Clinical findings for fungal infections caused by methylprednisolone injections.

BACKGROUND: Since September 18, 2012, public health officials have been investigating a large outbreak of fungal meningitis and other infections in patients who received epidural, paraspinal, or joint injections with contaminated lots of methylprednisolone acetate. Little is known about infections caused by Exserohilum rostratum, the predominant outbreak-associated pathogen. We describe the early clinical course of outbreak-associated infections. METHODS: We reviewed medical records for outbreak cases reported to the Centers for Disease Control and Prevention before November 19, 2012, from the six states with the most reported cases (Florida, Indiana, Michigan, New Jersey, Tennessee, and Virginia). Polymerase-chain-reaction assays and immunohistochemical testing were performed on clinical isolates and tissue specimens for pathogen identification. RESULTS: Of 328 patients without peripheral-joint infection who were included in this investigation, 265 (81%) had central nervous system (CNS) infection and 63 (19%) had non-CNS infections only. Laboratory evidence of E. rostratum was found in 96 of 268 patients (36%) for whom samples were available. Among patients with CNS infections, strokes were associated with an increased severity of abnormalities in cerebrospinal fluid (P<0.001). Non-CNS infections were more frequent later in the course of the outbreak (median interval from last injection to diagnosis, 39 days for epidural abscess and 21 days for stroke; P<0.001), and such infections developed in patients with and in those without meningitis. CONCLUSIONS: The initial clinical findings from this outbreak suggest that fungal infections caused by epidural and paraspinal injection of a contaminated glucocorticoid product can result in a broad spectrum of clinical disease, reflecting possible variations in the pathogenic mechanism and in host and exposure risk factors. (Funded by the Centers for Disease Control and Prevention.).

Fungal infections associated with contaminated methylprednisolone injections.

BACKGROUND: Fungal infections are rare complications of injections for treatment of chronic pain. In September 2012, we initiated an investigation into fungal infections associated with injections of preservative-free methylprednisolone acetate that was purchased from a single compounding pharmacy. METHODS: Three lots of methylprednisolone acetate were recalled by the pharmacy; examination of unopened vials later revealed fungus. Notification of all persons potentially exposed to implicated methylprednisolone acetate was conducted by federal, state, and local public health officials and by staff at clinical facilities that administered the drug. We collected clinical data on standardized case-report forms, and we tested for the presence of fungi in isolates and specimens by examining cultures and performing polymerase-chain-reaction assays and histopathological and immunohistochemical testing. RESULTS: By October 19, 2012, more than 99% of 13,534 potentially exposed persons had been contacted. As of July 1, 2013, there were 749 reported cases of infection in 20 states, with 61 deaths (8%). Laboratory evidence of Exserohilum rostratum was present in specimens from 153 case patients (20%). Additional data were available for 728 case patients (97%); 229 of these patients (31%) had meningitis with no other documented infection. Case patients had received a median of 1 injection (range, 1 to 6) of implicated methylprednisolone acetate. The median age of the patients was 64 years (range, 15 to 97), and the median incubation period (the number of days from the last injection to the date of the first diagnosis) was 47 days (range, 0 to 249); 40 patients (5%) had a stroke. CONCLUSIONS: Analysis of data from a large, multistate outbreak of fungal infections showed substantial morbidity and mortality. The infections were associated with injection of a contaminated glucocorticoid medication from a single compounding pharmacy. Rapid public health actions included prompt recall of the implicated product, notification of exposed persons, and early outreach to clinicians.

Agreement of Direct Antifungal Susceptibility Testing from Positive Blood Culture Bottles with the Conventional Method for Candida Species.

Early availability of antifungal susceptibilities can ensure timely institution of targeted therapy in candidemia, which can improve patient outcomes. This study prospectively determines the agreement between the results of direct testing of antifungal susceptibilities from blood culture bottles by disk diffusion and Etest and the results of standardized susceptibility testing methods; direct testing would allow susceptibility results to be available 1 to 2 days earlier. A total of 104 blood cultures with different Candida species (28% C. albicans, 27% C. parapsilosis, 26% C. tropicalis, etc.) were evaluated between January 2012 and May 2013 for agreement of fluconazole, voriconazole, and amphotericin B susceptibility results by disk diffusion. Agreement in MICs obtained by Etest was determined for fluconazole (21 isolates), voriconazole (28 isolates), amphotericin (29 isolates), and caspofungin (29 isolates). The kappa scores for categorical agreement were highest for fluconazole by disk diffusion (0.902, standard error [SE] = 0.076) and Etest (1.00, SE = 0.218) and for amphotericin B by disk diffusion (1.00, SE = 0.098). The Pearson correlation (r) of zone diameters was strongest for fluconazole (0.69) and amphotericin (0.70) and moderate for voriconazole (0.60), and the Pearson correlation of MICs was strongest for fluconazole (0.94) and caspofungin (0.88). However, the moderate correlation of amphotericin MICs with zone diameters (-0.42) precludes the use of amphotericin B disk diffusion for susceptibility testing. There were no very major errors; however, there were 1 (1%) major and 5 (4.8%) minor errors with disk diffusion and 4 (13.3%) minor errors with Etest. Thus, antifungal disk diffusion directly from blood culture bottles is a rapid and easy method for fluconazole and voriconazole susceptibility testing for timely tailoring of candidemia therapy.

Notes from the Field: Probable Mucormycosis Among Adult Solid Organ Transplant Recipients at an Acute Care Hospital - Pennsylvania, 2014-2015.

On September 17, 2015, the Pennsylvania Department of Health (PADOH) notified CDC of a cluster of three potentially health care-associated mucormycete infections that occurred among solid organ transplant recipients during a 12-month period at hospital A. On September 18, hospital B reported that it had identified an additional transplant recipient with mucormycosis. Hospitals A and B are part of the same health care system and are connected by a pedestrian bridge. PADOH requested CDC's assistance with an on-site investigation, which started on September 22, to identify possible sources of infection and prevent additional infections.

Valley fever: finding new places for an old disease: Coccidioides immitis found in Washington State soil associated with recent human infection.

We used real-time polymerase chain reaction and culture to demonstrate persistent colonization of soils by Coccidioides immitis, an agent of valley fever, in Washington State linked to recent human infections and located outside the endemic range. Whole-genome sequencing confirmed genetic identity between isolates from soil and one of the case-patients.

Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri, in 2011.

BACKGROUND: Mucormycosis is a fungal infection caused by environmentally acquired molds. We investigated a cluster of cases of cutaneous mucormycosis among persons injured during the May 22, 2011, tornado in Joplin, Missouri. METHODS: We defined a case as a soft-tissue infection in a person injured during the tornado, with evidence of a mucormycete on culture or immunohistochemical testing plus DNA sequencing. We conducted a case-control study by reviewing medical records and conducting interviews with case patients and hospitalized controls. DNA sequencing and whole-genome sequencing were performed on clinical specimens to identify species and assess strain-level differences, respectively. RESULTS: A total of 13 case patients were identified, 5 of whom (38%) died. The patients had a median of 5 wounds (range, 1 to 7); 11 patients (85%) had at least one fracture, 9 (69%) had blunt trauma, and 5 (38%) had penetrating trauma. All case patients had been located in the zone that sustained the most severe damage during the tornado. On multivariate analysis, infection was associated with penetrating trauma (adjusted odds ratio for case patients vs. controls, 8.8; 95% confidence interval [CI], 1.1 to 69.2) and an increased number of wounds (adjusted odds ratio, 2.0 for each additional wound; 95% CI, 1.2 to 3.2). Sequencing of the D1-D2 region of the 28S ribosomal DNA yielded Apophysomyces trapeziformis in all 13 case patients. Whole-genome sequencing showed that the apophysomyces isolates were four separate strains. CONCLUSIONS: We report a cluster of cases of cutaneous mucormycosis among Joplin tornado survivors that were associated with substantial morbidity and mortality. Increased awareness of fungi as a cause of necrotizing soft-tissue infections after a natural disaster is warranted.

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.

Exserohilum 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.

Notes from the field: Fatal gastrointestinal mucormycosis in a premature infant associated with a contaminated dietary supplement--Connecticut, 2014.

In October 2014, a hospital in Connecticut notified CDC and the Connecticut Department of Public Health of a fatal case of gastrointestinal mucormycosis in a preterm infant. The infant, born at 29 weeks' gestation and weighing 1,400 grams (about 3 pounds), had developed signs and symptoms initially consistent with necrotizing enterocolitis approximately 1 week after birth. Exploratory laparotomy revealed complete ischemia of the gastrointestinal tract from the esophagus to the rectum; a portion of necrotic cecum was sent for microscopic examination. Following surgery, the infant developed multiple areas of vascular occlusion, including a large clot in the aorta, findings not usually associated with necrotizing enterocolitis. The infant died soon after. Histopathology results from the resected cecum revealed an angioinvasive fungal infection consistent with mucormycosis. Gastrointestinal mucormycosis is an extremely rare fungal infection caused by mold in the order Mucorales. It occurs predominantly in low birth weight infants, patients with diarrhea and malnutrition, and those receiving peritoneal dialysis; mortality is 85%. Local investigation revealed that the infant had received a dietary supplement, ABC Dophilus Powder, for 7 days, beginning on day 1 of life.

Phaeohyphomycosis in transplant recipients: Results from the Transplant Associated Infection Surveillance Network (TRANSNET).

Transplant recipients are at a high risk for developing invasive fungal infections. The agents of phaeohyphomycosis are environmental molds found worldwide, and they cause a broad spectrum of disease including skin and subcutaneous lesions, pneumonia, central nervous system disease, fungemia, and disseminated disease. Using data from the Transplant Associated Infection Surveillance Network (TRANSNET), we evaluated patients with proven and probable phaeohyphomycosis. Centers collected data on demographics, co-morbid conditions, clinical features, treatment, and three-month mortality. Fifty-six patients with phaeohyphomycosis were identified from 15 centers, comprising 26 stem cell transplant (SCT) and 30 solid organ transplant (SOT) recipients. Median time to diagnosis post-transplant was 358 days (SCT 100 days; SOT 685 days; P = <.001). The most frequent pathogen was Alternaria species (32%). Disseminated disease was found in 55.4%. Cutaneous infection was more common in SOT (53.3% vs 23.1%; P = .021), while pulmonary disease was more common in SCT (57.7 vs. 26.7; P = .019). Voriconazole (44.6%) and amphotericin B preparations (37.5%) were the most common antifungal therapies. Overall mortality was 25% and was higher in SCT than in SOT (42% vs 10%; P = <.001). A wide variety of organisms encompass phaeohyphomycosis contributing to varying types of infection in transplant recipients. Site of infection, time to disease, and mortality varies significantly between SCT and SOT recipients. Lipid formulations of amphotericin B and voriconazole were the most common antifungals used to treat this disorder.

Utility of (1-3)-ß-D-glucan testing for diagnostics and monitoring response to treatment during the multistate outbreak of fungal meningitis and other infections.

BACKGROUND: The 2012 outbreak of fungal meningitis associated with contaminated methylprednisolone produced by a compounding pharmacy has resulted in >750 infections. An important question facing patients and clinicians is the duration of antifungal therapy. We evaluated (1-3)-ß-d-glucan (BDG) as a marker for monitoring response to treatment. METHODS: We determined sensitivity and specificity of BDG testing using the Fungitell assay, by testing 41 cerebrospinal fluid (CSF) specimens from confirmed cases of fungal meningitis and 66 negative control CSF specimens. We also assessed whether BDG levels correlate with clinical status by using incident samples from 108 case patients with meningitis and 20 patients with serially collected CSF. RESULTS: A cutoff value of 138 pg/mL provided 100% sensitivity and 98% specificity for diagnosis of fungal meningitis in this outbreak. Patients with serially collected CSF were divided into 2 groups: those in whom BDG levels declined with treatment and those in whom BDG remained elevated. Whereas most patients with a decline in CSF BDG had clinical improvement, all 3 patients with continually elevated BDG had poor clinical outcomes (stroke, meningitis relapse, or development of new disease). CONCLUSIONS: Our data suggest that measuring BDG in CSF is a highly sensitive test for diagnosis of fungal meningitis in this outbreak. Analysis of BDG levels in serially collected CSF demonstrated that BDG may correlate with clinical response. Routine measurement of BDG in CSF may provide useful adjunctive data for the clinical management of patients with outbreak-associated meningitis.

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum.

Exserohilum infections associated with contaminated steroid injections: a clinicopathologic review of 40 cases.

September 2012 marked the beginning of the largest reported outbreak of infections associated with epidural and intra-articular injections. Contamination of methylprednisolone acetate with the black mold, Exserohilum rostratum, was the primary cause of the outbreak, with >13,000 persons exposed to the potentially contaminated drug, 741 confirmed drug-related infections, and 55 deaths. Fatal meningitis and localized epidural, paraspinal, and peripheral joint infections occurred. Tissues from 40 laboratory-confirmed cases representing these various clinical entities were evaluated by histopathological analysis, special stains, and IHC to characterize the pathological features and investigate the pathogenesis of infection, and to evaluate methods for detection of Exserohilum in formalin-fixed, paraffin-embedded (FFPE) tissues. Fatal cases had necrosuppurative to granulomatous meningitis and vasculitis, with thrombi and abundant angioinvasive fungi, with extensive involvement of the basilar arterial circulation of the brain. IHC was a highly sensitive method for detection of fungus in FFPE tissues, demonstrating both hyphal forms and granular fungal antigens, and PCR identified Exserohilum in FFPE and fresh tissues. Our findings suggest a pathogenesis for meningitis involving fungal penetration into the cerebrospinal fluid at the injection site, with transport through cerebrospinal fluid to the basal cisterns and subsequent invasion of the basilar arteries. Further studies are needed to characterize Exserohilum and investigate the potential effects of underlying host factors and steroid administration on the pathogenesis of infection.

Real-time PCR assays for genotyping of Cryptococcus gattii in North America.

BACKGROUND: Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen. METHODS: We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human. RESULTS: Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA. CONCLUSIONS: The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.

Coccidioides immitis identified in soil outside of its known range - Washington, 2013.

Coccidioidomycosis ("valley fever") is caused by inhaling spores of the soil-dwelling fungi Coccidioides immitis or Coccidioides posadasii. Most infections are subclinical. When clinical manifestations do occur (typically 1-4 weeks after exposure), they are similar to those associated with influenza or community-acquired pneumonia. Disseminated disease is rare. Residual pulmonary nodules can lead to chronic lung disease. Fluconazole or other triazoles often are used for treatment, but mild cases often resolve without specific therapy. A total of 17,802 cases were reported in the United States in 2012.

Detection of fungal DNA in human body fluids and tissues during a multistate outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the major cause of an outbreak of fungal infections linked to injections of contaminated methylprednisolone acetate. Because almost 14,000 persons were exposed to product that was possibly contaminated with multiple fungal pathogens, there was unprecedented need for a rapid throughput diagnostic test that could detect both E. rostratum and other unusual agents of fungal infection. Here we report development of a novel PCR test that allowed for rapid and specific detection of fungal DNA in cerebrospinal fluid (CSF), other body fluids and tissues of infected individuals. The test relied on direct purification of free-circulating fungal DNA from fluids and subsequent PCR amplification and sequencing. Using this method, we detected Exserohilum rostratum DNA in 123 samples from 114 case-patients (28% of 413 case-patients for whom 627 samples were available), and Cladosporium DNA in one sample from one case-patient. PCR with novel Exserohilum-specific ITS-2 region primers detected 25 case-patients with samples that were negative using broad-range ITS primers. Compared to fungal culture, this molecular test was more sensitive: of 139 case-patients with an identical specimen tested by culture and PCR, E. rostratum was recovered in culture from 19 (14%), but detected by PCR in 41 (29%), showing a diagnostic sensitivity of 29% for PCR compared to 14% for culture in this patient group. The ability to rapidly confirm the etiologic role of E. rostratum in these infections provided an important contribution in the public health response to this outbreak.

Preliminary laboratory report of fungal infections associated with contaminated methylprednisolone injections.

In September 2012, the Centers for Disease Control and Prevention (CDC) initiated an outbreak investigation of fungal infections linked to injection of contaminated methylprednisolone acetate (MPA). Between 2 October 2012 and 14 February 2013, the CDC laboratory received 799 fungal isolates or human specimens, including cerebrospinal fluid (CSF), synovial fluid, and abscess tissue, from 469 case patients in 19 states. A novel broad-range PCR assay and DNA sequencing were used to evaluate these specimens. Although Aspergillus fumigatus was recovered from the index case, Exserohilum rostratum was the primary pathogen in this outbreak and was also confirmed from unopened MPA vials. Exserohilum rostratum was detected or confirmed in 191 specimens or isolates from 150 case patients, primarily from Michigan (n=67 patients), Tennessee (n=26), Virginia (n=20), and Indiana (n=16). Positive specimens from Michigan were primarily abscess tissues, while positive specimens from Tennessee, Virginia, and Indiana were primarily CSF. E. rostratum antifungal susceptibility MIC50 and MIC90 values were determined for voriconazole (1 and 2 µg/ml, respectively), itraconazole (0.5 and 1 µg/ml), posaconazole (0.5 and 1 µg/ml), isavuconazole (4 and 4 µg/ml), and amphotericin B (0.25 and 0.5 µg/ml). Thirteen other mold species were identified among case patients, and four other fungal genera were isolated from the implicated MPA vials. The clinical significance of these other fungal species remains under investigation. The laboratory response provided significant support to case confirmation, enabled linkage between clinical isolates and injected vials of MPA, and described significant features of the fungal agents involved in this large multistate outbreak.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

Histopathologic diagnosis of fungal infections in the 21st century.

Fungal infections are becoming more frequent because of expansion of at-risk populations and the use of treatment modalities that permit longer survival of these patients. Because histopathologic examination of tissues detects fungal invasion of tissues and vessels as well as the host reaction to the fungus, it is and will remain an important tool to define the diagnostic significance of positive culture isolates or results from PCR testing. However, there are very few instances where the morphological characteristics of fungi are specific. Therefore, histopathologic diagnosis should be primarily descriptive of the fungus and should include the presence or absence of tissue invasion and the host reaction to the infection. The pathology report should also include a comment stating the most frequent fungi associated with that morphology as well as other possible fungi and parasites that should be considered in the differential diagnosis. Alternate techniques have been used to determine the specific agent present in the histopathologic specimen, including immunohistochemistry, in situ hybridization, and PCR. In addition, techniques such as laser microdissection will be useful to detect the now more frequently recognized dual fungal infections and the local environment in which this phenomenon occurs.

Pseudo-outbreak of Lecanicillium and Acremonium species in orthopedic surgery patients.

Acremonium species cause a variety of human infections, while Lecanicillium species have not been reported as human pathogens. We describe a pseudo-outbreak involving both organisms, highlighting the role and limitations of molecular methods in the characterization of rare fungal isolates. Repeated isolation of these fungi from patient tissue samples raises concerns about exogenous contamination in the hospital environment.