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.     

A Real-Time PCR Assay for Rapid Detection and Quantification of Exserohilum rostratum,a Causative Pathogen of Fungal Meningitis Associated with Injection of Contaminated Methylprednisolone

A species-specific molecular beacon real-time PCR assay was developed for rapid diagnosis of Exserohilum rostratum infection. As low as 100 fg of E. rostratum DNA can be reliably detected in the presence of 50 ng of human DNA, with a dynamic linear quantification range from 20 ng to 200 fg.     

Immunotherapy of Fungal Infections

Fungal organisms are ubiquitous in the environment. Pathogenic fungi, although relatively few in the whole gamut of microbial pathogens, are able to cause disease with varying degrees of severity in individuals with normal or impaired immunity. The disease state is an outcome of the fungal pathogen’s interactions with the host immunity, and therefore, it stands to reason that deep/invasive fungal diseases be amenable to immunotherapy. Therefore, antifungal immunotherapy continues to be attractive as an adjunct to the currently available antifungal chemotherapy options for a number of reasons, including the fact that existing antifungal drugs, albeit largely effective, are not without limitations, and that morbidity and mortality associated with invasive mycoses are still unacceptably high. For several decades, intense basic research efforts have been directed at development of fungal immunotherapies. Nevertheless, this approach suffers from a severe bench-bedside disconnect owing to several reasons: the chemical and biological peculiarities of the fungal antigens, the complexities of host-pathogen interactions, an under-appreciation of the fungal disease landscape, the requirement of considerable financial investment to bring these therapies to clinical use, as well as practical problems associated with immunizations. In this general, non-exhaustive review, we summarize the features of ongoing research efforts directed towards devising safe and effective immunotherapeutic options for mycotic diseases, encompassing work on antifungal vaccines, adoptive cell transfers, cytokines, antimicrobial peptides (AMPs), monoclonal antibodies (mAbs), and other agents.     

Tracking a Hospital Outbreak of Carbapenem-Resistant Klebsiella pneumoniae with Whole-Genome Sequencing

The Gram-negative bacteria Klebsiella pneumoniae is a major cause of nosocomial infections, primarily among immunocompromised patients. The emergence of strains resistant to carbapenems has left few treatment options, making infection containment critical. In 2011, the U.S. National Institutes of Health Clinical Center experienced an outbreak of carbapenem-resistant K. pneumoniae that affected 18 patients, 11 of whom died. Whole-genome sequencing was performed on K. pneumoniae isolates to gain insight into why the outbreak progressed despite early implementation of infection control procedures. Integrated genomic and epidemiological analysis traced the outbreak to three independent transmissions from a single patient who was discharged 3 weeks before the next case became clinically apparent. Additional genomic comparisons provided evidence for unexpected transmission routes, with subsequent mining of epidemiological data pointing to possible explanations for these transmissions. Our analysis demonstrates that integration of genomic and epidemiological data can yield actionable insights and facilitate the control of nosocomial transmission.     

Tracing Melioidosis Back to the Source: Using Whole-Genome Sequencing To Investigate an Outbreak Originating from a Contaminated Domestic Water Supply

Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property''s groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens.     

Assessment of Whole-Genome Mapping in a Well-Defined Outbreak of Salmonella enterica Serotype Saintpaul

We investigated the use of whole-genome mapping and pulsed-field gel electrophoresis (PFGE) with isolates from an outbreak of Salmonella enterica serotype Saintpaul. PFGE and whole-genome mapping were concordant with 22 of 23 isolates. Whole-genome mapping is a viable alternative tool for the epidemiological analysis of Salmonella food-borne disease investigations.     

Tracking Nosocomial Klebsiella pneumoniae Infections and Outbreaks by Whole-Genome Analysis: Small-Scale Italian Scenario within a Single Hospital

Multidrug-resistant (MDR) Klebsiella pneumoniae is one of the most important causes of nosocomial infections worldwide. After the spread of strains resistant to beta-lactams at the end of the previous century, the diffusion of isolates resistant to carbapenems and colistin is now reducing treatment options and the containment of infections. Carbapenem-resistant K. pneumoniae strains have spread rapidly among Italian hospitals, with four subclades of pandemic clonal group 258 (CG258). Here we show that a single Italian hospital has been invaded by three of these subclades within 27 months, thus replicating on a small scale the “Italian scenario.” We identified a single clone responsible for an epidemic outbreak involving seven patients, and we reconstructed its star-like pattern of diffusion within the intensive care unit. This epidemiological picture was obtained through phylogenomic analysis of 16 carbapenem-resistant K. pneumoniae isolates collected in the hospital during a 27-month period, which were added to a database of 319 genomes representing the available global diversity of K. pneumoniae strains. Phenotypic and molecular assays did not reveal virulence or resistance determinants specific for the outbreak isolates. Other factors, rather than selective advantages, might have caused the outbreak. Finally, analyses allowed us to identify a major subclade of CG258 composed of strains bearing the yersiniabactin virulence factor. Our work demonstrates how the use of combined phenotypic, molecular, and whole-genome sequencing techniques can help to identify quickly and to characterize accurately the spread of MDR pathogens.     

Complement C3 Plays an Essential Role in the Control of Opportunistic Fungal Infections

The innate recognition of fungal pathogens is a crucial first step in the induction of protective antifungal immunity. Complement is thought to be one key component in this process, facilitating fungal recognition and inducing early inflammation. However, the roles of the individual complement components have not been examined extensively. Here we have used mice lacking C3 to examine its role in immunity to opportunistic fungal pathogens and show that this complement component is essential for resistance to infections with Candida albicans and Candida glabrata. We demonstrate that the absence of C3 impairs fungal clearance but does not affect inflammatory responses. We also show that the presence of C3 contributes to mortality in mice challenged with very high doses of Saccharomyces cerevisiae, although these effects were found to be mouse strain dependent.Over the last few decades, modern medical practice and acquired immunodeficiencies have contributed to a substantial increase in the incidence of infections with normally commensal or nonpathogenic fungi (22), prompting a renewed interest in expanding our understanding of the mechanisms underlying protective host immunity. Identification and characterization of the receptors involved in the innate recognition of these organisms are of particular importance, as these “pattern recognition receptors” (14) not only are responsible for mediating the recognition and uptake of fungi by phagocytes but also initiate and direct the resultant immune response (17). While numerous nonopsonic pattern recognition receptors for fungi have been well characterized, including several members of the C-type lectin and Toll-like receptor families (for recent reviews, see references 20, 21, and 32), less is known about the opsonic mechanisms of fungal recognition, such as those mediated by complement, despite the essential role of these systems in antifungal immunity (24).The complement system consists of more than 30 serum and cellular surface proteins and is activated through three main routes: the classical, alternative, and lectin pathways. Triggering of these three pathways initiates enzymatic cascades which converge on the third complement component, C3, whose activation leads ultimately to microbial opsonization, the release of chemotactic factors, including C3a and C5a, and the generation of a membrane attack complex (MAC) (18). Activation of the classical pathway occurs primarily following the binding of C1q to antibody-antigen complexes, and although involved in the adaptive arm of the immune system, this pathway is also triggered by natural antibodies, thereby contributing to the innate recognition of fungi (16). Initiation of the alternative pathway occurs through the spontaneous activation of C3 on microbial surfaces, and the lectin pathway is initiated by the binding of the mannose-binding lectin to carbohydrates on microbial surfaces (24).All three of these pathways are induced by opportunistic fungal pathogens, including Candida albicans (13, 15, 24), although the alternative pathway may be the most critical (7, 15). The protective role of complement in immunity to these pathogens has largely been determined in mice treated with cobra venom factor (CVF), which depletes serum complement by forming a potent C3 convertase, or in C5-deficient mouse strains, such as A/J or DBA2, where fungal opsonization remains intact but the animals lack the ability to generate C5a or the MAC (6, 7, 9, 10, 19). Why C5-deficient mice are susceptible is still unclear, but is likely to be due to aberrant inflammatory responses, as the MAC has little effect on the viability of fungal pathogens (15, 19). Although the roles of each of the pathways of complement activation have been relatively poorly examined, recent studies have started to address this issue, looking at the role of factor B, C2, C1q and mannose-binding lectin in the control of systemic candidiasis (11).C3 is a central component in all of the complement pathways, as described above, but it has not been studied in isolation with respect to fungal infection. Studying the role of this complement component in isolation is particularly relevant, given the alternative route of C5 activation by thrombin, which can substitute for C3-dependent C5 convertase activity (12, 30). Therefore, we undertook to specifically examine the role of C3 in the control of opportunistic fungal infections using C3-sufficient and C3-deficient mice.     

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.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.Epidural and intra-articular steroid injections are commonly administered for treatment of back, neck, and peripheral joint pain, especially in elderly patients. Infections develop in an estimated <1% to 2% of patients who receive spinal injections.^1–3 Introduction of skin flora into the injection site is the most common cause of infection, and Staphylococcus aureus is the most commonly implicated organism.^2,4–6 Fungal infections associated with spinal injections are exceedingly rare and are most frequently attributed to Aspergillus.^7–9 Although individual cases of infection are uncommon, outbreaks of infection associated with contaminated epidural or intra-articular injections are even more unusual. During the past decade, such examples include three outbreaks of Serratia marcescens infection associated with epidural or intra-articular injections,^10–12 an outbreak of Klebsiella pneumoniae and Enterobacter aerogenes bacteremia after sacroiliac (SI) joint steroid injections,¹³ an outbreak of Exophiala dermatitidis infection after epidural and intra-articular steroid injections,¹⁴ and an outbreak of Aspergillus meningitis after spinal anesthesia for caesarean section.⁷ The largest of these outbreaks involved 11 patients.¹⁰In September 2012, the Centers for Disease Control and Prevention (CDC) was alerted to multiple reports of fungal meningitis in patients who had recently received epidural steroid injections. The intensive, multistate, epidemiological and laboratory investigation that ensued identified an unprecedented outbreak of fungal infections associated with epidural, paraspinal, and peripheral joint injections, involving three contaminated lots of preservative-free methylprednisolone acetate (MPA) from a single compounding pharmacy. More than 17,000 vials from three contaminated lots were distributed to medical centers across the United States, and >13,000 people were injected with the potentially contaminated drug before discovery of the source of contamination.¹⁵ Through May 6, 2013, 741 cases and 55 deaths in 20 states have been associated with this outbreak, making it the largest recognized cluster of infections associated with epidural and intra-articular steroid injections. Most cases with a confirmed etiology have been attributed to Exserohilum rostratum. In few cases, other fungal or bacterial species have been identified by molecular studies or culture, either individually or in addition to Exserohilum.^16,17 Herein, we describe the pathological findings from 40 case patients from this outbreak with confirmed Exserohilum infection, correlate these findings with clinical and laboratory data, and use these data to provide insight into the pathogenesis of Exserohilum infections after injection of contaminated MPA.     

Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection

Salmonella enterica serovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis of S. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmental S. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of the S. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method for S. Enteritidis.