Ortiz B L, Díez S, Urán M E, Rivas J M, Romero M, Caicedo V, Restrepo A, McEwen J G
Biochemistry and Molecular Biology Units and Mycology Group, Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia.
Clin Diagn Lab Immunol. 1998 Nov;5(6):826-30. doi: 10.1128/CDLI.5.6.826-830.1998.
Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation of Escherichia coli DH5alpha, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 microgram/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic diagnosis of PCM and, by so doing, achieve high coverage rates. Furthermore, antigen reproducibility can now be ensured, thus facilitating inter- and intralaboratory standardization.
副球孢子菌病（PCM）是拉丁美洲最重要的地方性真菌病之一；通常通过观察和/或分离病原体巴西副球孢子菌以及多种免疫学方法进行诊断。尽管后者有效，但存在两种情况，即与其他真菌病原体的交叉反应以及批次间差异极大的抗原制备，阻碍了检测程序的适当标准化。为了克服这种缺乏可重复性的问题，利用分子生物学工具从这种真菌中制备了一种重组的27 kDa分子质量抗原；通过大肠杆菌DH5α发酵获得了大量这种抗原，该菌株能够表达真菌蛋白。后者通过Prep-Cell系统（伯乐公司）进行纯化；纯蛋白的回收率约为6%。一组160份人血清样本，包括64份在诊断时从患有各种临床类型PCM的患者身上采集的样本，加上分别来自组织胞浆菌病和曲霉病患者的15份血清样本、来自隐球菌病和结核病患者的各10份血清样本、来自球孢子菌病患者的6份血清样本以及来自健康受试者的40份血清样本，均采用纯化的27 kDa重组蛋白通过间接酶联免疫吸附试验进行检测。后者的使用浓度为1.0微克/孔；有三种血清稀释度（1:1000、1:2000和1:4000）。该实验至少重复两次。两个实验的平均灵敏度为73.4%；与健康受试者相比，PCM患者的特异性为87.5%，而其他真菌病患者的特异性为58.7%。检测到与曲霉病和组织胞浆菌病患者血清有重要的交叉反应。该检测的阳性预测值为90.4%。这些结果表明，使用重组抗原蛋白进行PCM的免疫诊断是可行的，并且这样做可以实现高覆盖率。此外，现在可以确保抗原的可重复性，从而便于实验室间和实验室内的标准化。
