Utility of Real-Time PCR for Detection of Exserohilum rostratum in Body and Tissue Fluids during the Multistate Outbreak of Fungal Meningitis and Other Infections

Abstract

bExserohilum rostratum was the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range and E. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection of E. rostratum in body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive for E. rostratum to 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-D-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data. Since September 2012, U.S. state and local health departments, the Centers for Disease Control and Prevention (CDC), and the Food and Drug Administration have been investigating the largest documented health care-associated outbreak in the United States of fungal meningitis and other infections developed after epidural, paraspinal, or joint injections with contaminated methylprednisolone acetate (MPA) from a single compounding pharmacy (1-3). The major cause of this outbreak was Exserohilum rostratum, a plant pathogen that rarely causes disease in humans (4). By using culture, PCR, and histopathology, this fungus was identified in cerebrospinal fluid (CSF), synovial fluid, abscess aspirate, fresh tissue, and formalin-fixed, paraffin-embedded tissue samples from 153 case patients, as well as in unopened vials of two implicated lots of MPA (3). Previously, we developed a method for extracting free circulating fungal DNA from different types of body fluids and tissues from patients in this outbreak and used two PCR tests, followed by DNA sequencing, to detect fungal DNA in patient specimens (5). Using this approach, we were able to detect fungal DNA in 28% of the case patients (3). Although the sensitivity of the PCR assay was considerably better than that of culture, which was ab...

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