Development of a Real-Time PCR Assay for Identification of Coccidioides immitis by Use of the BD Max System

Abstract

bRapid real-time PCR (RT-PCR) can be performed in a community hospital setting to identify Coccidioides species using the new Becton Dickinson molecular instrument BD Max. Following sample preparation, DNA extraction and PCR were performed on the BD Max using the BD Max extraction kit ExK-DNA-1 test strip and a master mix prepared by BioGX (Birmingham, AL). Sample preparation took 2 h, and testing on the BD Max took an additional 2 h. Method sensitivity and specificity were evaluated along with the limits of detection to confirm that this convenient method would provide medically useful information. Using serial dilutions, the lower limit of detection was determined to be 1 CFU/l. Testing with this method was validated using samples from various body sites, including bronchial alveolar lavage (BAL) fluid; sputum and lung tissue samples; and pleural and spinal fluids. Safety protocols were established, and specimen preparation processes were developed for the various types of specimens. The range for the cycle threshold (C T ) indicating adequate fluorescent signal to signify a positive result was established along with the acceptable range for the internal standard. Positive controls run with each batch were prepared by spiking a pooled BAL fluid specimen with a known dilution of Coccidioides immitis organism. Our experience with testing >330 patient samples shows that clinically relevant information can be available within 4 h using an RT-PCR method on the BD Max to identify Coccidioides spp., with sensitivity equivalent to culture. V alley fever caused by the dimorphic fungi Coccidioides immitis in the Central Valley of California and Coccidioides posadasiiin other arid areas of the southwestern United States continues to be an important illness in those areas (1-3). According to the Centers for Disease Control and Prevention, 4,431 cases of Valley fever were reported in California in 2012 (4). For most healthy residents of the Central Valley who contract this infection, Valley fever causes a rather mild influenza-like illness. However, the illness can be severe in some individuals, especially those with a compromised immune system. Without sensitive testing methods, it is usually not possible to distinguish the early symptoms of coccidioidomycosis from other causes of community-acquired pneumonia, which may lead to delayed or improper treatment (5).Current laboratory testing methods rely on lengthy, labor-intensive protocols using experienced and highly skilled laboratory personnel. Methods include traditional serology testing for immunodiffusion tube precipitin (TP) or complement fixation (CF) antibody, enzyme immunoassays, culture, and histopathology (6). Serology testing for Coccidioides is helpful but has limitations for diagnosing current, active infections, since antibodies may be slow to increase to detectable levels, especially in immunocompromised patients (7, 8). Culture-based methods require several days or weeks to grow sufficient fungi for identification by molecular methods, posing additional sa...

A Rapid and Automated Sample-to-Result Candida auris Real-Time PCR Assay for High-Throughput Testing of Surveillance Samples with the BD Max Open System

“…The highlights of this study are as follows: easy migration of the manual real-time PCR assay to an automated platform; the consistent performance of primers, probe, and master mix from our manual assay; comparable accuracy, sensitivity, and specificity; and easy integration in the laboratory workflow. These findings also confirm the suitability of the BD Max platform for direct detection of fungal pathogens from clinical specimens as reported earlier for Pneumocystis jirovecii and Coccidioides immitis (6,7). There was indication that the BD Max performed better than the manual assay for a few samples with high C T values, possibly due to the elimination of many hands-on steps and the use of purified DNA.…”

“…auris using the BD Max system, a fully integrated and automated platform amenable to direct detection of other fungal pathogens (6,7).…”

A rapid and automated sample-to-result Candida auris real-time PCR assay for high-throughput testing of surveillance samples with BD MAX™ open system

“…The highlights of this study are: easy migration of the manual real-time PCR assay to an automated platform, the consistent performance of primers, probe, and master mix from our manual assay, comparable accuracy, sensitivity and specificity, and easy integration in the laboratory workflow. These findings also confirm the suitability of BD MAX™ platform for direct detection of fungal pathogens from clinical specimens as reported earlier for Pneumocystis jirovecii and Coccidioides immitis (6, 7). There was indication that BD MAX™ performed better than the manual assay for a few samples with high Ct values possibly due to the elimination of many hands-on steps and the use of purified DNA.…”

“…However, the C. auris LDT is not amenable to automation and high-throughput screening, and adoption of the LDT has progressed slowly while the affected facilities and sample numbers continue to grow. Therefore, we developed and validated a real-time PCR assay for C. auris using BD MAX™ System, a fully integrated and automated platform amenable to direct detection of other fungal pathogens (6, 7).…”

Development of a Real-Time PCR Protocol Requiring Minimal Handling for Detection of Vancomycin-Resistant Enterococci with the Fully Automated BD Max System

“…A recent new device for fully automated molecular detection is the BD Max system, which combines nucleic acid extraction, PCR setup, and 5-color real-time PCR detection. The flexible programming allows user-developed assays to be run in an automated manner (21)(22)(23)(24)(25). However, those assays still require liquid handling and thus need a laboratory capable of performing molecular assays.…”