Alison J. Carey

Hospital-Acquired Infections in the NICU: Epidemiology for the New Millennium

Nosocomial infections are an important cause of morbidity and mortality in the preterm neonate. Extrinsic and intrinsic risk factors make the preterm neonate particularly susceptible to infection. This review focuses on two major pathogens that cause nosocomial infection, Candida and methicillin-resistant Staphylococcus aureus. The difficult diagnosis of meningitis in the neonate also is discussed.

The epidemiology of methicillin-susceptible and methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit, 2000―2007

Objective:To assess the epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) infections in a neonatal intensive care unit (NICU).Study Design:A retrospective chart review was conducted from 2000–2007; demographic and clinical characteristics of infected infants and crude mortality were assessed.Results:During the study period, there were 123 infections caused by MSSA and 49 infections caused by MRSA. Although the types of infections caused by MSSA and MRSA were similar, infants with MRSA infections were younger at clinical presentation than infants with MSSA infections (P=0.03). The overall rate of S. aureus infections was approximately 15–30 per 1000 patient-admissions. The rate of bacteremia and skin and soft tissue infections remained stable over time. Among extremely low birth weight infants (birth weight <1000 g), 4.8 and 1.8% developed an infection caused by MSSA or MRSA, respectively. Infections occurred in a bimodal distribution of birth weight; 53% of infections occurred in extremely low birth weight infants and 27% occurred among term infants birth weight ⩾2500 g, many of whom underwent surgical procedures.Conclusions:MSSA and MRSA remain significant pathogens in the NICU, particularly for extremely premature infants and term infants undergoing surgery. Further work should investigate infection control strategies that effectively target the highest risk groups and determine if vertical transmission of MRSA is responsible for the younger age at presentation of infection.

Changes in the Molecular Epidemiological Characteristics of Methicillin‐Resistant Staphylococcus aureus in a Neonatal Intensive Care Unit

OBJECTIVE To determine whether the molecular epidemiological characteristics of methicillin-resistant Staphylococcus aureus (MRSA) had changed in a level III neonatal intensive care unit (NICU). DESIGN Retrospective review of medical records. SETTING Level III NICU of a university-affiliated childrens hospital in New York, New York. PATIENTS Case patients were neonates hospitalized in the NICU who were colonized or infected with MRSA. METHODS Rates of colonization and infection with MRSA during the period from 2000 through 2008 were assessed. Staphylococcal chromosomal cassette (SCC) mecA analysis and genotyping for S. aureus encoding protein A (spa) were performed on representative MRSA isolates from each clonal pulsed-field gel electrophoresis pattern. RESULTS Endemic MRSA infection and colonization occurred throughout the study period, which was punctuated by 4 epidemiologic investigations during outbreak periods. During the study period, 93 neonates were infected and 167 were colonized with MRSA. Surveillance cultures were performed for 1,336 neonates during outbreak investigations, and 115 (8.6%) neonates had MRSA-positive culture results. During 2001-2004, healthcare-associated MRSA clones, carrying SCC mec type II, predominated. From 2005 on, most MRSA clones were community-associated MRSA with SCC mec type IV, and in 2007, USA300 emerged as the principal clone. CONCLUSIONS Molecular analysis demonstrated a shift from healthcare-associated MRSA (2001-2004) to community-associated MRSA (2005-2008).

Phosphatidylinositol 3-Kinase p110δ Isoform Regulates CD8+ T Cell Responses during Acute Viral and Intracellular Bacterial Infections

The p110δ isoform of PI3K is known to play an important role in immunity, yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8+ T cells, we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8+ T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8+ T cell activation, proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases, our study suggests caution in using these drugs in patients, as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8+ T cell immunity to microbial pathogens.

Rapid evolution of the CD8+ TCR repertoire in neonatal mice

Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8+ T cell (CTL) responses in neonates. TCRβ high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vβ family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366–374)–specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366–374)-specific CTL TCR repertoire revealed different Vβ gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257–264)-specific (OT-I) CD8+ T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8+ T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

Prevention of unplanned extubations in neonates through process standardization.

Objective:Unplanned extubation events (UPEs) in neonates are hazardous to patient safety. Our goal was to reduce UPE rate (#UPEs per 100 ventilator days) by 50% in 12 months at our 25-bed level III inborn unit.Study Design:Baseline data were gathered prospectively for 7 months. Three Plan-Do-Study-Act (PDSA) cycles targeting main causes of UPEs were developed over the next 20 months. Causes of UPEs were analyzed using Pareto charts; and a U control chart was created with QI Charts©. Standard rules for detecting special cause variation were applied.Result:Mean UPE rate decreased from 16.1 to 4.5 per 100 ventilator days, a 72% decrease, exceeding our goal. Analysis of U-chart demonstrated special cause variation, with eight consecutive points below the mean. Improvement was sustained throughout the study period.Conclusion:UPEs in neonates can be reduced with process standardization and frontline staff education, emphasizing vigilant endotracheal tube (ETT) maintenance.

Characterization of CD31 expression on murine and human neonatal T lymphocytes during development and activation

BackgroundCD31, expressed by the majority of the neonatal T-cell pool, is involved in modulation of T-cell receptor signaling by increasing the threshold for T-cell activation. Therefore, CD31 could modulate neonatal tolerance and adaptive immune responses.MethodsLymphocytes were harvested from murine neonates at different ages, human late preterm and term cord blood, and adult peripheral blood. Human samples were activated over a 5-day period to simulate acute inflammation. Mice were infected with influenza; lungs and spleens were harvested at days 6 and 9 post infection and analyzed by flow cytometry.ResultsCD31-expressing neonatal murine CD4+ and CD8a+ T cells increase over the first week of life. Upon in vitro stimulation, human infants’ CD4+ and CD8a+ T cells shed CD31 faster in comparison with adults. In the context of acute infection, mice infected at 3 days of age have an increased number of naive and activated CD31+ T lymphocytes at the site of infection at days 6 and 9 post infection, as compared with those infected at 7 days of age; however, the opposite is true in the periphery.ConclusionDifferences in trafficking of CD31+ cytotoxic T lymphocytes (CTLs) during acute influenza infection could modulate tolerance and contribute to a dampened adaptive immune response in neonates.

War on Staphylococcus aureus

Staphylococcus aureus has been a problem for both well and sick neonates for decades. As early as 1937, guidance for proper skin care of newborns was given to prevent staphylococcal infection.1 The first description of a penicillin-resistant S. aureus outbreak in the newborn nursery was in 1952,2 when aureomycin, the first of the tetracyclines, was used for the treatment of pustular dermatitis, conjunctivitis and pneumonia. However, prevention of these outbreaks has proven to be very difficult. In the early 1960s, artificial colonization was employed, whereby infants were purposely colonized with less virulent S. aureus to prevent colonization with the more virulent strains.3 Constantly evolving, hospital-associated (HA) methicillin-resistant S. aureus (MRSA) emerged in the 1970s in adult intensive care units, and gradually infiltrated the neonatal intensive care unit (NICU). However, community-associated (CA) MRSA spread across the globe in the 1990s, causing necrotizing soft tissue and bone infections in people of all ages and affecting neonates colonized from mothers or nursery environment. Over the past several decades, infection control policies have been developed to combat S. aureus, including decolonization strategies, hand hygiene, surveillance and isolation procedures. However, despite many years of devising plans against S. aureus, the NICU remains a haven for this formidable pathogen.

The Transcription Factor T-Bet Is Regulated by MicroRNA-155 in Murine Anti-Viral CD8+ T Cells via SHIP-1

We report here that the expression of the transcription factor T-bet, which is known to be required for effector cytotoxic CD8+ T lymphocytes (CTL) generation and effector memory cell formation, is regulated in CTL by microRNA-155 (miR-155). Importantly, we show that the proliferative effect of miR-155 on CD8+ T cells is mediated by T-bet. T-bet levels in CTL were controlled in vivo by miR-155 via SH2 (Src homology 2)-containing inositol phosphatase-1 (SHIP-1), a known direct target of miR-155, and SHIP-1 directly downregulated T-bet. Our studies reveal an important and unexpected signaling axis between miR-155, T-bet, and SHIP-1 in in vivo CTL responses and suggest an important signaling module that regulates effector CTL immunity.

Public Clonotypes and Convergent Recombination Characterize the Naïve CD8+ T-Cell Receptor Repertoire of Extremely Preterm Neonates

Respiratory support improvements have aided survival of premature neonates, but infection susceptibility remains a predominant problem. We previously reported that neonatal mice have a rapidly evolving T-cell receptor (TCR) repertoire that impairs CD8+ T cell immunity. To understand the impact of prematurity on the human CD8+ TCR repertoire, we performed next-generation sequencing of the complementarity-determining region 3 (CDR3) from the rearranged TCR variable beta (Vβ) in sorted, naïve CD8+ T cells from extremely preterm neonates (23–27 weeks gestation), term neonates (37–41 weeks gestation), children (16–56 months), and adults (25–50 years old). Strikingly, preterm neonates had an increased frequency of public clonotypes shared between unrelated individuals. Public clonotypes identified in preterm infants were encoded by germline gene sequences, and some of these clonotypes persisted into adulthood. The preterm neonatal naïve CD8+ TCR repertoire exhibited convergent recombination, characterized by different nucleotide sequences encoding the same amino acid CDR3 sequence. As determined by Pielou’s evenness and iChao1 metrics, extremely preterm neonates have less clonality, and a much lower bound for the number of unique TCR within an individual preterm neonate, which indicates a less rich and diverse repertoire, as compared to term neonates, children, and adults. This suggests that T cell selection in the preterm neonate may be less stringent or different. Our analysis is the first to compare the TCR repertoire of naïve CD8+ T cells between viable preterm neonates and term neonates. We find preterm neonates have a repertoire immaturity which potentially contributes to their increased infection susceptibility. A developmentally regulated, evenly distributed repertoire in preterm neonates may lead to the inclusion of public TCR CDR3β sequences that overlap between unrelated individuals in the preterm repertoire.