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The COVID-19 pandemic highlights the importance of understanding facilitators for disease transmission. Events such as Carnival, characterized by large gatherings and extensive social interactions, have the potential to become ‘super spreading events' for respiratory infections. This paper aims to assess the impact of large gatherings on virus transmission, providing crucial insights for the development of effective public health strategies.

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This survey was undertaken to obtain insight in the attitude of Dutch physicians towards pathogenicity, diagnostic- and therapeutic approach towards Dientamoeba fragilisin children. Physicians were invited by e-mail for a questionnaire. A total of 211 of 450 physicians (46.9%) completed the questionnaire, including 67 general practitioners (GPs) and 144 pediatricians. Of all respondents, 175 of 211 (82.9%) considered D fragilisa “potential pathogen”, when other causes of gastro-intestinal complaints are ruled out. Only 16 of 211 (7.6%) performed diagnostic tests regularly. Diagnostic tests were performed by 162 of 211 (77%) of respondents in children with diarrhea and abdominal pain in consideration of duration of symptoms. Fecal polymerase chain reaction (PCR) was diagnostic modality of preference. Eighty-nine of 142 (62.7%) prescribed metronidazole as antibiotic of first choice. This study shows heterogeneity in clinical practice amongst Dutch physicians regarding diagnostic- and therapeutic approach of D fragilisin children. Different attitude towards pathogenicity and inconsistent guidelines could be causative factors.

Background: Infection with SARS-CoV-2 causes corona virus disease (COVID-19). The most standard diagnostic method is reverse transcription-polymerase chain reaction (RT-PCR) on a nasopharyngeal and/or an oropharyngeal swab. The high occurrence of false-negative results due to the non-presence of SARS-CoV-2 in the oropharyngeal environment renders this sampling method not ideal. Therefore, a new sampling device is desirable. This proof-of-principle study investigated the possibility to train machine-learning classifiers with an electronic nose (Aeonose) to differentiate between COVID-19-positive and negative persons based on volatile organic compounds (VOCs) analysis. Methods: Between April and June 2020, participants were invited for breath analysis when a swab for RT-PCR was collected. If the RT-PCR resulted negative, the presence of SARS-CoV-2-specific antibodies was checked to confirm the negative result. All participants breathed through the Aeonose for five minutes. This device contains metal-oxide sensors that change in conductivity upon reaction with VOCs in exhaled breath. These conductivity changes are input data for machine learning and used for pattern recognition. The result is a value between − 1 and + 1, indicating the infection probability. Results: 219 participants were included, 57 of which COVID-19 positive. A sensitivity of 0.86 and a negative predictive value (NPV) of 0.92 were found. Adding clinical variables to machine-learning classifier via multivariate logistic regression analysis, the NPV improved to 0.96. Conclusions: The Aeonose can distinguish COVID-19 positive from negative participants based on VOC patterns in exhaled breath with a high NPV. The Aeonose might be a promising, non-invasive, and low-cost triage tool for excluding SARS-CoV-2 infection in patients elected for surgery.

While substantial efforts have been made to optimize and standardize fecal metabolomics for studies in adults, the development of a standard protocol to analyze infant feces is, however, still lagging behind. Here, we present the development of a hands-on and robust protocol for proton 1H NMR spectroscopy of infant feces. The influence of extraction solvent, dilution ratio, homogenization method, filtration, and duration of centrifugation on the biochemical composition of infant feces was carefully evaluated using visual inspection of 1H NMR spectra in combination with multivariate statistical modeling. The optimal metabolomics protocol was subsequently applied on feces from seven infants collected at 8 weeks, 4, and 9 months of age. Interindividual variation was exceeding the variation induced by different fecal sample preparation methods, except for filtration. We recommend extracting fecal samples using water with a dilution ratio of 1:5 feces-to-water to homogenize using bead beating and to remove particulates using centrifugation. Samples collected from infants aged 8 weeks and 4 months showed elevated concentrations of milk oligosaccharide derivatives and lactic acid, whereas short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) were higher in the 9 month samples. The established protocol enables hands-on and robust analyses of the infant gut metabolome. The wide-ranging application of this protocol will facilitate interlaboratory comparison of infants’ metabolic profiles and finally aid in a better understanding of infant gut health.

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.

Nonspecific adsorption of biomolecules to solid surfaces, a process called biofouling, is a major concern in many biomedical applications. Great effort has been made in the development of antifouling polymer coatings that are capable of repelling the nonspecific adsorption of proteins, cells, and micro-organisms. In this respect, we herein contribute to understanding the factors that determine which polymer brush results in the best antifouling coating. To this end, we compared five different monomers: two sulfobetaines, a carboxybetaine, a phosphocholine, and a hydroxyl acrylamide. The antifouling coatings were analyzed using our previously described bead-based method with flow cytometry as the read-out system. This method allows for the quick and automated analysis of thousands of beads per second, enabling fast analysis and good statistics. We report the first direct comparison made between a sulfobetaine with opposite charges separated by two and three methylene groups and a carboxybetaine bearing two separating methylene groups. It was concluded that both the distance between opposite charges and the nature of the anionic groups have a distinct effect on the antifouling performance. Phosphocholines and simple hydroxyl acrylamides are not often compared with the betaines. However, here we found that they perform equally well or even better, yielding the following overall antifouling ranking: HPMAA ≥ PCMA-2 ≈ CBMAA-2 > SBMAA-2 > SBMAA-3 ≫ nonmodified beads (HPMAA being the best).

Aim:We aimed to study the mucosal microbiota of the appendix in a prospective appendicitis cohort and to compare the fecal microbiota of patients and controls. We hypothesized that the microbiota may be associated with susceptibility to appendicitis. Patients & methods:The fecal microbiota of 99 patients and 106 controls were characterized using 16S–23S intergenic spacer profiling. Richness, diversity and community structure were compared. The appendiceal microbiota from 90 patients was analyzed according to the severity of appendicitis. Results:Overall fecal microbial richness and diversity were similar in patients and controls, yet richness and diversity within the group of Firmicutes, Actinobacteria, Fusobacteriaand Verrucomicrobiaphyla were lower in patients. Discriminant analyses could correctly classify patients and controls with fair accuracy. No differences were found according to severity in appendiceal or fecal microbiota. Conclusion:This study demonstrates differences in the composition of intestinal microbiota of appendicitis patients and healthy individuals.

Background: Comparative data collection in transborder areas can contribute to informed decision making processes when dealing with borderless health threats such as pandemics, and thus help minimize the negative health effects for its citizens. To examine the pandemic response over time and the impact of infectious disease control in a cross-border setting, a prospective longitudinal study was conducted in the border area between Germany, Belgium and the Netherlands. In the spring of 2021, a random sample of 26,925 adult citizens selected from governmental registries was invited to collect a blood sample at home for SARS-CoV-2 antibody testing and to fill in an online questionnaire on attitudes and behaviour towards infection prevention measures, cross-border mobility, social network and support, COVID-19 self-reported infection(s) and symptoms, vaccination, general self-reported health and socio-demographics. In autumn 2021, participants were invited for a follow-up round. An online tool was developed to coordinate fieldwork procedures, real-time monitoring of participation and consultation of antibody test results. Furthermore, a helpdesk in all three languages for participants’ support was set up. Results: In the first round, 6,006 citizens in the Meuse-Rhine Euroregion participated. 15.3% of the invited citizens on the Belgian side of the border participated. In the Netherlands and Germany this was respectively 27% and 23.7%. In the follow-up round 4,286 (71.4%) citizens participated for the second time. The participation rate was highest in the age group 50–69 years and lowest in > 80 in all sub regions of the Meuse-Rhine Euroregion. More women participated than men. Overall, more blood samples were returned than completed questionnaires. In total, 3,344 citizens in the Meuse-Rhine Euroregion completed all components of participation in both rounds. Conclusions: The collection of comparative data can help better assess the pandemic response and the impact of infectious disease control in a cross-border area. Recommendations for a longitudinal cross-border study include a centralized online environment, mapping out potential challenges related to national regulations in the preparation phase and organizing regional coordination centres to create more familiarity and trust towards the involved organisations.

Higher antibody levels, in particular antibodies directed against the receptor-binding domain (RBD) of the spike protein, protect against re-infection with SARS-CoV-2. Quantitative antibody response data provide insight into population immunity and are essential for decision-making on booster-vaccination strategies. We aimed to identify predictors for higher antibody responses such as gender, age, experienced COVID-19-compatible symptoms, disease severity and exposure to pre-determined risk factors associated with SARS-CoV-2 seropositivity.

The faecal, intestinal and mucosal microbiome, and manipulation of it, is the subject of intense categorisation, collation and study. Advances have been aided by novel culture-independent analytical techniques and a focus on how aberrations in the microbiome are associated with many diseases. Dramatic manipulation of the microbiome is possible using faecal microbiota transplantation (FMT). FMT has been shown to have a definite clinical role in treating recurrent Clostridium difficileinfection, and there is accumulating clinical data for a possible role in treating ulcerative colitis. FMT preparation can only take place in a laboratory with licensed facilities and equipment allowing safe treatment and processing of appropriate donor samples. The future of FMT treatment may involve diseases of extra-intestinal origin. This review clarifies recent developments in research and provides a how-to guide for interested clinicians.

Oral cholera vaccination is used to induce immune responses in the intestines to protect against cholera infection. However, oral vaccination may also affect immune responses in other mucosal tissues. To study this, tissue-specific homing potential and kinetics of B-cell responses were characterized after oral cholera vaccination. Healthy adult volunteers received two doses of Dukoral® and blood, saliva, nasal wash, and fecal samples were collected over time to detect vaccine-specific antibodies. Additionally, homing potential of lymphocytes to small intestine, colon, airways, skin, and periphery was measured by expression of Integrin β1 and β7, CCR9, CCR10, CCR7, and CLA. After vaccination, antibody responses to cholera toxin B (CTB) and Dukoral® were detected in serum and nasal wash. CTB-specific memory B cells in peripheral blood and tissue homing profiles of memory B cells peaked at day 18. IgA+memory B cells expressed markers that enable homing to the airways and colon, while IgA−memory B cells primarily expressed small-intestine-homing markers. These data show that oral cholera vaccination has a differential effect on immune responses in various mucosal sites, including the respiratory tract.

Oral cholera vaccination is used to induce immune responses in the intestines to protect against cholera infection. However, oral vaccination may also affect immune responses in other mucosal tissues. To study this, tissue-specific homing potential and kinetics of B-cell responses were characterized after oral cholera vaccination. Healthy adult volunteers received two doses of Dukoral® and blood, saliva, nasal wash, and fecal samples were collected over time to detect vaccine-specific antibodies. Additionally, homing potential of lymphocytes to small intestine, colon, airways, skin, and periphery was measured by expression of Integrin β1 and β7, CCR9, CCR10, CCR7, and CLA. After vaccination, antibody responses to cholera toxin B (CTB) and Dukoral® were detected in serum and nasal wash. CTB-specific memory B cells in peripheral blood and tissue homing profiles of memory B cells peaked at day 18. IgA+memory B cells expressed markers that enable homing to the airways and colon, while IgA−memory B cells primarily expressed small-intestine-homing markers. These data show that oral cholera vaccination has a differential effect on immune responses in various mucosal sites, including the respiratory tract.

Dietary Trp restriction linked to B cell development in BM and gut microbial composition. With aging, tryptophan metabolism is affected. Tryptophan has a crucial role in the induction of immune tolerance and the maintenance of gut microbiota. We, therefore, studied the effect of dietary tryptophan restriction in young wild-type (WT) mice (118-wk life span) and in DNA-repair deficient, premature-aged (Ercc1-/?7) mice (20-wk life span). First, we found that the effect of aging on the distribution of B and T cells in bone marrow (BM) and in the periphery of 16-wk-old Ercc1-/?7mice was comparable to that in 18-mo-old WT mice. Dietary tryptophan restriction caused an arrest of B cell development in the BM, accompanied by diminished B cell frequencies in the periphery. In general, old Ercc1-/?7mice showed similar responses to tryptophan restriction compared with young WT mice, indicative of age-independent effects. Dietary tryptophan restriction increased microbial diversity and made the gut microbiota composition of old Ercc1-/?7mice more similar to that of young WT mice. The decreased abundances of Alistipes and Akkermansia spp. after dietary tryptophan restriction correlated significantly with decreased B cell precursor numbers. In conclusion, we report that dietary tryptophan restriction arrests B cell development and concomitantly changes gut microbiota composition. Our study suggests a beneficial interplay between dietary tryptophan, B cell development, and gut microbial composition on several aspects of age-induced changes.

ABSTRACTStrong evidence suggests that the gut microbiota is altered in inflammatory bowel disease (IBD), indicating its potential role in noninvasive diagnostics. However, no clinical applications are currently used for routine patient care. The main obstacle to implementing a gut microbiota test for IBD is the lack of standardization, which leads to high interlaboratory variation. We studied the between-hospital and between-platform batch effects and their effects on predictive accuracy for IBD. Fecal samples from 91 pediatric IBD patients and 58 healthy children were collected. IS-pro, a standardized technique designed for routine microbiota profiling in clinical settings, was used for microbiota composition characterization. Additionally, a large synthetic data set was used to simulate various perturbations and study their effects on the accuracy of different classifiers. Perturbations were validated in two replicate data sets, one processed in another laboratory and the other with a different analysis platform. The type of perturbation determined its effect on predictive accuracy. Real-life perturbations induced by between-platform variation were significantly greater than those caused by between-laboratory variation. Random forest was found to be robust to both simulated and observed perturbations, even when these perturbations had a dramatic effect on other classifiers. It achieved high accuracy both when cross-validated within the same data set and when using data sets analyzed in different laboratories. Robust clinical predictions based on the gut microbiota can be performed even when samples are processed in different hospitals. This study contributes to the effort to develop a universal IBD test that would enable simple diagnostics and disease activity monitoring.

ABSTRACTMolecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice.

Low-frequency (LF) electromagnetic fields (EMFs) are abundantly present in modern society, and the potential biological consequences of exposure to these fields are under intense debate. Immune cells are suggested as possible target cells, though a clear mechanism is lacking. Considering their crucial role in innate immune activation, we selected an ex vivo exposure set-up with human neutrophils to investigate a possible correlation between neutrophil extracellular trap (NET) formation and LF EMF exposure. Our study shows that formation of NETs is enhanced by LF EMF exposure. Enhanced NET formation leads to increased antimicrobial properties as well as damage to surrounding cells. We found that LF-EMF-induced NET formation is dependent on the NADPH oxidase pathway and production of reactive oxygen species. Additionally, LF EMF exposure does not influence autophagy and PAD4 activity. Our study provides a mechanism by which exposure to LF EMFs could influence the innate immune system.

There is a need for detailed data on early antibody responses against SARS-CoV-2 as this may contribute to the prediction of the clinical course of COVID-19 and the optimization of convalescent plasma treatment. This study aims to gain insight into developing antibodies to SARS-CoV-2 in health care workers (HCWs) infected in the first wave of the SARS-CoV-2 pandemic in the Netherlands.

ABSTRACTAuthoritative guidelines state that the diagnosis of ventilator-associated pneumonia (VAP) can be established using either endotracheal aspirate (ETA) or bronchoalveolar lavage fluid (BALF) analysis, thereby suggesting that their results are considered to be in accordance. Therefore, the results of ETA Gram staining and semiquantitative cultures were compared to the results from a paired ETA-BALF analysis. Different thresholds for the positivity of ETAs were assessed. This was a prospective study of all patients who underwent bronchoalveolar lavage for suspected VAP in a 27-bed university intensive care unit during an 8-year period. VAP was diagnosed when =2% of the BALF cells contained intracellular organisms and/or when BALF quantitative culture revealed =104CFU/ml of potentially pathogenic microorganisms. ETA Gram staining and semiquantitative cultures were compared to the results from paired BALF analysis by Cohen's kappa coefficients. VAP was suspected in 311 patients and diagnosed in 122 (39%) patients. In 288 (93%) patients, the results from the ETA analysis were available for comparison. Depending on the threshold used and the diagnostic modality, VAP incidences varied from 15% to 68%. For the diagnosis of VAP, the most accurate threshold for positivity of ETA semiquantitative cultures was moderate or heavy growth, whereas the optimal threshold for BALF Gram staining was =1 microorganisms per high power field. The Cohen's kappa coefficients were 0.22, 0.31, and 0.60 for ETA and paired BALF Gram stains, cultures, and BALF Gram stains, respectively. Since the ETA and BALF Gram stains and cultures agreed only fairly, they are probably not interchangeable for diagnosing VAP.

The gold standard for the diagnosis of a peanut allergy is an oral food challenge (OFC), but it is a time-consuming, patient-unfriendly, and expensive test. The in vitro directbasophil activation test (BAT) for peanuts was shown to be a promising diagnostic tool for replacing the OFC.

Viral upper respiratory tract infections have been described as an important factor in the development of otitis media (OM), although it is unclear whether they facilitate bacterial OM or can directly cause OM. To clarify the role of viral infections in OM, we compared the relative contribution of viruses and bacteria with the induction of inflammatory cytokine responses in the middle ear of children suffering from OM.

ABSTRACTThe worldwide prevalence of highly resistant Gram-negative rods (HR-GNR) is increasing rapidly. Reliable typing methods are needed to detect and control outbreaks and to monitor the effectiveness of infection control programs in endemic situations. In this study, we investigated the performance of the DiversiLab typing method in comparison with the amplified fragment length polymorphism (AFLP) typing method. Six hundred fifty-three HR-GNR isolates, which were obtained during a 6-month prospective survey in 18 Dutch hospitals, were typed by AFLP and DiversiLab. Subsequently, the sensitivity and specificity of DiversiLab were calculated, using AFLP as the reference method. In addition, results were compared by means of epidemiological linkage, and Cohen's kappa for agreement was calculated. DiversiLab considered significantly more isolates (275) to belong to a cluster than AFLP (198) (P< 0.001). In direct comparison, the sensitivity was 83.8%, and the specificity was 78.6%. When epidemiological linkage was included in the analysis, DiversiLab considered eight isolates as secondary cases, which were considered unique in AFLP. Only two secondary cases, according to AFLP, were missed by DiversiLab. This results in a kappa for agreement of 0.985. In daily practice, a typing method has to be used in combination with epidemiological information. When this was done, DiversiLab was shown to be a reliable method for the typing of HR-GNR. This, in combination with the ease of use and the speed, makes DiversiLab an appropriate method for screening in routine clinical practice. When a cluster is suspected and the consequences of these findings are substantial, a confirmatory analysis should be performed.

Comparable diets were found to modulate levels of specific and natural humoral immunity in different manners over two generations of genetically selected hens for a high or low Ab response. These diets were based on ingredients that were grown organically (diet A) or conventionally (diet B). Here we report the effects of these diets on cellular immune parameters such as monocyte reactivity measured by NO production, proliferation of whole blood leucocytes and PBMC with the T- and B-cell mitogens, ConA and LPS respectively. Furthermore we measured the in vitromodulatory effects of water soluble extracts of the two diets on T-cell proliferation of whole blood cultures. In both generations a feed change enhanced monocyte reactivity in all birds with the high line birds being most sensitive. Whole blood assays showed the most pronounced diet effects on T-cell reactivity. The low line birds of the first generation showed the greatest effect, but in the second generation all lines were affected by the diets. In the PBMC the greatest effects were found in the control values, with the effects differing in each generation. The present results together with the results found in the whole blood cultures, suggest dietary effects on the intrinsic reactivity of peripheral lymphocytes as well as in vivoeffects, the first with quickly measurable effects and the last activity reflected in our later measurements. These results suggest that a feed change with only minor nutritional differences will induce immunomodulatory effects, and each diet has a unique effect on cellular parameters of innate and adaptive immunity.

The effect of differences in the composition of nutrients of two nutritionally complete layer diets on parameters from innate and adaptive immunity of chickens were examined. The diets were based on ingredients grown either organically or conventionally. As individual differences in nutrient sensitivity have been reported and as the immune system was used as a sensory organ to detect possible effects, layer hens divergently selected for high and low specific antibody (Ab) responses to SRBC, i.e. low line hens and high line hens, reflecting a genetically based differential immune competence were used. The parental line of these hens was randomly bred as the control line, and was used as well. To examine maternal and/or epigenetic effects on nutrient sensitivity, two subsequent generations were studied. In addition, the second generation was challenged with keyhole limpet haemocyanin (KLH). The most pronounced dietary effects were found in the low line birds of the first generation: specific Ab titres to NCD vaccine were significantly elevated in one of the two diets. In the second generation, significant differences were found in Ab and complement responses to the KLH inoculation. Immune competence of the selection lines was not affected. In the second generation control line hens showed the most pronounced effects of dietary treatment in immune responsiveness, with significant effects on specific Ab vaccine titres as well as in innate parameters. The results suggest that small nutritional differences due to the use of different sources of raw ingredients have immunomodulatory effects on innate and adaptive humoral immune parameters. The data indicate the importance of dietary components displaying the capacity to modulate the immune system.

A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617Fis somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617Fallele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2was blocked with a peptide nucleic acid oligonucleotide. JAK2V617Fpatient DNA diluted in JAK2wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n= 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617Fallele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F.

Objectives.The objectives of this study were to determine the incidence density and the occurrence of horizontal spread of highly resistant gram-negative rods (HR-GNRs) in Dutch hospitals. The factors that influence these outcome measures were also investigated.Methods.All patients with HR-GNRs, as determined by sample testing, who were hospitalized in 1 of 18 hospitals during a 6-month period (April through October 2007) were included in this study. For all available isolates, the species was identified, susceptibility was determined (including the presence of extended-spectrum β-lactamases [ESBLs]), and molecular typing was performed. On the basis of a combination of species identification, molecular typing, and epidemiological data, the occurrence of nosocomial transmission was determined.Results.The mean incidence density of patients with HR-GNRs was 55 per 100,000 patient-days (cumulative incidence, 39 per 10,000 patients admitted). A facility being a university hospital was a statistically significant (P= .03) independent determinant of a higher incidence of patients with HR-GNRs. The majority of HR-GNR isolates were ESBL producers. The adjusted transmission index—the ratio between secondary and primary cases—in the participating hospitals ranged from 0.0 to 0.2. The overall adjusted transmission index of HR-GNRs was 0.07. No determinants for a higher transmission index were identified.Discussion.The nosocomial transmission rate of HR-GNRs was relatively low in all hospitals where well-established transmission-based precautions were used. The incidence density of patients with HR-GNRs was higher in university hospitals, probably due to the patient population and the complexity of the care provided.

The effects of a restricted elimination diet in children with attention-deficit hyperactivity disorder (ADHD) have mainly been investigated in selected subgroups of patients. We aimed to investigate whether there is a connection between diet and behaviour in an unselected group of children.

ABSTRACTDirect detection of Streptococcus pneumoniaeDNA in blood adds to culture results in the etiological diagnosis of patients with community-acquired pneumonia (CAP). Quantification of the amount of DNA, the bacterial DNA load (BDL), provides a measurement of DNAemia that may increase the understanding of the clinical relevance of S. pneumoniaeDNA in blood. We evaluated the S. pneumoniaeBDL as a diagnostic tool in adult patients with CAP. The BDL was determined in whole-blood samples collected simultaneously with blood for culture from 45 adult patients with CAP. After DNA extraction, S. pneumoniaeDNA was detected with specific real-time PCR amplification, and the BDL was calculated with a standard curve. PCR and microbiological results were compared, and the BDL was related to clinical and laboratory parameters. S. pneumoniaeDNA was detected in 10/13 patients with positive blood cultures and in 67% of patients with microbiologically confirmed pneumococcal pneumonia. The positive predictive values of the receiver operating characteristic curves for the BDLs for pneumococcal infection (100%) and pneumococcal bacteremia (69%) were higher than those for the level of C-reactive protein (CRP; 43% and 23%, respectively) and the white blood cell count (WBC; 42% and 35%, respectively); the negative predictive values of these three parameters were in the same range (±90 and ±97%, respectively). The BDL was higher in patients presenting with systemic inflammatory response syndrome and in patients with bacteremia. Positive correlations were observed for the BDL with WBC, CRP level, and length of stay. We conclude that the BDL supports the diagnosis of S. pneumoniaeinfection in patients with CAP and provides a putative marker of the severity of disease.

Direct detection of Streptococcus pneumoniae DNA in blood adds to culture results in the etiological diagnosis of patients with community-acquired pneumonia (CAP). Quantification of the amount of DNA, the bacterial DNA load (BDL), provides a measurement of DNAemia that may increase the understanding of the clinical relevance of S. pneumoniae DNA in blood. We evaluated the S. pneumoniae BDL as a diagnostic tool in adult patients with CAP. The BDL was determined in whole-blood samples collected simultaneously with blood for culture from 45 adult patients with CAP. After DNA extraction, S. pneumoniae DNA was detected with specific real-time PCR amplification, and the BDL was calculated with a standard curve. PCR and microbiological results were compared, and the BDL was related to clinical and laboratory parameters. S. pneumoniae DNA was detected in 10/13 patients with positive blood cultures and in 67% of patients with microbiologically confirmed pneumococcal pneumonia. The positive predictive values of the receiver operating characteristic curves for the BDLs for pneumococcal infection (100%) and pneumococcal bacteremia (69%) were higher than those for the level of C-reactive protein (CRP; 43% and 23%, respectively) and the white blood cell count (WBC; 42% and 35%, respectively); the negative predictive values of these three parameters were in the same range (±90 and ±97%, respectively). The BDL was higher in patients presenting with systemic inflammatory response syndrome and in patients with bacteremia. Positive correlations were observed for the BDL with WBC, CRP level, and length of stay. We conclude that the BDL supports the diagnosis of S. pneumoniae infection in patients with CAP and provides a putative marker of the severity of disease.

Integrons play an important role in the dissemination of resistance genes among bacteria. Nearly 70% of highly resistant gram-negative bacteria isolated at a tertiary care hospital harbored an integron. Epidemiologic analysis suggests that horizontal gene transfer is an important mechanism of resistance spread and has a greater contribution than cross-transmission to levels of resistance in settings where highly resistant gram-negative bacteria are endemic.

A common method to study protein complexes is immunoprecipitation (IP), followed by mass spectrometry (thus labeled: IP-MS). IP-MS has been shown to be a powerful tool to identify protein–protein interactions. It is, however, often challenging to discriminate true protein interactors from contaminating ones. Here, we describe the preparation of antifouling azide-functionalized polymer-coated beads that can be equipped with an antibody of choice via click chemistry. We show the preparation of generic immunoprecipitation beads that target the green fluorescent protein (GFP) and show how they can be used in IP-MS experiments targeting two different GFP-fusion proteins. Our antifouling beads were able to efficiently identify relevant protein–protein interactions but with a strong reduction in unwanted nonspecific protein binding compared to commercial anti-GFP beads.

ADHD is a neuropsychiatric disorder characterized by chronic hyperactivity, inattention and impulsivity, which affects about 5 of schoolage children. ADHD persists into adulthood in at least 15 of cases. It is highly heritable and familial influences seem strongest for ADHD persisting into adulthood. However, most of the genetic research in ADHD has been carried out in children with the disorder. The gene that has received most attention in ADHD genetics is SLC6A3DAT1encoding the dopamine transporter. In the current study we attempted to replicate in adults with ADHD the reported association of a 10–6 SLC6A3haplotype, formed by the 10repeat allele of the variable number of tandem repeat VNTR polymorphism in the 3′ untranslated region of the gene and the 6repeat allele of the VNTR in intron 8 of the gene, with childhood ADHD. In addition, we wished to explore the role of a recently described VNTR in intron 3 of the gene. Two hundred sixteen patients and 528 controls were included in the study. We found a 9–6 SLC6A3haplotype, rather than the 10–6 haplotype, to be associated with ADHD in adults. The intron 3 VNTR showed no association with adult ADHD. Our findings converge with earlier reports and suggest that age is an important factor to be taken into account when assessing the association of SLC6A3with ADHD. If confirmed in other studies, the differential association of the gene with ADHD in children and in adults might imply that SLC6A3plays a role in modulating the ADHD phenotype, rather than causing it. © 2008 WileyLiss, Inc.

Objective.To determine the incidence density of highly resistant organisms (HROs) and the relative contribution of horizontal spread in a setting of endemicity.Methods.Prospective surveillance was performed among hospitalized patients during an 18-month period. Enterobacteriaceae, non-fermentative gram-negative bacilli, Staphylococcus aureus, Streptococcus pneumoniae,and Enterococcus faecium—all considered highly resistant, according to Dutch guidelines—were included. Epidemiological linkage and nosocomial transmission were determined on the basis of molecular typing and hospital admission data.Results.From 119 patients, we recovered a total of 170 unique HRO isolates, as follows: Escherichia coli,96 isolates; Klebsiellaspecies, 11 isolates; Enterobacterspecies, 8 isolates; Proteusspecies, 9 isolates; Citrobacterspecies, 5 isolates; Pseudomonasspecies, 5 isolates; Aci-netobacterspecies, 3 isolates; Morganellaspecies, 2 isolates; Salmonellaspecies, 1 isolate; Serratiaspecies, 1 isolate; S. pneumoniae,20 isolates; and S. aureus,9 isolates. No vancomycin-resistant E. faeciumwas found. The incidence density was 4.3 HRO isolates per 10,000 patient-days. The majority of HRO isolates were unique, and nosocomial transmission was observed 4 times for highly resistant gram-negative bacilli (case reproduction rate, 0.05) and 4 times for penicillin-nonsusceptible S. pneumoniae(case reproduction rate, 0.29). A stay on the intensive care unit was the main determinant for the recovery of an HRO.Conclusion.Nosocomial transmission of HROs was observed 8 times during the 18-month period. The intensive care unit was identified as the main reservoir of horizontal spread of HROs. This study shows that nosocomial transmission of HROs is largely preventable using transmission precautions.

ABSTRACTUsing 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficiletyping techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdCgene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile(BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdCgene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

ABSTRACTDirect detection of bacterial DNA in blood offers a fast alternative to blood culture and is presumably unaffected by the prior use of antibiotics. We evaluated the performance of two real-time PCR assays for the quantitative detection of Staphylococcus aureusbacteremia and for Enterococcus faecalisbacteremia directly in blood samples, without prior cultivation. Whole-blood samples for PCR were obtained simultaneously with blood cultures from patients admitted to the intensive care unit of our hospital. After the extraction of DNA from 200 µl of blood, real-time PCR was performed for the specific detection and quantification of S. aureusand E. faecalisDNA. The sensitivity for bacteremia of the S. aureusPCR was 75% and that of the E. faecalisPCR was 73%, and both tests had high specificity values (93 and 96%, respectively). PCR amplification reactions were positive for S. aureusfor 10 (7%) blood samples with negative blood cultures, and 7 (4%) PCR reactions were positive for E. faecalis. The majority of these PCR results were likely (50%) or possibly (42%) related to infection with the specific microorganism, based on clinical data and radiological and microbiological investigations. PCR results were concordant for 95% of paired whole-blood samples, and blood culture results were concordant for 97% of the paired samples. We conclude that the detection of S. aureusand E. faecalisDNA in blood by real-time PCR enables a rapid diagnosis of bacteremia and that a positive DNAemia is related to proven or possible infection with the specific microorganism in the majority of patients with negative blood cultures.

Background:Saliva has been studied for the presence of subgingival pathogens in periodontitis patients. With the anaerobic culture technique, the discrepancy between salivary recovery and subgingival presence has been significant, which makes this approach not suitable for practical use in the microbial diagnosis of periodontitis patients. The real‐time polymerase chain reaction (PCR) technique represents a very sensitive technique to detect and quantify bacterial pathogens. The aim of the study was to compare the presence and numbers of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia, and Micromonas microsin subgingival plaque and mouthwash samples by the anaerobic culture and real‐time PCR techniques. Methods:Pooled subgingival plaque samples and 10‐ml mouthwash samples were collected from 21 adult patients with periodontitis and analyzed by quantitative anaerobic culture and real‐time PCR for A. actinomycetemcomitans, P. gingivalis, T. forsythensis, P. intermedia, and M. micros. Results:The detection frequency of A. actinomycetemcomitans, P. gingivalis, and T. forsythensisin subgingival plaque was identical by culture and real‐time PCR and was higher for P. intermediaand M. microsby real‐time PCR. The highest detection frequencies for the target bacteria were found in mouthwash samples by real‐time PCR. The additional value of the real‐time PCR to detect target bacteria was 38% for P. gingivalis, 73% for T. forsythensis, 77% for P. intermedia, and 71% for M. micros. The sensitivity to detect target species in mouthwash by real‐time PCR was 100% for all test species except for P. intermedia(93.8%). Conclusions:Rapid detection and quantification of periodontal pathogens in mouthwash samples are possible by real‐time PCR. The procedure is significantly less time‐consuming than subgingival sampling with paper points. This approach to detect major periodontal pathogens in mouthwash samples may simplify microbial diagnosis in periodontitis patients and may be used to monitor periodontal treatment.

Background:The patient environment is increasingly considered a major source of transmission of nosocomial bacteria to patients. In May 2019, a cluster of 3 patients with multiresistant Enterobacter cloacaewas discovered in the hematology-oncology department of the Maastricht University Medical Center (built in 1991). The strains had an identical antibiogram: ESBL-positive, ciprofloxacin R, cotrimoxazole R, meropenem S, and colistin S. One neutropenic patient had a positive blood culture for this strain, resistant to the empiric treatment with piperacillin-tazobactam, but the patient recovered after switching the antibiotic regimen to meropenem. All strains were determined to be identical by amplified-fragment length polymorphism and whole-genome multi-locus sequencing typing (genotype A). New cases occurred, despite the introduction of contact isolation of positive and contact patients. Therefore, weekly point-prevalence screening was introduced, in which more newly colonized patients were identified in the subsequent weeks. Attention to hand hygiene was enforced, and the hypothesis of contamination from “wet” environmental locations was tested by performing cultures of sinks and shower drains. In June and July, 47 of 241 environmental cultures (19.5%) were positive for E. cloacaewith an identical antibiogram, among which some were typed as genotype A. To diminish the environmental contamination, all siphons of sinks were replaced, and disinfection of sinks and shower drains was intensified using chlorine and soda on a daily basis. Replacement of shower drains was not possible. After this intervention, the incidence of newly colonized patients declined gradually. A change in the regimen of selective gut decontamination in hematology patients was considered as an alternative intervention, but with the decrease in new patient cases, this was not implemented. A final round of environmental cultures at the end of August revealed 8 positive cultures, of which 5 were positive for genotype A. In retrospect, this finding could be explained by the fact that the cleaning team did not follow the intensified instructions for disinfection. From week 29, genotype A E. cloacaewas no longer cultured in weekly patient screenings. Based on this observation, it is important that in (re)building plans for hospitals, a master plan for the prevention of nosocomial transmission from environment to patients is incorporated.Funding:NoneDisclosures:None

AbstractAlthough blood component transfusion is currently regarded to be safe, adverse events may occur in recipients of these products. Among those, blood borne viral, bacterial and parasitic infections are best known. For detection of bacterial contamination in platelet concentrates various methods are available or under investigation. One of these methods, real-time polymerase chain reaction (PCR) with particular focus on real-time 16S rDNA detection, will be discussed in this review.

Objectives: During treatment with selective decontamination of the digestive tract (SDD), four multidrug-resistant (MDR) strains, three different Escherichia coli and one Klebsiella pneumoniae, were isolated from four patients not known as carriers of such MDR strains before their admission to the intensive care unit (ICU) in the Academic Medical Center (AMC) in Amsterdam. These isolates were extended-spectrum β-lactamase (ESBL)-positive. We investigated whether this was due to interspecies transfer of resistance genes.Methods: The MDR strains were typed by amplified fragment length polymorphism (AFLP) analysis. The plasmids from these strains were characterized by restriction fragment length polymorphism and the resistance genes were characterized by PCR and sequence analysis.Results: The strains were genetically unrelated and contained identical plasmids with ESBL genes.Conclusions: We identified an outbreak of plasmid-mediated ESBL genes during SDD treatment in the ICU. The use of third-generation cephalosporins in SDD is associated with the emergence of ESBLs. We conclude that identification of emerging MDR Gram-negative bacteria and recognition of resistance plasmid transfer during SDD treatment are crucial for optimal application of this regimen in ICUs.

Glucocorticoids (GCs) are commonly used to treat a variety of immune diseases. However, the efficacy of treatment is greatly influenced by an individual variation in sensitivity to GCs, which is caused by differences in the glucocorticoid receptor (GR). The variable receptor profile results from variations in the GR gene, or alternative splicing of the gene coded. We investigated the evolution of the GR gene by comparing genomic GR sequences of vertebrates. Exon length and amino acid sequence are conserved among all classes of vertebrates studied, which indicates strong evolutionary pressure on conservation of this gene. Interestingly, teleostean fishes have two different GR proteins. One of the duplicate fish GR genes has a nine-amino-acid insert in the DNA binding region that results from alternative splicing. The duplicate GR genes and products of alternative splicing in teleostean fishes are differentially expressed in vivoand show different transactivation capacity in vitro.The presence of two GR genes appears to be a result of divergence of receptors rather than of ligands. Teleostean fishes express different, evolutionarily related, functional GR proteins within a single organism. Hereby, teleostean fishes present a model that facilitates investigation of the molecular basis of cortisol resistance and different regulatory functions of cortisol.

ABSTRACTRapid identification of microorganisms in blood cultures is required to optimize empirical treatment at an early stage. Fluorescence in situ hybridization (FISH) can reduce the time to identification of microorganisms in growth-positive blood cultures. In this study, we evaluated the performance, time to identification, and potential clinical benefits of FISH compared to those of conventional culture methods in routine practice. After Gram staining, blood culture fluids were simultaneously further identified with FISH and with conventional culture methods. Results and points in time of FISH and culture identification (provisional and final identifications) were collected and compared. For 91% of microorganisms, the genus or family name was identified, and for 79%, the species name could be attributed. The sensitivity and specificity of the individual probes exceeded 95%, except for the Enterobacteriaceaeprobe (sensitivity, 89%). Cross-hybridization was obtained with the Klebsiella pneumoniaeprobe for Klebsiella oxytoca. The time gains of FISH and final culture identification were more than 18 h for bacteria and 42 h for yeasts. With FISH, Staphylococcus aureuswas differentiated from coagulase-negative staphylococci 1.4 h faster than by provisional identification (P< 0.001). In conclusion, FISH allows rapid and reliable identification of the majority of microorganisms in growth-positive blood cultures. The substantial time gain of identification with FISH may allow same-day adjustment of antimicrobial therapy, and FISH is especially useful if no provisional identification is obtained. With further extension of the number of probes and a reduction in turnaround time, FISH will become a very useful diagnostic tool in the diagnosis of bloodstream infections.

ABSTRACTThe increasing demand for molecular diagnostics in clinical microbiology laboratories necessitates automated sample processing. In the present study, we evaluated the performance of the MagNA Pure LC total nucleic acid isolation kit (M extraction) in comparison with the manual method (Si extraction) according to Boom et al. (R. Boom, C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990) for the detection of viral DNA by competitive quantitative PCR. Reconstruction experiments with HindIII-digested phage lambda DNA and HaeIII-digested fX174 DNA showed that the recovery of DNA from phosphate-buffered saline, cerebrospinal fluid, EDTA-anticoagulated plasma, and EDTA-anticoagulated whole blood by M extraction is, on average, 6.6-fold lower compared to Si extraction. PCR signals of spiked PCR control DNAs for Epstein-Barr virus and varicella-zoster virus were also between 1.9- and 14.2-fold lower after M extraction compared to Si extraction, also suggesting impaired DNA recovery. M extraction of spiked cytomegalovirus strain AD 169 in whole blood showed a 5- to 10-fold reduction in PCR sensitivity compared to Si extraction. This reduction of PCR sensitivity was also observed when clinical whole blood samples were processed by M extraction. Before implementing M extraction, the clinical consequences of the reduced recovery should first be considered, especially when maximal sensitivity is required.

ABSTRACTAmplified fragment length polymorphism (AFLP) was used for typing avian mycoplasma species. Forty-four avian mycoplasma strains were successfully typed into eight distinct groups, with each representing a different species. Homology of AFLP patterns of 35% or less was used as a cutoff value to differentiate avian mycoplasma strains into different species.

ObjectiveTo investigate in a randomized controlled trial the effects of mutual support groups in rheumatic diseases on social network size, social skills, loneliness, daily functioning, and life satisfaction as well as to identify patients perceptions of the support group.MethodsParticipants were 112 patients with chronic rheumatic disorders affecting the joints. Data were collected with selfreport questionnaires and group interviews.ResultsEffects have been found on social skills only. More specifically, mutual support groups at postintervention decreased distress in expressing negative feelings toward other people. This effect did not persist at the 6month followup evaluation, but at that time an increase in frequency in making ones wishes known to others was found. In patients who attended at least 5 of all 10 sessions, an increase in expressing positive feelings toward others was found at followup. Mutual support groups were evaluated positively.ConclusionMutual support groups are recommended for patients experiencing difficulties in social interactions.