Kathleen Chiotos

Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes

Background: Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression. Methods: Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bear- ing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes. Results: AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers. Conclusion: Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.

Carbapenem-Resistant Enterobacteriaceae Infections in Children.

Carbapenem-resistant Enterobacteriaceae (CRE) are an emerging global public health threat. Infections due to CRE are associated with significant morbidity and mortality. Few therapeutic options are available for treatment of these infections, and optimal antibiotic treatment regimens are unclear. Along with the rapidly increasing prevalence of CRE in the USA and worldwide, several studies have described the epidemiology of CRE in the adult population. While CRE are now also reported sporadically in children, there is a significant lack of data on the epidemiology, risk factors, treatment, and outcomes in this population. This article provides a comprehensive review of what is known to date about CRE, including clinical and molecular epidemiology, microbiologic diagnosis, antibiotic treatment options, and outcomes. In particular, this review will focus on the available data on CRE in the pediatric population.

Blood Volume Required for Detection of Low Levels and Ultralow Levels of Organisms Responsible for Neonatal Bacteremia by Use of Bactec Peds Plus/F, Plus Aerobic/F Medium, and the BD Bactec FX System: an In Vitro Study

ABSTRACT We used an in vitro technique to investigate blood volumes required to detect bacteremia and fungemia with low concentrations of an organism. At 1 to 10 CFU/ml, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Listeria monocytogenes, Candida albicans, and Candida parapsilosis isolates were detected in volumes as low as 0.5 ml. Detection of Streptococcus agalactiae and detection of bacteremia at <1 CFU/ml were unreliable.

Multicenter Study of the Risk Factors for Colonization or Infection with Carbapenem-Resistant Enterobacteriaceae in Children

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) are increasingly identified in children in the United States, but data on the epidemiology of CRE in this population are limited. The objectives of this study were to characterize the risk factors for colonization or infection with CRE and describe the microbiologic characteristics of pediatric CRE isolates. We performed a multicenter matched case-control study from January 2011 to October 2015 at three tertiary care pediatric centers. Case patients were hospitalized children with CRE isolated from clinical cultures and were matched in a 2:1 ratio to control patients with carbapenem-susceptible Enterobacteriaceae (CSE). Risk factors for colonization or infection with CRE were then evaluated using a multivariable conditional logistic regression. Additionally, we comprehensively reported the antimicrobial susceptibility pattern for CRE isolates. Sixty-three case patients were identified and matched to 126 control patients. On multivariable analysis, antipseudomonal antibiotic exposure within the previous 3 months (odds ratio [OR], 5.20; 95% confidence interval [CI], 1.71 to 15.9; P = 0.004), prior surgery (OR, 6.30; 95% CI, 1.83 to 21.6; P = 0.003), and mechanical ventilation (OR, 12.4; 95% CI, 1.26 to 122; P = 0.031) were identified as risk factors for colonization or infection with CRE. Pediatric CRE isolates demonstrated relatively low rates of resistance to amikacin (5%) and ciprofloxacin (25%). Our findings support an important role for antibiotic stewardship interventions limiting the unnecessary use of antipseudomonal antibiotics as a strategy to prevent widespread emergence of CRE in children. Future studies should further characterize molecular determinants of antibiotic resistance among pediatric CRE isolates.

Increased 30-Day Mortality Associated With Carbapenem-Resistant Enterobacteriaceae in Children.

Abstract In this multicenter study, we identified an increased risk of 30-day mortality among hospitalized children with carbapenem-resistant Enterobacteriaceae (CRE) isolated from clinical cultures compared with those with carbapenem-susceptible Enterobacteriaceae. We additionally report significant variation in antibiotic treatment for children with CRE infections with infrequent use of combination therapy.

Use of Carbapenems, Polymyxins, and Tigecycline in United States Children’s Hospitals, 2010–2014

Abstract We characterized use of the carbapenems, polymyxins, and tigecycline in United States children’s hospitals between 2010 and 2014. We found substantial variability in use across hospitals and overall decreased use over time. Most polymyxin and tigecycline use occurred in cystic fibrosis patients, and appendectomy was a common indication for carbapenem therapy.

What is this chocolate milk in my circuit? A cause of acute clotting of a continuous renal replacement circuit: Questions.

Introduction One of the greatest problems associated with continuous renal replacement therapy (CRRT) is the early clotting of filters. A literature search revealed three case reports of lipemic blood causing recurrent clotting and reduced CRRT circuit survival time in adult patients, but no reports of cases in children.

Management of invasive fungal infections in the pediatric intensive care unit

Invasive candidiasis (IC) are the most common invasive fungal infections in pediatric intensive care unit patients due to commonly present risk factors including central venous catheters, antibiotic exposure, recent surgery, total parenteral nutrition, and prior colonization with Candida species. These infections are economically costly and have been associated with an attributable mortality of approximately 10%. As a result, investigators have attempted to identify cohorts of pediatric intensive care unit patients at highest risk of development of IC who may benefit from prophylactic, early empiric, or pre-emptive therapy. This review article will consider these prediction models and therapeutic approaches. Additionally, the management of confirmed IC, including anti-fungal therapeutic options, evaluation for disseminated disease, and management of central venous catheters, will be reviewed.

Management of bacterial severe sepsis and septic shock

Bacterial sepsis is a leading cause of pediatric morbidity and mortality worldwide. Early diagnosis, a coordinated and aggressive approach to initial resuscitation, and timely and appropriate antibiotic therapy are paramount to improving outcomes of these dangerous infections. The basic tenants of initial and ongoing resuscitation include rapid isotonic intravenous fluid boluses with reassessment for physiologic response, empiric broad-spectrum antibiotics directed to cover suspected sources of infection, source control, vasoactive infusions, supportive critical care and monitoring of response to therapy. In addition to resuscitation of bacterial sepsis, this article will review approaches to empiric antibiotic choice in septic shock, and detail definitive management of infections caused by several specific organisms, including Staphylococcus aureus , group A Streptococcus, Pseudomonas aeruginosa, Mycobacterium tuberculosis , and Clostridium difficile . Lastly, management of several common pediatric infections, including community acquired bacterial pneumonia and bacterial meningitis, will be reviewed.