(A) Representative immunoblot reactions of PCM patients' sera (1∶1,000) with rPlp43 and Endo H-deglycosylated (EH-) gp43.

<p>The overall results are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003111#pntd-0003111-t001" target="_blank">Table 1</a> for sera groups 1, 2, 3, and individual sera 99M, 100M, and101M. On the right, CBB-stained SDS-PAGE gel showing the amount of antigen used in the reactions. Results with Endo H-deglycosylated gp43r3 were similar to (EH-)gp43 and are not shown. (<b>B</b>) <b>PCR amplification of </b><i>HSP70</i><b> using primers for the </b><i>P. lutzii</i><b> gene as shown in an agarose gel.</b> Partial <i>HSP70</i> PCR (529 bp) amplification shows that clinical isolates Pb51M and Pb52M are <i>P. lutzii</i>. DNA extracted from Pb01 (<i>P. lutzii</i>) and Pb18 (<i>P. brasiliensis</i>) were used as species control. Crt-, in the absence of DNA.</p

Overall results of the I.D. and immunoblot reactivity of recombinant <i>P. lutzii</i> (rPlp43), native or recombinant gp43 (gp43, gp43r3) and their Endo H-deglycosylated forms (EH-gp43, EH-gp43r3) with PCM patients' sera from Brazilian Southeastern (S) and Midwestern (M) regions (Table S1).

<p>*Sera groups: 1, 1S–50S and 82M–95M; 2, 51M–81M; 3, 96M–98M.</p>#<p>ID titers with purified gp43 or Pb339 culture supernantant are specified in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003111#pntd.0003111.s002" target="_blank">Table S2</a>.</p><p>Overall results of the I.D. and immunoblot reactivity of recombinant <i>P. lutzii</i> (rPlp43), native or recombinant gp43 (gp43, gp43r3) and their Endo H-deglycosylated forms (EH-gp43, EH-gp43r3) with PCM patients' sera from Brazilian Southeastern (S) and Midwestern (M) regions (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003111#pntd.0003111.s001" target="_blank">Table S1</a>).</p

Amino acid sequence alignment and molecular models show differences between Plp43 (from Pb01) and gp43 (from Pb18).

<p>Differences in amino acid residues can be seen. Known sequence features are indicated: SP (signal peptide), MAb3e epitope (D/purple), T-cell P10 epitope (grey shade with boxed antigenic core; A/navy), N-glycosylation site (N-gly, C/green), and NEP glucanase active site (Glu, B). The sequences corresponding to primers gp01S and gp01AS used to amplify Pl<i>P43</i> ORF are shown. In the molecular models below, arrows (1–4) point to remarkable differences between Plp43 and gp43; caps letters indicate the motifs above specified. Yellow, beta-sheets; magenta, alpha-helix.</p

Recombinant rPlp43 is not glycosylated and seems to be enzymatically active.

<p>(<b>A</b>) Heterologous expression of rPlp43 in <i>P. pastoris</i> culture supernatants from methanol-induced recombinant yeasts containing the Pl<i>P43</i> insert or not (EV, empty vector). (<b>B</b>) SDS-PAGE profile of rPlp43 and control gp43 before (−) and after (+) treatment with Endo H. (<b>C</b>) Glucanase activity against PNPG in culture supernatants from <i>P. pastoris</i> expressing rPlp43 when compared with that, at equivalent total protein amount, of yeasts containing empty vector (EV), wild type <i>P. pastoris</i> and <i>S. cerevisiae</i>. Purified gp43 was used as protein negative control at equivalent protein amount to rPlp43, as estimated in SDS-PAGE gels. BSA was also included as negative control at higher amounts. PBS+rPlp43 (without substrate) and PNPG+enzyme buffer (without substrate) were used as negative controls. Migration of standard molecular masses is indicated.</p

Immunoblot reactivity of anti-gp43 monoclonal antibodies with gp43 and rPlp43.

<p>Reactivity of rPlp43 was tested with five different anti-gp43 MAbs (indicated) in immunoblot, using gp43 as positive control. On the right, CBB-stained SDS-PAGE gel showing the amount of antigen used in the assay. The results were the same for HisPlp43 expressed as inclusion bodies in bacteria.</p

Brazilian guidelines for the clinical management of paracoccidioidomycosis

<div><p>Abstract Paracoccidioidomycosis is a systemic fungal disease occurring in Latin America that is associated with rural environments and agricultural activities. However, the incidence and prevalence of paracoccidiodomycosis is underestimated because of the lack of compulsory notification. If paracoccidiodomycosis is not diagnosed and treated early and adequately, the endemic fungal infection could result in serious sequelae. While the Paracoccidioides brasiliensis ( P. brasiliensis ) complex has been known to be the causal agent of paracoccidiodomycosis, a new species, Paracoccidioides lutzii ( P. lutzii ), has been reported in Rondônia, where the disease has reached epidemic levels, and in the Central West and Pará. Accurate diagnoses and availability of antigens that are reactive with the patients’ sera remain significant challenges. Therefore, the present guidelines aims to update the first Brazilian consensus on paracoccidioidomycosis by providing evidence-based recommendations for bedside patient management. This consensus summarizes etiological, ecoepidemiological, molecular epidemiological, and immunopathological data, with emphasis on clinical, microbiological, and serological diagnosis and management of clinical forms and sequelae, as well as in patients with comorbidities and immunosuppression. The consensus also includes discussion of outpatient treatments, severe disease forms, disease prevalence among special populations and resource-poor settings, a brief review of prevention and control measures, current challenges and recommendations.</p></div