Proteogenomic analysis of chemo-refractory high-grade serous ovarian cancer.

To improve the understanding of chemo-refractory high-grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one discovery and two validation cohorts across two biospecimen types (formalin-fixed paraffin-embedded and frozen). We identified a 64-protein signature that predicts with high specificity a subset of HGSOCs refractory to initial platinum-based therapy and is validated in two independent patient cohorts. We detected significant association between lack of Ch17 loss of heterozygosity (LOH) and chemo-refractoriness. Based on pathway protein expression, we identified 5 clusters of HGSOC, which validated across two independent patient cohorts and patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic vulnerabilities.

Dissecting the contribution of host genetics and the microbiome in complex behaviors.

The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2-/- model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2-/- mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2-/- mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them.

exRNA Atlas Analysis Reveals Distinct Extracellular RNA Cargo Types and Their Carriers Present across Human Biofluids.

To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.

Monitoring Both Extended and Tryptic Forms of Stable Isotope-Labeled Standard Peptides Provides an Internal Quality Control of Proteolytic Digestion in Targeted Mass Spectrometry-Based Assays.

Targeted mass spectrometry (MS)-based proteomic assays, such as multiplexed multiple reaction monitoring (MRM)-MS assays, enable sensitive and specific quantification of proteotypic peptides as stoichiometric surrogates for proteins. Efforts are underway to expand the use of MRM-MS assays in clinical environments, which requires a reliable strategy to monitor proteolytic digestion efficiency within individual samples. Towards this goal, extended stable isotope-labeled standard (SIS) peptides (hE), which incorporate native proteolytic cleavage sites, can be spiked into protein lysates prior to proteolytic (trypsin) digestion, and release of the tryptic SIS peptide (hT) can be monitored. However, hT measurements alone cannot monitor the extent of digestion and may be confounded by matrix effects specific to individual patient samples; therefore, they are not sufficient to monitor sample-to-sample digestion variability. We hypothesized that measuring undigested hE, along with its paired hT, would improve detection of digestion issues compared to only measuring hT. We tested the ratio of the SIS pair measurements, or hE/hT, as a quality control (QC) metric of trypsin digestion for two MRM assays: a direct-MRM (398 targets) and an immuno-MRM (126 targets requiring immunoaffinity peptide enrichment) assay, with extended SIS peptides observable for 54% (216) and 62% (78) of the targets, respectively. We evaluated the quantitative bias for each target in a series of experiments that adversely affected proteolytic digestion (e.g., variable digestion times, pH, and temperature). We identified a subset of SIS pairs (36 for the direct-MRM, 7 for the immuno-MRM assay) for which the hE/hT ratio reliably detected inefficient digestion that resulted in decreased assay sensitivity and unreliable endogenous quantification. The hE/hT ratio was more responsive to a decrease in digestion efficiency than a metric based on hT measurements alone. For clinical-grade MRM-MS assays, this study describes a ready-to-use QC panel and also provides a road map for designing custom QC panels.

Testicular germ cell tumors arise in the absence of sex-specific differentiation.

In response to signals from the embryonic testis, the germ cell intrinsic factor NANOS2 coordinates a transcriptional program necessary for the differentiation of pluripotent-like primordial germ cells toward a unipotent spermatogonial stem cell fate. Emerging evidence indicates that genetic risk factors contribute to testicular germ cell tumor initiation by disrupting sex-specific differentiation. Here, using the 129.MOLF-Chr19 mouse model of testicular teratomas and a NANOS2 reporter allele, we report that the developmental phenotypes required for tumorigenesis, including failure to enter mitotic arrest, retention of pluripotency and delayed sex-specific differentiation, were exclusive to a subpopulation of germ cells failing to express NANOS2. Single-cell RNA sequencing revealed that embryonic day 15.5 NANOS2-deficient germ cells and embryonal carcinoma cells developed a transcriptional profile enriched for MYC signaling, NODAL signaling and primed pluripotency. Moreover, lineage-tracing experiments demonstrated that embryonal carcinoma cells arose exclusively from germ cells failing to express NANOS2. Our results indicate that NANOS2 is the nexus through which several genetic risk factors influence tumor susceptibility. We propose that, in the absence of sex specification, signals native to the developing testis drive germ cell transformation.

Deconvolution of cancer cell states by the XDec-SM method.

Proper characterization of cancer cell states within the tumor microenvironment is a key to accurately identifying matching experimental models and the development of precision therapies. To reconstruct this information from bulk RNA-seq profiles, we developed the XDec Simplex Mapping (XDec-SM) reference-optional deconvolution method that maps tumors and the states of constituent cells onto a biologically interpretable low-dimensional space. The method identifies gene sets informative for deconvolution from relevant single-cell profiling data when such profiles are available. When applied to breast tumors in The Cancer Genome Atlas (TCGA), XDec-SM infers the identity of constituent cell types and their proportions. XDec-SM also infers cancer cells states within individual tumors that associate with DNA methylation patterns, driver somatic mutations, pathway activation and metabolic coupling between stromal and breast cancer cells. By projecting tumors, cancer cell lines, and PDX models onto the same map, we identify in vitro and in vivo models with matching cancer cell states. Map position is also predictive of therapy response, thus opening the prospects for precision therapy informed by experiments in model systems matched to tumors in vivo by cancer cell state.

Perillyl alcohol reduces parasite sequestration and cerebrovascular dysfunction during experimental cerebral malaria.

Cerebral malaria (CM) is a severe immunovasculopathy which presents high mortality rate (15-20%), despite the availability of artemisinin-based therapy. More effective immunomodulatory and/or antiparasitic therapies are urgently needed. Experimental Cerebral Malaria (ECM) in mice is used to elucidate aspects involved in this pathology since manifests many of the neurological features of CM. In the present study, we evaluated the potential mechanisms involved in the protection afforded by perillyl alcohol (POH) in mouse strains susceptible to CM caused by Plasmodium berghei ANKA (PbA) infection through intranasal preventive treatment. Additionally, to evaluate the interaction of POH with the cerebral endothelium using an in vitro model of human brain endothelial cells (HBEC). Pharmacokinetic approaches demonstrated constant and prolonged levels of POH in the plasma and brain after a single intranasal dose. Treatment with POH effectively prevented vascular dysfunction. Furthermore, treatment with POH reduced the endothelial cell permeability and PbA s in the brain and spleen. Finally, POH treatment decreased the accumulation of macrophages and T and B cells in the spleen and downregulated the expression of endothelial adhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain. POH is a potent monoterpene that prevents cerebrovascular dysfunction in vivo and in vitro, decreases parasite sequestration, and modulates different processes related to the activation, permeability, and integrity of the blood brain barrier (BBB), thereby preventing cerebral oedema and inflammatory infiltrates.

Internal Standard Triggered-Parallel Reaction Monitoring Mass Spectrometry Enables Multiplexed Quantification of Candidate Biomarkers in Plasma.

Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Previously, mass spectrometry-based approaches have been used to fill this gap but feature poor quantitative performance and were generally limited to hundreds of proteins. Here, we demonstrate the capability of an internal standard triggered-parallel reaction monitoring (IS-PRM) assay to greatly expand the numbers of candidates that can be tested with improved quantitative performance. The assay couples immunodepletion and fractionation with IS-PRM and was developed and implemented in human plasma to quantify 5176 peptides representing 1314 breast cancer biomarker candidates. Characterization of the IS-PRM assay demonstrated the precision (median % CV of 7.7%), linearity (median R2 > 0.999 over 4 orders of magnitude), and sensitivity (median LLOQ < 1 fmol, approximately) to enable rank-ordering of candidate biomarkers for validation studies. Using three plasma pools from breast cancer patients and three control pools, 893 proteins were quantified, of which 162 candidate biomarkers were verified in at least one of the cancer pools and 22 were verified in all three cancer pools. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

Memory like NK cells display stem cell like properties after Zika virus infection.

NK cells have been shown to display adaptive traits such as memory formation akin to T and B lymphocytes. Here we show that Zika virus infection induces memory like NK cells that express CD27. Strikingly, these cells exhibit stem-like features that include expansion capacity, self-renewal pathway, differentiation into effector cells, longer telomeres and gene signature associated with hematopoietic stem cell (HSC) progenitors. This subset shared transcriptional and epigenetic changes with memory CD8 T cells, stem cells and stem like T cells. These NK cells with memory and stem cell features, which we term "NK memory stem cells", demonstrated greater antiviral potential than CD27- or naïve CD27+ NK when adoptively transferred to Zika infected mice. Our results also suggest a role for the transcription factor TCF-1 in memory and stemness features of this NK subset. This study defines a unique TCF1hi CD27+ NK subset with memory capacity and stem cell features that play a role in antiviral immunity.

Costimulatory CD226 Signaling Regulates Proliferation of Memory-like NK Cells in Healthy Individuals with Latent Mycobacterium tuberculosis Infection.

It is now widely accepted that NK cells can acquire memory, and this makes them more effective to protect against some pathogens. Prior reports indicate memory-like NK cells (mlNKs) in murine model of Mycobacterium tuberculosis (Mtb) as well as in healthy individuals with latent TB infection (LTBI). The increased expression of CD226 was evident in mlNKs from LTBI+ people after stimulation with γ-irradiated Mtb (γ-Mtb). We thus evaluated the contribution of costimulatory CD226 signaling in the functionality of mlNKs in LTBI+ people. We found that blockade of CD226 signaling using the antibody- or CRISPR/Cas9-mediated deletion of the CD226 gene in NK cells diminished the proliferation of mlNKs from LTBI+ people. Blocking CD226 signaling also reduced the phosphorylation of FOXO1 and cMyc expression. Additionally, cMyc inhibition using a chemical inhibitor reduced proliferation by mlNKs from LTBI+ people. Moreover, blocking CD226 signaling reduced glycolysis in NK cells, and the inhibition of glycolysis led to reduced effector function of mlNKs from LTBI+ people. Overall, our results provide a role for CD226 signaling in mlNK responses to Mtb.

Infections after spine instrumentation: effectiveness of short antibiotic treatment in a large multicentre cohort.

BACKGROUND AND OBJECTIVES: Available information about infection after spine instrumentation (IASI) and its management are scarce. We aimed to analyse DAIR (debridement, antibiotics and implant retention) prognosis and evaluate effectiveness of short antibiotic courses on early forms. METHODS: Multicentre retrospective study of patients with IASI managed surgically (January 2010-December 2016). Risk factors for failure were analysed by multivariate Cox regression and differences between short and long antibiotic treatment were evaluated with a propensity score-matched analysis. RESULTS: Of the 411 IASI cases, 300 (73%) presented in the first month after surgery, 48 in the second month, 22 in the third and 41 thereafter. Infections within the first 2 months (early cases) occurred mainly to older patients, with local inflammatory signs and predominance of Enterobacteriaceae, unlike those in the later periods. When managed with DAIR, prognosis of early cases was better than later ones (failure rate 10.4% versus 26.1%, respectively; P = 0.02). Risk factors for DAIR failure in early cases were female sex, Charlson Score, large fusions (>6 levels) and polymicrobial infections (adjusted HRs of 2.4, 1.3, 2.6 and 2.26, respectively). Propensity score matching proved shorter courses of antibiotics (4-6 weeks) as effective as longer courses (failure rates 11.4% and 10.5%, respectively; P = 0.870). CONCLUSIONS: IASIs within the first 2 months could be managed effectively with DAIR and shorter antibiotic courses. Clinicians should be cautious when faced with patients with comorbidities, large fusions and/or polymicrobial infections.

Measurement uncertainty of ß-lactam antibiotics results: estimation and clinical impact on therapeutic drug monitoring.

Background Despite that measurement uncertainty data should facilitate an appropriate interpretation of measured values, there are actually few reported by clinical laboratories. We aimed to estimate the measurement uncertainty of some ß-lactam antibiotics (ß-LA), and to evaluate the impact of reporting the measurement uncertainty on clinicians' decisions while guiding antibiotic therapy. Methods Measurement uncertainty of ß-LA (aztreonam [ATM], cefepime [FEP], ceftazidime [CAZ], and piperacillin [PIP]) values, obtained by an UHPLC-MS/MS based-method, was estimated using the top-down approach called the single laboratory validation approach (EUROLAB guidelines). Main uncertainty sources considered were related to calibrators' assigned values, the intermediate precision, and the bias. As part of an institutional program, patients with osteoarticular infections are treated with ß-LA in continuous infusion and monitored to assure values at least 4 times over the minimal inhibitory concentration (4×MIC). We retrospectively evaluated the impact of two scenarios of laboratory reports on clinicians' expected decisions while monitoring the treatment: reports containing only the ß-LA values, or including the ß-LA coverage intervals (ß-LA values and their expanded measurement uncertainties). Results The relative expanded uncertainties for ATM, FEP, CAZ and PIP were lower than 26.7%, 26.4%, 28.8%, and 25.5%, respectively. Reporting the measurement uncertainty, we identified that clinicians may modify their decision especially in cases where 4×MIC values were within the ß-LA coverage intervals. Conclusions This study provides a simple method to estimate the measurement uncertainty of ß-LA values that can be easily applied in clinical laboratories. Further studies should confirm the potential impact of reporting measurement uncertainty on clinicians' decision-making while guiding antibiotic therapy.

Impact of ß-Lactam and Daptomycin Combination Therapy on Clinical Outcomes in Methicillin-susceptible Staphylococcus aureus Bacteremia: A Propensity Score-matched Analysis.

BACKGROUND: Mortality rates from Staphylococcus aureus bacteremia are high and have only modestly improved in recent decades. We compared the efficacies of a ß-lactam in combination with daptomycin (BL/D-C) and ß-lactam monotherapy (BL-M) in improving clinical outcomes in methicillin-susceptible S. aureus (MSSA) bacteremia. METHODS: A retrospective cohort study of MSSA bacteremia was performed in a tertiary hospital from January 2011 to December 2017. Patients receiving BL/D-C and BL-M were compared to assess 7-, 30-, and 90-day mortality rates. A 1:2 propensity score matching analysis was performed. Differences were assessed using Cox regression models. RESULTS: Of the 514 patients with MSSA bacteremia, 164 were excluded as they had received combination therapies other than BL/D-C, had pneumonia, or died within 48 hours of admission. Of the remaining 350 patients, 136 and 214 received BL/D-C and BL-M, respectively. BL/D-C patients had higher Pitt scores and persistent bacteremia more often than BL-M patients. In the raw analysis, there were no differences in mortality rates between groups. After propensity score matching, there were no significant differences between the BL/D-C (110 patients) and BL-M (168 patients) groups for all-cause mortality rates at 7 days (8.18% vs 7.74%; P = 1.000), 30 days (17.3% vs 16.1%; P = .922), and 90 days (22.7% vs 23.2%; P = 1.000), even in a subanalysis of patients with high-risk source of infection and in a subgroup excluding catheter-related bacteremia. CONCLUSIONS: BL/D-C failed to reduce mortality rates in patients with MSSA bacteremia. Treatment strategies to improve survival in MSSA bacteremia are urgently needed.

Comparative Antibiofilm Efficacy of Meropenem Alone and in Combination with Colistin in an In Vitro Pharmacodynamic Model by Extended-Spectrum-ß-Lactamase-Producing Klebsiella pneumoniae.

We compared the efficacies of meropenem alone and in combination with colistin against two strains of extended-spectrum-ß-lactamase-producing Klebsiella pneumoniae, using an in vitro pharmacodynamic model that mimicked two different biofilm conditions. Meropenem monotherapy achieved remarkable efficacy (even a bactericidal effect) under all conditions, whereas colistin was almost inactive and resistance emerged. The addition of colistin to meropenem produced no relevant benefits, in contrast to experiences with other microorganisms.

Predicting clone genotypes from tumor bulk sequencing of multiple samples.

Motivation: Analyses of data generated from bulk sequencing of tumors have revealed extensive genomic heterogeneity within patients. Many computational methods have been developed to enable the inference of genotypes of tumor cell populations (clones) from bulk sequencing data. However, the relative and absolute accuracy of available computational methods in estimating clone counts and clone genotypes is not yet known. Results: We have assessed the performance of nine methods, including eight previously-published and one new method (CloneFinder), by analyzing computer simulated datasets. CloneFinder, LICHeE, CITUP and cloneHD inferred clone genotypes with low error (<5% per clone) for a majority of datasets in which the tumor samples contained evolutionarily-related clones. Computational methods did not perform well for datasets in which tumor samples contained mixtures of clones from different clonal lineages. Generally, the number of clones was underestimated by cloneHD and overestimated by PhyloWGS, and BayClone2, Canopy and Clomial required prior information regarding the number of clones. AncesTree and Canopy did not produce results for a large number of datasets. Overall, the deconvolution of clone genotypes from single nucleotide variant (SNV) frequency differences among tumor samples remains challenging, so there is a need to develop more accurate computational methods and robust software for clone genotype inference. Availability and implementation: CloneFinder is implemented in Python and is available from https://github.com/gstecher/CloneFinderAPI. Supplementary information: Supplementary data are available at Bioinformatics online.

Clinical Use of Colistin in Biofilm-Associated Infections.

Biofilm is an adaptive bacterial strategy whereby microorganisms become encased in a complex glycoproteic matrix. The low concentration of oxygen and nutrients in this environment leads to heterogeneous phenotypic changes in the bacteria, with antimicrobial tolerance being of paramount importance. As with other antibiotics, the activity of colistin is impaired by biofilm-embedded bacteria. Therefore, the recommendation for administering high doses in combination with a second drug, indicated for planktonic infections, remains valid in this setting. Notably, colistin has activity against metabolically inactive biofilm-embedded cells located in the inner layers of the biofilm structure. This is opposite and complementary to the activity of other antimicrobials that are able to kill metabolically active cells in the outer layers of the biofilm. Several experimental models have shown a higher activity of colistin when used in combination with other agents, and have reported that this can avoid the emergence of colistin-resistant subpopulations. Most experience of colistin in biofilm-associated infections comes from patients with cystic fibrosis, where the use of nebulized colistin allows high concentrations to reach the site of the infection. However, limited clinical experience is available in other scenarios, such as osteoarticular infections or device-related central nervous system infections caused by multi-drug resistant microorganisms. In the latter scenario, the use of intraventricular or intrathecal colistin also permits high local concentrations and good clinical results. Overall, the efficacy of intravenous colistin seems to be poor, but its association with a second antimicrobial significantly increases the response rate. Given its activity against inner bioflm-embedded cells, its possible role in combination with other antibiotics, beyond last-line therapy situations, should be further explored.

Potentiating Hsp104 activity via phosphomimetic mutations in the middle domain.

Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase separation by TDP-43, FUS and α-synuclein connected to devastating neurodegenerative disorders. Subtle missense mutations in the Hsp104 MD can enhance activity, indicating that post-translational modification of specific MD residues might also potentiate Hsp104. Indeed, several serine and threonine residues throughout Hsp104 can be phosphorylated in vivo. Here, we introduce phosphomimetic aspartate or glutamate residues at these positions and assess Hsp104 activity. Remarkably, phosphomimetic T499D/E and S535D/E mutations in the MD enable Hsp104 to counter TDP-43, FUS and α-synuclein aggregation and toxicity in yeast, whereas T499A/V/I and S535A do not. Moreover, Hsp104T499E and Hsp104S535E exhibit enhanced ATPase activity and Hsp70-independent disaggregase activity in vitro. We suggest that phosphorylation of T499 or S535 may elicit enhanced Hsp104 disaggregase activity in a reversible and regulated manner.

Endocarditis associated with vertebral osteomyelitis and septic arthritis of the axial skeleton.

PURPOSE: The relationship between infective endocarditis (IE) and osteoarticular infections (OAIs) are not well known. We aimed to study the characteristics of patients with IE and OAIs, and the interactions between these two infections. METHODS: An observational study (1993-2014) which includes two cohorts: (1) patients with IE (n = 607) and (2) patients with bacteremic OAIs (n = 458; septic arthritis of peripheral and axial skeleton, and vertebral and peripheral osteomyelitis). These two cohorts were prospectively collected, and we retrospectively reviewed the clinical and microbiological variables. RESULTS: There were 70 cases of IE with concomitant OAIs, representing 11.5% of IE cases and 15% of bacteremic OAI cases. Among cases with IE, the associated OAIs mainly involved the axial skeleton (n = 54, 77%): 43 were vertebral osteomyelitis (61%), mainly caused by "less virulent" bacteria (viridans and bovis streptococci, enterococci, and coagulase-negative staphylococci), and 15 were septic arthritis of the axial skeleton (21%), which were mainly caused by Staphylococcus aureus. OAIs with involvement of the axial skeleton were associated with IE (adjusted OR = 2.2; 95% CI 1.1-4.3) independently of age, sex, and microorganisms. CONCLUSIONS: Among patients with IE, the associated OAIs mainly involve the axial skeleton. Transesophageal echocardiography should be carefully considered in patients presenting with these bacteremic OAIs.

Beta-lactams in continuous infusion for Gram-negative bacilli osteoarticular infections: an easy method for clinical use.

Continuous infusion (CI) of beta-lactams could optimize their pharmacokinetic/pharmacodynamic indices, especially in difficult-to-treat infections. PURPOSE: To validate an easy-to-use method to guide beta-lactams dosage in CI (formula). METHODS: A retrospective analysis was conducted of a prospectively collected cohort (n = 24 patients) with osteoarticular infections caused by Gram-negative bacilli (GNB) managed with beta-lactams in CI. Beta-lactams dose was calculated using a described formula (daily dose = 24 h × beta-lactam clearance × target "steady-state" concentration) to achieve concentrations above the MIC. We correlated the predicted concentration (Cpred = daily dose/24 h × beta-lactam clearance) with the patient's observed concentration (Cobs) measured by UPLC-MS/MS (Spearman's coefficient). RESULTS: The most frequent microorganism treated was P. aeruginosa (21 cases; 9 MDR). Beta-lactams in CI were ceftazidime (n = 14), aztreonam (7), and piperacillin/tazobactam (3), mainly used in combination (12 with colistin, 5 with ciprofloxacin) and administered without notable side effects. The plasma Cobs was higher overall than Cpred; the Spearman correlation between both concentrations was rho = 0.6 (IC 95%: 0.2-0.8) for all beta-lactams, and rho = 0.8 (IC 95%: 0.4-1) for those treated with ceftazidime. CONCLUSIONS: The formula may be useful in clinical practice for planning the initial dosage of beta-lactams in CI, while we await a systematic therapeutic drug monitoring. The use of beta-lactams in CI was safe.