Erin McElvania TeKippe

The NLRP3 inflammasome functions as a negative regulator of tumorigenesis during colitis-associated cancer

Colitis-associated cancer (CAC) is a major complication of inflammatory bowel diseases. We show that components of the inflammasome are protective during acute and recurring colitis and CAC in the dextran sulfate sodium (DSS) and azoxymethane + DSS models. Mice lacking the inflammasome adaptor protein PYCARD (ASC) and caspase-1 demonstrate increased disease outcome, morbidity, histopathology, and polyp formation. The increased tumor burden is correlated with attenuated levels of IL-1β and IL-18 at the tumor site. To decipher the nucleotide-binding domain, leucine-rich-repeat-containing (NLR) component that is involved in colitis and CAC, we assessed Nlrp3 and Nlrc4 deficient mice. Nlrp3−/− mice showed an increase in acute and recurring colitis and CAC, although the disease outcome was less severe in Nlrp3−/− mice than in Pycard−/− or Casp1−/− animals. No significant differences were observed in disease progression or outcome in Nlrc4−/− mice compared with similarly treated wild-type animals. Bone marrow reconstitution experiments show that Nlrp3 gene expression and function in hematopoietic cells, rather than intestinal epithelial cells or stromal cells, is responsible for protection against increased tumorigenesis. These data suggest that the inflammasome functions as an attenuator of colitis and CAC.

Granuloma Formation and Host Defense in Chronic Mycobacterium tuberculosis Infection Requires PYCARD/ASC but Not NLRP3 or Caspase-1

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1β secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3−/−, Casp-1−/−, and WT mice showed no differences in pulmonary IL-1β production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard−/− mice. Decreased survival of Pycard−/− animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.

Optimizing Identification of Clinically Relevant Gram-Positive Organisms by Use of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used as a method for the rapid identification of microorganisms. This study evaluated the Bruker Biotyper (MALDI-TOF MS) system for the identification of clinically relevant Gram-positive organisms. We tested 239 aerobic Gram-positive organisms isolated from clinical specimens. We evaluated 4 direct-smear methods, including “heavy” (H) and “light” (L) smears, with and without a 1-μl direct formic acid (FA) overlay. The quality measure assigned to a MALDI-TOF MS identification is a numerical value or “score.” We found that a heavy smear with a formic acid overlay (H+FA) produced optimal MALDI-TOF MS identification scores and the highest percentage of correctly identified organisms. Using a score of ≥2.0, we identified 183 of the 239 isolates (76.6%) to the genus level, and of the 181 isolates resolved to the species level, 141 isolates (77.9%) were correctly identified. To maximize the number of correct identifications while minimizing misidentifications, the data were analyzed using a score of ≥1.7 for genus- and species-level identification. Using this score, 220 of the 239 isolates (92.1%) were identified to the genus level, and of the 181 isolates resolved to the species level, 167 isolates (92.2%) could be assigned an accurate species identification. We also evaluated a subset of isolates for preanalytic factors that might influence MALDI-TOF MS identification. Frequent subcultures increased the number of unidentified isolates. Incubation temperatures and subcultures of the media did not alter the rate of identification. These data define the ideal bacterial preparation, identification score, and medium conditions for optimal identification of Gram-positive bacteria by use of MALDI-TOF MS.

Increased Prevalence of Anellovirus in Pediatric Patients with Fever

The Anelloviridae family consists of non-enveloped, circular, single-stranded DNA viruses. Three genera of anellovirus are known to infect humans, named TTV, TTMDV, and TTMV. Although anelloviruses were initially thought to cause non-A-G viral hepatitis, continued research has shown no definitive associations between anellovirus and human disease to date. Using high-throughput sequencing, we investigated the association between anelloviruses and fever in pediatric patients 2–36 months of age. We determined that although anelloviruses were present in a large number of specimens from both febrile and afebrile patients, they were more prevalent in the plasma and nasopharyngeal (NP) specimens of febrile patients compared to afebrile controls. Using PCR to detect each of the three species of anellovirus that infect humans, we found that anellovirus species TTV and TTMDV were more prevalent in the plasma and NP specimens of febrile patients compared to afebrile controls. This was not the case for species TTMV which was found in similar percentages of febrile and afebrile patient specimens. Analysis of patient age showed that the percentage of plasma and NP specimens containing anellovirus increased with age until patients were 19–24 months of age, after which the percentage of anellovirus positive patient specimens dropped. This trend was striking for TTV and TTMDV and very modest for TTMV in both plasma and NP specimens. Finally, as the temperature of febrile patients increased, so too did the frequency of TTV and TTMDV detection. Again, TTMV was equally present in both febrile and afebrile patient specimens. Taken together these data indicate that the human anellovirus species TTV and TTMDV are associated with fever in children, while the highly related human anellovirus TTMV has no association with fever.

Comparison of the Next-Generation Xpert MRSA/SA BC Assay and the GeneOhm StaphSR Assay to Routine Culture for Identification of Staphylococcus aureus and Methicillin-Resistant S. aureus in Positive-Blood-Culture Broths

ABSTRACT A bloodstream infection with Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a serious condition that carries a high mortality rate and is also associated with significant hospital costs. The rapid and accurate identification and differentiation of methicillin-susceptible S. aureus (MSSA) and MRSA directly from positive blood cultures has demonstrated benefits in both patient outcome and cost-of-care metrics. We compare the next-generation Xpert MRSA/SA BC (Xpert) assay to the GeneOhm StaphSR (GeneOhm) assay for the identification and detection of S. aureus and methicillin resistance in prospectively collected blood culture broths containing Gram-positive cocci. All results were compared to routine bacterial culture as the gold standard. Across 8 collection and test sites, the Xpert assay demonstrated a sensitivity of 99.6% (range, 96.4% to 100%) and a specificity of 99.5% (range, 98.0% to 100%) for identifying S. aureus, as well as a sensitivity of 98.1% (range, 87.5% to 100%) and a specificity of 99.6% (range, 98.3% to 100%) for identifying MRSA. In comparison, the GeneOhm assay demonstrated a sensitivity of 99.2% (range, 95.2% to 100%) and a specificity of 96.5% (range, 89.2% to 100%) for identifying S. aureus, as well as a sensitivity of 94.3% (range, 87.5% to 100%) and a specificity of 97.8% (range, 96.1% to 100%) for identifying MRSA. Five of six cultures falsely reported as negative for MRSA by the GeneOhm assay were correctly identified as positive by the Xpert assay, while one culture falsely reported as negative for MRSA by the Xpert assay was correctly reported as positive by the GeneOhm assay.

Diagnostic Assays for Identification of Microorganisms and Antimicrobial Resistance Determinants Directly from Positive Blood Culture Broth

The detection of blood stream infections is one of the most important functions of the clinical microbiology laboratory. Sepsis is a clinical emergency, and mortality increases if commencement of appropriate antimicrobial therapy is delayed. Automated blood culture systems are the most sensitive approach for detection of the causative agent of sepsis. Several laboratory methods have been developed to expedite identification of organisms directly from positive blood culture broth. The principle and analytical performance characteristics of these methods are described in this review.

Primary osteomyelitis caused by hypervirulent Klebsiella pneumoniae

Klebsiella pneumoniae is the most clinically relevant species of this genus, known to cause both community-acquired and nosocomial infections worldwide. In the past two decades, a distinct hypervirulent strain of K pneumoniae, characterised by its hypermucoviscous phenotype, has emerged as a clinically significant pathogen responsible for highly invasive infections. We present a case of osteomyelitis due to hypervirulent K pneumoniae reported in the USA. Genomic testing of the K pneumoniae isolate was performed due to the striking clinical presentation of the infection as well as the hypermucoid nature of the isolates, raising the suspicion for possible infection with the hypervirulent strain. Whole-genome sequencing and additional PCR testing demonstrated the isolate to be a K1 serotype, sequence type 23 strain expressing rmpA and rmpA2. Given the multiple reports of this pathogen causing invasive infections, clinicians should be aware of the possible presentation of metastatic and severe infection, including osteomyelitis, due to the hypervirulent strain of K pneumoniae not typical of classic K pneumoniae variants. In this Grand Round, we review the clinical features of hypervirulent K pneumoniae and its link to invasive infections, and discuss the need for improved awareness and identification of the pathogen.

Diagnosis of Bloodstream Infections in Children

ABSTRACT Identification of bloodstream infections is among the most critical tasks performed by the clinical microbiology laboratory. While the criteria for achieving an adequate blood culture specimen in adults have been well described, there is much more ambiguity in pediatric populations. This minireview focuses on the available pediatric literature pertaining to the collection of an optimal blood culture specimen, including timing, volume, and bottle selection, as well as rapid diagnostic approaches and their role in the management of pediatric bloodstream infections.Identification of bloodstream infections is among the most critical tasks performed by the clinical microbiology laboratory. While the criteria for achieving an adequate blood culture specimen in adults have been well described, there is much more ambiguity in pediatric populations. This minireview focuses on the available pediatric literature pertaining to the collection of an optimal blood culture specimen, including timing, volume, and bottle selection, as well as rapid diagnostic approaches and their role in the management of pediatric bloodstream infections.

Two Cases of Kerstersia gyiorum Isolated from Sites of Chronic Infection

ABSTRACT Kerstersia gyiorum is infrequently associated with human infection. We report the isolation of Kerstersia gyiorum from two patients: the first, a patient with chronic ear infections, and the second, a patient with a chronic leg wound. Both isolates were resistant to ciprofloxacin, which has not been previously reported.

Low Utility of Pediatric Isolator Blood Culture System for Detection of Fungemia in Children: a 10-Year Review

ABSTRACT The use of the Wampole Isolator 1.5-ml pediatric blood culture tube for the detection of fungemia in children was assessed by a 10-year retrospective review at two pediatric hospitals, The Hospital for Sick Children in Toronto, Canada, and the Childrens Medical Center of Dallas, Texas. Over this period, a total of 9,442 pediatric Isolator specimens were processed, with yeast or yeast-like organisms recovered in 297 (3.1%) of the specimens (151 [1.6%] unique clinical episodes) and filamentous or dimorphic fungi recovered in 31 (0.3%) of the specimens (25 unique clinical episodes). Only 18 of the 151 clinical episodes of fungemia attributable to yeast were not detected by automated blood culture systems. The majority of isolated yeast were Candida spp., which were usually detected by automated systems, whereas the most common non-Candida yeast was Malassezia furfur, which the automated system failed to detect. Filamentous or dimorphic fungi were detected in 25 episodes, of which only 9 (36%) episodes were deemed clinically significant after chart review, indicating that in the majority of cases (16/25, 64%) fungal isolation represented contamination. In five of the nine clinically significant episodes, the isolated fungus (Histoplasma capsulatum, Coccidioides immitis/posadasii, Fusarium oxysporum, Aspergillus spp., and Bipolaris spp.) was also identified in other clinical specimens. Over the 10-year study period, the use of the pediatric Isolator system, at the discretion of the treating physician, only rarely provided useful clinical information for the diagnosis of fungemia in children, with the exception of M. furfur and possibly endemic mycoses.