Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
Department of Biomedical Sciences, University at Albany, Albany, New York, United States of America.
PLoS Negl Trop Dis. 2021 Sep 16;15(9):e0009765. doi: 10.1371/journal.pntd.0009765. eCollection 2021 Sep.
Coccidioidomycosis (Valley fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.
球孢子菌病（谷热）是一种肺部和全身性真菌病，其发病率不断增加，流行地区不断扩大。在临床实验室中，区分病原体粗球孢子菌和波氏球孢子菌仍然存在问题，因为常规 PCR 和卫星分型方案并不简单。因此，我们开发了 Cy5 和 FAM 标记的 TaqMan 探针，用于快速区分培养物和临床标本中的粗球孢子菌和波氏球孢子菌的双实时 PCR 检测。编码富含脯氨酸抗原 2 的 RRA2 基因是第一组引物和探针的来源，该基因特异性针对球孢子菌属。使用粗球孢子菌 Contig 2.2（GenBank：AAEC02000002.1）设计第二组引物和探针。由于 Contig 中有 86bp 的缺失，第二组引物/探针不能扩增相应的波氏球孢子菌 DNA。该检测方法具有很高的灵敏度，检测限为 0.1pg gDNA/PCR 反应，相当于大约十个粗球孢子菌或波氏球孢子菌的基因组拷贝。该检测方法具有高度特异性，与广泛的真菌和细菌病原体无交叉反应。对 1995 年至 2020 年提交的真菌分离株和原发性标本进行回顾性分析，证实了 168 株分离株和 4 株原发性标本为人类球孢子菌病病例中的波氏球孢子菌，30 株为粗球孢子菌，而来自两只动物（恒河猴和犀牛）的 8 株原发性标本均确认为波氏球孢子菌。对 15 份脑脊液（CSF）和胸腔液样本的初步分析显示，两种样本的血清学检测与实时 PCR 检测呈正相关。该球孢子菌属实时 PCR 双检测方法可快速区分临床标本中的粗球孢子菌和波氏球孢子菌，并进一步增强球孢子菌病的治疗和监测。
