Heterogeneity among septic shock patients in a set of immunoregulatory markers.

Immune activation is a regular feature of sepsis, but the incidence and nature of the ensuing inflammation-resolving and immunosuppressive component is less well understood. In this study, we compared immunoregulatory markers on blood leukocytes from patients with Gram-negative or Gram-positive sepsis or septic shock, and compared this to blood from patients with severe virosis or healthy controls. To this end, blood from 32 patients with sepsis, including ten cases with shock, and 12 patients with severe virosis were analysed by flow cytometry for the expression levels of monocyte HLA-DR, CD11c, CD14 and CD40, and for frequencies of CD163(+)-suppressive monocytes, HLA-DR(+) or CD40(+)-activated T cells and Tregs. Plasma cytokine levels were analysed as a functional measurement. Signs of immunosuppression dominated in the septic shock and Gram-positive sepsis groups, whereas monocyte activation was common in Gram-negative sepsis patients without shock. However, the main finding was the large inter-individual variation of immune activation and immunosuppression, with no correlation to prognosis among the shock patients. The pronounced inter-individual variation in the analysed monocyte and lymphocyte markers forms a strong argument that, when immunomodulatory treatment is considered in a sepsis patient, it should be personalised and guided by a detailed immune status assessment.

Wnt5a inhibits human monocyte-derived myeloid dendritic cell generation.

Wnt5a is a non-canonical Wnt protein that is expressed at elevated levels in inflammatory conditions. Its role in inflammation remains unclear, although it is known that Wnt5a is expressed at a higher level in monocyte-derived myeloid dendritic cells (Mo-mDCs) than in monocytes and macrophages. The function of Wnt5a in dendritic cells (DCs) remains relatively unexplored. Here, we found that under Mo-mDC culture conditions, Wnt5a inhibited the generation of CD14(⁺/low) Mo-mDCs while promoting the generation of CD14⁺/⁺⁺ CD16⁺ monocytes. We could further show that stimulation of monocytes with rWnt5a induced a rapid IL-6 production and that the rWnt5a treated Mo-mDC differentiation was restored upon blocking of IL-6. Also, conditioned media from Wnt5a stimulated human breast cancer cells producing IL-6, specifically inhibited Mo-mDC differentiation. These observations are strengthened by our finding that patients with sepsis, a disease involving elevated Wnt5a and IL-6 levels, also showed a significant increase in the CD14⁺ CD16⁺⁺/CD14⁺/⁺⁺ CD16⁺ monocyte populations, which was accompanied by a significant decrease in circulating mDCs. We finally show that under typical Mo-mDC culture conditions, monocytes isolated from patients with sepsis as compared to healthy controls, preferentially differentiated into CD14CD14⁺/⁺⁺ HLA-DR⁺⁺ cells. We suggest that Wnt5a is a possible candidate mediator for the CD14⁺/⁺⁺ CD16⁺ monocyte accumulation seen in patients with infectious disease and cancer.

Reducing blood culture contamination by a simple informational intervention.

Compared to truly negative cultures, false-positive blood cultures not only increase laboratory work but also prolong lengths of patient stay and use of broad-spectrum antibiotics, both of which are likely to increase antibiotic resistance and patient morbidity. The increased patient suffering and surplus costs caused by blood culture contamination motivate substantial measures to decrease the rate of contamination, including the use of dedicated phlebotomy teams. The present study evaluated the effect of a simple informational intervention aimed at reducing blood culture contamination at Skåne University Hospital (SUS), Malmö, Sweden, during 3.5 months, focusing on departments collecting many blood cultures. The main examined outcomes of the study were pre- and postintervention contamination rates, analyzed with a multivariate logistic regression model adjusting for relevant determinants of contamination. A total of 51,264 blood culture sets were drawn from 14,826 patients during the study period (January 2006 to December 2009). The blood culture contamination rate preintervention was 2.59% and decreased to 2.23% postintervention (odds ratio, 0.86; 95% confidence interval, 0.76 to 0.98). A similar decrease in relevant bacterial isolates was not found postintervention. Contamination rates at three auxiliary hospitals did not decrease during the same period. The effect of the intervention on phlebotomists' knowledge of blood culture routines was also evaluated, with a clear increase in level of knowledge among interviewed phlebotomists postintervention. The present study shows that a relatively simple informational intervention can have significant effects on the level of contaminated blood cultures, even in a setting with low rates of contamination where nurses and auxiliary nurses conduct phlebotomies.

Lactobacillus plantarum 299v reduces colonisation of Clostridium difficile in critically ill patients treated with antibiotics.

BACKGROUND: The incidence of Clostridium difficile-associated disease (CDAD) in hospitalised patients is increasing. Critically ill patients are often treated with antibiotics and are at a high risk of developing CDAD. Lactobacillus plantarum 299v (Lp299v) has been found to reduce recurrence of CDAD. We investigated intensive care unit (ICU) patients with respect to the impact of Lp299v on C. difficile colonisation and on gut permeability and parameters of inflammation and infection in that context. METHODS: Twenty-two ICU patients were given a fermented oatmeal gruel containing Lp299v, and 22 received an equivalent product without the bacteria. Faecal samples for analyses of C. difficile and Lp299v were taken at inclusion and then twice a week during the ICU stay. Other cultures were performed on clinical indication. Infection and inflammation parameters were analysed daily. Gut permeability was assessed using a sugar probe technique. RESULTS: Colonisation with C. difficile was detected in 19% (4/21) of controls but in none of the Lp299v-treated patients (P<0.05). CONCLUSIONS: Enteral administration of the probiotic bacterium Lp299v to critically ill patients treated with antibiotics reduced colonisation with C. difficile.

Low prevalence of nosocomial Clostridium difficile transmission, as determined by comparison of arbitrarily primed PCR and epidemiological data.

An increased prevalence of patients with C. difficile-associated diarrhoea in a hospital setting suggested the possible existence of an endemic occurrence. A study was therefore designed to determine clonal relatedness among 173 isolates of C. difficile, collected consecutively during 1995 from 147 patients (89 inpatients and 58 outpatients) and to estimate the probability of nosocomial transmission. Arbitrarily primed PCR (AP-PCR) with three different primers, AP1, AP2 and CLD1, was used for fingerprinting and identified 21, 92 and 70 types, respectively. Overall DNA analysis of the combined AP-PCR data yielded 140 types, of which 130 were unique, whereas 10 types occurred repeatedly in 36 isolates from 33 patients; seven isolates were non-typeable by one of the primers. Epidemiological data confirmed that in eight of the 33 patients there was a high probability of nosocomial transmission. Despite a high prevalence of C. difficile among hospitalized patients, a low frequency of nosocomial transmission was suggested by high resolution molecular typing of bacterial isolates in conjunction with traditional epidemiological methods.

Comparison of AP-PCR typing and PCR-ribotyping for estimation of nosocomial transmission of Clostridium difficile.

We recently attempted to clarify an increased incidence of Clostridium difficile-associated diarrhoea (CDAD) in our hospital by arbitrarily primed polymerase chain reaction (AP-PCR) typing of isolates from 147 consecutive patients collected during a 12 month period (Wullt et al. J Hosp Infect 1999;43:265-273). In the present study we compared the results based on previous AP-PCR data with those based on recent PCR ribotyping of the same isolates and re-analysis of a subset of isolates by AP-PCR typing. The pattern of PCR ribotypes was similar among inpatients and outpatients. A cluster of three closely related PCR ribotypes, related to those of the serogroup H and A8 type strains, dominated and comprised 31% of inpatient and 28% of outpatient C. difficile isolates. The apparent nosocomial transmission rate among inpatients with CDAD was only 9% by AP-PCR typing compared with 18 or 36% by PCR ribotyping depending on the definition used (proportion of patients sharing C. difficile type and ward within two or 12 months). Corresponding rates for all CDAD patients were 5% by AP-PCR and 11 or 21% by PCR ribotyping. Thus, most CDAD patients apparently became ill due to their endogenous strain of C. difficile. Because of the low concordance between the two typing methods the proportion of patients fulfilling the criteria for nosocomial transmission by both methods was only 1%. Re-examination of isolates from patients with recurrences revealed a reproducibility problem with AP-PCR typing. We conclude, that of these two PCR-based options for typing of C. difficile PCR ribotyping offers a superior experimental robustness compared with AP-PCR typing.

IgG antibody response to toxins A and B in patients with Clostridium difficile infection.

IgG antibodies against Clostridium difficile toxins A and B were followed in controls and in patients with an initial C. difficile infection (CDI). Of the 50 CDI patients, 38 were cured and 12 developed recurrence. Compared to controls, patients had significantly lower anti-toxin A and B IgGs at inclusion, but the subsequent levels rose slightly regardless of clinical outcome. The results imply that the general serum reactivity against toxins A and B in the population reduces the risk of CDI, which suggests implications for vaccine strategies.

A head-to-head comparison of hydrogen peroxide vapor and aerosol room decontamination systems.

OBJECTIVE: New technologies have emerged in recent years for the disinfection of hospital rooms and equipment that may not be disinfected adequately using conventional methods. There are several hydrogen peroxide-based area decontamination technologies on the market, but no head-to-head studies have been performed. DESIGN: We conducted a head-to-head in vitro comparison of a hydrogen peroxide vapor (HPV) system (Bioquell) and an aerosolized hydrogen peroxide (aHP) system (Sterinis). SETTING: The tests were conducted in a purpose-built 136-m(3) test room. METHODS: One HPV generator and 2 aHP machines were used, following recommendations of the manufacturers. Three repeated tests were performed for each system. The microbiological efficacy of the 2 systems was tested using 6-log Tyvek-pouched Geobacillus stearothermophilus biological indicators (BIs). The indicators were placed at 20 locations in the first test and 14 locations in the subsequent 2 tests for each system. RESULTS: All BIs were inactivated for the 3 HPV tests, compared with only 10% in the first aHP test and 79% in the other 2 aHP tests. The peak hydrogen peroxide concentration was 338 ppm for HPV and 160 ppm for aHP. The total cycle time (including aeration) was 3 and 3.5 hours for the 3 HPV tests and the 3 aHP tests, respectively. Monitoring around the perimeter of the enclosure with a handheld sensor during tests of both systems did not identify leakage. CONCLUSION: One HPV generator was more effective than 2 aHP machines for the inactivation of G. stearothermophilus BIs, and cycle times were faster for the HPV system.

Mutations in fusA associated with posttherapy fusidic acid resistance in Clostridium difficile.

In silico, we identified fusA (2,067 bp) in Clostridium difficile 630. Sequencing of fusA in posttherapy fusidic acid-resistant C. difficile isolates from 12 patients with C. difficile-associated diarrhea (CDAD) identified fusA mutations, one or two nonsynonymous substitutions, or in one case a deletion of one codon associated with resistance. Five of these mutations have previously been described in fusA of fusidic acid-resistant Staphylococcus aureus, but seven were novel fusA mutations. Fusidic acid monotherapy for CDAD seemed to rapidly select conserved resistant mutants.

Frequent emergence of resistance in Clostridium difficile during treatment of C. difficile-associated diarrhea with fusidic acid.

Samples from patients with Clostridium difficile-associated diarrhea (CDAD) that were randomized to fusidic acid (n = 59) or metronidazole (n = 55) therapy for 7 days were cultured for Clostridium difficile in feces on days 1, 8 to 13, and 35 to 40. Of the patients who were culture positive only before treatment, 77% (36/47) were permanently cured (no treatment failure and no clinical recurrence), compared to 54% (22/41) of those with persistence of C. difficile at one or both follow-ups (P = 0.03). A similar association between bacterial persistence and a worse outcome of therapy was seen in both treatment groups. Resistance to fusidic acid was found in 1 of 88 pretherapy isolates available, plus in at least 1 subsequent isolate from 55% (11/20) of patients who remained culture-positive after fusidic acid therapy. In 10 of these 11 patients, the resistant follow-up isolate(s) belonged to the same PCR ribotype as the susceptible day 1 isolate, confirming frequent emergence of resistance to fusidic acid during treatment. Despite this, 5 of these 11 patients were permanently cured with fusidic acid, relative to 5 of 9 patients with susceptible C. difficile at follow-up (P = 1.0). None of the 36 PCR ribotypes of C. difficile identified was associated with any particular clinical outcome or emergence of fusidic acid resistance. In conclusion, culture positivity for C. difficile was common after both fusidic acid and metronidazole therapy and was associated with treatment failure or recurrence of CDAD. Development of resistance in C. difficile was frequent in patients given fusidic acid, but it was without apparent negative impact on therapeutic efficacy in the actual CDAD episode.