Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens

Abstract

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.

Risk of Exposure to Coccidioides spp. in the Temblor Special Recreation Management Area (SRMA), Kern County, CA

“…Even though an alignment of all PCR amplicons obtained with the diagnostic primer pair EC3/EC100 revealed slight genetic differences among them, the short branches displayed in the phylogenetic tree that was compiled did not allow us to conclude without doubt that both species of Coccidioides are present in the soils of the Temblor SRMA. C. posadasii and C. immitis have been detected in some clinical samples outside their known endemic areas; however, travel history of these patients could not be verified without doubt [ 40 , 41 , 42 ]. Although C. immitis is traditionally thought to be the only species of Coccidioides in the Central Valley of California, this could change in the future with changing climate conditions.…”

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis

“…Centre 4 used a duplex qPCR targeting the single copy proline-rich antigen 2 (PRA2) gene, able to differentiate C. immitis and C. posadasii. 68 Centre 5 performed a broad-range fungal cPCR with subsequent identification by sequencing. 78 The methods are summarised in Table 4.…”

“…They were applied on various sample types, such as formalin‐fixed paraffin‐embedded and fresh biopsies, 4,32,40,41 blood, 28,42–45 serum, 29,33,43,45–47 bronchoalveolar lavage fluid (BAL), 28,45,46,48 cerebrospinal fluid, 28,45,47,49 bone marrow, 28,43,48,49 pleural fluid 28,48 and urine 50 . They target multicopy ribosomal DNAs 28,29,33,36,39,43–45,47,49,51–59 (rDNAs) or species‐specific genes, 27,41,42,46,48,60–73 mostly single copy, except for the region identified in the NCBI database as a ‘copia‐like retrotransposon’ 1,37,38,74,75 with approximately 60 copies per Coccidioides genome 74 …”

Coccidioides Species: A Review of Basic Research: 2022

“…The use of PCR to detect fungal DNA in soil has been a major step forward since it is a very sensitive test for the detection of the organism. Several tests are available that use a variety of targets including the internal transcribed spacer 2 region, a transposon, and other genes [ 45 , 46 , 47 , 48 , 49 ]. The sensitivity of the tests is largely dependent on the number of repeats of the gene target in the genome.…”

“…There is one FDA-approved test for identification of C. immitis and C. posadasii DNA that distinguishes between the two species that has also been approved for direct testing of clinical specimens without culture ( ) (accessed 10 August 2022). Several other tests are also available for detection of Coccidioides DNA in clinical specimens [ 45 , 46 , 47 , 48 ], one of which also distinguishes between the two species [ 49 ].…”