Standardizing the approach to late onset sepsis in neonates through antimicrobial stewardship: a quality improvement initiative.

BACKGROUND: Antibiotics are the most prescribed medication in the neonatal intensive care unit (NICU) and there is marked variation in their use. While they are vital for treatment of infections, they put infants at risk for infections with drug resistant organisms, alteration in their microbiome and several other morbidities. Specific guidelines for neonates are often lacking and our NICU is not compliant with late onset sepsis (LOS) guidelines. OBJECTIVE: By January 2019, there will be >75% compliance with our LOS bundle for any infant admitted to Tampa General Hospital's (TGH) NICU undergoing a LOS evaluation at >72 h of life. The bundle includes documented reason for LOS evaluation, appropriate initial antibiotic selection, appropriate initial evaluation considered, and appropriate de-escalation of antibiotics. STUDY DESIGN: The project was implemented in the NICU at TGH, the academic medical center affiliated with the University of South Florida in Tampa, FL. The multidisciplinary antimicrobial stewardship (ASP) team responsible for the project consists of a neonatology attending, three neonatology fellows, a pediatric infectious disease attending, and two NICU pharmacists. The project was started in January 2017 and all data were collected prospectively. We implemented multiple Plan-Do-Study-Act cycles in a stepwise manner; outcome measures included compliance with the LOS bundle and ASP team recommendations. Our process measures were the documented reason for sepsis evaluation, appropriate initial evaluation considered, appropriate antibiotic selection and appropriate antibiotic de-escalation. Patient length of stay was the balancing measure studied. RESULTS: During this 20-month initiative, there were 232 infants who underwent LOS evaluation and there were 98 true positive cultures from blood (28%), urine (35%), and cerebrospinal fluid (3%). Commonly documented rationales for treatment of culture negative sepsis were clinical pneumonia (38%) and necrotizing enterocolitis (38%). Common indications for LOS evaluations were increased respiratory support (51%) and abdominal distension (17%). There was improvement in appropriate initial antibiotic selection (70% vs. 94%); appropriate consideration of initial evaluation (63% vs. 94%, respectively); appropriate de-escalation of antibiotics (86% vs. 100%, respectively) and increase in LOS bundle compliance (44% vs. 87%, respectively). The overall antibiotic utilization rate and length of treatment did not change significantly. CONCLUSIONS: Developing and engaging a NICU ASP team improves compliance with late onset sepsis guidelines through the implementation of a LOS bundle of care.

STAT5A and STAT5B have opposite correlations with drug response gene expression.

INTRODUCTION: STAT5A and STAT5B are important transcription factors that play a key role in regulation of several important physiological processes including proliferation, survival, mediation of responses to cytokines and in regulating gender differences in drug response genes such as the hepatic cytochrome P450s (CYPs) that are responsible for a large majority of drug metabolism reactions in the human body. STAT5A and STAT5b have a high degree of sequence homology and have been reported to have largely similar functions. Recent studies have, however, indicated that they can also often have distinct and unique roles in regulating gene expression. OBJECTIVE: In this study, we evaluated the association of STAT5A and STAT5B mRNA expression levels with those of several key hepatic cytochrome P450s (CYPs) and hepatic transcription factors (TFs) and evaluated the potential roles of STAT5A and 5b in mediating gender differences in these CYPs and TFs. METHODS: Expression profiling for major hepatic CYP isoforms and transcription factors was performed using RNA sequencing (RNA-seq) in 102 human liver samples (57 female, 45 male). Real time PCR gene expression data for selected CYPs and TFs was available on a subset of 50 human liver samples (25 female, 25 male) and was used to validate the RNA-seq findings. RESULTS: While STAT5A demonstrated significant negative correlation with expression levels of multiple hepatic transcription factors (including NR1I2 and HNF4A) and DMEs such as CYP3A4 and CYP2C19, STAT5B expression was observed to demonstrate positive associations with several CYPs and TFs analyzed. As STAT5A and STAT5B have been shown to be important in regulation of gender differences in CYPs, we also analyzed STAT5A and 5b associations with CYPs and TFs separately in males and females and observed gender dependent differential associations of STATs with several CYPs and TFs. Results from the real time PCR validation largely supported our RNA-seq findings. CONCLUSIONS: Using both RNA sequencing and real time PCR, we examined the association of STAT5A and STAT5B mRNA expression with CYP and TF gene expression. While STAT5A demonstrated significant negative correlations with expression levels of multiple hepatic TFs (including NR1I2 and HNF4α) and CYPs (eg. CYP3A4, CYP2C19), STAT5B expression was observed to demonstrate positive association with most of the CYPs/TFs analyzed suggesting that STAT5A and STAT5b have potentially different and distinct roles in regulating expression of hepatic drug response genes. Further studies are needed to elucidate the potential roles of STAT5A and 5b in regulation of CYPs/TFs and the potential implications of these findings.

microRNA-34a is associated with expression of key hepatic transcription factors and cytochromes P450.

microRNA (miRNA) mediated regulation of gene expression has emerged as a significant mechanism contributing to variation in gene expression. In this study, we evaluated the potential role of miRNAs in regulating expression of hepatic cytochromes P450 and their transcriptional regulatory genes. We screened the Targetscan database for high scoring miRNA binding site predictions in selected hepatic DMEs and transcription factors. Expression profiling for candidate miRNAs (n=22) and their target genes (n=20) was performed in 50 human liver samples (25 female, 25 male). Significant negative correlations were observed between expression levels of several CYPs/hepatic transcription factors and the hepatic miRNAs studied. Interestingly, hepatic miR-34a demonstrated significant negative correlation with expression levels of multiple hepatic transcription factors (including NR1I2 and HNF4α) and DMEs (CYP3A4, CYP2C19). miR-34a expression was also significantly higher in males than in females in congruence with previous observations of higher CYP3A4 expression in females versus males. A mediation analysis revealed that miR-34a was involved in significant mediation of the association observed between CYP2C19 and several hepatic transcription factors (HNF4α, NR1I2). miR-34a may thus play a key regulatory role and be a key contributory factor to the inter-individual variability observed in expression of key drug metabolizing genes in humans.

Using PharmGKB to train text mining approaches for identifying potential gene targets for pharmacogenomic studies.

The main objective of this study was to investigate the feasibility of using PharmGKB, a pharmacogenomic database, as a source of training data in combination with text of MEDLINE abstracts for a text mining approach to identification of potential gene targets for pathway-driven pharmacogenomics research. We used the manually curated relations between drugs and genes in PharmGKB database to train a support vector machine predictive model and applied this model prospectively to MEDLINE abstracts. The gene targets suggested by this approach were subsequently manually reviewed. Our quantitative analysis showed that a support vector machine classifiers trained on MEDLINE abstracts with single words (unigrams) used as features and PharmGKB relations used for supervision, achieve an overall sensitivity of 85% and specificity of 69%. The subsequent qualitative analysis showed that gene targets "suggested" by the automatic classifier were not anticipated by expert reviewers but were subsequently found to be relevant to the three drugs that were investigated: carbamazepine, lamivudine and zidovudine. Our results show that this approach is not only feasible but may also find new gene targets not identifiable by other methods thus making it a valuable tool for pathway-driven pharmacogenomics research.

Prediction of CYP3A4 enzyme activity using haplotype tag SNPs in African Americans.

The CYP3A locus encodes hepatic enzymes that metabolize many clinically used drugs. However, there is marked interindividual variability in enzyme expression and clearance of drugs metabolized by these enzymes. We utilized comparative genomics and computational prediction of transcriptional factor binding sites to evaluate regions within CYP3A that were most likely to contribute to this variation. We then used a haplotype tagging single-nucleotide polymorphisms (htSNPs) approach to evaluate the entire locus with the fewest number of maximally informative SNPs. We investigated the association between these htSNPs and in vivo CYP3A enzyme activity using a single-point IV midazolam clearance assay. We found associations between the midazolam phenotype and age, diagnosis of hypertension and one htSNP (141689) located upstream of CYP3A4. 141689 lies near the xenobiotic responsive enhancer module (XREM) regulatory region of CYP3A4. Cell-based studies show increased transcriptional activation with the minor allele at 141689, in agreement with the in vivo association study findings. This study marks the first systematic evaluation of coding and noncoding variation that may contribute to CYP3A phenotypic variability.

Pharmacogenetic pathway analysis of docetaxel elimination.

The purpose of this study was to evaluate the affinity of docetaxel for 14 transporter proteins and assess the functional significance of 17 variants in five genes involved in drug elimination. Among the transfected models investigated, OATP1B3 (SLCO1B3) was identified as the most efficient influx transporter for docetaxel. None of the observed genotypes (SLCO1B3, ABCB1, and ABCC2) was related with docetaxel clearance in 92 white patients (P > 0.17). However, the simultaneous presence of the CYP3A4*1B and CYP3A5*1A alleles was associated with a 64% increase in docetaxel clearance (P = 0.0015), independent of both sex and CYP3A activity (as determined using the erythromycin breath test). This haplotype was also associated with increased midazolam clearance in another population (P = 0.0198). An analysis of the CYP3A locus among CEPH-HapMap samples revealed that CYP3A4*1B is present exclusively among a subset of CYP3A5 expressors. Therefore, future studies should first stratify the population on the basis of CYP3A5 genotype and then compare CYP3A activity between individuals with and without the CYP3A4*1B allele.

ADME transcriptome in Hispanic versus White donor livers: evidence of a globally enhanced NR1I3 (CAR, constitutive androstane receptor) gene signature in Hispanics.

Previous studies have found that, compared with Whites, Hispanic donor livers had elevated expression of CYP2 enzymes, gene products regulated by the constitutive androstane receptor (CAR). The objectives of the current study were to determine (1) the CAR activation signature in human liver (2) whether other drug detoxification (absorption, distribution, metabolism and excretion (ADME)) genes were differentially expressed in Hispanic versus White livers, and (3) the extent of overlap in the CAR and Hispanic liver transcriptomes. The CAR transcriptome (ADME genes differentially expressed following phenobarbital versus vehicle treatment of human hepatocytes) and the Hispanic liver transcriptome (ADME genes differentially expressed in Hispanic versus White livers) were identified using Affymetrix oligonucleotide arrays. Quantitative real-time polymerase chain reaction (PCR) was used to verify candidate genes in a larger sample size. Comparison of the CAR and Hispanic liver ADME transcriptomes revealed a significant association between the gene changes. Sixty-four per cent of the ADME genes induced more than twofold by phenobarbital were also induced in Hispanics, and 14% of the ADME genes repressed more than twofold by phenobarbital were repressed in Hispanics. In conclusion, compared with Whites, Hispanic donor livers have increased expression of many genes that are transcriptionally regulated by CAR. This result has practical implications to the drug treatment of Hispanic patients.

Genetic polymorphism of CYP2D6 in North Indian subjects.

OBJECTIVES: CYP2D6 polymorphism of clinical relevance occurs with variable frequency in different ethnic groups. Since this polymorphism has not been studied in a North Indian population, the present study was undertaken. METHODS: One hundred healthy unrelated North Indian subjects received 30 mg dextromethorphan (DM) orally at bed-time. The amounts of DM and its metabolite, dextrorphan (DR), excreted in 8 h urine were estimated by high performance liquid chromatography. Metabolic ratio (DM/DR excreted in 8 h) was used as an index of the metabolic status of an individual. RESULTS: The analysis of the data by frequency distribution histogram, probit and NTV plots demonstrated bimodal distribution of the North Indian subjects with respect to hepatic CYP2D6. Out of 100 subjects, 97 were extensive metabolizers (EMs), whereas three were poor metabolizers (PMs). EMs and PMs excreted 29.82 and 2.67 micromol DR (mean value) and 2.59 and 8.82 micromol DM (mean value) in 8 h, respectively. MR and log MR was 197- and 2.2-fold higher in PMs versus EMs. The antimode value of zero was determined by visual observation in frequency distribution histogram and inflection point in probit plot. CONCLUSION: From this study, it can be concluded that the PM phenotype of CYP2D6 occurs with a frequency of 3% (95% confidence interval of 0.33%-6.33%) in North Indians.

Gonadotrophin-induced oocyte maturation in the catfish, Heteropneustes fossilis (Bloch), requires steroidogenesis in both interrenal and ovary.

Intact or hypophysectomized catfish, Heteropneustes fossilis, were administered a single injection of ovulating doses of ovine luteinizing hormone (LH: 200 micrograms/100 g body wt) or partially-purified salmon gonadotrophin (SG-G100: 100 micrograms/100 g body wt). Identical groups of catfish were injected with a suboptimal dose of LH (20 micrograms/100 g body wt) or with porcine adrenocorticotrophin (ACTH: 0.25 IU/100 g body wt). At short intervals after hormone administration, plasma and/or ovarian tissue were analyzed for cortisol (F), testosterone (T), and estradiol-17 beta (E2) by radioimmunoassay. Following administration of ovulatory doses of gonadotrophins, plasma levels of the three steroids increased in a sequential manner; high levels were recorded between 15 and 45 min for F and between 45 and 90 min for T and E2. In gonadotrophin-injected catfish, the ovarian content of T and E2 increased during the first 45 min and then declined up to 90 min even as their titers in the plasma were still increasing. When ovarian pieces containing yolky oocytes were incubated in vitro with LH (50 micrograms/ml), levels of T and E2 in the culture medium increased in a sequential manner similar to that observed following in vivo administration of gonadotrophin. No significant change was observed in the levels of any of the three steroids in catfish injected with a suboptimal dose of LH. In catfish treated with ACTH, plasma F levels increased 40-fold, whereas T and E2 levels did not change; ACTH administration had no effect on oocyte maturation. These results suggest that gonadotrophin, at doses sufficient to evoke oocyte maturation, acts at two loci, the interrenal and the ovary. The results also suggest that the failure of ACTH to induce oocyte maturation is due to its inability to act on the ovary.

Circannual and circadian variations in plasma levels of steroids (cortisol, estradiol-17 beta, estrone, and testosterone) correlated with the annual gonadal cycle in the catfish, Heteropneustes fossilis (Bloch).

Circannual and circadian variations in plasma levels of steroids were estimated by radioimmunoassay in the female and male catfish, Heteropneustes fossilis, over two consecutive annual reproductive cycles. In the female catfish, testosterone (T), estradiol-17 beta (E2), and estrone (E1) were detectable in the plasma only during the reproductively active (preparatory through spawning) period and their levels increased during vitellogenesis. In the fully gravid catfish, when vitellogenesis was nearly complete, levels of E2 declined but those of T continued to increase suggesting a product-precursor relationship between the two steroids. Plasma cortisol (F) was detectable throughout the year and exhibited three peaks coinciding with summer, monsoon, and winter; the first and second peaks coincided with vitellogenesis and spawning, respectively. In the male catfish, changes in plasma T and F levels closely paralleled the seasonal recrudescence and activity of testes and seminal vesicles. After spawning, gonads regressed and levels of sex steroids declined sharply. In the absence of natural spawning due to scanty monsoon rains, as during the second year of this study, gonadal regression was delayed and the sex steroids persisted in the plasma well beyond the normal spawning season. In addition, the first two peaks of F levels merged to form a plateau extending from the preparatory period until the late spawning period. The three sex steroids (T, E2, and E1) exhibited identical circadian rhythms; a major peak occurred at the onset of the dark phase (20:00 hr) and a minor peak was generally observed 4 hr after the onset of the light phase (12:00 hr). The amplitude of rhythms was greatest during the prespawning and the spawning periods. Cortisol peak levels generally alternated with those of sex steroids. Steroid rhythms show rather precise correlations with environmental factors such as photoperiod, temperature, and rainfall as well as with seasonal reproductive activity in both sexes of catfish.