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V I E W P O I N T S

What Can We Learn and What Do We Need toKnow Amidst the Iatrogenic Outbreak ofExserohilum RostratumMeningitis?

Dimitrios P. Kontoyiannis,1,2 David S. Perlin,2,3 Emmanuel Roilides,2,4,5 and Thomas J. Walsh2,5,6,7

1Department of Infectious Diseases, University of Texas MD Anderson Cancer Center, Houston, 2Exserohilum Meningitis Research Consortium, and3Public Health Research Institute at the International Center for Public Health, New Jersey Medical School, Newark; 4Third Department of Pediatrics,Aristotle University, Hippokration General Hospital, Thessaloniki, Greece; 5Transplantation-Oncology Infectious Diseases Program, Division of InfectiousDiseases, 6Department of Pediatrics, and 7Department of Microbiology and Immunology, Weill Cornell Medical Center, New York, New York

The tragedy of the ongoing epidemic of meningitis caused by Exserohilum rostratum brings into focus the epi-demiology, risk factors, pathogenesis, diagnosis, and treatment of a multitude of opportunistic mold infectionsof the central nervous system. Herein we provide our perspective regarding the translational research objectivesof this infection that are needed to make an impact on this important healthcare crisis.

Keywords. Exserohilum rostratum; meningoencephalitis; antifungals; risk factors; pathogenesis.

Contamination of a steroid injection preparation hasrecently resulted in a multistate (19 states) epidemic of Ex-serohilum rostratum meningitis (ERM) or arthritis [1, 2].More than 700 cases of spinal and paraspinal infectionshave been encountered, and 46 patients have died (www.cdc.gov/HAI/outbreaks, last updated 8 April 2013). Newcases are continuing to be discovered each day with noapparent end in sight. More than 13 000 patients from20 states are estimated to have received the contaminat-ed product. ERM has been associated with hemorrhagicand ischemic infarcts, mycotic aneurysms, painfularachnoiditis, epidural abscess, phlegmon, discitis, andvertebral osteomyelitis at or near the site of injection.This is a historically unprecedented public health threatof unmeasured dimensions as the natural history of thisserious infection and its immunopathogenesis, treat-ment, and prognosis are unknown. The purpose of this

commentary is to discuss critical questions that mayguide the science and management of these devastatinginfections.

VIRULENCE FACTORSANDCENTRALNERVOUS SYSTEM INFECTION

Exserohilum rostratum is a thermophilic dematiaceous(melanized) mold that is an uncommon cause of humandisease [3]. Little is known about immune predisposi-tion of the host, if any, for ERM. Local impairment ofimmune defenses by concentrated corticosteroids [4] islikely a key determinant of disease in a closed spacesuch as the cerebrospinal uid (CSF).

In understanding hostpathogen interactions, theeffector-to-target-ratio, measured as the number ofphagocytic cells to number of fungal elements, is a keyfactor. Because we do not know the concentration oforganisms within the contaminated solution of methyl-prednisolone, we are not able to accurately quantify thevirulence of this organism in humans. Given that mul-tiple vials were injected with no prior indication thatthe vials displayed visible growth, the inoculum mayhave been relatively low, thus possibly accounting forthe relatively low infection/exposure rate of >600 pa-tients among 13 000 exposed patients. Quantication

Received 19 November 2012; accepted 12 April 2013; electronically published 6May 2013.

Correspondence: Thomas J. Walsh, MD, Transplantation-Oncology InfectiousDiseases Program, Weill Cornell Medical Center of Cornell University, 1300 YorkAve, Rm A421, New York, NY 10065 ([email protected]).

Clinical Infectious Diseases 2013;57(6):8539 The Author 2013. Published by Oxford University Press on behalf of the InfectiousDiseases Society of America. All rights reserved. For Permissions, please e-mail:[email protected]: 10.1093/cid/cit283

Exserohilum RostratumMeningitis CID 2013:57 (15 September) 853

of organisms in existing vials by quantitative polymerase chainreaction (PCR) and culture would be valuable in understandingvirulence in humans.

The predominance of a single fungus infection in the settingof fungal outbreaks raises important questions in fungal patho-genesis. For example, trauma victims following the devastatingtornado in Joplin, Missouri, in 2011 had necrotizing mucormy-cosis due to the uncommon Mucorales Apophysomyces trapezi-formis [5]. Although tornado victims were exposed to soil anddirt containing myriad bacteria and fungi, this unusual patho-gen emerged. Perhaps unique geoclimatic conditions of atornado (eg, turbulence), locally traumatized tissue, and a pre-viously dormant organism conferred an advantage in virulence.Similarly, vials of methylprednisolone were contaminated byuncommon species of molds. Among those organisms, only E.rostratum grew at 37C and only this organism caused centralnervous system (CNS) infection. Incubation of E. rostratum in astarvation mode (older lots in the presence of sparse nutrientmedium with concentrated methylprednisolone) could result ina growth advantage and/or improved immune evasion strategies.

Binding by fungi of human sterols is well described. Cortico-steroids in an apparent receptor/ligand system in Aspergillusfumigatus can accelerate growth [6] andmetabolism. Ng and col-leagues [6] observed an approximately 35% increased growthrate in A. fumigatus and Aspergillus avus exposed to pharmaco-logical doses of hydrocortisone. If this corticosteroid-induced en-hancement of growth and metabolism occurs in E. rostratum, itmay coincide with enhanced melanin biogenesis [7, 8], secretedhydrolytic enzymes, toxin production [9], and more neutrophil-associated inammatory injury (Figure 1). If E. rostratum

possesses corticosterone-binding protein, as observed inCandida species, this may also support corticosteroid-enhancedaccelerated growth. In Coccidioides species, where progesteroneand 17--estradiol promote growth and endospore release [10],this response correlated with the presence of a high-afnitycytosol binding protein receptor for estradiol and a low-afnityreceptor for testosterone in Coccidioides species [11]. Thesereceptor/ligand interactions are of course consistent with theincreased risk of accelerated coccidioidomycosis during preg-nancy. The converse applies in Paracoccidioides brasiliensis,where paracoccidioidomycosis predominates in males in asso-ciation with 17--estradiol-induced inhibition of the mycelial-to-yeast transformation [1214].

Melanin content in E. rostratums cell wall is a prime candi-date for tness advantage [6, 7]. Fungal melanin serves as atype of reactive armor that quenches neutrophil-derived reac-tive oxygen species, such as superoxide, hydrogen peroxide,and hydroxyl radicals. Melanin also inhibits phagocytosis ofconidia and protects lamentous fungi, such as Exophiala(Wangiella) dermatitidis and Aspergillus nidulans, from hydro-lytic enzymes. Unlike the melanin of Cryptococcus neoformans,which is synthesized through a phenolic pathway, the melaninof dematiaceous molds, such as E. rostratum, is elaboratedthrough an indole-dependent pentaketide pathway.

The fundamental molecular studies by Paolo et al [15] andFeng et al [16] of the dematiaceous mold E. dermatitidisprovide insight into possible contributions of melanin to thepathogenesis and antifungal resistance of E. rostratum. Cell wallpigmentation in E. dermatitidis depends on WdPKS1, whichencodes a polyketide synthase required for biosynthesis of the

Figure 1. Pathogenesis model of Exserohilum rostratum meningitis. Abbreviations: H2O2, hydrogen peroxide; O2, oxygen; OH, hydroxyl radical; ROS,reactive oxygen species.

854 CID 2013:57 (15 September) Kontoyiannis et al

precursor for dihydroxynaphthalene melanin. Organisms withdisrupted WdPKS1 or absent gene are more vulnerable to neu-trophil killing and are less virulent in mice. They also havethinner cell walls, lack an electron-opaque layer, and are moresusceptible to killing by voriconazole, amphotericin B, defensinNP-1, heat, and lysing enzymes than the wild-type melanizedstrain. Small molecules targeting fungal polyketide synthasewould provide potentially novel therapeutic advances againstE. rostratum and other dematiaceous molds.

That E. rostratum is able to grow at 40C is a potential viru-lence factor that is shared with other neurotropic dematiaceousmolds, including Ochroconis gallopava and Cladophialophorabantiana. Both organisms are well known to cause cerebralphaeohyphomycosis [1719]. What accounts for this propensi-ty to infect the CNS is not well understood. Whether there istropism of specic strains within a genus or a general charac-teristic within a fungal order for the brain and for their procliv-ity to create specic clinical manifestations (eg, meningitis vsmeningoencephalitis) remains to be understood. An alternativehypothesis is that E. rostratum may be neurotrophic; that is,when the organism reaches the CNS, the tissue microenviron-ment affords a favorable nutritive environment.

Could polymorphisms in genes encoding innate host defensemolecules have created a heightened risk in a subpopulation ofthe >13 000 patients who received the contaminated steroidalproduct? A haplotype of several polymorphisms may haveheightened the risk for infection. Although neutropenia is knownto be associated with greater risk of mortality in phaeohyphomy-cosis [20], the role of neutrophils and other immune cells in hostdefenses against E. rostratum is not known. The answers to theseand other questions will likely reveal a more intricate interactionof fungal pathogenesis and innate host defenses (Figure 1).

DIAGNOSIS ANDMONITORING OFTHERAPEUTIC RESPONSE

Patients who present with a history of corticosteroid injectionand new onset of neurological symptoms and who have a leu-kocytosis of 5 white blood cells per high-powered eld areassumed to have ERM warranting antifungal therapy. Cultureof E. rostratum in CSF may be delayed or absent.

Development of assays for rapid diagnosis and monitoringof therapeutic response is critically needed. The Centers forDisease Control and Prevention has developed a PCR systemusing panfungal primers; the amplicon is then sequenced foridentication at the species level. However, this assay is usedcurrently for epidemiologic and forensic documentation ratherthan for immediate bedside care.

In addressing this critical need, Zhao et al [21] from the NewJersey Public Health Research Institute, in collaboration withWeill Cornell Medical Center, developed a quantitative PCR

system using beacon probes specic for E. rostratum for rapiddiagnosis, monitoring of therapeutic response, and environ-mental microbiologic surveillance. A key feature of the assay issensitive detection (50100 fg) of target nucleic acid even in thepresence of excess human DNA. The primers and probes forthis assay have been placed in public domain for availability toclinics, hospitals, and reference laboratories.

The cell wall carbohydrate polymer (13)--D-glucan alsohas potential utility as a biomarker for diagnosis and monitor-ing of therapeutic response of ERM. Aspergillus species andother molds, with exception of the Mucorales, broadly expressthis molecule. Candida species, but not Cryptococcus neofor-mans, also express (13)--D-glucan. Although serum (13)--D-glucan is used successfully in diagnosis of invasive fungalinfections in high-risk patients [22], little has been knownabout its role in fungal meningitis. Our laboratory demonstrat-ed proof of concept of the expression of (13)--D-glucan inCSF during fungal meningitis in the diagnosis and monitoringof therapeutic response in experimental hematogenousCandida meningoencephalitis (HCME) [23]. As an extensionof these laboratory studies, we have since documented expres-sion of CSF (13)--D-glucan in pediatric patients withHCME and CNS aspergillosis [24]. Consistent with these nd-ings, 3 of 5 patients with presumed Exserohilum CNS infectionhad CSF samples with (13)--D-glucan levels >31 pg/mL,suggesting its utility for diagnosis and monitoring of ERM [25].

As patients are being treated for ERM, there is understand-able reluctance in discontinuation of therapy. Discontinuationof therapy based solely upon improvement of symptoms andCSF pleocytosis may not be reliable. Use of CSF PCR and(13)--D-glucan may provide more objective microbiologicevidence of eradication of ERM.

PHARMACOLOGY, PHARMACODYNAMICS,AND THERAPEUTIC INTERVENTIONS

Currently, voriconazole, a lipophilic triazole that highly pene-trates the CNS, is recommended as the initial antifungal agenton the basis of published minimum inhibitory concentrations(MICs) of a collection of Exserohilum isolates [2628]. However,the correlations between in vitro activity dened by MICs andclinical outcome are difcult to evaluate for this dematiaceousmold. The MICs of voriconazole against these fungal speciesvary widely, ranging from 0.03 g/mL to 1.0 g/mL. Other iso-lates may have an MIC of 2 g/mL. For organisms with MICsof 12 g/mL, sustained CNS concentrations may be difcultto achieve without reaching toxic plasma concentrations.Because reduction of voriconazole dosage may lead to clinicalprogression, alternative therapeutic approaches are needed.

The currently recommended dosage of voriconazole 6 mg/kgintravenously every 12 hours is intended to achieve plasma and


CSF exposures that would exceed the MICs of 12 g/mL of in-fecting strains. Although this dosage is routinely used as aloading dose in adults, little is known about maintenance ad-ministration of voriconazole at 6 mg/kg intravenously. Because6 mg/kg intravenously every 12 hours exceeds the Michaelis-Menten saturation of voriconazoles metabolism, the plasmaconcentrations will predictably display nonlinear plasma phar-macokinetics, resulting in precipitously high circulating con-centrations. Thus, some patients receiving this regimen haveshown serum trough levels exceeding 10 g/mL in associationwith dose-limiting visual hallucinations, photosensitization,and incapacitating systemic symptoms.

Liposomal amphotericin B (LAmB) is recommended as analternative to voriconazole. Studies of plasma pharmacokineticsof LAmB at 7.5 mg/kg/day demonstrate an area under the con-centrationtime curve that would exceed the MIC of 1 g/mLfor this organism [29]. Although the MICs of amphotericin Bare within safely achievable concentrations, this higher dosemay not be predictive of CNS response [30]. Moreover, dose-limiting nephrotoxicity, especially in older patients and thosewith diabetes mellitus or other underlying renal impairment,are requiring a lower dosage of LAmB or alternative antifungaloptions. Whether a lower and less nephrotoxic dosage ofLAmB at 5 mg/kg/day may also be effective against ERM insuch patients is not known.

Other antifungal triazoles, such as posaconazole and itraco-nazole, may provide alternative options for single-agenttherapy. Posaconazole has in vitro activity within achievableplasma concentrations that exceed the MICs of several dematia-ceous molds, including Bipolaris species, C. bantiana, Curvu-laria species, E. dermatitidis, and Rhinocladiella mackenziei [31].Studies in clinical CNS cryptococcosis, experimental phaeohy-phomycosis, and clinical chromoblastomycosis have shown ac-tivity of posaconazole [3234]. Posaconazole also has favorablein vitro activity against E. rostratum with median MICs of0.003 g/mL. However, little is known about the in vivo anti-fungal activity and CNS pharmacology of posaconazole againstthis organism or other mold pathogens.

Itraconazole also has in vitro activity against dematiaceousmolds, including E. rostratum with MICs that are similar tothose of posaconazole. The activity of itraconazole against coc-cidioidal meningitis [35] provides a foundation for understand-ing its potential utility in treatment of ERM.

Isavuconazole is an investigational triazole that is structurallysimilar to voriconazole [36]. Isavuconazole has in vitro activityagainst E. rostratum; however, the MICs are approximately4 g/mL. Although these MIC values are higher than those ofthe hydrophobic triazoles (posaconazole and itraconazole), theCSF penetration of isavuconazole is approximately 50% incomparison to being almost undetectable for itraconazole. Al-though isavuconazole is active in vitro against E. rostratum,

there is a paucity of data on CNS pharmacology of isavucona-zole. Flucytosine (5-uorocytosine) has a high level of CNS pen-etration but has minimal in vitro activity against E. rostratum.

The pharmacokinetic and pharmacodynamic exposuresneeded for successful treatment of ERM using voriconazole, li-posomal amphotericin B, posaconazole, itraconazole, or isavu-conazole are not known. One should underscore that CSFlevels do not necessarily correlate with therapeutic response.Concentrations of antifungal agents needed to treat infected pa-renchymal brain tissue are not necessarily reected by CSFlevels. For example, amphotericin B and itraconazole, whichare active in treatment of CNS mycoses, have minimal CSFlevels [37, 38].

Combination antifungal therapy against ERM also may bebenecial but has not been dened either in vitro or in vivo.The rationale for the original recommendation of the combina-tion of voriconazole and LAmB was based on providing abroad spectrum against possible polymicrobial infection.However, for patients who are not able to tolerate the higherdosages of voriconazole, the combination of a triazole with anechinocandin warrants further exploration. While the echino-candins are active in vitro against E. rostratum, they optimallywould be used in combination with an antifungal triazole.

The commonly held belief that echinocandins have no activi-ty in the CNS is not accurate. Although echinocandins do notpenetrate an intact bloodbrain barrier, they do have efcacy intreatment of CNS infections where the bloodbrain barrier isdisrupted. Laboratory studies demonstrate successful treatmentof experimental HCME with echinocandins in association withdetectable drug levels in CNS tissue [39]. These ndings arecompatible with that of an earlier case of HCME successfullytreated with caspofungin and were predictive of the results of arandomized trial of caspofungin in treatment of neonatal can-didemia and HCME [40, 41].

Optimal duration of antifungal therapy of ERM also isunknown. Early indications suggest that the time to eradicationof E. rostratum may be measured in months rather than weeks.As little is known about the outcome of ERM following discon-tinuation of antifungal therapy, consideration should be givento the possibility that relapse may occur and that some patientsmay require life-long suppression of ERM. That life-long anti-fungal therapy may be needed for a CNS mycosis is exempliedin the management of meningeal coccidioidomycosis, wherechronic suppression with uconazole or itraconazole is consid-ered standard of care [35]. If voriconazole is used for chronicsuppression, the potential development of cutaneous melano-mas and squamous cell carcinomas need to be considered.

Chronic administration of voriconazole of ERM is challeng-ing. Therapeutic drug monitoring of serum voriconazole is war-ranted to ascertain stability and attainment of target serumconcentrations. Because voriconazole achieves an approximately


50% CSF/plasma ratio, CSF concentrations are not necessary.Assuming a voriconazole MIC of 1.0 g/mL, serum proteinbinding of 50%, and 50% CSF penetration, one would need aminimum serum trough concentration of 4.0 g/mL. Some pa-tients clearly do not tolerate this sustained concentration ofvoriconazole. Other patients have been found to have unremit-ting decline of their serum concentrations over time on thesame dosage. This decline of voriconazole serum concentrationsover time also has been observed in oncology patients [42].Moriyama et al demonstrated that this process is likely relatedto autoinduction [43]. The approaches to such patients includeincrease of dosage and addition of a CYP2C19 inhibitor, suchas omeprazole. However, when such measures do not overcome

the autoinduction effect, alternative antifungal agents arerequired.

Intrathecal or intraventricular administration has been sug-gested as adjunctive therapy but has little data to support itsusage. The potential for introduction of bacterial pathogensthrough an Ommaya reservoir is high and the possibility ofinducing chemical arachnoiditis via the intrathecal route isprohibitive in patients who already have arachnoidal patholo-gy. Moreover, penetration of intrathecally administered anti-microbial agents beyond the subarachnoid space into CNSparenchymal lesions is relatively unpredictable. By compari-son, delivery of antifungal agents via the circulation providesmore predictable CSF and parenchymal levels without the

Table 1. Scientic Objectives Needed to Understand and Advance the Care of Patients With Exserohilum rostratum Meningitis

Pathogenesis

Whole genome sequencing Effect of glucocorticosteroids on growth and virulence, including toxin secretion and regulation of melanization

Thermotolerance

Development of physiologically and medically relevant models of acute and subacute infections in and innovative minihost models to studyCNS tropism of Exserohilum rostratum

Quantification of organisms in vials and relation to outcome in patients

Host defense Analysis for host immunogenetics for increased susceptibility to ERM through whole genome or candidate gene strategies

Innate host responses to E. rostratum and cognate receptors of relevant effector immune cells (TLR, dectin-1) Innate host response to E. rostratum and role of melanin Effect of glucocorticosteroids on CNS host response, including microglial cells through functional genomic, transcriptomic, and proteomicresponses

Role of phagocytic and immunoregulatory cells Functional genomic and transcriptional messenger RNA and proteomic responses of peripheral blood mononuclear cells, neutrophils, andlymphocytes

Effects of corticosteroids on innate host response Augmentation and immunoregulation of innate host response

Molecular detection and monitoring of therapeutic response

Application and study of developed qPCR system Application and study of (13)--D-glucan

Development of sensitive neuroimaging strategies for screening and therapeutic monitoring

Predictive models for discontinuation of therapy of ERM based on composite end points that use radiologic and non-culture-based diagnosticsCNS pharmacology, pharmacokinetics, and pharmacodynamics of antifungal agents

Development of relevant animal models of ERM to study CNS pharmacology, pharmacokinetics, and pharmacodynamics of availableantifungal agents

Combination antifungal therapy to improve therapeutic index and efficacy Bridging studies to patients for optimization of dose-exposure-response relations and explore combination antifungal therapy to improvetherapeutic index and efficacy

Study of the clinical impact of voriconazole and therapeutic drug monitoring on outcome of ERM and long-term drug-related toxicity Study of the clinical impact of liposomal amphotericin B on outcome of ERM and long-term drug-related toxicity

Clinical epidemiology

Prospective registries to determine the burden of ERM and impact of type/timing antifungal use on clinical presentation, diagnosis, andlong-term outcome

Predictive models for development and complications of ERM

Abbreviations: CNS, central nervous system; ERM, Exserohilum rostratummeningitis; qPCR, quantitative polymerase chain reaction; TLR, Toll-like receptor.


risks of bacterial infection, CSF dural leaks, and chemicalarachnoiditis.

PROGNOSIS ANDOUTCOME

As ERM is treated, patterns of persistence, recurrence, and de-velopment of new symptoms and signs may emerge. The newonset of lumbosacral arachnoiditis, epidural abscesses, and par-aspinal infections are being reported in patients receiving vori-conazole. Development of predictive population-based riskmodels for assessment of prognosis and outcome is needed. Inthe absence of such models, quantitative serial molecular diag-nostic assays, such as quantitative PCR, nucleic acid sequence-based amplication, and (13)--D-glucan in CSF, may in-dicate patients who are at risk for persistent or refractoryinfection.

Why would ERM persist despite antifungal therapy? Melani-zation of E. rostratum may contribute to evasion of hostdefense and increase resistance. In addition to subtherapeuticconcentrations of voriconazole in CSF, we do not know if vori-conazole truly kills E. rostratum in the CNS. Fungistatic activityof voriconazole coupled with melanin-driven evasion of innatehost defenses may be critical factors contributing to persistenceor relapse of ERM.

FUTURE DIRECTIONSAND RESEARCHNEEDS

Improved understanding of the pathogenesis and host defensesmay permit development of new approaches for immune aug-mentation in patients with ERM (Table 1). Critically needed inthis public health crisis are denitive animal models for charac-terization of the antifungal pharmacokinetics, pharmacody-namics, molecular detection, therapeutic monitoring, and hostdefense that will provide a scientic foundation for treatingthese patients. During the bioterrorist anthrax attacks in theUnited States in 2001, the availability of data from the primatemodel of inhalational anthrax was critical in guiding the use ofciprooxacin over penicillin. Such model systems are urgentlyneeded to meet this public health threat.

Providing a rapid emergency regulatory process with expe-dited scientic review for approval of new diagnostic systems orprotocols for Investigational New Drug applications could ac-celerate availability for new strategies in the battle against ERM.Whole genome sequencing of the organism may reveal new un-derstanding of its virulence, pathogenesis, and targets fortherapy. Understanding host genetic risk factors for ERM mayhelp to dene the subpopulation among all patients exposed tothe contaminated methylprednisolone solution. More workalso is needed to delineate the nature of potential exposure risk,populations at highest risk of disease and adverse outcomes, andprophylaxis or preemptive treatment strategies. Development of

a Bayesian predictive model may allow identication of the pa-tients at highest risk for early intervention. This public healthcrisis also should be a catalyst for better regulation and oversightof the manufacturing process of such products and enforcementof safe practices by compounding pharmacies so that such tragicepidemics may be prevented [44].

Notes

Financial support. T. J. W. is a Scholar of the Henry Schueler Founda-tion and a Scholar of Pediatric Infectious Diseases of the Sharpe FamilyFoundation, and also receives support from the Save Our Sick Kids Founda-tion (R34HL117352 and 1$01AI103315-01A1). D. P. K. acknowledges theFrances King Black Endowed Professorship for Cancer Research.

Potential conicts of interest. T. J. W. has received research grants forexperimental and clinical antimicrobial pharmacotherapeutics from Astel-las, Novartis, Merck, ContraFect, and Pzer and has served as a consultantto Astellas, ContraFect, Drais, iCo, Novartis, Pzer, Methylgene, SigmaTau,and Trius. D. P. K. has received research grants from Merck, Pzer, Astellas,and Gilead and serves on the advisory board of Merck and on the speakersbureau of Gilead. D. S. P. has received research support from Astellas,Merck, bioMrieux, Myconostica, and Pzer. E. R. reports no potentialconicts.

All authors have submitted the ICMJE Form for Disclosure of PotentialConicts of Interest. Conicts that the editors consider relevant to thecontent of the manuscript have been disclosed.

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