Laboratory Testing and Results from the Outbreak

CDC laboratory-confirmed results found on this page are from three New England Compounding Center (NECC) preservative-free methylprednisolone acetate (MPA) lots recalled on September 26, 2012. ¹

For laboratory results on NECC products other than preservative-free MPA, see CDC's non-MPA recall website and FDA's website.

CDC's Fungus Reference Laboratory has the capacity and technology to examine fungal isolates under the microscope and to confirm their identification using DNA sequencing methods.

CDC Laboratory Results

Human Tissue and Fluid Samples ^{2, 3, 4}

CDC and State health laboratories are testing tissue and fluid samples from patients with probable or confirmed fungal infection. CDC scientists are using DNA-based methods and other advanced testing to determine whether the samples contain fungi and other microbial pathogens, and if so, what type.

As of March 11, 2013, Exserohilum rostratum has been identified in 151 case-patients and continues to be the predominant fungal infection in this outbreak. One patient, the index case, was infected with Aspergillus fumigatus, and 22 additional species were identified: Aspergillus species (4 cases), Cladosporium spp. (6), Alternaria sp. (4), Bipolaris sp. (1), Chaetomium sp. (1), coelomycete fungus (1), Epicoccum nigrum (1), Paecilomyces (1), Penicillium sp. (1), Scopulariopsis brevicaulis (1), and Stachybotrys chartarum (1).

Early in the outbreak, patient specimens were primarily cerebrospinal fluid and fluid from joints (knee, etc.). Recently, fluids from epidural abscesses (accumulations of pus around the spine) and tissues have been the focus of laboratory testing.

The numbers reported here do not reflect all cases of fungal meningitis, joint infections, and epidural abscesses because laboratory methods are not sensitive enough to detect all positive samples. Many patients with potential fungal infections will have a negative laboratory result. ^{2, 3}

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Table 1. CDC Laboratory Identification of Exserohilum rostratum from 151 Case-Patients*

Source	Number of E. rostratum Identified	Fungus Description
Cerebrospinal Fluid	82	Exserohilum is a common mold found in soil and on plants, especially grasses, and thrives in warm and humid climates. Exserohilum rarely causes infections in people, however Exserohilum rostratum has been recognized as a human pathogen. More
Tissue	46
Joint fluids	4
Other specimens	2
Cultured Isolates†	49

* Multiple specimens were received from some case-patients
† Fungal cultures isolated from human specimens that were received from State Health Department laboratories.

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Antifungal Susceptibility Testing ⁵

Fifty isolates of Exserohilum rostratum were cultured from patient specimens and tested for their susceptibility to a variety of antifungal drugs. Susceptibility to amphotericin B was determined using the Epsilometer test (Etest) method according to the manufacturer’s instructions [Photo 1]. Susceptibility to voriconazole, posaconazole, itraconazole, fluconazole, and the investigational drug isavuconazole was tested by broth microdilution according to methods defined by the Clinical and Laboratory Standards Institute (CLSI) (M38-A2) ⁵. Exserohilum rostratum drug susceptibility results are shown in Table 2 below.

Table 2. CDC Antifungal Susceptibility Testing

	Minimum Inhibitory Concentration (in µg/ml) at 48-72h
Antifungal agent (no. of isolates tested)	Range (lowest to highest value)	MIC50	MIC90	Mode (most frequent value)
Voriconazole (50)	1-4	1	2	1-2
Fluconazole (50)	16-256	64	64	64
Itraconazole (50)	0.25-4	0.5	1	0.5
Posaconazole (49)	0.25-1	0.5	1	0.5
Isavuconazole (50)	2-4	4	4	4
Amphotericin B (48)	0.032-2	0.25	0.5	0.38

The clinical relevance of MIC testing of this fungal pathogen remains uncertain, and breakpoints with proven relevance have yet to be identified or approved by CLSI or any regulatory agency.

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CDC's PCR test for detection of fungal DNA

CDC has developed a novel test using PCR and DNA sequencing to detect fungal DNA in cerebrospinal fluid (CSF), other body fluids, and tissues from patients in this outbreak. To assess the performance of this PCR test, CDC tested 627 samples from 413 case-patients. The main results of the validation study are as follows:

• The PCR test detected Exserohilum rostratum DNA in 123 samples from 114 case-patients (28% of the 413 case-patients for whom samples were available). The test also detected Cladosporium sp. DNA in one case-patient.
• PCR was more sensitive than culture. Of 139 case-patients who had a specimen tested by both PCR and culture, E. rostratum DNA was detected by PCR in 41 (29%) but was only recovered from culture in 19 (14%). In total, 33% of case-patients had fungi detected in specimens by either culture or PCR.
• There were no false-positive PCR results (100% specificity). Among 136 specimens from patients who did not meet the case definition, all were negative.

The full results of the study performed to validate this PCR test are available here. The results suggest that the test is a useful tool to quickly detect fungi in body fluids and tissues, and it can detect fungal DNA in specimens that may not have shown any fungus in culture.

However, PCR for fungal detection is still a research test that has not been cleared or approved by the FDA and should not be used for diagnosis, treatment, or assessment of patient health or management. In addition, for both PCR and culture, a negative result does not rule out infection.

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Product Samples: Three Lots of Preservative-Free MPA

Tests at CDC and FDA laboratories on the preservative-free MPA vials have confirmed the presence of Exserohilum rostratum in unopened vials from two of the three recalled lots. ¹ These laboratory test results strengthen the link between preservative-free MPA vials and the outbreak.

The types of fungi found in patient specimens and in the recalled MPA vials are common in the environment, but were not known to cause meningitis prior to this outbreak.

• As of October 22, CDC laboratories have confirmed the presence of Exserohilum rostratum [JPG] in lots #08102012@51 BUD 2/6/2013 and #06292012@26 BUD 12/26/2012. This fungus is the same genus and species as the one found in laboratory-confirmed cases of human infection.
• As of January 22, 2013, CDC laboratories have recovered Cladosporium cladosporioides in one unopened vial from lot#0812012@51 sent from the state of Tennessee. This fungus has also been recovered from five patient isolates sent to CDC for confirmation, and from one CSF sample sent to CDC for testing.
• As of February 8, 2013, CDC laboratories confirmed the presence of Exserohilum rostratum, Bacillus subtilis and Bacillus pumilus in one unopened vial of lot 08102012@51 that was cultured locally in Idaho and submitted to CDC through the Idaho state public health laboratory. The Bacillus organisms are bacteria.
• As of February 8, 2013, CDC laboratories reconfirmed Paecilomyces formosus from 20 unopened vials and Exserohilum rostratum from one unopened vial, both from lot 05212012@68 cultured and identified at the Wadsworth Center New York State public health laboratory.
• CDC also identified non-human pathogens Rhodotorula laryngis and Rhizopus stolonifer in lot #08102012@51, BUD 2/6/2013; and Rhodotorula laryngis in lot #06292012@26, BUD 12/26/2012. The fungi Rhodotorula laryngis and Rhizopus stolonifer are not known to cause disease in humans; they cannot grow at human body temperature.

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Table 3. CDC Laboratory-Confirmed Organisms From Recalled Lots of Preservative-Free MPA

Product(s)	Fungi Isolated	Description
MPA (PF) Lot #06292012@26 Beyond Use Date 12/26/2012 MPA (PF) Lot #08102012@51 Beyond Use Date 2/6/2013	Exserohilum rostratum	Known to cause human disease
MPA (PF) Lot #08102012@51 Beyond Use Date 2/6/2013	Cladosporium cladosporioides	Does not grow at human body temperature
MPA (PF) Lot #06292012@26 Beyond Use Date 12/26/2012 MPA (PF) Lot #08102012@51 Beyond Use Date 2/6/2013	Rhodotorula laryngis	Does not grow at human body temperature Not known to cause human disease
MPA (PF) Lot #08102012@51 Beyond Use Date 2/6/2013	Rhizopus stolonifer	Does not grow at human body temperature Not known to cause human disease

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