Fungal Disease Following Contaminated Steroid Injections

Abstract

This Commentary highlights the article by Ritter et al. that reported the pathology associated with the recent fungal outbreak associated with contaminated methylprednisolone acetate injections.

CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.

The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.

CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

The recent outbreak of fungal disease associated with contaminated methylprednisolone acetate (MPA) injections forced the practices of compounding pharmacies into the spotlight. The causative organism in most of the cases was Exserohilum rostratum, a black mold that is otherwise uncommonly seen as a pathogen. This epidemic forced public health officials to confront an iatrogenic outbreak of unprecedented proportion of a fungal disease for which there is no specific diagnostic test, no animal model, little data to inform selection of optimal antifungal therapy, and minimal information for determining duration of therapy.¹ Central to the ability of the research community to develop rational approaches to prevention, diagnosis, and treatment is an understanding of disease pathogenesis.

In this issue of The American Journal of Pathology, Ritter et al² report on the pathology associated with this outbreak while providing clinical and laboratory correlation. Their findings support the presumed entry of fungi via cerebrospinal fluid (CSF) penetration from the injection site, with transport via the CSF and basilar artery invasion. They also demonstrate outstanding sensitivity of immunohistochemistry (IHC) for fungal detection in tissue. This study significantly advances our understanding of E. rostratum pathogenesis.

The Outbreak

In September 2012, the Tennessee Department of Health reported the first cases of fungal meningitis to the Centers for Disease Control and Prevention (CDC). These cases occurred in patients who had received epidural steroid injections mainly for back pain, and the source of the outbreak was rapidly traced to three lots of preservative-free methylprednisolone acetate produced at the New England Compounding Center (Framingham, MA). By the time the affected lots were recalled 8 days later, >13,000 people had been exposed during the preceding four months.³ Subsequently, these lots were shown to be contaminated with E. rostratum as well as with several other bacteria and fungi.⁴ The high mortality associated with this multistate outbreak necessitated very rapid mobilization of the CDC with the organization of committees to define case definitions, optimal approaches to therapy, and the establishment of research priorities. The response on the part of public health officials was nothing short of heroic.

Early on in the epidemic, the predominant clinical syndromes were meningitis and stroke. As the epidemic evolved, epidural abscesses, paravertebral phlegma, sacroilitis, and peripheral joint infections emerged as more common manifestations.¹ Though the pace has slowed, additional cases continue to be reported, with numbers standing at 749 cases and 63 deaths in 20 states as of July 1, 2013 (CDC, http://www.cdc.gov/hai/outbreaks/meningitis-map-large.html, last accessed August 6, 2013). A report of a patient who has relapsed off therapy following presumed cure now has appeared,⁵ highlighting the consequences of limited knowledge.

One positive consequence of the outbreak has been an increased awareness by the medical community and the general public of the existence of large scale compounding centers that prepare medications for distribution to wide areas without the degree of regulation placed on manufacturers. Fortunately, this attention has resulted in the introduction of legislation in the US Senate that would give the US Food and Drug Administration (FDA) broader regulatory powers (The New York Times, http://www.nytimes.com/2013/05/23/us/politics/senate-committee-approves-bill-on-compounding.html?_r=0, last accessed June 20, 2013).⁶ Action cannot come soon enough, as evidenced by the recent appearance of a new multistate outbreak of skin and soft tissue infections associated with injection of MPA produced at a compounding pharmacy in Tennessee, where again, multiple bacterial and fungal species are being isolated (CDC, http://www.cdc.gov/hai/outbreaks/TN-pharmacy/index.html, last accessed June 20, 2013).

Black Molds and E. rostratum

Phaeohyphomycosis, defined as “condition of fungi with dark hyphae,” is a commonly used term that lumps together diseases caused by >100 species of fungi in 60 genera spanning several orders.⁷ This usage reflects the low frequency with which each of these organisms causes disease. Though genomic sequencing for most of these organisms has not been done, the potential evolutionary divergence is huge. However, knowledge regarding pathogenesis of one is carried to the others, albeit with a degree of necessity. The very significant morbidity and mortality from the current epidemic provides the impetus to dissect the clinical and pathological features of E. rostratum in great detail.

The dark color of the hyphal walls is due to melanin. Although some black molds are more geographically restricted, Exserohilum spp. are broadly distributed. E. rostratum is isolated from soil and marine environments.⁸ The organism is a phytopathogen, particularly for grasses, and its use for biocontrol of weeds has been studied.⁹ Three of the approximately 35 species of Exserohilum have been reported as causes of human disease (E. rostratum, E. longirostratum, and E. mcginnisii) though more recent multilocus sequence analysis suggests that all three may be conspecific.¹⁰

Pathogenesis of E. rostratum

The collection of samples from 40 patients in the report by Ritter et al² with analysis by one group represents a unique resource, as earlier studies analyzed no more than two new cases each with heavy reliance on repeated literature review. As Ritter et al² observe, several features are consistent with what has been seen previously, whereas others extend our understanding of the spectrum of disease, particularly since these cases are the first reports of E. rostratum meningitis. Based on prior cases, the portals of entry include the respiratory tract, producing disease of the sinuses and lungs, and traumatic inoculation, resulting in skin or corneal disease, as well as osteomyelitis. Here, host barriers obviously were breached by direct inoculation of the organisms, which is consistent with prior routes. But, as discussed later in this section, some questions remain as to how the epidural injections caused meningitis.

Ritter et al² describe the spectrum of histopathology previously reported and note that their cases demonstrate the full range of inflammatory responses. Interestingly, in tissue samples from patients with only epidural or paraspinal infections at the injection site, tissue more commonly had lymphohistiocytic inflammation, whereas in patients with meningitis, the pathology of the injection site tissues was more necrosuppurative, suggesting a more indolent process in the former group. The overall fungal burden in tissue from these patients also was lower. These patients also apparently presented later, though statistical analysis for this comparison is not provided. Whether these outcomes, which may represent qualitatively different immune responses, reflect differences in the fungal inoculum, host differences, or other factors remains unanswered.

As Ritter et al² note, the pathogenesis of E. rostratum meningitis is similar to that of other fungal pathogens in terms of angioinvasion with resultant thrombosis and infarction. The degree to which vascular invasion was seen in this sample collection is striking. An interesting feature is that fungi in vessels and dense connective tissue of injection sites were not necessarily accompanied by inflammation, though it is unclear whether or not these areas were relatively avascular when present in connective tissue.

Ritter et al² also note an apparent predilection for the base of the brain despite inoculation in the lumbar or cervicothoracic spine. The mold that most commonly causes CNS disease is Aspergillus fumigatus. As they point out, this feature has been seen in Aspergillus meningitis associated with epidural steroid injections but not when CNS Aspergillus infection occurs hematogenously. However, following either hematogenous spread or extension from a contiguous focus, the most common manifestation of CNS aspergillosis is abscess formation. Meningitis, which occurs less commonly, mainly results from involvement of adjacent brain tissue.¹¹ CNS disease due to other agents of phaeohyphomycosis, which are thought to share the same portals of entry as E. rostratum, mostly has been reported as cerebritis or abscess.¹² Thus, the absence of apparent basilar predilection is less surprising in these instances. Involvement of the basilar meninges with or without arterial invasion has been described for other fungal pathogens, including Coccidioides immitis and Cryptococcus neoformans,^13,14 organisms for which meningitis is a manifestation of disseminated disease, though these pathogens typically produce a lymphocytic meningitis. Similar tendency is seen in tuberculous meningitis, as well as meningitis due to other bacterial pathogens. As suggested in the present report, this distribution is not well understood but likely results from the dynamics of CSF circulation, as studies with radionucleotide imaging after lumbar inoculation not only show rapid appearance in the basal cisterns but accumulation in this area before moving through the communicating pathways.¹⁵ Thus, the basilar predominance may be less reflective of properties of the organism than that of the physiology of the host.

Very interesting and important are the twin observations noting there was no evidence for either direct invasion from the meninges into underlying parenchyma of the brain or spinal cord, except at the injection site, and no dissemination to organs outside the CNS.² Thus, though hyphae invaded blood vessels extensively, and the authors present EM evidence consistent with degradation of vessel walls, they apparently did not penetrate the pia mater or establish disease distally. The host-pathogen relationships that limited deeper and metastatic spread remain unknown.

As Ritter et al² discuss, an unanswered question is whether meningitis resulted from direct inoculation via epidural puncture, fungal invasion from the epidural/paraspinal space, or via lymphatic flow. The incidence of dural puncture as a complication of epidural injection is very difficult to determine, though a recent study reported rates ranging from 0.5% to 1.3%, depending on the location.¹⁶ Even when performed under fluoroscopic guidance, this complication may go unrecognized, though the use of contrast can aid in detection, and its occurrence may be underreported.¹⁷ In the analysis of the clinical data from the Tennessee cases, two factors that improved the fitness of the model for assessment of risk factors for disease were a translaminar approach and the use of contrast material (adjusted odds ratios, 2.01 and 0.23, respectively). However, they did not retain significance in the multivariate analysis.³ Should these factors remain significant in the larger data set, they could suggest a significant role for dural puncture.

The variety of fungal morphological forms seen in tissue is also interesting. Specifically, Ritter et al² detected conidia (spores) raising the possibility that in addition to vegetative growth, the organism undergoes sporulation in tissue. Prior descriptions of the organism in tissue refer either to hyphal forms or to fungal elements. I have not seen conidia described previously. However, the alternative possibility is that conidia were present in the contaminated MPA and did not germinate. Provision of more detail regarding the frequency of this observation and the tissue location (injection site versus other) might further clarify these possibilities. Whether or not sporulation occurs in vivo, as happens with some fungal pathogens but not others, has implications for the development of animal models, as well as for pathogenesis and treatment. Persistence of spores in tissue could result in maintenance of a potential focus of infection that likely would be refractory to antifungal therapy.

Nonculture-Based Diagnostics

A major clinical challenge for management of systemic mycoses is the difficulty both in making a diagnosis of invasive fungal disease and in identifying the specific causative organism. The problem is made more urgent by the low sensitivity of fungal culture and requirement for long periods for growth in combination with the correlation between earlier diagnosis and improved therapeutic outcome. The sensitivity of culture in the present epidemic has been reported as 14%.¹⁸ IHC in the present study was outstanding with 100% fungal detection in tissue from the 40 cases. As noted by Ritter et al,² the IHC strategy using polyclonal antibody with broad fungal cross-reactivity followed by two monoclonal antibodies to exclude more common fungal pathogens does not allow specific attribution of disease to E. rostratum. Therefore, an important caveat is that for 14 of the cases, E. rostratum could not be directly confirmed by culture or PCR. The appearance of many phaeohyphomycosis agents in tissue is similar. More specific reagents will likely be made in response to this outbreak. The obvious disadvantages to reliance on IHC, including the need for tissue as well as the time for sample processing, make availability of molecular diagnostics highly desirable.

Molecular methods, particularly PCR, have been explored for fungal diagnostics for the past two decades. PCR-based methods have potential to provide specific diagnoses within very short time frames, but their development and implementation into clinical diagnostics have been hampered by difficulties in identifying fungal DNA in complex clinical samples as well as by environmental contamination.¹⁹ CDC scientists developed a PCR protocol for investigation of this outbreak, which was less sensitive than IHC for detecting fungi in tissue. The strategy used for PCR development used broad-spectrum fungal primers that amplify a segment of ribosomal DNA genes that includes the internal transcribed spacer 2 region, a second PCR using primers specific for E. rostratum, and the use of amplified products for species confirmation by sequence analysis.¹⁸ This approach proved critically important, as multiple fungi were isolated from clinical samples, as well as from the contaminated lots of MPA, and the causative organism in the majority of cases previously had been found only rarely in human disease. Diagnostically, PCR is most useful outside the context of an outbreak investigation when organisms are detectable in biological fluids such as blood or, in this case, CSF. Though the reported limit of detection for this PCR is approximately 1 pg/mL of DNA (or an estimated 23 genomes/mL of E. rostratum), the assay was positive in CSF samples from only 24% of case patients with meningitis tested at the CDC. Ritter et al² report positivity on one or more tissues from 50% of patients from whom samples were available, which is also quite low. The authors attribute the low sensitivity to difficulties in DNA extraction from melanized fungi. In a recently published CDC preliminary laboratory report from this epidemic, Lockhart et al⁴ proposed that one likely reason was improper sample handling, particularly early in the outbreak. This suggestion would be supported by the somewhat better apparent performance of the assay in the present study on formalin-fixed, paraffin-embedded samples compared to those from fresh tissue, a result that would not necessarily be predicted, though both types of specimens were available only for a very small number of samples. In response to the outbreak, investigators at the Public Health Research Institute Center (Newark, NJ) have already developed a molecular beacon real-time PCR assay specific for E. rostratum.²⁰ Though the sensitivity for clinical isolates is reportedly approximately 100 fg of fungal DNA, even in the presence of human DNA, the performance characteristics of this assay in clinical samples have not yet been reported. Accurate determination of the performance of either assay will require further study.

Unanswered Questions

The epidemic remains ongoing and clinical follow up is not complete for all of the cases. This information will be important for assessment of optimal therapy and definition of the role of surgical approaches to management. Little is known about the biology of E. rostratum itself. However, regarding the intersection of clinical and epidemiological features with pathology and pathogenesis, several additional intriguing questions remain. First, as raised by Ritter et al,² the organisms were inoculated in steroids, which are immunosuppressive drugs. In addition, as has been discussed recently in the context of this outbreak, many fungi have sterol receptors and exposure can increase their growth.²¹ There have been rare case reports of dermal E. rostratum disease believed to be related to topical or intramuscular steroids.²² Did the presence of steroids participate in disease pathogenesis in these patients, and if so, how?

A second set of questions relates to the case attack rate: of more than 13,000 exposed, 745 have developed disease. Some factors associated with the injected solutions, such as the specific lot as well as the interval between production and administration, have been identified as risk factors and likely relate to fungal burden.³ Did additional clinical factors, such as the technique used for corticosteroid injection or the use of contrast during fluoroscopy, play a role? Did underlying morbidities predispose patients to disease? Did additional host factors, such as genetic polymorphisms in immune molecules, enhance susceptibility?

In the meantime, the scientific community is discussing and undertaking the broad ranging types of study needed to equip the field with tools to develop diagnostic and treatment strategies for invasive disease due to E. rostratum.²¹ Perhaps the disease has earned its own name – invasive exserohilosis?