Jennifer Dien Bard

Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma

Aberrations of the Wnt canonical pathway (WCP) are known to contribute to the pathogenesis of various types of cancer. We hypothesize that these defects may exist in mantle cell lymphoma (MCL). Both the upstream and downstream aspects of WCP were examined in MCL cell lines and tumors. Using WCP-specific oligonucleotide arrays, we found that MCL highly and consistently expressed Wnt3 and Wnt10. beta-catenin, a transcriptional factor that is a downstream target of WCP, is localized to the nucleus and transcriptionally active in all 3 MCL cell lines examined. By immunohistochemistry, 33 (52%) of 64 MCL tumors showed nuclear localization of beta-catenin, which significantly correlated with the expression of the phosphorylated/inactive form of GSK3beta (p-GSK3beta; P = .011, Fisher). GSK3beta inactivation is directly linked to WCP stimulation, since addition of recombinant sFRP proteins (a naturally occurring decoy for the Wnt receptors) resulted in a significant decrease in p-GSK3beta. Down-regulation of DvL-2 (an upstream signaling protein in WCP) by siRNA or selective inhibition of beta-catenin using quercetin significantly decreased cell growth in MCL cell lines. To conclude, WCP is constitutively activated in a subset of MCL and it appears to promote tumorigenesis in MCL.

Multicenter Evaluation of BioFire FilmArray Meningitis/Encephalitis Panel for Detection of Bacteria, Viruses, and Yeast in Cerebrospinal Fluid Specimens

ABSTRACT Rapid diagnosis and treatment of infectious meningitis and encephalitis are critical to minimize morbidity and mortality. Comprehensive testing of cerebrospinal fluid (CSF) often includes Gram stain, culture, antigen detection, and molecular methods, paired with chemical and cellular analyses. These methods may lack sensitivity or specificity, can take several days, and require significant volume for complete analysis. The FilmArray Meningitis/Encephalitis (ME) Panel is a multiplexed in vitro diagnostic test for the simultaneous, rapid (∼1-h) detection of 14 pathogens directly from CSF specimens: Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, varicella-zoster virus, and Cryptococcus neoformans/Cryptococcus gattii. We describe a multicenter evaluation of 1,560 prospectively collected CSF specimens with performance compared to culture (bacterial analytes) and PCR (all other analytes). The FilmArray ME Panel demonstrated a sensitivity or positive percentage of agreement of 100% for 9 of 14 analytes. Enterovirus and human herpesvirus type 6 had agreements of 95.7% and 85.7%, and L. monocytogenes and N. meningitidis were not observed in the study. For S. agalactiae, there was a single false-positive and false-negative result each, for a sensitivity and specificity of 0 and 99.9%, respectively. The specificity or negative percentage of agreement was 99.2% or greater for all other analytes. The FilmArray ME Panel is a sensitive and specific test to aid in diagnosis of ME. With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship are anticipated.

IL-21 contributes to JAK3/STAT3 activation and promotes cell growth in ALK-positive anaplastic large cell lymphoma.

Interleukin (IL)-21 has been reported to both stimulate cell growth and promote survival in benign lymphoid cells and several types of hematopoietic neoplasms. It induces JAK3/STAT3 signaling, a biologically important cellular pathway activated in most cases of anaplastic lymphoma kinase (ALK)-expressing anaplastic large cell lymphoma (ALK(+)ALCL). Therefore, we hypothesize that IL-21 may contribute to JAK3/STAT3 activation and cell growth in ALK(+)ALCL. By reverse transcription-PCR, we found consistent expression of IL-21 receptor (IL-21R) in all ALK(+)ALCL cell lines and frozen tumors examined. IL-21 was also consistently expressed in ALK(+)ALCL tumors, although its mRNA was detectable in only one of three cell lines tested. By immunohistochemistry, we examined 10 paraffin-embedded ALK(+)ALCL tumors; all cases were positive for both IL-21 and IL-21R in these neoplastic cells. IL-21 signaling is biologically significant in ALK(+)ALCL since the addition of recombinant IL-21 enhanced the activation of JAK3/STAT3 and significantly increased cell growth in ALK(+)ALCL cell lines. However, small interfering RNA down-regulation of IL-21R significantly decreased both STAT3 activation and cell growth. IL-21R expression is not linked to nucleophosmin-ALK since forced expression of nucleophosmin-ALK and small interfering RNA down-regulation of nucleophosmin-ALK did not significantly change the expression of either IL-21R or IL-21. Our findings thus support the enhancement of JAK3/STAT3 activation and cell growth in ALK(+)ALCL via IL-21 signaling. These results further support the concept that constitutive activation of STAT3 in these tumors is multifactorial.

Performance of the Verigene Gram-positive blood culture assay for direct detection of Gram-positive organisms and resistance markers in a pediatric hospital.

ABSTRACT The performance characteristics of the Verigene Gram-positive blood culture (BC-GP) assay were evaluated in pediatric patients. Concordance of the BC-GP assay was 95.8%, with significant decreases in turnaround time for identification and resistance detection. BC-GP is highly accurate and can be integrated into the routine workflow of the microbiology laboratory.

Pediatric Carbapenem-resistant Enterobacteriaceae in Los Angeles, California, a High-prevalence Region in the United States

Background: Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are on the rise worldwide but are not well described in pediatric populations. This study characterizes the clinical, phenotypic and genotypic characteristics of CRE infections at a free-standing US children’s hospital. Methods: CRE were defined as any clinical Enterobacteriaceae isolate non-susceptible to either imipenem or meropenem and resistant to ceftriaxone, cefotaxime and ceftazidime determined by routine antimicrobial susceptibility testing. The modified Hodge test was performed to screen for the production of carbapenemase. Clinical data were reviewed, and molecular characterization of phylogenetic and resistance-associated traits was performed. Results: CRE isolates were recovered from sterile and non-sterile sites in 10 patients, 6 weeks to 24 years of age, between 2011 and 2013. Co-morbidities included hematologic, genetic and urologic abnormalities. Two patients had traveled abroad (India, Lebanon) before CRE recovery. Carbapenemase determinants were detected in 5 cases, including KPC-3 in 2 Klebsiella pneumoniae (ST258 and ST18) and 1 Escherichia coli (ST131), and NDM-1 in 1 K. pneumoniae (ST37) and 1 E. coli (ST101) isolate. Additional resistance determinants were detected, including CTX-M-15, SHV-11, TEM-1, CMY-2, CMY-4 and CMY-42. Four patients died, including 2 of 3 patients with CRE bacteremia. There was no evidence of epidemiologic or molecular relatedness between any 2 cases. Conclusions: This report documents the appearance of highly resistant Gram-negative pathogens in a vulnerable patient population at a pediatric tertiary referral center in a major US metropolitan area. Detailed understanding of the distribution and spread of CRE is essential for the timely detection and containment of these perilous pathogens.

Roseomonas mucosa Isolated from Bloodstream of Pediatric Patient

ABSTRACT We report a case of catheter-related bacteremia associated with Roseomonas mucosa isolated from an immunocompromised pediatric patient with a history of multiple episodes of urinary tract infection and bacteremia.

Neurobrucellosis: Unexpected Answer From Metagenomic Next-Generation Sequencing

A diagnosis of brucellosis can be difficult because routine culture and serological methods exhibit variable sensitivity and specificity. We present the use of a metagenomic next- generation sequencing assay to diagnose a case of neurobrucellosis from cerebrospinal fluid, resulting in the institution of appropriate antibiotic treatment and a favorable clinical outcome.

First Report of Treatment of Anaerobiospirillum succiniciproducens Bloodstream Infection with Levofloxacin

ABSTRACT The full extent of the clinical spectrum and optimal therapy of Anaerobiospirillum succiniciproducens infections remains to be determined. We describe the first case of bloodstream infection (BSI) due to A. succiniciproducens in an asymptomatic elderly male with poor dentition that was treated with levofloxacin.

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.

Molecular and Clinical Diagnosis of Group A Streptococcal Pharyngitis in Children

ABSTRACT Group A Streptococcus (GAS) pharyngitis is a very common condition causing significant morbidity in children. Accurate diagnosis followed by appropriate antimicrobial therapy is recommended to prevent postinfectious sequelae. Diagnosis of GAS pharyngitis by a rapid antigen detection test (RADT) or culture in the absence of discriminating clinical findings remains challenging. Validation of new sensitive rapid diagnostic tests is therefore a priority. The performance of a loop-mediated isothermal amplification (LAMP) assay (illumigene assay) for the diagnosis of GAS pharyngitis was compared with that of a RADT and standard culture in 361 pediatric throat swab samples. Discrepant results were resolved using an alternate molecular assay. Test results were correlated with clinical presentations in patients positive by either method. The closest estimate of the true prevalence of GAS pharyngitis was 19.7% (71/361 samples). The illumigene assay alone detected 70/71 GAS-positive samples; RADT and culture detected 35/71 and 55/71 samples, respectively. RADT followed by culture confirmation of RADT-negative specimens detected 58/71 cases. The illumigene assay increased identification among children eligible for testing by American College of Physicians (ACP)/American Academy of Family Physicians (AAFP) criteria from 31 to 39 positive cases, five of which were false positives. Analysis of clinical data in GAS-positive patients indicated that a significantly greater proportion of children with McIsaac scores of ≥4 tested positive by the illumigene assay versus RADT and culture. Overall, the illumigene assay was much more sensitive and was similarly specific for GAS detection, compared to culture alone, RADT alone, or the ACP/AAFP RADT/culture algorithm. Combining high sensitivity with rapidly available results, the illumigene GAS assay is an appropriate alternative to culture for the laboratory diagnosis of GAS pharyngitis in patients for whom testing is clinically indicated.