Evaluation of the Accelerate Pheno System: Results from Two Academic Medical Centers.

Rapid diagnostic tests are needed to improve patient care and to combat the problem of antimicrobial resistance. The Accelerate Pheno system (Accelerate Diagnostics, Tucson, AZ) is a new diagnostic device that can provide rapid bacterial identification and antimicrobial susceptibility test (AST) results directly from a positive blood culture. The device was compared to the standard of care at two academic medical centers. There were 298 blood cultures included in the study, and the Accelerate Pheno system provided a definitive identification result in 218 instances (73.2%). The Accelerate Pheno system provided a definitive and correct result for 173 runs (58.1%). The Accelerate Pheno system demonstrated an overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 94.7%, 98.9%, 83.7%, and 99.7%, respectively. An AST result was available for analysis in 146 instances. The overall category agreement was 94.1% with 12 very major errors, 5 major errors, and 55 minor errors. After a discrepancy analysis, there were 5 very major errors and 4 major errors. The Accelerate Pheno system provided an identification result in 1.4 h and an AST result in 6.6 h; the identification and AST results were 41.5 h and 48.4 h faster than those with the standard of care, respectively. This study demonstrated that the Accelerate Pheno system is able to provide fast and accurate organism identification and AST data. A limitation is the frequency with which cultures required the use of alternative identification and AST methods.

The Added Cost of Rapid Diagnostic Testing and Active Antimicrobial Stewardship: Is It Worth It?

Rapid diagnostic testing reduces the turnaround time for pathogen identification in the clinical microbiology laboratory, but the impact on patient care and hospital costs is a matter of speculation. Patel et al. (J. Clin. Microbiol. 55:60-67, 2017, https://doi.org/10.1128/JCM.01452-16) investigate the impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in conjunction with active antimicrobial stewardship to determine if implementation is indeed worth the added costs.

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

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 Children's 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.

Diagnosis of Bloodstream Infections in Children.

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.

Lack of clinical utility of urine gram stain for suspected urinary tract infection in pediatric patients.

Urinary tract infection (UTI) is one of the most common infections in children. Urine culture remains the gold standard for diagnosis, but the utility of urine Gram stain relative to urinalysis (UA) is unclear. We reviewed 312 pediatric patients with suspected UTI who had urine culture, UA, and urine Gram stain performed from a single urine specimen. UA was considered positive if ≥10 leukocytes per oil immersion field were seen or if either nitrates or leukocyte esterase testing was positive. Urine Gram stain was considered positive if any organisms were seen. Sensitivity, specificity, and positive and negative predictive values were calculated using urine culture as the gold standard. Thirty-seven (12%) patients had a culture-proven UTI. Compared to urine Gram stain, UA had equal sensitivity (97.3% versus 97.5%) and higher specificity (85% versus 74%). Empirical therapy was prescribed before the Gram stain result was known in 40 (49%) patients and after in 42 (51%) patients. The antibiotics chosen did not differ between the two groups (P=0.81), nor did they differ for patients with Gram-negative rods on urine Gram stain compared to those with Gram-positive cocci (P=0.67). From these data, we conclude that UA has excellent negative predictive value that is not enhanced by urine Gram stain and that antibiotic selection did not vary based on the urine Gram stain result. In conclusion, the clinical utility of urine Gram stain does not warrant the time or cost it requires.

Detection of intracellular parasites by use of the CellaVision DM96 analyzer during routine screening of peripheral blood smears.

Conventional microscopy is the gold standard for malaria diagnosis. The CellaVision DM96 is a digital hematology analyzer that utilizes neural networks to locate, digitize, and preclassify leukocytes and characterize red blood cell morphology. This study compared the detection rates of Plasmodium and Babesia species on peripheral blood smears utilizing the CellaVision DM96 with the rates for a routine red blood cell morphology scan. A total of 281 slides were analyzed, consisting of 130 slides positive for Plasmodium or Babesia species and 151 negative controls. Slides were blinded, randomized, and analyzed by CellaVision and microscopy for red cell morphology scans. The technologists were blinded to prior identification results. The parasite detection rate was 73% (95/130) for CellaVision and 81% (105/130) for microscopy for positive samples. The interobserver agreement between CellaVision and microscopy was fair, as Cohen's kappa coefficient equaled 0.36. Pathologist review of CellaVision images identified an additional 15 slides with parasites, bringing the total number of detectable positive slides to 110 of 130 (85%). Plasmodium ovale had the lowest rate of detection at 56% (5 of 9); Plasmodium malariae and Babesia spp. had the highest rate of detection at 100% (3/3 and 6/6, respectively). The detection rate by CellaVision was 100% (23/23) when the parasitemia was ≥2.5%. The detection rate for <0.1% parasitemia was 63% (15/24). Technologists appropriately classified all negative specimens. The percentage of positive specimens detectable by CellaVision (73%) approaches results for microscopy on routine scan of peripheral blood smears for red blood cell morphology.

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.

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.

De Novo meningitis caused by Propionibacterium acnes in a patient with metastatic melanoma.

Propionibacterium acnes is a known cause of postneurosurgical meningitis; however, it is rarely implicated in de novo meningitis. Herein we report a case of a 49-year-old male with de novo meningitis caused by P. acnes with metastatic melanoma as the only identified risk factor for his infection.

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.

Renal abscess caused by a Providencia stuartii isolate biochemically misidentified as Pasteurella.

Providencia stuartii is associated with urinary tract infection (UTI) in catheterized patients. Here we report an abscess containing P. stuartii in a patient with a history of UTI, renal stones, and stent placement. This organism was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing following biochemical identification as Pasteurella.

Characterization of NLRP12 during the in vivo host immune response to Klebsiella pneumoniae and Mycobacterium tuberculosis.

The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1ß production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12(-/-) mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12(-/-) mice showed a slight decrease in IL-1ß and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12(-/-) mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12(-/-) mice. Likewise, Nlrp12(-/-) mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.

Two cases of Kerstersia gyiorum isolated from sites of chronic infection.

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.

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.

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.

The clinical and environmental determinants of airway transcriptional profiles in allergic asthma.

RATIONALE: Gene expression profiling of airway epithelial and inflammatory cells can be used to identify genes involved in environmental asthma. METHODS: Airway epithelia and inflammatory cells were obtained via bronchial brush and bronchoalveolar lavage (BAL) from 39 subjects comprising three phenotypic groups (nonatopic nonasthmatic, atopic nonasthmatic, and atopic asthmatic) 4 hours after instillation of LPS, house dust mite antigen, and saline in three distinct subsegmental bronchi. RNA transcript levels were assessed using whole genome microarrays. MEASUREMENTS AND MAIN RESULTS: Baseline (saline exposure) differences in gene expression were related to airflow obstruction in epithelial cells (C3, ALOX5AP, CCL18, and others), and to serum IgE (innate immune genes and focal adhesion pathway) and allergic-asthmatic phenotype (complement genes, histone deacetylases, and GATA1 transcription factor) in inflammatory cells. LPS stimulation resulted in pronounced transcriptional response across all subjects in both airway epithelia and BAL cells, with strong association to nuclear factor-κB and IFN-inducible genes as well as signatures of other transcription factors (NRF2, C/EBP, and E2F1) and histone proteins. No distinct transcriptional profile to LPS was observed in the asthma and atopy phenotype. Finally, although no consistent expression changes were observed across all subjects in response to house dust mite antigen stimulation, we observed subtle differences in gene expression (e.g., GATA1 and GATA2) in BAL cells related to the asthma and atopy phenotype. CONCLUSIONS: Our results indicate that among individuals with allergic asthma, transcriptional changes in airway epithelia and inflammatory cells are influenced by phenotype as well as environmental exposures.

The inflammasome component NLRP3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells.

The inflammasome is a proteolysis complex that generates the active forms of the proinflammatory cytokines interleukin (IL)-1ß and IL-18. Inflammasome activation is mediated by NLR proteins that respond to microbial and nonmicrobial stimuli. Among NLRs, NLRP3 senses the widest array of stimuli and enhances adaptive immunity. However, its role in antitumor immunity is unknown. Therefore, we evaluated the function of the NLRP3 inflammasome in the immune response using dendritic cell vaccination against the poorly immunogenic melanoma cell line B16-F10. Vaccination of Nlrp3(-/-) mice led to a relative 4-fold improvement in survival relative to control animals. Immunity depended on CD8(+) T cells and exhibited immune specificity and memory. Increased vaccine efficacy in Nlrp3(-/-) hosts did not reflect differences in dendritic cells but rather differences in myeloid-derived suppressor cells (MDSC). Although Nlrp3 was expressed in MDSCs, the absence of Nlrp3 did not alter either their functional capacity to inhibit T cells or their presence in peripheral lymphoid tissues. Instead, the absence of Nlrp3 caused a 5-fold reduction in the number of tumor-associated MDSCs found in host mice. Adoptive transfer experiments also showed that Nlrp3(-/-) MDSCs were less efficient in reaching the tumor site. Depleting MDSCs with an anti-Gr-1 antibody increased the survival of tumor-bearing wild-type mice but not Nlrp3(-/-) mice. We concluded that Nlrp3 was critical for accumulation of MDSCs in tumors and for inhibition of antitumor T-cell immunity after dendritic cell vaccination. Our findings establish an unexpected role for Nlrp3 in impeding antitumor immune responses, suggesting novel approaches to improve the response to antitumor vaccines by limiting Nlrp3 signaling.

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-1beta 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-1beta 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.