Detailed Phenotypic and Functional Characterization of a Rare, Antibody-Dependent SLAM-Associated Protein Expression Pattern.

SLAM-associated protein (SAP) is an adaptor molecule that facilitates critical effector functions in immune cells, and its deficiency causes X-linked lymphoproliferative disease type 1 in which effector responses directed against EBV are severely compromised. The primary objective of this study was to phenotypically and functionally characterize a rare, CD8 T cell-restricted bimodal SAP expression pattern observed in healthy, human donors with the widely used 1C9-SAP mAb clone. We initially observed this pattern during the clinical validation of our flow cytometry-based assay to diagnose X-linked lymphoproliferative disease type 1 in our laboratory. For this validation study, we used multiparameter flow cytometry to identify cytosolic SAP expression in lymphocyte subsets, and CD8 T cells from the donors displaying the rare SAP expression pattern mentioned above were separately further evaluated by intracellular cytokine and CD107a staining to examine polyfunctionality following PMA/ionomycin and HLA class I allele-restricted EBV peptide epitope-induced T cell activation. Our data revealed that SAP 1C9-hi CD8 T cells clearly displayed higher polyfunctional responses versus SAP 1C9-lo CD8 T cells following PMA/ionomycin stimulation. Furthermore, polyfunctional EBV-specific CD8 T cell responses segregated with the SAP 1C9-hi CD8 T cells and not the SAP 1C9-lo CD8 T cells. Additionally, and rather intriguingly, short- and long-term T cell stimulation selectively diminished the signal for the 1C9-hi subset. Overall, our data suggest that although rare, this unique SAP expression pattern merits further evaluation as it has the potential to provide some insight into fundamental processes as they might relate to host-pathogen dynamics.

Ideal and Actual Impact of Rapid Diagnostic Testing and Antibiotic Stewardship on Antibiotic Prescribing and Clinical Outcomes in Children With Positive Blood Cultures.

BACKGROUND: Implementing matrix-assisted laser desorption ionization-time of flight and multiplex polymerase chain reaction has been associated with decreased mortality and hospital length of stay in adults, but the impact in pediatrics is less understood. METHODS: This pre-post quasi-experimental study compared antibiotic prescribing for positive blood cultures in patients ≤21 years of age collected in 2012 (preintervention) and in 2015 (after matrix-assisted laser desorption ionization-time of flight/multiplex polymerase chain reaction). Time to effective and optimal antimicrobial therapy was evaluated using Cox proportional hazards regression. Time to ideal optimal therapy was estimated as the earliest potential initiation of optimal therapy. Antibiotic use and clinical outcomes were measured. RESULTS: There were 242 and 192 positive monomicrobial blood cultures in 2012 and 2015, respectively. Postintervention, time to optimal therapy (73.8 vs. 48.8 hours; P < 0.001) and organism identification (55.6 vs. 29.5 hours; P < 0.001) were reduced, and patients were more likely to receive optimal therapy by 7 days (hazard ratio, 1.85; P < 0.001). In the ideal scenario in 2015, there was an 8.8-hour delay in initiating optimal therapy based on the time that sufficient microbiologic data were available. Postintervention, time to effective therapy (2.8 vs. 2.7 hours; P = 0.782) and clinical outcomes did not differ. Unnecessary antibiotic duration for probable contaminants (skin flora) (43.1 vs. 29.7 hours; P = 0.027), vancomycin for methicillin-sensitive Staphylococcus aureus (54.0 vs. 41.3 hours; P = 0.008) and nonpenicillin/ampicillin antibiotics for group A Streptococcus, group B Streptococcus and Enterococcus faecalis (87.2 vs. 33.4 hours; P < 0.001) were reduced postintervention. CONCLUSIONS: Rapid diagnostics reduced time to optimal antimicrobial therapy and unnecessary antibiotic use without worse clinical outcomes.

Utilizing the electronic health record to construct antibiograms for previously healthy children with urinary tract infections.

Traditional antibiograms can guide empiric antibiotic therapy, but they may miss differences in resistance across patient subpopulations. In this retrospective descriptive study, we constructed and validated antibiograms using International Classification of Disease, Tenth Revision (ICD-10) codes and other discrete data elements to define a cohort of previously healthy children with urinary tract infections. Our results demonstrate increased antibiotic susceptibility. This methodology may be modified to create other syndrome-specific antibiograms.

Caution Needed: Molecular Diagnosis of Pediatric Group A Streptococcal Pharyngitis.

Among throat swabs processed in the microbiology laboratory as back-up for negative rapid antigen detection test results, we found a significant increase in the proportion that tested positive for group A streptococci after changing from throat culture to a molecular test.For group A streptococcus testing, our hospital laboratory replaced throat cultures with a stand-alone molecular diagnostic test that takes no more than 1 hour to perform. The prevalence of positive laboratory test results increased significantly (P < .0001) after the change to molecular testing, probably because of the extreme sensitivity of the molecular test.

Differential recognition of the multiple banded antigen isoforms across Ureaplasma parvum and Ureaplasma urealyticum species by monoclonal antibodies.

Two separate species of Ureaplasma have been identified that infect humans: Ureaplasma parvum and Ureaplasma urealyticum. Most notably, these bacteria lack a cell wall and are the leading infectious organism associated with infection-related induction of preterm birth. Fourteen separate representative prototype bacterial strains, called serovars, are largely differentiated by the sequence of repeating units in the C-terminus of the major surface protein: multiple-banded antigen (MBA). Monoclonal antibodies that recognise single or small groups of serovars have been previously reported, but these reagents remain sequestered in individual research laboratories. Here we characterise a panel of commercially available monoclonal antibodies raised against the MBA and describe the first monoclonal antibody that cross-reacts by immunoblot with all serovars of U. parvum and U. urealyticum species. We also describe a recombinant MBA expressed by Escherichia coli which facilitated further characterisation by immunoblot and demonstrate immunohistochemistry of paraffin-embedded antigens. Immunoblot reactivity was validated against well characterised previously published monoclonal antibodies and individual commercial antibodies were found to recognise all U. parvum strains, only serovars 3 and 14 or only serovars 1 and 6, or all strains belonging to U. parvum and U. urealyticum. MBA mass was highly variable between strains, consistent with variation in the number of C-terminal repeats between strains. Antibody characterisation will enable future investigations to correlate severity of pathogenicity to MBA isoform number or mass, in addition to development of antibody-based diagnostics that will detect infection by all Ureaplasma species or alternately be able to differentiate between U. parvum, U. urealyticum or mixed infections.

Longitudinal Characterization of Herpes Simplex Virus (HSV) Isolates Acquired From Different Sites in an Immune-Compromised Child: A New HSV Thymidine Kinase Mutation Associated With Resistance.

BACKGROUND: Herpes simplex virus resistance to acyclovir is well described in immune-compromised patients. Management of prolonged infection and recurrences in such patients may be problematic. METHODS: A patient with neuroblastoma developed likely primary herpes gingivostomatitis shortly after starting a course of chemotherapy, with spread to the eye during treatment with acyclovir. Viral isolates were serially obtained from separate sites after treatment was begun and tested for susceptibility to acyclovir and foscarnet by plaque reduction and plating efficiency assays. The thymidine kinase and DNA polymerase genes from each isolate were sequenced. RESULTS: Initial isolates from a throat swab, an oral lesion, and conjunctiva were resistant to acyclovir within 13 days of treatment. Subsequent isolates while on foscarnet were initially acyclovir-susceptible, but reactivation of an acyclovir-resistant isolate was subsequently documented while on acyclovir suppression. Genotypic analysis identified a previously unreported UL23 mutation in some resistant isolates. None of the amino acid changes identified in UL30 were associated with resistance. CONCLUSIONS: Phenotypic and genotypic antiviral resistance of herpes simplex isolates may vary from different compartments and over time in individual immune-compromised hosts, highlighting the importance of obtaining cultures from all sites. Phenotypic resistance testing should be considered for isolates obtained from at-risk patients not responding to first-line therapy. Empiric combination treatment with multiple antivirals could be considered in some situations.

Outbreak of life-threatening coxsackievirus B1 myocarditis in neonates.

In the summer and fall of 2007, we observed a unique cluster of cases of severe coxsackievirus B1 (CVB1) infection among Chicago area neonates. Eight neonates had closely related strains of CVB1 that were typed at the Centers of Disease Control and Prevention; 2 other neonates had CVB infections, 1 of which was further identified as serotype CVB1. All had severe myocarditis; 1 neonate underwent heart transplantation, and 1 died of severe left ventricular dysfunction.

A day in the life of a nebulizer: surveillance for bacterial growth in nebulizer equipment of children with cystic fibrosis in the hospital setting.

BACKGROUND: Cystic fibrosis (CF) is characterized by chronic lung infection. Minimizing exposure to pathogens is important. Treating a CF pulmonary exacerbation includes nebulizer therapies, but little is known about pathogen exposure from nebulizer equipment in CF. OBJECTIVE: To assess microbial growth in nebulizer equipment used by hospitalized CF patients. HYPOTHESIS: The small-volume nebulizer would not support the growth of the important CF pathogens: Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia. METHODS: During a 6-month period, we prospectively enrolled 30 patients who were admitted for pulmonary exacerbation of CF and were prescribed an aerosolized bronchodilator 4 times daily. Bronchodilator was administered via disposable small-volume nebulizer, prior to airway clearance. The nebulizer was not cleaned or disinfected between treatments, but instead was replaced after 24 hours. Sputum or throat cultures were obtained prior to admission or on the day of admission, and standard culture techniques were used for CF microbes. After the first bronchodilator treatment, a sample was taken from the residual fluid inside the nebulizer cup. The second, third, and fourth samples were taken from the nebulizer cup after it was filled with a unit dose of the bronchodilator but prior to administering the bronchodilator. At the 24th hour, the nebulizer was filled with 3 mL of sterile water, from which the fifth sample was obtained, then the nebulizer was disposed of. RESULTS: On respiratory culture, ten patients had Pseudomonas aeruginosa, 5 had both P. aeruginosa and S. aureus, 6 had only S. aureus, and 1 had both S. aureus and H. influenzae. Three had other organisms, 4 had normal flora, and 1 had no culture data. Of the 150 nebulizer sample cultures, only 3 showed bacterial growth. Bacillus species, Corynebacterium, coagulase-negative Staphylococcus, and Candida albicans were isolated at low colony counts. CONCLUSIONS: We suspect that the organisms identified were caused by skin contamination of the samples rather than contamination of the nebulizer cup. We conclude that there is a low risk of microbial contamination with CF pathogens from the interior of a disposable nebulizer over a 24 hour period.

Pasteurella aerogenes hamster bite peritonitis.

Pasteurella multocida has been reported to cause peritoneal dialysis-associated peritonitis after a cat bite or scratch to the catheter. We report a teenager with hamster bite peritonitis caused by P. aerogenes, an organism predominantly isolated from swine.