%), but its sensitivity is relatively low (90%), leading to false-negative results. the aim of this study was to determine the profiles of antibodies in sera from patients with proven PCM and with negative results in the ID test (IDneg) versus positive results in the ID test (IDpos). We analyzed 46 sera from patients with active PCM for total immunoglobulin G (IgG) and IgG subclass responses to Paracoccidioides brasiliensis gp43 antigen (treated or not treated with sodium metaperiodate) by enzyme-linked immunosorbent assay and immunoblotting. Immunoblotting showed that both IDneg and IDpos sera recognized predominantly the gp43 fraction of the P. brasiliensis antigen used in the ID test. IDneg sera contain low-avidity antibodies, low levels of specific IgG (total) and IgGI, and high levels of IgG2 compared with IDpos sera. the antibodies present in IDpos sera were predominantly directed against carbohydrate epitopes, since treatment with sodium metaperiodate resulted in a significant decrease in antibody reactivity. These data suggest that the lack of reactivity of sera from PCM patients in the ID test may be related to the production of low-avidity IgG2 antibodies directed against carbohydrate epitopes.UNICAMP, Fac Med Sci, Dept Clin Pathol, BR-13083970 Campinas, SP, BrazilUNIFESP, Discipline Cellular Biol, São Paulo, SP, BrazilUNIFESP, Discipline Cellular Biol, São Paulo, SP, BrazilWeb of Scienc

Low-level Laser Therapy to the Mouse Femur Enhances the Fungicidal Response of Neutrophils against Paracoccidioides brasiliensis

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. the goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. the aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. the number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. the highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. the kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)National Institute of Health (US NIH)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Microbiol & Immunol, Alfenas, MG, BrazilFed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Biochem, Alfenas, MG, BrazilState Univ Campinas UNICAMP, Inst Biol, Dept Struct & Funct Biol, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, São Paulo, SP, BrazilMassachusetts Gen Hosp, Wellman Ctr Photomed, Boston, MA 02114 USAHarvard Univ, Sch Med, Dept Dermatol, Boston, MA 02115 USAMIT, Harvard Mit Div Hlth Sci & Technol, Cambridge, MA 02139 USAFed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Pathol & Parasitol, Alfenas, MG, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, São Paulo, SP, BrazilCNPq: 486135/2012-8CNPq: 304827/2012-6FAPEMIG: CBB-PPM-00119-14National Institute of Health (US NIH): R01AI050875CAPES: AEX-9765-14-0Web of Scienc

Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients’ sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost

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

Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

Sporothrix infections are emerging as an important human and animal threat among otherwise healthy patients, especially in Brazil and China. Correct identification of sporotrichosis agents is beneficial for epidemiological surveillance, enabling implementation of adequate public-health policies and guiding antifungal therapy. In areas of limited resources where sporotrichosis is endemic, high-throughput detection methods that are specific and sensitive are preferred over phenotypic methods that usually result in misidentification of closely related Sporothrix species. We sought to establish rolling circle amplification (RCA) as a low-cost screening tool for species-specific identification of human-pathogenic Sporothrix. We developed six species-specific padlock probes targeting polymorphisms in the gene encoding calmodulin. BLAST-searches revealed candidate probes that were conserved intraspecifically; no significant homology with sequences from humans, mice, plants or microorganisms outside members of Sporothrix were found. The accuracy of our RCA-based assay was demonstrated through the specificity of probe-template binding to 25 S. brasiliensis, 58 S. schenckii, 5 S. globosa, 1 S. luriei, 4 S. mexicana, and 3 S. pallida samples. No cross reactivity between closely related species was evident in vitro, and padlock probes yielded 100% specificity and sensitivity down to 3 × 10(6) copies of the target sequence. RCA-based speciation matched identifications via phylogenetic analysis of the gene encoding calmodulin and the rDNA operon (kappa 1.0; 95% confidence interval 1.0-1.0), supporting its use as a reliable alternative to DNA sequencing. This method is a powerful tool for rapid identification and specific detection of medically relevant Sporothrix, and due to its robustness has potential for ecological studies

Current Progress on Epidemiology, Diagnosis, and Treatment of Sporotrichosis and Their Future Trends

Sporotrichosis, a human and animal disease caused by Sporothrix species, is the most important implantation mycosis worldwide. Sporothrix taxonomy has improved in recent years, allowing important advances in diagnosis, epidemiology, and treatment. Molecular epidemiology reveals that S. brasiliensis remains highly prevalent during the cat-transmitted sporotrichosis outbreaks in South America and that the spread of S. brasiliensis occurs through founder effects. Sporothrix globosa and S. schenckii are cosmopolitan on the move, causing major sapronoses in Asia and the Americas, respectively. In this emerging scenario, one-health approaches are required to develop a creative, effective, and sustainable response to tackle the spread of sporotrichosis. In the 21st century, it has become vital to speciate Sporothrix, and PCR is the main pillar of molecular diagnosis, aiming at the detection of the pathogen DNA from clinical samples through multiplex assays, whose sensitivity reaches remarkably three copies of the target. The treatment of sporotrichosis can be challenging, especially after the emergence of resistance to azoles and polyenes. Alternative drugs arising from discoveries or repositioning have entered the radar of basic research over the last decade and point to several molecules with antifungal potential, especially the hydrazone derivatives with great in vitro and in vivo activities. There are many promising developments for the near future, and in this review, we discuss how these trends can be applied to the Sporothrix-sporotrichosis system to mitigate the advance of an emerging and re-emerging disease

Negative Immunodiffusion Test Results Obtained With Sera Of Paracoccidioidomycosis Patients May Be Related To Low-avidity Immunoglobulin G2 Antibodies Directed Against Carbohydrate Epitopes.

Immunodiffusion (ID) is the serologic test most frequently used for the diagnosis and posttherapy follow-up of patients with paracoccidioidomycosis (PCM). The ID test is highly specific (100%), but its sensitivity is relatively low (90%), leading to false-negative results. The aim of this study was to determine the profiles of antibodies in sera from patients with proven PCM and with negative results in the ID test (IDneg) versus positive results in the ID test (IDpos). We analyzed 46 sera from patients with active PCM for total immunoglobulin G (IgG) and IgG subclass responses to Paracoccidioides brasiliensis gp43 antigen (treated or not treated with sodium metaperiodate) by enzyme-linked immunosorbent assay and immunoblotting. Immunoblotting showed that both IDneg and IDpos sera recognized predominantly the gp43 fraction of the P. brasiliensis antigen used in the ID test. IDneg sera contain low-avidity antibodies, low levels of specific IgG (total) and IgG1, and high levels of IgG2 compared with IDpos sera. The antibodies present in IDneg sera were predominantly directed against carbohydrate epitopes, since treatment with sodium metaperiodate resulted in a significant decrease in antibody reactivity. These data suggest that the lack of reactivity of sera from PCM patients in the ID test may be related to the production of low-avidity IgG2 antibodies directed against carbohydrate epitopes.10802-

CHARACTERIZATION OF GLYCOPROTEIN GP43, THE MAJOR LAMININ-BINDING PROTEIN OF PARACOCCIDIOIDES-BRASILIENSIS

We have demonstrated that laminin mediates the adhesion of P. brasiliensis to monolayers of epithelial cells through specific binding to the surface glycoprotein gp43. This binding seems to be related to the fungal pathogenesis. We now report the confirmation of these findings by scanning electron microscopy and show that some isolates that do not secrete gp43 do express the protein as seen by studying whole cell extracts. These results confirm the ability of these strains to produce paracoccidioidomycosis but should not be used for serological purposes since the absence of gp43 in exoantigens may lead to false negative results.FED UNIV RIO DE JANEIRO,INST BIOFIS CARLOS CHAGAS FILHO,BR-21949900 RIO JANEIRO,BRAZILWeb of Scienc

Tacrolimus Increases the Effectiveness of Itraconazole and Fluconazole against Sporothrix spp.

Calcineurin inhibitors – such as the clinically used drug tacrolimus – are active against important fungal pathogens, particularly when combined with azoles. However, tacrolimus has not been tested against sporotrichosis, an endemic subcutaneous mycosis with worldwide distribution. Here, we evaluated the activity of tacrolimus and cyclosporine A in vitro – as monotherapy and in combination with itraconazole or fluconazole – against yeasts of Sporothrix brasiliensis and S. schenckii, the main sporotrichosis agents in Brazil. We also analyzed the effect of tacrolimus treatment on intracellular neutral lipid levels, which typically increase after azole treatment. Tacrolimus inhibited the growth of yeasts from S. brasiliensis and S. schenckii reference isolates, with minimum inhibitory concentration (MIC) values (required for ≥50% growth inhibition) of 1 and 2 mg/L, respectively. Importantly, the combination of tacrolimus and azoles exhibited high synergy toward reference Sporothrix isolates. Tacrolimus combined with itraconazole significantly increased neutral lipid accumulation in S. brasiliensis, but not in S. schenckii. Clinical isolates of S. brasiliensis and S. schenckii were more sensitive to tacrolimus as monotherapy than feline-borne isolates, however, synergy between tacrolimus and azoles was only observed for feline-borne isolates. Cyclosporine A was effective against S. brasiliensis and S. schenckii as monotherapy (MIC = 1 mg/L), but exhibited no synergy with itraconazole and fluconazole. We conclude that tacrolimus has promising antifungal activity against sporotrichosis agents, and also increases the activity of the current anti-sporotrichosis therapy (itraconazole and fluconazole) in combination assays against S. brasiliensis feline-borne isolates