Evaluation of a rapid direct assay for identification of bacteria and the mec A and van genes from positive-testing blood cultures

We performed the first evaluation of a DNA strip assay (GenoType blood culture; Hain Lifescience, Nehren, Germany) for the detection of the most relevant bacterial sepsis pathogens directly from positive BACTEC blood culture bottles (Becton Dickinson, Heidelberg, Germany). The test comprises two panels, one for the direct species identification of important gram-positive cocci and the other for gram-negative rods. Additionally, detection of the mec A and the van genes are implemented. The GenoType assay was validated regarding its analytical sensitivity with blood cultures spiked with reference strains. Approximately 10(4) CFU per ml were detected. Analytical specificity was calculated with a test panel of 212 reference strains. Of the strains tested, 99% were correctly identified. Additionally, 279 consecutive blood cultures signaled positive by BACTEC were processed directly, in comparison to conventional methods. The GenoType assays were performed according to Gram stain morphology. A total of 243 (87.1%) of the 279 organisms isolated were covered by specific probes. A total of 152 organisms were gram-positive cocci, of which 148 (97.4%) were correctly identified by the GenoType assay. Ninety-one organisms were gram-negative rods, of which 89 (97.8%) were correctly identified. Concerning mec A gene detection, GenoType assay correctly detected 12 of 13 methicillin-resistant Staphylococcus aureus isolates. One Enterococcus faecium isolate with a positive van A gene isolated was correctly differentiated by the assay. All results were available 4 h after the results of microscopic analysis. The evaluated GenoType blood culture assay showed fast and reliable results in detecting the most important sepsis pathogens and the mec A and van genes directly from positive blood culture bottles.     

The 16189 variant of mitochondrial DNA occurs more frequently in C282Y homozygotes with haemochromatosis than those without iron loading

Background:Patients with hereditary haemochromatosis (HH) are usually homozygous for the C282Y mutation in the HFE gene. They have variable expression of iron overload and present with a variety of complications, including liver disease, diabetes, arthropathy, fatigue, and cardiomyopathy. The mitochondrial 16189 variant is associated with diabetes, dilated cardiomyopathy, and low body fat at birth, and might contribute to genetic predisposition in further multifactorial disorders. The objective of this study was to determine the frequency of the 16189 variant in a range of patients with haemochromatosis, who had mutations in the HFE gene.

Methods:Blood DNA was analysed for the presence of the 16189 variant in British, French, and Australian C282Y homozygotes and controls, with known iron status, and in birth cohorts.

Results:The frequency of the mitochondrial 16189 variant was found to be elevated in individuals with haemochromatosis who were homozygous for the C282Y allele, compared with population controls and with C282Y homozygotes who were asymptomatic (42/292 (14.4%); 102/1186 (8.6%) (p = 0.003); and 2/64 (3.1%) (p = 0.023), respectively).

Conclusions:Iron loading in C282Y homozygotes with HH was exacerbated by the presence of the mitochondrial 16189 variant.

     

Monocyte response to LPS after exposure to corticosteroids and chloroquine with implications for systemic lupus erythematosus

Essential part of a response to infection is early pathogen recognition and adequate initiation of innate immunity. One of the hallmarks of systemic lupus erythematosus (SLE) is reduced resistance to infection despite overall hyperactivity of the immune system. Immunosuppressive drugs (high-dose corticosteroids and cytotoxic agents) are independent risk factors for infection in SLE, with bacteria as predominant cause. To investigate whether less aggressive immunomodulatory treatment may still affect recognition and response to Gram-negative bacteria, we measured TLR4 expression in monocytes of untreated SLE patients and patients on chloroquine and low-dose steroid therapy and examined the drugs' influence on monocyte TLR4 expression in peripheral blood mononuclear cell (PBMC) culture. Additionally, we determined whether induction of monocyte NF-κB signalling, TNF-α and IL-6 production with lipopolysaccharide (LPS), a TLR4 ligand, can be altered with dexamethasone, chloroquine or both. There was no statistically significant difference in TLR4 expression between patients with SLE and controls, even though treated SLE patients tended to have lower frequency of TLR4(+) monocytes and TLR4 mean fluorescence intensity than healthy controls. However, neither dexamethasone nor chloroquine had major influence on TLR4 expression in vitro or suppressed LPS-induced NF-κB activation in monocytes, although dexamethasone decreased TNF-α and IL-6 production. Therefore, even if low-dose steroids or chloroquine do not seem to affect TLR4 expression and signalling, steroids might decrease cytokine production in response to LPS.     

Nosocomial urinary tract infection in the intensive care unit: when should Pseudomonas aeruginosa be suspected? Experience of the French national surveillance of nosocomial infections in the intensive care unit,Rea-Raisin

Individual and ward risk factors for P. aeruginosa-induced urinary tract infection in the case of nosocomial urinary tract infection in the intensive care unit were determined with hierarchical (multilevel) logistic regression. The 2004-2006 prospective French national intensive care unit nosocomial infection surveillance dataset was used and 3252 patients with urinary tract infection were included; 16% were infected by P. aeruginosa. Individual risk factors were male sex, duration of stay, antibiotics at admission and transfer from another intensive care unit. Ward risk factors were patient turnover and incidence of P. aeruginosa-infected patients.     

A common variant of the interleukin 6 receptor (IL-6r) gene increases IL-6r and IL-6 levels, without other inflammatory effects

Interleukin-6 (IL-6) is a key inflammatory cytokine, signalling to most tissues by binding to a soluble IL-6 receptor (sIL-6r), making a complex with gp130. We used 1273 subjects (mean age 68 years) from the InCHIANTI Italian cohort to study common variation in the IL-6r locus and associations with interleukin 6 receptor (IL-6r), IL-6, gp130 and a battery of inflammatory markers. The rs4537545 single nucleotide polymorphism (SNP) tags the functional non-synonymous Asp358Ala variant (rs8192284) in IL-6r (r(2)=0.89, n=343). Individuals homozygous for the rs4537545 SNP minor allele (frequency 40%) had a doubling of IL-6r levels (132.48 pg/ml, 95% CI 125.13-140.27) compared to the common allele homozygous group (68.31 pg/ml, 95% CI 65.35-71.41): in per allele regression models, the rs4537545 SNP accounted for 20% of the variance in sIL-6r, with P=5.1 x 10(-62). The minor allele of rs4537545 was also associated with higher circulating IL-6 levels (P=1.9 x 10(-4)). There was no association of this variant with serum levels of gp130 or with any of the studied pro- and anti-inflammatory markers. A common variant of the IL-6r gene results in major changes in IL-6r and IL-6 serum levels, but with no apparent effect on gp130 levels or on inflammatory status in the general population.     

Identification of seven novel germline mutations in the human E-cadherin (CDH1) gene

Hereditary diffuse gastric cancer (HDGC) is a cancer predisposition syndrome caused by germline mutation of the gene encoding the tumour-suppressor E-cadherin (CDH1). We describe the search for CDH1 mutations in 36 new diffuse gastric cancer families. All 16 CDH1 exons, neighbouring intronic sequence and an essential promoter region were screened by DNA sequencing. We detected nine different mutations, seven of which were novel. Of the seven novel mutations, five were identified in families who met the IGCLC clinical criteria for HDGC. Two mutations resulted in a premature stop codon and truncation of the protein. Three mutations affected splice sites; two of the splice-site mutations were shown by RT-PCR to disturb normal CDH1 splicing, while the third splice-site mutation was present in two unrelated HDGC families. The remaining two mutations resulted in amino acid substitutions and impaired the ability of E-cadherin protein to form cellular aggregates and suppress invasion in vitro. Together with the occurrence of extra-gastric tumours such as lobular breast and colorectal cancer, these findings further extend the types of CDH1 mutations and the spectrum of tumours associated with HDGC.     

The impact of a nap opportunity during the night shift on the performance and alertness of 12-h shift workers

The purpose of this workplace evaluation was to assess the effects on performance, alertness and subsequent sleep of strategic napping on 12-h overnight shifts. In a counterbalanced crossover design, 24 male aircraft maintenance engineers working in a forward rotating 12-h shift pattern volunteered to take part in the study for two work weeks. During the experimental week, each subject was given the opportunity to take a 20-min nap at work between 01:00 and 03:00 h on each of their two overnight shifts. On the control week no naps were taken on the night shifts. A computerized neurobehavioural test battery was employed to assess performance and subjective levels of fatigue at the beginning and end of each night shift, and pre- and postnap. Subjects were also asked to rate how near they had come to falling asleep while driving to and from work. The results revealed that taking a single 20-min nap during the first night shift significantly improved speed of response on a vigilance task measured at the end of the shift compared with the control condition. On the second night shift there was no effect of the nap on performance. Taking a short nap during either night shift had no significant effect on subjective ratings of fatigue, the level of sleepiness reported while driving to and from work, or subsequent sleep duration and sleep quality. Overall the results suggest some promise for a short duration nap taken in the workplace to counteract performance deficits associated with the first night shift.     

Time to pregnancy as a function of male and female serum concentrations of 2,2'4,4'5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE)

BACKGROUND: Persistent organochlorine pollutants (POP) may affect both the female and male reproductive system in animals as well as in humans. METHODS: Blood samples were collected from pregnant women and their partners from Greenland, Warsaw and Kharkiv, and from a cohort of Swedish fishermen's wives. Blood samples were analysed for 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE). Information on the participants' fertility, measured as time to pregnancy (TTP), was collected. In total, 778 men and 1505 women were included in the analyses. RESULTS: The data from Warsaw, Kharkiv and the Swedish fishermen's wives indicated no effect of either male or female exposure to POP on TTP. However, among men and women from Greenland, there seemed to be an association between serum concentrations of CB-153 and p,p'-DDE and prolonged TTP. Due to the strong intra-individual correlation between CB-153 and p,p'-DDE in the Greenlandic population, it was not possible to determine whether the risk was associated with CB-153 or p, p'-DDE or was an interaction between the two compounds. CONCLUSIONS: The overall results of the present study create a somewhat ambiguous pattern, but give some support to the idea that dietary POP exposure might be harmful for couple fertility.     

The challenge of diagnosing atopic diseases: outcomes in Cuban children depend on definition and methodology

BACKGROUND: Prevalences of childhood asthma and other atopic diseases are increasing worldwide, and so is the number of diagnostic methods and definitions used. We determined the occurrence of atopic diseases in Cuban children with a range of diagnostic approaches commonly used or proposed in epidemiological studies, and compared the different outcome measures. METHODS: A total of 398 Cuban schoolchildren between 5 and 13 years of age were diagnosed by International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire, clinical examination, pre- and post-exercise spirometry, and skin prick testing. All results were considered separately, as well as jointly by using scores and definitions as described in the literature. RESULTS: Using questionnaire-based approaches, 21-39% of the children were positive for asthma, 9-19% for atopic dermatitis, and 15-46% for rhinoconjunctivitis. With spirometry, 7% of the children had asthma. Definitions based on a combination of questionnaire and spirometry results yielded asthma rates of 5%. Of all children, 6% wheezed on clinical examination, and only one child showed clinical signs of atopic dermatitis. Eleven percent of the children had a positive skin prick test. In total, 254 children (64%) had an atopic disease as based on the ISAAC questionnaire, and 263 (66%) based on all approaches used. CONCLUSION: Diagnostic outcomes on atopic diseases vary considerably depending on definition and methodology. Our results clearly demonstrate the need for consensus on diagnosing asthma and other atopic diseases in epidemiological studies. Based on the most commonly used ISAAC questionnaire, our data suggest prevalences of atopic diseases in Cuban children that rival those found in some other Latin American countries and developed nations with the highest prevalences in the world.     

Intrathecal hyperbaric bupivacaine 3 mg + fentanyl 10 microg for outpatient knee arthroscopy with tourniquet

BACKGROUND: Combination of local anesthetic and opioid enables the use of less spinal anesthetic and increases the success of anesthesia. Intrathecal opioid does not prolong motor recovery and thus should not delay discharge home. We hypothesized that 3 mg of hyperbaric bupivacaine with 10 microg of fentanyl permits fast-tracking or shorter stay in post anesthesia care unit (PACU), and earlier discharge home, compared with 4 mg of hyperbaric bupivacaine. METHODS: In this double-blind study, 100 outpatients undergoing knee arthroscopy received randomly either 4 mg of bupivacaine (B4) or 3 mg of bupivacaine + 10 microg fentanyl (B3F) intrathecally. The volume of 0.8 ml was injected at the L2/3 interspace over a 2-min period. A lateral decubitus position was maintained for 10 min. The sensory block was recorded by using thermal stimuli, and motor block was assessed according to a modified Bromage scale. Fast-tracking criteria were complete recovery of motor block, sensory block Th12 or lower and stable vital signs. RESULTS: One block (1%) failed. Motor recovery was faster in the B3F group: 60% of the patients recovered in 80 min or less compared with 28% in group B4 (P = 0.002). The PACU-time was shorter: 36 (10-103) vs. 55 (10-140) min, respectively (P = 0.005). Seventeen (B3F) vs. nine patients (B4) could bypass PACU (NS). Time to discharge home was similar in both groups. In the B3F group, 75% of the patients developed pruritus. CONCLUSION: Both solutions produced reliable spinal anesthesia for outpatient knee arthroscopy. The PACU-time was shorter in the bupivacaine-fentanyl group, but both groups reached home-readiness equally.