Apparent Intracellular Helicobacter pylori Detected by Immunohistochemistry: The Missing Link in Eradication Failure.

BACKGROUND: Helicobacter pylori is primarily an extracellularly living bacterium. However, seemingly intracellular occurrence can often be detected by immunohistochemical stains. Considering antimicrobial resistance, we investigated the impact of the apparent intracellular H. pylori (aiHp) on treatment failure of first-line triple therapies. METHODS: Gastric biopsies of 814 patients infected with H. pylori naive to treatment were analyzed before and after eradication therapy by immunohistochemistry. Of these, 373 received treatment consisting of amoxicillin, clarithromycin, and proton pump inhibitor (AC/PPI). Availability of polymerase chain reaction-based clarithromycin susceptibility test results from pretreatment gastric biopsies was a precondition for matching 52 aiHp to 52 non-aiHp cases within the AC/PPI group. RESULTS: AiHp were detected mostly in low counts predominantly in corpus biopsies, rarely in antrum biopsies (95.2% vs 24.6%); they were found in 497 (61%) of all patients and in 192 of 373 patients (51.5%) in the AC/PPI group. The eradication rate in aiHp versus non-aiHp cases was 44.4% versus 72.9% in the entire sample and 45.3% versus 66.8% in the AC/PPI group. Among the 104 paired patients, respective values were 46.2% versus 78.8%; in clarithromycin-susceptible cases, 60.6% versus 91.9%. Both aiHp and resistance to clarithromycin proved to be highly significant (P ≤ .001) and independent predictors of eradication failure. Twelve of 13 aiHp cases with a clarithromycin-sensitive strain who failed eradication developed resistance to the antibiotic. CONCLUSIONS: AiHp found by immunohistochemical staining especially in corpus biopsies proved to be a risk factor for failure of first-line triple therapies; occurrence of aiHp should be considered with regard to therapy options.

Education of medical personnel optimizes filling volume of blood culture bottles without negatively affecting microbiology testing.

BACKGROUND: Anemia is a risk factor for adverse outcomes, which can be aggravated by unnecessary phlebotomies. In blood culture testing, up to 30 ml of blood can be withdrawn per sample, even though most manufacturers recommend blood volumes of 10 ml or less. After assessing the filling volume of blood culture bottles at our institution, we investigated whether an educational intervention could optimize filling volume of blood culture bottles without negatively affecting microbiology testing. METHODS: We weighed 10,147 blood cultures before and 11,806 blood cultures after a six-month educational intervention, during which employees were trained regarding correct filling volume via lectures, handouts, emails, and posters placed at strategic places. RESULTS: Before the educational intervention, only 31% of aerobic and 34% of anaerobic blood cultures were filled correctly with 5-10 ml of blood. The educational intervention increased the percentage of correctly filled bottles to 43% (P < 0.001) for both aerobic and anaerobic samples without negatively affecting results of microbiologic testing. In addition, sample volume was reduced from 11.0 ± 6.5 to 9.4 ± 5.1 ml (P < 0.001) in aerobic bottles and from 10.1 ± 5.6 to 8.8 ± 4.8 ml (P < 0.001) in anaerobic bottles. CONCLUSION: Education of medical personnel is a simple and effective way to reduce iatrogenic blood loss and possibly moderate the extent of phlebotomy-induced anemia.

Review: Diagnosis of Helicobacter pylori infection.

Endoscopic imaging of the stomach is improving. In addition to narrow band imaging, other methods, for example, blue light imaging and linked color imaging, are now available and can be combined with artificial intelligence systems to obtain information on the gastric mucosa and detect early gastric cancer. Immunohistochemistry is only recommended as an ancillary stain in case of chronic active gastritis without Helicobacter pylori detection by standard staining, and recommendations to exclude false negative H. pylori results have been made. Molecular methods using real-time PCR, droplet digital PCR, or amplification refractory mutation system PCR have shown a high accuracy, both for detecting H. pylori and for clarithromycin susceptibility testing, and can now be used in clinical practice for targeted therapy. The most reliable non-invasive test remains the 13 C-urea breath test. Large data sets show that DOB values are higher in women and that the cut-off for positivity could be decreased to 2.74 DOB. Stool antigen tests using monoclonal antibodies are widely used and may be a good alternative to UBT, particularly in countries with a high prevalence of H. pylori infection. Attempts to improve serology by looking at specific immunodominant antigens to distinguish current and past infection have been made. The interest of Gastropanel® which also tests pepsinogen levels was confirmed.

Risk factors for development of spontaneous bacterial peritonitis and subsequent mortality in cirrhotic patients with ascites.

BACKGROUND: Patients with ascites are at risk for developing spontaneous bacterial peritonitis (SBP) - a severe complication associated with high mortality. We aimed to identify risk factors for SBP development and mortality to optimize stratification for primary prophylaxis and therapeutic strategies to improve survival. METHODS: 575 patients with cirrhosis and ascites undergoing paracentesis at a tertiary care hospital were included in this retrospective cohort study. Demographical, clinical and laboratory parameters were recorded at first paracentesis and during follow-up. Multivariate logistic regression analysis was used to identify independent predictors of SBP development and mortality. RESULTS: Child-Pugh stage C (OR: 3.323; P = 0.009), ascitic fluid polymorph-nuclear cell (PMN) count (OR: 1.544; P = 0.028) and low serum sodium (OR: 0.917; P = 0.029) emerged as independent risk factors for SBP development. SBP-naïve patients undergoing paracentesis and presenting with PMN-counts ≥100 cells/µl, or hyponatraemia <125 mM were at highest risk for developing SBP. Increases in MELD and CRP levels indicated SBP development, while no changes where observed in a matched control group with sterile ascites at multiple paracenteses. MELD score (OR: 1.565; P = 0.001) and CRP (OR: 1.067; P = 0.037) were identified as independent risk factors for 30-day mortality after SBP diagnosis. In particular SBP patients with MELD≥22, CRP ≥3.5 mg/dl and development of grade III/IV hepatic encephalopathy showed highest mortality. CONCLUSIONS: Low serum sodium levels, Child-Pugh stage C and elevated ascites PMN counts (≥100 cells/µl) indicate a significant risk for SBP development. SBP-related mortality is highest in patients with MELD≥22 and elevated CRP levels.

Bacterial adherence to different components of total hip prosthesis in patients with prosthetic joint infection.

PURPOSE: The purpose of our study was to evaluate and quantify the bacterial adherence to the different components of total hip prosthesis. METHODS: The bacterial load of 80 retrieved hip components from 24 patients was evaluated by counting of colony-forming units (CFU) dislodged from component surfaces using the sonication culture method. RESULTS: Micro-organisms were detected in 68 of 80 explanted components. The highest bacterial load was detected on the polyethylene liners, showing a significant difference in distribution of CFU between the liner and metal components (stem and cup). Staphylococcus epidermidis was identified as the pathogen causing the highest CFU count, especially from the polyethylene liner. CONCLUSIONS: Results of our study confirm that sonicate culture of the retrieved liners and heads, which revealed the highest bacterial loads, are reliable and sufficient for pathogen detection in the clinical diagnostic routine.

Helicobacter pylori resistance to antibiotics in Europe and its relationship to antibiotic consumption.

OBJECTIVE: Resistance to antibiotics is the major cause of treatment failure of Helicobacter pylori infection. A study was conducted to assess prospectively the antibacterial resistance rates of H pylori in Europe and to study the link between outpatient antibiotic use and resistance levels in different countries. DESIGN: Primary antibiotic resistance rates of H pylori were determined from April 2008 to June 2009 in 18 European countries. Data on yearly and cumulative use over several years of systemic antibacterial agents in ambulatory care for the period 2001-8 were expressed in Defined Daily Doses (DDD) per 1000 inhabitants per day. The fit of models and the degree of ecological association between antibiotic use and resistance data were assessed using generalised linear mixed models. RESULTS: Of 2204 patients included, H pylori resistance rates for adults were 17.5% for clarithromycin, 14.1% for levofloxacin and 34.9% for metronidazole, and were significantly higher for clarithromycin and levofloxacin in Western/Central and Southern Europe (>20%) than in Northern European countries (<10%). Model fit improved for each additional year of antibiotic use accumulated, but the best fit was obtained for 2005. A significant association was found between outpatient quinolone use and the proportion of levofloxacin resistance (p=0.0013) and between the use of long-acting macrolides only and clarithromycin resistance (p=0.036). CONCLUSION: In many countries the high rate of clarithromycin resistance no longer allows its empirical use in standard anti-H pylori regimens. The knowledge of outpatient antibiotic consumption may provide a simple tool to predict the susceptibility of H pylori to quinolones and to macrolides and to adapt the treatment strategies.

Neutralization of antimicrobial substances in new BacT/Alert FA and FN Plus blood culture bottles.

Time to detection (TTD) in automated blood culture systems is delayed for sensitive microorganisms in the presence of antimicrobial substances and has been associated with worse outcomes for sepsis patients on inadequate empirical therapy. While resin addition removes antimicrobial substances to various degrees from blood culture media, media formulations and the blend of resins may influence performance. The BacT/Alert 3D system (bioMérieux) was investigated using the new resin-containing medium types FA Plus (aerobic) and FN Plus (anaerobic). TTD was compared between control and test bottles containing relevant bacteria or Candida albicans, with and without defined concentrations of antimicrobials. Failure of neutralization was defined as a negative blood culture on day 3. In general, growth delay was nonlinear, concentration dependent, bottle type specific, and reciprocally associated with MICs. Substance-specific serum drug concentrations corresponding to a predefined, clinically relevant 3-h delay of TTD were calculated. Where appropriate, a time interval allowing for drug elimination below this critical level was obtained by pharmacokinetic modeling. Clarithromycin, clindamycin, gentamicin, linezolid, tigecycline, vancomycin, and fluconazole were neutralized. For ciprofloxacin and piperacillin-tazobactam, which were only incompletely neutralized in combination with the most sensitive test strains, a maximum waiting time for blood draw of 1 h was determined based on pharmacokinetics. One or more test strains did not grow in bottles containing either amoxicillin-clavulanate, cefepime, cefotaxime, meropenem, or metronidazole, and we thus recommend particular caution in timing of blood draws if patients have been pretreated with these agents.

Use of a commercial PCR-based line blot method for identification of bacterial pathogens and the mecA and van genes from BacTAlert blood culture bottles.

In this study, the PCR-based DNA strip assay GenoType BC for the identification of bacteria and the resistance genes mecA, vanA, vanB, vanC1, and vanC2/3 directly from positive BacTAlert blood culture bottles was evaluated in a multicenter study. Of a total of 511 positive blood cultures, correct identification percentages for Gram-negative bacteria, Gram-positive bacteria, and the mecA gene were 96.1%, 89.9%, and 92.9%, respectively. Results were available 4 h after growth detection.

Staphylococcus aureus fibronectin-binding protein specifically binds IgE from patients with atopic dermatitis and requires antigen presentation for cellular immune responses.

BACKGROUND: Staphylococcus aureus superinfections occur in more than 90% of patients with atopic dermatitis (AD) and aggravate skin inflammation. S aureus toxins lead to tissue damage and augment T-cell-mediated skin inflammation by a superantigen effect. OBJECTIVE: To characterize IgE-reactive proteins from S aureus. METHODS: A genomic S aureus library was screened with IgE from patients with AD for DNA clones coding for IgE-reactive antigens. One was identified as fibronectin-binding protein (FBP). Recombinant FBP was expressed in Escherichia coli, purified, and tested for specific IgE reactivity in patients with AD. Its allergenic activity was studied in basophil activation experiments and T-cell cultures. The in vivo allergenic activity was investigated by sensitizing mice. RESULTS: Using IgE from patients with AD for screening of a genomic S aureus library, an IgE-reactive DNA clone was isolated that coded for FBP. Recombinant FBP was expressed in E coli and purified. It reacted specifically with IgE from patients with AD and exhibited allergenic activity in basophil degranulation assays. FBP showed specific T-cell reactivity requiring antigen presentation and induced the secretion of proinflammatory cytokines from PBMCs. Mice sensitized with FBP mounted FBP-specific IgE responses, showed FBP-specific basophil degranulation as well as FBP-specific T-cell proliferation, and mixed T(h)2/T(h)1 cytokine secretion. CONCLUSION: Evidence is provided that specific humoral and cellular immune responses to S aureus antigens dependent on antigen presentation represent a novel mechanism for S aureus-induced skin inflammation in AD. Furthermore, FBP may be used for the development of novel diagnostic and therapeutic strategies for S aureus infections.

Helicobacter pylori eradication rates in children upon susceptibility testing based on noninvasive stool polymerase chain reaction versus gastric tissue culture.

BACKGROUND AND OBJECTIVES: In children with clarithromycin-resistant Helicobacter pylori, clarithromycin-containing therapies often fail. The present study aimed to assess the outcome of tailored therapy upon noninvasive versus invasive H pylori susceptibility testing. PATIENTS AND METHODS: A retrospective cohort study was conducted in a pediatric outpatient clinic located in a region where H pylori clarithromycin resistance is highly prevalent. Between June 2007 and September 2009, 96 infected children (mean age 10.8 years), naïve to H pylori eradication treatment, were prescribed triple eradication therapies. These therapies were individually tailored upon susceptibility testing performed either noninvasively using stool polymerase chain reaction (stool PCR group) or invasively using endoscopy, biopsy, and culturing of gastric biopsies (gastric biopsy group). Eradication was defined by negative results upon noninvasive testing including stool PCR at least 5 weeks after the end of treatment. RESULTS: H pylori was eradicated in 43 of 55 stool PCR group versus 30 of 41 gastric biopsy group children (78.2% vs 73.2%, P = 0.63). Of those H pylori strains with pretherapeutic clarithromycin susceptibility, 78.8% were eradicated in the stool PCR group and 69.2% in the gastric biopsy group (P = 0.41) following clarithromycin-containing therapy; clarithromycin resistance was acquired by 4.1% of strains in the former group versus 12% in the latter (P = 0.33). CONCLUSIONS: Stool PCR is as effective as the invasive approach of H pylori susceptibility testing for targeting resistance-guided eradication treatments in children. Furthermore, stool PCR is a useful tool for tracking the emergence of clarithromycin resistance following eradication treatment.

Increased temperature enhances the antimicrobial effects of daptomycin, vancomycin, tigecycline, fosfomycin, and cefamandole on staphylococcal biofilms.

Implant-related infections are serious complications of trauma and orthopedic surgery and are most difficult to treat. The bacterial biofilms of 34 clinical Staphylococcus sp. isolates (Staphylococcus aureus, n = 14; coagulase-negative staphylococci, n = 19) were incubated with daptomycin (DAP; 5, 25, or 100 mg/liter), vancomycin (VAN; 5, 25, or 100 mg/liter), tigecycline (TGC; 1, 5, or 25 mg/liter), fosfomycin (FOM; 100, 250, or 1,000 mg/liter), and cefamandole (FAM; 50, 100, or 500 mg/liter) for 24 h at three different ambient temperatures: 35°C, 40°C, and 45°C. To quantify the reduction of the biomass, the optical density ratio (ODr) of stained biofilms and the number of growing bacteria were determined. Increasing the temperature to 45°C or to 40°C during incubation with FAM, FOM, TGC, VAN, or DAP led to a significant but differential reduction of the thickness of the staphylococcal biofilms compared to that at 35°C (P < 0.05). Growth reduction was enhanced for DAP at 100 mg/liter at 35°C, 40°C, and 45°C (log count reductions, 4, 3.6, and 3.3, respectively; P < 0.05). A growth reduction by 2 log counts was detected for FAM at a concentration of 500 mg/liter at 40°C and 45°C (P = 0.01). FOM at 1,000 mg/liter reduced the bacterial growth by 1.2 log counts (not significant). The antibacterial activity of antimicrobial agents is significantly but differentially enhanced by increasing the ambient temperature and using high concentrations. Adjuvant hyperthermia may be of value in the treatment of biofilm-associated implant-related infections.

Stool polymerase chain reaction for Helicobacter pylori detection and clarithromycin susceptibility testing in children.

BACKGROUND & AIMS: This study was undertaken in a pediatric gastroenterology clinic to retrospectively evaluate a real-time polymerase chain reaction (PCR) for the detection and clarithromycin susceptibility testing of Helicobacter pylori using stool specimens. METHODS: All consecutive children who underwent a gastroscopy between March 2006 and February 2009 and also having been examined by stool PCR were enrolled. Rapid urease test, histology, and culture were the reference methods for the detection of H pylori and E-test for susceptibility testing, respectively. RESULTS: A total of 143 children (mean age, 10.8 y; range, 2.8-17.9; males:females, 1:1.5) were evaluable. Sensitivity, specificity, and test accuracy for the detection of H pylori were 83.8%, 98.4%, and 90.2%, respectively. Sensitivity, specificity, and accuracy for the detection of clarithromycin resistance were 89.2%, 100%, and 94.0%, respectively. CONCLUSIONS: Stool PCR was a reliable and useful noninvasive tool for detection and clarithromycin susceptibility testing of H pylori in a pediatric population with a high prevalence of clarithromycin-resistant strains.

Time trends of Helicobacter pylori resistance to antibiotics in children living in Vienna, Austria.

BACKGROUND: Increase of antibiotic resistance is a worldwide problem. Within the 4 years before the turn of the millennium Helicobacter pylori strains isolated in children living in Vienna, Austria, showed a primary clarithromycin and metronidazole resistance of 20% and 16%, respectively. The aim of this retrospective follow-up survey was to assess the further development and current antimicrobial resistance status. METHODS: Children having undergone upper endoscopy between March 2002 and March 2008 at the same two co-operating pediatric gastroenterology units which had also been collaborating on the prior assessment were included. H. pylori infection was diagnosed by rapid urease test, histology, and culture. If the latter was positive, susceptibility testing to amoxicillin, clarithromycin and metronidazole by E-test followed. From March 2004 onwards, susceptibility to levofloxacin, tetracycline and rifampin was additionally assessed. RESULTS: Out of 897 children, 153 had a proven infection with H. pylori and no history of prior eradication treatment. Their median age was 11.5 years (range 0.5-20.9 years). Primary resistance to clarithromycin and metronidazole were 34% and 22.9%, respectively; dual resistance was found in 9.8% of the strains; 0.9% was resistant to tetracycline and rifampin, respectively. No case of amoxicillin resistance was detected. The only independent risk factor for clarithromycin resistance turned out to be the origin of a child from Austrian parents. CONCLUSIONS: In the last decade, the rate of primary resistance of H. pylori to clarithromycin continued to rise. No significant change was found regarding primary resistance to metronidazole or dual resistance to metronidazole and clarithromycin, respectively.

Effects of azithromycin in combination with vancomycin, daptomycin, fosfomycin, tigecycline, and ceftriaxone on Staphylococcus epidermidis biofilms.

Staphylococcal biofilms on surgical implants are the underlying cause of a lack of response to antimicrobial treatment. We investigated the effects of vancomycin (VAN), daptomycin (DAP), fosfomycin (FOS), tigecycline (TGC), and ceftriaxone (CRX), alone and in combination with azithromycin (AZI), on established biofilms of Staphylococcus epidermidis. Biofilms were studied using the static microtiter plate model with established S. epidermidis biofilms, with an initial inoculum of 10(6)/ml in 96-well polystyrene flat-bottom microtiter plates. Biofilms were inoculated with VAN, DAP, FOS, TGC, or CRX at two concentrations, alone or in combination with AZI (2, 512, or 1,024 mg/liter). To assess the reduction in biomass, the optical density ratio (ODr), calculated as (optical density [OD] of the treated biofilm)/(OD of the untreated biofilm, taken as 1), was used. For antibacterial efficacy, the viable bacterial count was used. Reductions in the biofilm ODr were observed for VAN (15 and 40 mg/liter) and FOS (200 mg/liter) only (ODr [mean +/- standard deviation] for VAN at 15 and 40 mg/liter, 0.77 +/- 0.32 and 0.8 +/- 0.35, respectively; ODr for FOS at 200 mg/liter, 0.78 +/- 0.26; P < 0.05), but not for DAP (2 and 5 mg/liter), TGC (0.2 and 2 mg/liter), or CRX (600 and 2,400 mg/liter). The addition of AZI had no further effect on the ODr, but a significant reduction of bacterial growth was achieved with high doses of AZI plus TGC or AZI plus CRX (a 3-log count reduction for AZI at 1,024 mg/liter plus CRX at 600 mg/liter and for AZI at 512 or 1,024 mg/liter plus CRX at 2,400 mg/liter; a 2-log count reduction for AZI at 512 or 1,024 mg/liter plus TGC at 2 mg/liter [P < 0.05]). No significant reduction in bacterial growth was observed for FOS (50 and 200 mg/liter), DAP (2 and 5 mg/liter), or TGC (0.2 mg/liter) in combination with AZI. None of the antibiotics at either concentration reduced the bacterial count of the biofilms when used alone. Thus, the use of a combination of AZI plus TGC, FOS, or CRX at high concentrations has little effect on biofilm density but significantly reduces bacterial growth.

A novel noninvasive genotyping method of Helicobacter pylori using stool specimens.

BACKGROUND & AIMS: The source(s) of the infection and the route(s) of transmission of Helicobacter pylori have not yet been clarified. This is to introduce a noninvasive protocol allowing molecular typing of H pylori using stool specimens. METHODS: The genotyping method is based on 2 H pylori-specific biprobe real-time polymerase chain reaction assays using fragments of the glmM and the recA genes as target sequences. Discrimination between strains results from differences in the melting temperature during melting curve analysis. In case of identical melting temperatures in both assays, sequence analysis of the glmM amplicon was performed to confirm strain identity. The method was validated using gastric biopsy specimens and stool specimens of 97 unrelated individuals suffering from abdominal pain and stool specimens of members of 10 families in Austria (infected index child and family members) and 8 African households. RESULTS: Of the 97 patients, 27 were infected as shown by culture, histology, and rapid urease test. The sensitivity of each of the assays was 100% in gastric biopsy specimens and 92.2% in stool specimens; the specificity was 100%. The discriminatory capacity of the method was 100%. Clonal identities were found in 9 of 10 (90%) European and 7 of 8 (87.5%) African households. In 2 African households, 2 different clonal lineages each were found. CONCLUSIONS: The genotyping protocol introduced allows for both accurate detection and discrimination of H pylori strains in stool samples. Large-scale studies using this protocol may contribute to the clarification of the transmission pathways of infection with H pylori.

Substantial diagnostic impact of blood culture independent molecular methods in bloodstream infections: Superior performance of PCR/ESI-MS.

This study analyzed the performance of different molecular technologies along with blood culture (BC) in the diagnosis of bloodstream infections (BSI) in patients from internal medicine wards - including intensive care units (ICUs) - and the emergency room. Patients with systemic inflammatory response syndrome were prospectively included. BCs and EDTA whole blood were obtained simultaneously. The latter was analyzed by PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS; IRIDICA BAC BSI assay, Abbott) and by SeptiFast (Roche). Cases were classified as BSI according to adapted European Centre for Disease Prevention and Control criteria. Out of 462 analyzed episodes, 193 with valid test results fulfilled the inclusion criteria and were further evaluated. Sixty-nine (35.8%) were classified as BSI. PCR/ESI-MS showed a significantly better overall performance than BC (p = 0.004) or SeptiFast (p = 0.034). Only in patients from the ICU the performance of SeptiFast was comparable to that of PCR/ESI-MS. Mainly due to the negative effect of antimicrobial pre-treatment on BC results, the cumulative performance of each of the molecular tests with BC was significantly higher than that of BC alone (p < 0.001). SeptiFast and in particular the broad-range pathogen detection system PCR/ESI-MS proved to be an essential addition to BC-based diagnostics in BSI.

Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition.

BACKGROUND: Pathogen identification in clinical routine is based on the cultivation of microbes with subsequent morphological and physiological characterisation lasting at least 24 hours. However, early and accurate identification is a crucial requisite for fast and optimally targeted antimicrobial treatment. Molecular biology based techniques allow fast identification, however discrimination of very closely related species remains still difficult. RESULTS: A molecular approach is presented for the rapid identification of pathogens combining PCR amplification with microarray detection. The DNA chip comprises oligonucleotide capture probes for 25 different pathogens including Gram positive cocci, the most frequently encountered genera of Enterobacteriaceae, non-fermenter and clinical relevant Candida species. The observed detection limits varied from 10 cells (e.g. E. coli) to 10(5) cells (S. aureus) per mL artificially spiked blood. Thus the current low sensitivity for some species still represents a barrier for clinical application. Successful discrimination of closely related species was achieved by a signal pattern recognition approach based on the k-nearest-neighbour method. A prototype software providing this statistical evaluation was developed, allowing correct identification in 100 % of the cases at the genus and in 96.7 % at the species level (n = 241). CONCLUSION: The newly developed molecular assay can be carried out within 6 hours in a research laboratory from pathogen isolation to species identification. From our results we conclude that DNA microarrays can be a useful tool for rapid identification of closely related pathogens particularly when the protocols are adapted to the special clinical scenarios.

SeptiFast versus blood culture in clinical routine - A report on 3 years experience.

BACKGROUND: In recent years a multiplex real-time PCR (SeptiFast) has been introduced, allowing detection of 25 common blood pathogens considerably faster than conventional blood culture. METHODS: SeptiFast was applied routinely in addition to blood culture in cases of critically ill patients with fever and other signs of severe systemic infections. In this study data of 470 episodes were retrospectively analysed to assess the impact of various parameters, such as clinical indications, assigning ward and antimicrobial treatment on test outcome using a multivariate logistic model. RESULTS: After exclusion of microorganisms classified as contaminants, the concordance between SeptiFast and blood culture was 85.5%. SeptiFast detected 98 out of 120, while blood culture merely found 63 out of 120 potential pathogens. In comparison to blood culture, SeptiFast showed considerably higher positivity rates in sepsis, pneumonia and febrile immunosuppression and a lower rate in endocarditis. The highest positivity and concordance between tests was shown in patients from the emergency room (P = 0.007). CONCLUSIONS: The results obtained in this study are similar to those from prospective settings confirming the robustness of the SeptiFast assay in routine use. Our data suggest that SeptiFast is a valuable add-on to blood culture and may increase the diagnostic efficiency of a microbiological laboratory.

Detection of fungal pathogens by a new broad range real-time PCR assay targeting the fungal ITS2 region.

PURPOSE: The rise in the incidence of fungal infections and the expanding spectrum of fungal pathogens make early and broad detection of fungal pathogens essential. In the present study, a panfungal real-time PCR assay for the broad-range detection of fungal DNA (Fungi assay) in a wide variety of clinical specimens was developed. METHODOLOGY: Our in-house, HybProbe real-time PCR assay targets the ITS2 region of fungal DNA. The applicability was evaluated by testing 105 clinical samples from 98 patients with suspected fungal infection. Samples included tissue biopsies, paraffin embedded tissues, aspirates, EDTA-anticoagulated blood, cerebrospinal fluids and bronchoalveolar lavages. RESULTS: Fungal pathogens were identified by the Fungi assay in 47 samples. In all of these cases, conventional methods and clinical data were also indicative for a fungal infection. Five samples were interpreted false negative. blast analyses of the amplicons derived from 11 samples revealed the presence of environmental fungal species while other tests and clinical data did not suggest a fungal infection. This fact might indicate contaminated samples. The remaining 42 samples were negative by the Fungi assay as well as the conventional methods and were therefore regarded as true negatives. Thus, sensitivity was 90.4 % and specificity 79.2 %. CONCLUSION: The Fungi assay improved the targeted diagnosis of fungal infections allowing pathogen identification in samples that were histologically positive but culture negative. For reliable diagnosis, results have to be interpreted in context with conventional methods and clinical data.

Sonication culture improves microbiological diagnosis of modular megaprostheses.

Modular megaprostheses are known for high infection rates followed by high rates of revisions. Microbial biofilms growing adherently on prosthetic surfaces may inhibit the detection of the pathogens causing prosthetic joint infections. We sought to answer the following questions: Does sonication culture (SC) improve the microbiological diagnosis of periprosthetic infections of megaprostheses compared to conventional tissue culture (TC)? Which pathogens were detected on the surface of megaprostheses with either SC or TC and do the findings help to identify low-grade infections? Included were 31 patients with modular megaprostheses, whose implant had been explanted due to suspected joint infection or revision surgery. SCs were performed according to the protocol by Trampuz et al. The diagnosis of infection was evaluated according to the definition of the Musculoskeletal Infection Society. The sensitivity of SC was 91.3% compared to 52.2% for TC and the specificity was 100% for SC and TC (p = 0.004). Under preoperative antibiotic therapy, the sensitivity of SC was 83.3% while the sensitivity of TC was 50%. Without preoperative antibiotic therapy the sensitivity of SC was 100% compared to 54.5% for TC. In nine cases, SCs detected microorganisms, while TC was negative. Detected bacteria were Staphylococcus epidermidis in four, Micrococcus species in one, Finegoldia magna in one, Brevibacterium casei in one, Pseudomonas fluorescens in one, and Enterococcus faecium in one. SC is a reliable method for dislodging pathogens from orthopedic implants. The SC of modular megaprostheses showed significantly higher pathogen detection than the periprosthetic TC, especially for low virulence pathogens. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1383-1387, 2017.