Diagnosing viral gastro-enteritis using the fully automated sample-in, result-out STARlet All in one system (AIOS).

The STARlet All-In-One system is a modular platform that integrates the complete molecular diagnostic workflow from nucleic acid extraction of clinical samples to PCR set-up and amplification. The platform was evaluated in comparison with laboratory developed tests (LDT) on fecal samples from patients with suspected viral gastro-enteritis. In a retrospective study, 72 positive samples were analysed, including all pathogens detected by the Seegene Allplex™ GI-virus assay, adenovirus, astrovirus, norovirus GI and GII, sapovirus, and rotavirus. Concordant results were obtained for 69 samples (96 %). Three discordant results were observed, one norovirus GII positive that gave an invalid result in the AIOS and two samples that were negative in the AIOS. One adenovirus positive that was subtyped as a genotype 2 virus, which is not associated with gastro-enteritis, and a sapovirus. In the prospective part of the study, 661 fecal samples were included. A total of 61 positive samples were detected, of which 60 were also detected by the AIOS. One norovirus GII positive sample (CT 35.2) was tested negative in the AIOS. Two additional sapovirus positive samples, CT 37 and 38, were detected by the AIOS but not by the LDT. The STARlet All-In-One platforms results in an automated molecular workflow with reduced hands-on time and enables running assays during out of office hours. Application of the Seegene Allplex™ GI-virus assay showed excellent concordance to the current diagnostic LDT. In a prospective comparison, only three discordant results were observed, all with CT values over 35 and therefore unlikely of clinical relevance.

Genetic glucocorticoid receptor variants differ between ethnic groups but do not explain variation in age of diabetes onset, metabolic and inflammation parameters in patients with type 2 diabetes.

Aims: The effect of excess glucocorticoid receptor (GR) stimulation through glucocorticoid medication or cortisol on glucose metabolism is well established. There are genetic GR variants that result in increased or decreased GR stimulation. We aimed to determine the prevalence of genetic GR variants in different ethnic groups in a cohort of patients with type 2 diabetes, and we aimed to determine their association with age of diabetes onset and metabolic and inflammation parameters. Methods: A cross-sectional analysis was performed in a multiethnic cohort (n = 602) of patients with established type 2 diabetes. Polymorphisms in the GR gene that have previously been associated with altered glucocorticoid sensitivity (TthIIII, ER22/23EK N363S, BclI and 9ß) were determined and combined into 6 haplotypes. Associations with age of diabetes onset, HbA1c, hs-CRP and lipid values were evaluated in multivariate regression models. Results: The prevalence of the SNPs of N363S and BclI was higher in Dutch than in non-Dutch patients. We observed a lower prevalence of the SNP 9ß in Dutch, South(East) Asian and Black African patients versus Turkish and Moroccan patients. We did not detect an association between SNPs and diabetes age of onset or metabolic parameters. We only found a trend for lower age of onset and higher HbA1c in patients with 1 or 2 copies of haplotype 3 (TthIIII + 9ß). Conclusions: The prevalence of genetic GR variants differs between patients of different ethnic origins. We did not find a clear association between genetic GR variants and age of diabetes onset or metabolic and inflammation parameters. This indicates that the clinical relevance of GR variants in patients with established type 2 diabetes is limited.

Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC.

BACKGROUND: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC. METHODS: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates. RESULTS: Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons. CONCLUSIONS: In this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.

RT-PCR detection of respiratory pathogens in newborn children admitted to a neonatal medium care unit.

BACKGROUND: This study aimed to determine the prevalence of respiratory pathogens among newborns admitted to a neonatal medium care unit (NMCU) and to identify clinical predictors. METHODS: A 1-y observational study was performed of neonates admitted to an NMCU in Amsterdam, The Netherlands. Nasopharyngeal samples were collected for the detection of respiratory viruses and bacteria by real-time PCR (RT-PCR). Cycle threshold (Ct) values were provided to estimate viral load. Predictors for the presence of study pathogens were identified. RESULTS: From October 2010 through September 2011, 334 neonates (median age 1.3 d, 53.6% male) were included. Overall, 37 respiratory pathogens were detected in 34 children (10.2%): parainfluenza-1 (n = 9), human rhinovirus (n = 7), parainfluenza-3 (n = 6), respiratory syncytial virus (RSV, n = 6), Streptococcus pneumoniae (n = 3), adenovirus (n = 2), human coronavirus (n = 2), influenza A (n = 1), and bocavirus (n = 1). Neonates with higher viral loads (Ct <35; n = 11) were more often clinically ill than those with lower viral loads (Ct ≥35; n = 23). Two variables significantly contributed to the detection of study pathogens: age (odds ratio (OR) 1.21 for each day older; 95% confidence interval 1.12-1.30) and rhinorrhea (OR 6.71; 95% confidence interval 1.54-29.21). CONCLUSION: Respiratory pathogens seem to play a role in neonates admitted to an NMCU. The influence of respiratory pathogen detection on clinical management remains to be determined.

Characterization of 2009 H1N1 pandemic influenza in a population of Dutch children with influenza-like signs and symptoms.

AIM: To determine causative respiratory pathogens and describe epidemiological and clinical characteristics in a paediatric population with influenza-like illness during the 2009 H1N1-pandemic. METHODS: Observational study of 412 children visiting an outpatient clinic of a Dutch teaching hospital. RESULTS: From August to December 2009, 412 children were tested at the clinic; 32% proved H1N1-positive, confirmed by reverse-transcriptase-polymerase-chain-reaction (RT-PCR). Pathogens were detected in 65% of samples. Influenza A(H1N1) (n = 132), human rhinovirus (n = 55), respiratory syncytial virus (n = 45) and adenovirus (n = 34) were mostly identified. Co-infections were seen in 34 children (8.3%). Mean age was 6.8 and 4.2 years in H1N1-positive and H1N1-negative cases, respectively (p < 0.01). H1N1-positive outpatient children reported fever, cough and rhinorrhoea more frequently than their H1N1-negative counterparts. Of 72 hospitalized children, 31% proved H1N1-positive; all showed a relatively mild clinical illness. None of the children had been admitted to an intensive care unit or died. Oseltamivir treatment was initiated in 72 children and discontinued in 42 (63%) when RT-PCR results turned negative. CONCLUSION: The 2009 H1N1-pandemic showed a mild clinical course in a Dutch paediatric outpatient clinic population. Respiratory pathogens were detected in the majority of children with influenza-like illness and influenza A(H1N1) virus was identified in one-third. Testing symptomatic children during an influenza pandemic has effectively limited the use of oseltamivir.

Low attack rate of novel influenza A (H1N1) virus infection among healthcare workers: a prospective study in a setting with an elaborated containment plan.

PURPOSE: This study aimed to determine incidence rates of novel influenza A (H1N1) infection among healthcare personnel with different exposure risks during the 2009 H1N1 pandemic. METHODS: From August 2009 until April 2010, 66 healthcare workers from a 410 bed teaching hospital in Amsterdam were monitored. The following three different exposure groups were created: a high- (n = 26), intermediate- (n = 20), and low-risk group (n = 20). Throat swabs were collected each week and analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in order to detect the H1N1 virus. Blood was drawn at study enrollment and once monthly thereafter, and serum specimens were tested with an H1N1-specific hemagglutination-inhibition serologic assay. Influenza-like signs and symptoms were assessed weekly. RESULTS: One of 26 high-risk group participants proved H1N1 positive once by RT-PCR. This corresponds to an incidence rate in the high-risk group of 5.7/1,000 person weeks (95% CI 0-17/1,000). None of the intermediate- and low-risk group participants proved H1N1 positive by RT-PCR. Significant antibody titer rises in convalescent sera were demonstrated in three participants: one was a confirmation of the case that had proved H1N1 positive by RT-PCR; the others occurred in two asymptomatic participants belonging to the low- and high-risk groups. An influenza-like illness was assumed in four participants from the high- (n = 1), intermediate- (n = 1) and low-risk (n = 2) groups; these findings were not confirmed by positive results from either diagnostic test. CONCLUSIONS: This study demonstrates a low incidence rate of influenza A (H1N1) infection among healthcare workers during the 2009 H1N1 pandemic in a setting with high hygiene standards.

Germline polymorphisms in patients with advanced nonsmall cell lung cancer receiving first-line platinum-gemcitabine chemotherapy: a prospective clinical study.

BACKGROUND: The authors assessed the impact of germline polymorphisms on clinical outcome in patients with advanced nonsmall cell lung cancer (NSCLC) who received platinum-gemcitabine (PG) chemotherapy. METHODS: In total, 137 patients with stage IIIB/IV NSCLC were included who received first-line PG chemotherapy (74% of patients received cisplatin, and 26% received carboplatin). Twenty-three germline polymorphisms that were identified in peripheral blood samples were analyzed for progression-free survival (PFS), treatment response, overall survival (OS), and toxicity. RESULTS: The median PFS was 5.8 months, the median OS was 10.2 months, and 44 patients (32%) had a partial treatment response. Carriers of the excision repair cross-complementation group 1 (ERCC1) mutant thymine (T) allele had a lower treatment response rate (29% vs 52%; P = .02), shorter PFS (adjusted hazard ratio [HR], 1.60; P = .04), and shorter OS (adjusted HR, 1.54; P = .05) compared with carriers of the wild-type cytosine/cytosine (CC) genotype. The xeroderma pigmentosum group A member 10 (XPD10) mutant adenine (A) allele (adjusted HR, 0.64; P = .04) and the x-ray cross-complementing group 1 (XRCC1) mutant guanine (G) allele (adjusted HR, 0.51; P = .02) also were independent predictors of OS. Carriers of the mutant adenosine triphosphate-dependent DNA helicase Q1 (RECQ1) C allele or the mutant cytidine deaminase (CDA) C allele were more likely to experience severe leukocytopenia (26% vs 10% [P = .03] and 28% vs 11% [P = .02], respectively) compared with wild-type genotype carriers. Patients who carried the homozygous mutant glutathione S-transferase π 1(GSTP1) GG genotype were at considerable risk for severe platinum-associated polyneuropathy (18% vs 3% in wild-type vs heterozygous mutant patients, respectively; P = .01). CONCLUSIONS: To the authors' knowledge, this is the first prospective study to date in patients with advanced NSCLC describing predictive germline polymorphisms not only for the clinical activity of PG chemotherapy (ERCC1, XPD10) but also for its toxicity (GSTP1, RECQ1, CDA). Nonplatinum-containing chemotherapy in carriers of the ERCC1 T allele or the XPD10 G allele should be studied prospectively.

Relationship between single nucleotide polymorphisms and haplotypes in DPYD and toxicity and efficacy of capecitabine in advanced colorectal cancer.

PURPOSE: To explore the effect of dihydropyrimidine dehydrogenase (DPD) single nucleotide polymorphisms (SNP) and haplotypes on outcome of capecitabine. EXPERIMENTAL DESIGN: Germline DNA was available from 568 previously untreated patients with advanced colorectal cancer participating in the CAIRO2 trial, assigned to capecitabine, oxaliplatin, and bevacizumab ± cetuximab. The coding region of dihydropyrimidine dehydrogenase gene (DPYD) was sequenced in 45 cases with grade 3 or more capecitabine-related toxicity and in 100 randomly selected controls (cohort). Most discriminating (P < 0.1) or frequently occurring (>1%) nonsynonymous SNPs were analyzed in all 568 patients. SNPs and haplotypes were associated with toxicity, capecitabine dose modifications, and survival. RESULTS: A total of 29 SNPs were detected in the case-cohort analysis, of which 8 were analyzed in all 568 patients. Of the patients polymorphic for DPYD IVS14+1G>A, 2846A>T, and 1236G>A, 71% (5 of 7), 63% (5 of 8), and 50% (14 of 28) developed grade 3 to 4 diarrhea, respectively, compared with 24% in the overall population. All patients polymorphic for IVS14+1G>A developed any grade 3 to 4 toxicity, including one possibly capecitabine-related death. Because of toxicity, a mean capecitabine dose reduction of 50% was applied in IVS14+1G>A and 25% in 2846A>T variant allele carriers. Patients were categorized into six haplotype groups: one predicted for reduced (10%), and two for increased risks (41% and 33%) for severe diarrhea. Individual SNPs were not associated with overall survival, whereas one haplotype was associated with overall survival [HR (95% CI) = 0.57 (0.35-0.95)]. CONCLUSIONS: DPYD IVS14+1G>A and 2846A>T predict for severe toxicity to capecitabine, for which patients require dose reductions. Haplotypes assist in selecting patients at risk for toxicity to capecitabine.

Adult outpatient experience of the 2009 H1N1 pandemic: clinical course, pathogens, and evaluation of case definitions.

OBJECTIVES: The aim was to describe causative agents and clinical characteristics in adult outpatients with upper airway symptoms during the 2009 H1N1 pandemic and to evaluate case definitions that are used in clinical practice. METHODS: From August through December 2009, 964 symptomatic adult outpatients were included. RT-PCR was used to detect the following pathogens: influenza A (H1N1) and B, parainfluenza 1-4, adenovirus, respiratory syncytial virus, human rhinovirus, human metapneumovirus, human coronavirus (OC43, 229E, NL63), Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella species. The Dutch GHOR, American CDC and WHO, and British HPA case definitions were evaluated. RESULTS: A respiratory pathogen was detected in 41% of tested patient samples; influenza A (H1N1) and human rhinovirus were both detected in 16%. Clinical presentation of influenza cases was significantly more serious when compared to rhinovirus or negative-tested cases. Test characteristics were almost similar for all 4 case definitions, with an average sensitivity of 66%, specificity of 70%, positive predictive value of 34% and negative predictive value of 90%. CONCLUSIONS: Influenza A (H1N1) and human rhinovirus were the major pathogens responsible for respiratory disease. The 2009 H1N1 pandemic in Amsterdam followed a mild course. Test characteristics of 4 different clinical case definitions seemed comparable but rather useless.