Immunization with a whole cell vaccine reduces pneumococcal nasopharyngeal density and shedding, and middle ear infection in mice.

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. WCVs would be beneficial in low and middle-income settings where pneumococcal carriage in children is high.

Reptile-associated Salmonella urinary tract infection: a case report.

We present an 18-year-old woman with a urinary tract infection caused by Salmonella Oranienburg. S. Oranienburg was isolated from her pet snake and confirmed as the source of infection using whole genome sequencing. Our case demonstrates the risk of acquiring reptile-associated salmonellosis and stretches the need for awareness to prevent these infections.

Diagnostic work-up of urinary tract infections in pregnancy: study protocol of a prospective cohort study.

INTRODUCTION: Symptoms of urinary tract infections in pregnant women are often less specific, in contrast to non-pregnant women where typical clinical symptoms of a urinary tract infection are sufficient to diagnose urinary tract infections. Moreover, symptoms of a urinary tract infection can mimic pregnancy-related symptoms, or symptoms of a threatened preterm birth, such as contractions. In order to diagnose or rule out a urinary tract infection, additional diagnostic testing is required.The diagnostic accuracy of urine dipstick analysis and urine sediment in the diagnosis of urinary tract infections in pregnant women has not been ascertained nor validated. METHODS AND ANALYSIS: In this single-centre prospective cohort study, pregnant women (≥16 years old) with a suspected urinary tract infection will be included. The women will be asked to complete a short questionnaire regarding complaints, risk factors for urinary tract infections and baseline characteristics. Their urine will be tested with a urine dipstick, urine sediment and urine culture. The different sensitivities and specificities per test will be assessed. Our aim is to evaluate and compare the diagnostic accuracy of urine dipstick analysis and urine sediment in comparison with urine culture (reference test) in pregnant women. In addition, we will compare these tests to a predefined 'true urinary tract infection', to distinguish between a urinary tract infection and asymptomatic bacteriuria. ETHICS AND DISSEMINATION: Approval was requested from the Medical Ethics Review Committee of the Academic Medical Centre; an official approval of this study by the committee was not required. The outcomes of this study will be published in a peer-reviewed journal.

Diapers as Promising Alternative Collection Method for Urine Specimens in Nursing Home Residents: A Noninferiority Study.

OBJECTIVE: There is unmet need for an easy, noninvasive urine collection method to diagnose urinary tract infections (UTIs) in nursing home residents suffering from urinary incontinence or cognitive impairments. UTIs are highly prevalent in nursing home residents, and urine specimen collection can be difficult. The objective of this study was to assess if urine specimens collected from super-absorbing incontinence pads (adult diapers) are a reliable collection method for UTI diagnosis. DESIGN: This was a paired noninferiority laboratory study, in which pairing refers to UTI diagnostics performed directly using clinical urine specimens (reference specimen) and indirectly using urine extracted from diapers (diaper specimen). SETTING AND PARTICIPANTS: In this study, remnants of 250 clinical urine specimens were used to assess noninferiority in diagnosing UTIs, based on a 1-sided type I error of 2.5%, a power of 90%, and a noninferiority margin of 15%. METHODS: Urine specimens were poured on super-absorbing disposable adult diapers and extracted after 3 hours, to use for dipstick urinalysis and bacterial culture. UTIs were defined as presence of leukocytes and a positive bacterial culture. Noninferiority was assessed by calculating a Wald-type test statistic. RESULTS: Noninferiority was established for diagnosing UTIs in diaper specimens, and for each of its components (dipstick leukocyte detection and bacterial culture positivity). Positive bacterial cultures were found in 72 (29.0%) diaper specimens compared with 65 (26.2%) reference specimens (difference -2.8%, 97.5% CI -7.1% to 1.5%). Leukocytes were present in 162 (64.8%) diaper specimens, compared with 175 (70.0%) reference specimens (difference -5.7%, 97.5% CI: -10.6% to -0.7%). CONCLUSION AND IMPLICATIONS: Our results on diagnosing UTIs, by dipstick analysis and bacterial cultures, using super-absorbing adult diapers are promising. Before translation into clinical practice, further studies are needed to evaluate the risk of bacterial contamination by wearing adult diapers, possibly resulting in overdiagnosis of UTI.

Pharmacokinetic/pharmacodynamic target attainment of ciprofloxacin in adult patients on general wards with adequate and impaired renal function.

Limited prospective data on pharmacokinetic/pharmacodynamic (PK/PD) target attainment of ciprofloxacin in patients with adequate and impaired renal function (eGFR <30 mL/min/1.73m2) are available in the literature. We aimed to investigate whether the PK/PD target (AUC/MIC ≥125) is attained in patients with adequate and impaired renal function receiving regular and reduced ciprofloxacin doses. This prospective observational cohort study included adult patients on general wards treated with ciprofloxacin. Three blood samples per patient were obtained for ciprofloxacin concentration measurement. Individual AUCs were calculated using a population PK model developed by non-linear mixed-effects modelling. Forty patients were included, of whom eight had impaired renal function and were treated with a guideline-recommended reduced dose. Using the clinical breakpoint MIC of the most isolated bacteria (Escherichia coli, 0.25 mg/L), AUC0-24/MIC ≥125 was attained in 13/32 (41%) patients with adequate renal function receiving regular doses and in 1/8 (13%) patients with impaired renal function receiving reduced doses. Median drug exposure (AUC0-24) for patients with impaired renal function was 19.0 [interquartile range (IQR) 14.2-23.3] mg/L•h, which was statistically significantly lower than that for patients with adequate renal function [29.3 (IQR 25.0-36.0) mg/L•h] (P < 0.01). AUC0-24/MIC ≥125 is not attained in the majority of adult patients on general wards for clinically relevant bacteria with MICs at or just below the clinical breakpoint. The risk of not attaining the target appears to be highest in patients with impaired renal function receiving guideline-recommended reduced doses, as drug exposure is significantly lower in these patients.

Reduced CETP glycosylation and activity in patients with homozygous B4GALT1 mutations.

The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying the effects of defective glycosylation on plasma lipids in patients with B4GALT1-CDG, caused by a mutation in B4GALT1 with defective N-linked glycosylation. We studied plasma lipids, cholesteryl ester transfer protein (CETP) glyco-isoforms with isoelectric focusing followed by a western blot and CETP activity in three known B4GALT1-CDG patients and compared them with 11 age- and gender-matched, healthy controls. B4GALT1-CDG patients have significantly lowered non-high density lipoprotein cholesterol (HDL-c) and total cholesterol to HDL-c ratio compared with controls and larger HDL particles. Plasma CETP was hypoglycosylated and less active in B4GALT1-CDG patients compared to matched controls. Our study provides insight into the role of protein glycosylation in human lipoprotein homeostasis. The hypogalactosylated, hypo-active CETP found in patients with B4GALT1-CDG indicates a role of protein galactosylation in regulating plasma HDL and LDL. Patients with B4GALT1-CDG have large HDL particles probably due to hypogalactosylated, hypo-active CETP.

Sensitivity of point-of-care testing C reactive protein and procalcitonin to diagnose urinary tract infections in Dutch nursing homes: PROGRESS study protocol.

INTRODUCTION: Suspected urinary tract infection (UTI) ranks among the most common reasons for antibiotic use in nursing homes. However, diagnosing UTI in this setting is challenging because UTI often presents with non-specific symptomatology. Moreover asymptomatic bacteriuria is common in elderly, which complicates attribution of causality to detection of bacteria in urine. These diagnostic challenges contribute to overuse of antibiotics and emergence of antimicrobial resistance in nursing homes. Given the diagnostic challenges, there is a need for point-of-care (POC) diagnostic tests to support clinical rules for diagnosing UTI. Procalcitonin (PCT) and C reactive protein (CRP) are inflammatory blood markers that have been proven useful to support diagnosis and monitoring of (bacterial) respiratory tract infections and sepsis. While limited studies suggest their usefulness in supporting UTI diagnosis, their utility has not been studied in elderly populations for this purpose. METHODS AND ANALYSIS: In a 24-month matched prospective study, 'PROGRESS' will assess and compare the sensitivity of rapid POC measurements of blood CRP and PCT levels to support clinical rules for diagnosing UTI in nursing home residents. The primary outcome measure is sensitivity of the POC tests to identify patients with true UTI based on the predefined definition, as derived from receiver operating curves. ETHICS AND DISSEMINATION: This study will be conducted in accordance with Good Clinical Practice guidelines and the principles of the Declaration of Helsinki. The study protocol is approved by the Medical Ethical Committee of Amsterdam UMC location VUmc with reference number 2017.350 and National Central Committee on Research involving Human Subjects with reference number NL62067.029.17. TRIAL REGISTRATION NUMBER: NTR6467.

N-Glycosylation Defects in Humans Lower Low-Density Lipoprotein Cholesterol Through Increased Low-Density Lipoprotein Receptor Expression.

BACKGROUND: The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. METHODS: We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. RESULTS: We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. CONCLUSIONS: Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

Barriers and facilitators and the need for a clinical guideline for microbiological diagnostic testing in the hospital: a qualitative and quantitative study.

The appropriate use of microbiological investigations is an important cornerstone of antibiotic stewardship programmes, but receives relatively limited attention. This study aimed to identify influencing factors in performing microbiological diagnostic tests and to assess the need for a clinical guideline. We performed a qualitative (focus group) and quantitative (online questionnaire survey) study among medical specialists and residents to identify physicians' considerations in performing microbiological diagnostic tests and to assess the need for a diagnostic guideline. The questionnaire consisted of 14 statements, divided into three categories: knowledge, influencing factors and presence of guidelines. The questionnaire was sent to physicians of the departments of internal medicine, intensive care, paediatrics and pulmonology in five hospitals in the Netherlands. Sub-analyses for medical specialists versus residents and for paediatric versus non-paediatric departments were performed. We included 187 completed questionnaires in our analyses. The physicians reported having adequate knowledge on methods, time-to-result and accuracy, but inadequate knowledge on costs of the tests. Patients' clinical condition, comorbidity, local guidelines and accuracy of tests were appraised as the four most important influencing factors to perform tests. Over 70% (132/187) of physicians reported being interested in a guideline for microbiological diagnostic testing. Fifteen physicians (8.0%) provided additional comments. This study identifies the influencing factors to microbiological testing and shows the demand for a clinical guideline among physicians. IMPORTANCE: Microbiological diagnostic tests are an important cornerstone within antibiotic stewardship programmes [1-5]. These programmes aim to ameliorate the appropriate use of antibiotics and thus improve clinical outcomes of infectious diseases, whilst reducing the emergence of antimicrobial resistance [6]. However, inappropriate microbiological testing is a widely recognised problem [7-12], and influencing factors to testing have not been studied in the past. Our research shows the demand for a clinical guideline among physicians, and it identifies their influencing factors to testing. These results can be used to create a clinical guideline for microbiological diagnostic testing, thus supporting antibiotic stewardship programmes and reducing antimicrobial resistance.

High epidemic burden of RSV disease coinciding with genetic alterations causing amino acid substitutions in the RSV G-protein during the 2016/2017 season in The Netherlands.

BACKGROUND: We found amino acid substitutions in the Gglycoprotein of respiratory syncytial virus (RSV) A during the 2016/2017 epidemic in The Netherlands. OBJECTIVES: We evaluated whether these alterations led to increased RSV incidence and disease burden. STUDY DESIGN: We sequenced the gene encoding the G-protein of prospectively collected clinical specimens from secondary care adult patients testing positive for RSV during the 2016/2017 and 2017/2018 epidemic RSV season. We evaluated associations between genetic, clinical and epidemiological data. RESULTS: We included 49 RSV strains. In 2016/2017 28 strains were included, 20 community acquired RSV-A, 5 hospital acquired RSV-A and 3 community acquired RSV-B. In 2017/2018 21 strains were included, 8 community acquired RSV-A and 13 community acquired RSV-B. G-proteins of 10 out of the 20 community acquired 2016/2017 RSV-A strains shared a set of eight novel amino acid substitutions of which seven in mucin-like regions 1 and 2 and one in the heparin binding domain. This genetic variant was no longer detected among 2017/2018 RSV-A strains. Among patients carrying the novel RSV-A strain-type, 30% died. CONCLUSIONS: A set of eight amino acid substitutions was found in 50% of the 2016/2017 community acquired RSV-A G-proteins. This combination of substitutions was globally never observed before. The appearance of this new strain-type coincided with an increased RSV peak in The Netherlands and was associated with higher disease severity. The transient character of this epidemic strain-type suggests rapid clearance of this lineage in our study community.