Figure 1.
Electron microscopy of P. lutzii yeast phase showing multiple budding cells.
Figure 2.
Map of the Brazilian regions, highlighting the Central - West region where P. lutzii predominates and the South and Southeast region where P. brasiliensis predominates.
Table 1.
Paracoccidioides lutzii isolates used in the study.
Figure 3.
A) Immunodiffusion tests with different CFAs from 12 strains of Paracoccidioides lutzii.
1 = EPM 147; 2 = EPM 148; 3 = EPM 196; 4 = EPM 201; 5 = EPM 205; 6 = EPM 206; 7 = EPM 208; 8 = EPM 212; 9 = EPM 213; 10 = EPM 223; 11 = EPM 209; 12 = EPM 226. In the center well, S* = P. lutzii serum (gold standard). P. lutzii strains EPM 227 and EPM 228 were positive (not shown). B) Immunodiffusion test showing that S* = P. lutzii gold standard serum do not react with CFA from P. brasiliensis Pb18 and B339. C) Immunodiffusion test using Ag Exo (crude exoantigen of P. lutzii) and Ag TCA (crude exoantigen of P. lutzii precipitated with trichloroacetic acid) versus P. lutzii serum (S* = P. lutzii gold standard serum and S = suspected P. lutzii serum). Observe the weak band in both cases. D) immunodiffusion test showing that S* (P. lutzii gold standard serum) do not react with Ag Exo, Ag TCA and CFA from P. brasiliensis B339 antigen. E) Immunodiffusion test of 12 sera from patients with PCM due to P. lutzii using P. lutzii CFA. In the center well is the CFA antigen (Ag), and different PCM sera are in the peripheral wells (1 to 12). F) Immunodiffusion test showing that S* (P. lutzii gold standard serum) do not react with Exo or CFA of P. brasiliensis B339; and SPb (serum of PCM patient due to P. brasiliensis) do not react with Exo or CFA of P. lutzii.
Figure 4.
Receiver operating characteristics (ROC) curve for the CFA, EXO and TCA antigens derived from Paracoccidioides lutzii EPM208 isolate.
The ROC is plotted between the true-positive rate (sensitivity) on the y-axis, and the false-positive rate (100-specificity) on the x-axis. The area under the curve (AUC) represents the accuracy of the ID test which was 1.0, IC 0.95–1.0, p<0.0001 for the CFA, 0.74±0.06, IC 0.68–0.87, p<0.0001 for the EXO and 0.58±0.04, IC 0.47–0.69, p = 0.0641 for the TCA antigens. The higher AUC above 0.5, the better the test.
Figure 5.
A) Individual serum ELISA titers (1/T) of 28 sera from patients with PCM due to P. lutzii (blue) compared to individual serum ELISA titers (1/T) of 28 sera from patients with PCM due to P. brasiliensis (red) reacting with Ag CFA-Pl EPM 208 (12.5 µg/ml).
The P. lutzii sera presented ID + for P. lutzii CFA antigen and ID − for P. brasiliensis exoantigen (AgPbB339). The group of P. brasiliensis sera presented ID + for AgPbB339 antigen and ID − for P. lutzii CFA antigen. (*) represents P. lutzii gold standard serum and (**) represents sera that had a very weak cross reaction with P. brasiliensis exoantigen. B) Individual serum ELISA titers (1/T) of 28 sera from patients with PCM due to P. lutzii (blue) compared to individual serum ELISA titers (1/T) of 28 sera from patients with PCM due to P. brasiliensis (red) reacting with Ag Exo Pb B339 (10 µg/ml). The P. lutzii sera presented ID + for P. lutzii CFA antigen and ID − for P. brasiliensis exoantigen (AgPbB339). The group of P. brasiliensis sera presented ID + for AgPbB339 antigen and ID − for P. lutzii CFA antigen. (*) represents P. lutzii gold standard serum and (**) represents sera that had a very weak cross reaction with P. brasiliensis exoantigen. The same group of sera was used in A and B. C) ELISA median curves from sera from patients with PCM due to P. lutzii, PCM due to P. brasiliensis, and heterologous sera such as histoplasmosis, aspergillosis, sporotrichosis, and normal human serum. Antigen used was P. lutzii CFA (Ag CFA-Pl EPM 208 at 12.5 µg/ml). D) ELISA individual serum titers (1/T) of patients with PCM due to P. lutzii, P. brasiliensis, and heterologous sera such as Histoplasmosis, Aspergillosis, Sporotrichosis and normal human sera. By the end titer we could distinguish sera of PCM patients due to P. lutzii from sera of PCM due to P. brasiliensis and other mycotic diseases. Antigen used was P. lutzii CFA (Ag CFA-Pl EPM 208 at 12.5 µg/ml). Bars indicate the median.
Figure 6.
A) Western blot assays showing the reactivity of sera from patients with PCM due to P. lutzii versus P. lutzii-CFA antigen.
Hc, Asp, Sp and NHS: representative blots of heterologous sera and normal human sera (15 sera each). (*) = represents P. lutzii gold standard serum. B) Reactivity of sera from patients with PCM due to P. brasiliensis versus P. lutzii-CFA antigen. We could distinguish P. brasiliensis from P. lutzii sera, as only sera from patients with PCM due to P. lutzii recognize antigenic molecules from P. lutzii-CFA. C) Western blot assays showing the reactivity of sera from patients with PCM due to P. brasiliensis versus P. brasiliensis-CFA (strain B339). Gp43 is basically recognized by PCM sera due to P. brasiliensis. D) Reactivity of sera due to P. lutzii versus P. brasiliensis-CFA (strain B339).