Broad-Range Direct Detection and Identification of Fungi by Use of the PLEX-ID PCR-Electrospray Ionization Mass Spectrometry (ESI-MS) System

Abstract

The PLEX-ID system is a novel technology that couples PCR amplification and electrospray ionization-mass spectrometry to identify pathogens directly in clinical specimens. The analytical performance of the PLEX-ID Broad Fungal assay was compared with that of traditional culture identification by using 91 characterized fungal culture isolates (64 manufacturer-claimed and 27 nonclaimed organisms) and directly by using 395 respiratory specimens. Discordant results were resolved by D2 large-subunit ribosomal DNA fungal sequencing. Environmental studies were performed to monitor for potential contamination. The PLEX-ID Broad Fungal assay correctly identified 95.6% (87/91) and 81.3% (74/91) of the culture isolates to the genus and species levels, respectively. Of the manufacturer-claimed organisms, 100% (64/64) and 92.2% (59/64) were correctly identified to the genus and species levels, respectively. Direct analysis of respiratory specimens resulted in 67.6% (267/395) and 66.6% (263/395) agreement with culture results to the genus and species levels, respectively, with 16.2% (64/395) of the results discordant with culture and 16.2% (64/395) not detected by the system. The majority (>95%) of the isolates not detected directly by the PLEX-ID system ultimately grew in low quantities in culture (<20 colonies). In 20.3% (35/172) of the respiratory specimens where no growth was observed in culture, the PLEX-ID system identified a fungus, suggesting a potential increase in sensitivity over culture in some instances. The PLEX-ID system provides a rapid method for the detection of a broad array of fungi directly in respiratory specimens and has the potential of impacting turnaround times and patient care by reducing the need to wait for the growth of an organism in culture. Invasive fungal infections (IFIs) have increased in parallel with the escalating number of immunosuppressed patients seen in health care facilities over the past several decades. IFIs are a major cause of morbidity and mortality in high-risk populations, including those individuals who are at the extremes of age, who are undergoing solid organ or hematopoietic stem cell transplantation, who are undergoing prescribed immunosuppressive therapy, or who have an immunosuppressive disease (e.g., AIDS) (1). The most common fungal causes of IFIs are Candida species, Aspergillus species, and Cryptococcus species. However, the spectrum of fungal agents causing IFIs has broadened to include more atypical agents, including opportunistic yeasts (e.g., Trichosporon species and Rhodotorula species), the mucormycetes (e.g., Lichtheimia, Rhizopus, and Mucor species), the hyalohyphomycetes (e.g., Fusarium and Scedosporium species), the phaeohyphomycetes (e.g., Alternaria, Scedopsorium, and Bipolaris species), and the causes of endemic mycoses (e.g., Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis/C. posadasii) (1, 2). Early diagnosis and prompt therapy of IFIs are instrumental in the successful treatment of immunosuppressed populations, but convent...

Identification of Mycobacterium Species and Mycobacterium tuberculosis Complex Resistance Determinants by Use of PCR-Electrospray Ionization Mass Spectrometry

“…The line probe assays are the NAAT method which comes closest to providing this information, but at least two distinct strips/assays are needed at this time. Another advantage of the PCR-ESI-MS method is the ability to test for a wide variety of organisms on the same platform, including bacteria, viruses, fungi, and parasites (6,10,23,24). This can be accomplished by some of the current NAAT tests (PCR and sequencing) but generally requires the performance of a variety of PCR assays and the use of multiple sequencing targets.…”

“…The MDR-TB assay utilizes signal thresholds (cutoffs) designed to limit reporting of irreproducible detections. Cutoffs are applied to two measurements, the level and the Q score, as previously described (9,10). The level is an indication of the amount of the amplicon present in the sample in comparison to a calibrant, and the Q score is a rating between 0 (low) and 1 (high) which represents the strength of the data supporting identification.…”

“…The overall work flow to process 12 samples (one MDR-TB assay plate) on the PCR-ESI-MS system from start to finish is approximately 6 h, with approximately 1 h of hands-on time. Following a strict unidirectional workflow, the overall turnaround time (TAT) to reporting of results was 24 h, since the plates were placed on the PCR-ESI-MS system at the end of the work day and the data were analyzed the following morning, as previously described (10).…”

“…AB-1720; Thermo Scientific) at 175°C for 1.5 s (ThermoSci ALPS 50V; Thermo Scientific), centrifuged at 1,800 ϫ g for 1 min, and loaded on to the Eppendorf Mastercycler proS thermocycler (Eppendorf, Hamburg, Germany. The PCR was carried out using the manufacturer's recommended thermocycler protocol as previously reported (10).…”

“…From the stock lysates, a 1:20 dilution was performed by adding 20 l of lysate into 180 l of sterile water. The diluted lysates were used as the template for the PCRs (10).…”