Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations.

OBJECTIVES: To investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. METHODS: During a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. RESULTS: In 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a ß-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82-100%) and 92% ((67/73); 95% CI, 83-97%), respectively. CONCLUSIONS: The prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.

The Effect of Door Openings on Numbers of Colony Forming Units in the Operating Room during Hip Revision Surgery.

BACKGROUND: The aim of this study was to investigate the effect of door opening rates on air quality in the operation room during hip revision surgery by measuring the number of colony forming units per cubic meter (CFU/m(3)). METHODS: During 70 hip revision operations the number of CFU/m(3) was measured at four time points. Factors that may influence air quality were recorded, including the number of persons present, duration of surgery, and door opening rates. The measured CFU/m(3) was dichotomized as either acceptable (≤20 CFU/m(3)) or not acceptable (>20 CFU/m(3)). To determine whether door openings were associated with CFU/m(3) values, we used generalized linear mixed models to model the dichotomized repeatedly measured CFU/m(3) values. RESULTS: The median number of door openings per operation was eight (range, 0-72), the median duration of surgery was 145 min (range, 60-285), and the median number of persons present during surgery was eight (range, 5-10). Adjusted for number of persons in the operation room and duration of surgery, the number of door openings per operation was associated (odds ratio [OR] 1.05 [95% confidence interval {CI} 1.02-1.09]; p = 0.003) with an unacceptable number of CFU/m(3). CONCLUSIONS: In conclusion, a substantial association between number of door openings and an unacceptable number (>20) of CFU/m(3) was found. Adjusted for number of persons in the operation room and duration of surgery, every door opening increased the odds of unacceptable CFU/m(3) values by 5%. Number of persons present during surgery and duration of surgery were not related to CFU/m(3).

Impaired Porphyromonas gingivalis-Induced Tumor Necrosis Factor Production by Dendritic Cells Typifies Patients With Rheumatoid Arthritis.

OBJECTIVE: The prevalence of periodontitis is increased in patients with rheumatoid arthritis (RA), and the severity of periodontitis can affect the level of arthritis. Porphyromonas gingivalis is one of the main bacteria involved in periodontitis. Our aim was to determine if there are differences in the innate immune response against P gingivalis between healthy controls and RA patients. METHODS: Monocyte-derived dendritic cells (DCs) from healthy controls, RA patients, and patients with psoriatic arthritis (PsA) were stimulated with P gingivalis, a range of other bacteria, and Toll-like receptor agonists. Cytokine production was determined, and blocking studies were performed to determine which receptors were involved in differential recognition of P gingivalis. Effects on T cell cytokines were also determined in cultures of peripheral blood mononuclear cells (PBMCs). RESULTS: Upon stimulation with P gingivalis, RA patient DCs produced less tumor necrosis factor as compared to healthy control DCs, which was not observed in PsA patients or upon stimulation with other bacteria. In addition, P gingivalis-mediated activation of RA patient PBMCs showed a clear reduction of interferon-γ production. Among the various possible underlying mechanisms investigated, only blockade of CR3 abolished the difference between RA patients and healthy controls, suggesting the involvement of CR3 in this process. CONCLUSION: Immune cells from RA patients display a reduced response to P gingivalis, which has functional consequences for the immune response. This may result in prolonged survival of P gingivalis, possibly driving autoantibody formation and a self-perpetuating loop of chronic inflammation. The possible role of CR3 in this process warrants further investigation.

Differences in expression of uroplakin III, cytokeratin 7, and cyclooxygenase-2 in canine proliferative urothelial lesions of the urinary bladder.

The expression of immunohistochemical markers that have been used in diagnosis and/or prognostication of urothelial tumors in humans (uroplakin III [UPIII], cytokeratin 7 [CK7], cyclooxygenase-2 [COX-2], and activated caspase 3) was evaluated in a series of 99 canine proliferative urothelial lesions of the urinary bladder and compared to the lesion classification and grade as defined by the World Health Organization / International Society of Urologic Pathology consensus system. There were significant associations between tumor classification and overall UPIII pattern (P = 1.49 × 10(-18)), loss of UPIII (P = 1.27 × 10(-4)), overall CK7 pattern (P = 4.34 × 10(-18)), and COX-2 pattern (P = 8.12 × 10(-25)). In addition, there were significant associations between depth of neoplastic cell infiltration into the urinary bladder wall and overall UPIII pattern (P = 1.54 × 10(-14)), loss of UPIII (P = 2.07 × 10(-4)), overall CK7 pattern (P = 1.17 × 10(-13)), loss of CK7 expression (P = .0485), and COX-2 pattern (P = 8.23 × 10(-21)). There were no significant associations between tumor classification or infiltration and caspase 3 expression pattern.

Microbial profiling does not differentiate between childhood recurrent acute otitis media and chronic otitis media with effusion.

OBJECTIVES: Otitis media (OM) is one of the most frequent diseases of childhood, with a minority of children suffering from recurrent acute otitis media (rAOM) or chronic otitis media with effusion (COME), both of which are associated with significant morbidity. We investigated whether the microbiological profiling could be used to differentiate between these two conditions. METHODS: Children up to five years of age, with rAOM (n = 45) or COME (n = 129) and scheduled for tympanostomy tube insertion were enrolled in a prospective study between 2008 and 2009. Middle ear fluids (n = 119) and nasopharyngeal samples (n = 173) were collected during surgery for bacterial culture and PCR analysis to identify Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis, and to detect 15 distinct respiratory viruses. RESULTS: The occurrence of bacterial and viral pathogens in middle ear fluids did not significantly differ between patients suffering from rAOM and COME. In both patient cohorts, H. influenzae and rhinovirus were the predominant pathogens in the middle ear and nasopharynx. Nasopharyngeal carriage with two or three bacterial pathogens was associated with the presence of bacteria in middle ear fluid (P = 0.04). The great majority of the bacteria isolated from middle ear fluid were genetically identical to nasopharyngeal isolates from the same patient. CONCLUSIONS: Based on these results, we propose that the common perception that rAOM is associated with recurrent episodes of microbiologically mediated AOM, whereas COME is generally a sterile inflammation, should be reconsidered.

Performance and clinical significance of direct antimicrobial susceptibility testing on urine from hospitalized patients.

BACKGROUND: Urinary tract infections (UTIs) are common infections in the community and the hospital. With increasing antimicrobial resistance, specifically in the Gram-negative uropathogens, reliable, rapid antimicrobial susceptibility data would be useful to guide antimicrobial treatment. Direct antimicrobial susceptibility testing (DST) of urine with microscopic evidence of Gram-negative bacterial infection and its clinical significance was investigated in this study. METHODS: DST was performed by Kirby-Bauer disk diffusion method using undiluted urine as a non-standardized inoculum. Urine specimens with Gram-negative bacteria on microscopy were included. DST results from growth of Gram-negative bacteria were compared to routine antimicrobial susceptibility testing by Phoenix automated system (AST). Errors were scored as 'very major error' if susceptible by DST but resistant by AST and as 'major error' if resistant by DST but susceptible by AST. All other discrepancies were defined as 'minor error'. Discrepancies were resolved by determination of minimum inhibitory concentrations (MICs) using Etests. After discrepancy analysis, errors were scored as above using the Etest as the reference method. For analysis, specimens were divided into 3 categories: category A: 1 isolate found by DST as well as by routine culture; category B: 1 isolate detected by DST, but more than 1 isolate found on routine culture; category C: more than 1 isolate found by both DST and routine culture. The clinical significance of DST was determined prospectively by investigating the potential impact of DST on antimicrobial therapy. RESULTS: One hundred and sixteen urine specimens were included. For DST and AST there was agreement in 96% of 1152 comparisons in category A (n = 100), 88% of 41 comparisons in category B (n = 4), and 88% of 110 comparisons in category C (n = 12). The 64 discrepancies included 18 very major errors, 7 major errors, and 39 minor errors. Eight very major errors and 11 minor errors were not investigated because the isolates were not available. After Etest MIC determination for the 45 remaining discrepancies, DST showed 1 very major error, 1 major error, and 8 minor errors in category A, none in category B, and 5 major errors and 4 minor errors in category C. Antimicrobial therapy for UTI was prescribed for 53 patients. For 4 patients (8%) therapy was adjusted based on DST because of antimicrobial resistance and for 12 patients (23%) antimicrobial treatment could have been streamlined. CONCLUSIONS: DST on urine is reliable in monobacterial Gram-negative infections. With increasing antimicrobial resistance, DST can make an important contribution to patient management and reduce the use of broad-spectrum antimicrobials.

Selective digestive tract decontamination and selective oropharyngeal decontamination and antibiotic resistance in patients in intensive-care units: an open-label, clustered group-randomised, crossover study.

BACKGROUND: Previously, we assessed selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) on survival and prevention of bacteraemia in patients in intensive-care units. In this analysis, we aimed to assess effectiveness of these interventions for prevention of respiratory tract colonisation and bacteraemia with highly resistant microorganisms acquired in intensive-care units. METHODS: We did an open-label, clustered group-randomised, crossover study in 13 intensive-care units in the Netherlands between May, 2004, and July, 2006. Participants admitted to intensive-care units with an expected duration of mechanical ventilation of more than 48 h or an expected stay of more than 72 h received SOD (topical tobramycin, colistin, and amphotericin B in the oropharynx), SDD (SOD antibiotics in the oropharynx and stomach plus 4 days' intravenous cefotaxime), or standard care. The computer-randomised order of study regimens was applied by an independent clinical pharmacist who was masked to intensive-care-unit identity. We calculated crude odds ratios (95% CI) for rates of bacteraemia or respiratory tract colonisation with highly resistant microorganisms in patients who stayed in intensive-care units for more than 3 days (ie, acquired infection). This trial is registered at http://isrctn.org, number ISRCTN35176830. FINDINGS: Data were available for 5927 (>99%) of 5939 patients, of whom 5463 (92%) were in intensive-care units for more than 3 days. 239 (13%) of 1837 patients in standard care acquired bacteraemia after 3 days, compared with 158 (9%) of 1758 in SOD (odds ratio 0·66, 95% CI 0·53-0·82), and 124 (7%) of 1868 in SDD (0·48, 0·38-0·60). Eight patients acquired bacteraemia with highly resistant microorganisms during SDD, compared with 18 patients (with 19 episodes) during standard care (0·41, 0·18-0·94; rate reduction [RR] 59%, absolute risk reduction [ARR] 0·6%) and 20 during SOD (0·37, 0·16-0·85; RR 63%, ARR 0·7%). Of the patients staying in intensive-care units for more than 3 days, we obtained endotracheal aspirate cultures for 881 (49%) patients receiving standard care, 886 (50%) receiving SOD, and 828 (44%) receiving SDD. 128 (15%) patients acquired respiratory tract colonisation with highly resistant microorganisms during standard care, compared with 74 (8%) during SDD (0·58, 0·43-0·78; RR 38%, ARR 5·5%) and 88 (10%) during SOD (0·65, 0·49-0·87; RR 32%, ARR 4·6%). Acquired respiratory tract colonisation with Gram-negative bacteria or cefotaxime-resistant and colistin-resistant pathogens was lowest during SDD. INTERPRETATION: Widespread use of SDD and SOD in intensive-care units with low levels of antibiotic resistance is justified. FUNDING: None.

Ecological effects of selective decontamination on resistant gram-negative bacterial colonization.

RATIONALE: Selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) eradicate gram-negative bacteria (GNB) from the intestinal and respiratory tract in intensive care unit (ICU) patients, but their effect on antibiotic resistance remains controversial. OBJECTIVES: We quantified the effects of SDD and SOD on bacterial ecology in 13 ICUs that participated in a study, in which SDD, SOD, or standard care was used during consecutive periods of 6 months (de Smet AM, Kluytmans JA, Cooper BS, Mascini EM, Benus RF, van der Werf TS, van der Hoeven JG, Pickkers P, Bogaers-Hofman D, van der Meer NJ, et al. N Engl J Med 2009;360:20-31). METHODS: Point prevalence surveys of rectal and respiratory samples were performed once monthly in all ICU patients (receiving or not receiving SOD/SDD). Effects of SDD on rectal, and of SDD/SOD on respiratory tract, carriage of GNB were determined by comparing results from consecutive point prevalence surveys during intervention (6 mo for SDD and 12 mo for SDD/SOD) with consecutive point prevalence data in the pre- and postintervention periods. MEASUREMENTS AND MAIN RESULTS: During SDD, average proportions of patients with intestinal colonization with GNB resistant to either ceftazidime, tobramycin, or ciprofloxacin were 5, 7, and 7%, and increased to 15, 13, and 13% postintervention (P < 0.05). During SDD/SOD resistance levels in the respiratory tract were not more than 6% for all three antibiotics but increased gradually (for ceftazidime; P < 0.05 for trend) during intervention and to levels of 10% or more for all three antibiotics postintervention (P < 0.05). CONCLUSIONS: SOD and SDD have marked effects on the bacterial ecology in an ICU, with rising ceftazidime resistance prevalence rates in the respiratory tract during intervention and a considerable rebound effect of ceftazidime resistance in the intestinal tract after discontinuation of SDD.

Prevalence, molecular characterization, and phenotypic confirmation of extended-spectrum beta-lactamases in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca at the Radboud University Nijmegen Medical Centre in The Netherlands.

The prevalence and molecular types of extended-spectrum beta-lactamases (ESBLs) were determined during a 1-year period in unselected clinical nonduplicate isolates of Escherichia coli (n = 1,738), Klebsiella pneumoniae (n = 436), and Klebsiella oxytoca (n = 208), cultured at the University Medical Centre Nijmegen, The Netherlands. Isolates identified as ESBL producer by the Phoenix automated system were collected prospectively and subjected to molecular analysis for the most common ESBLs TEM, SHV, and CTX-M, as well as OXA and GES. Both the Etest ESBL and double-disk synergy test were performed as confirmatory tests. The estimated prevalence of ESBLs was 2.1% in E. coli, 5.2% in K. pneumoniae, and 2.4% in K. oxytoca. TEM-12 and -26, SHV-5 and -12, and CTX-M groups 1 and 9 were the most frequent ESBLs found. Isolates identified as ESBLs by the Phoenix were confirmed by polymerase chain reaction (PCR) in only 42%. In ESBL PCR-positive E. coli and K. pneumoniae, both confirmatory tests were positive in 95% of the isolates. In 28% of the Etest and 13% of the double-disk synergy test-positive isolates, PCR could not detect any ESBL gene. In these cases, other resistance mechanisms may play a role. Confirmatory tests were unreliable for K. oxytoca. A previously described mutation in the K1 enzyme was detected in one ceftazidime-resistant K. oxytoca. The prevalence of ESBLs in The Netherlands is increasing. The predominant molecular types of ESBLs detected were comparable to other studies. Phoenix ESBL results need to be confirmed as advocated by ESBL detection guidelines.

Two cases of Mycobacterium microti-induced culture-negative tuberculosis.

We present descriptions of two human Mycobacterium microti infections: one of a patient with pulmonary disease and one of a patient with osteomyelitis of the hip. Both patients had acid-fast bacilli and a positive Mycobacterium tuberculosis complex PCR from clinical specimens, but mycobacterial cultures remained negative. The microbiological diagnosis was established by molecular methods.

Treatment of knee prosthesis infections: evaluation of 15 patients over a 5-year period.

Our objective was to evaluate different treatment alternatives for total knee arthroplasty (TKA) infection and to compare outcomes depending on adherence to a current treatment algorithm. All patients treated for a first episode of TKA infection between January 2000 and July 2005 were included. Patient records were reviewed and data were extracted retrospectively. Fifteen patients were followed up for a median of 25 months. The cure rate in patients with two-stage exchange of knee prosthesis was higher than in patients who had débridement without implant removal (100 vs 37%, p = 0.03). Cure rates were not different between these two surgical approaches in ten patients who were treated according to a current treatment algorithm. Success rates for treatment of TKA infections varied considerably with the treatment strategy chosen. Our results support the use of existing algorithms to select patients who are eligible for débridement with retention of the prosthesis or need two-stage exchange of knee implants.

Classification of canine urinary bladder urothelial tumours based on the World Health Organization/International Society of Urological Pathology consensus classification.

One hundred canine urinary bladder urothelial (transitional cell) tumours, including roughly equal numbers of benign and malignant forms, were retrospectively categorized in accordance with the newly described human consensus classification of the World Health Organization/International Society of Urological Pathology (WHO/ISUP). The tumours were reviewed and classified by three veterinary pathologists from Michigan State University and two human pathologists from the Armed Forces Institute of Pathology (AFIP). The current human WHO/ISUP classification system was considered to be readily applicable to the dog. Canine tumours, however, differed from human tumours in that the great majority showed extensive glandular differentiation (or metaplasia) and hyperplastic lesions tended to be more florid than those seen in human beings. The various diagnoses and grades assigned to the tumours were highly consistent between all reviewing pathologists. This paper presents the salient features of the new WHO and ISUP consensus classification and provides illustrations of the various tumour types that were directly applicable to the dog.

Legionella pneumophila in commercial bottled mineral water.

Sixty-eight commercial bottled mineral waters (64 brands, 68 different 'best-before dates') were tested for the presence of bacteria and fungi. Six samples were Legionella antigen positive and six were Legionella pneumophila PCR positive. Two samples were both Legionella antigen and L. pneumophila PCR positive. Legionella cultures were negative. Although the PCR might have detected only dead Legionella cells, the PCR has been described to detect specifically viable but not culturable (VBNC) L. pneumophila cells as well. Whether VBNC bacteria may be present in bottled mineral waters and the risk for infection this may pose for severely immunocompromised patients should be investigated.

Molecular diagnosis of lymphogranuloma venereum in patients with genital ulcer disease.

The detection of herpes, chancroid, and syphilis in genital ulcers is done by PCR. This is not so for lymphogranuloma venereum (LGV). We report on the use of a PCR with digestion that differentiates the LGV biovar from the trachoma biovar. Our findings suggest that the clinical description of LGV in current textbooks is incomplete.

Association between HIV-1 infection, the etiology of genital ulcer disease, and response to syndromic management.

BACKGROUND: Reports on the effect of HIV-1 infection on healing rates of ulcers are conflicting. GOAL: The goal was to determine the etiology and response to treatment of genital ulcer disease (GUD) in relation to HIV-1 infection. STUDY DESIGN: This was a cohort study of patients with GUD treated with local syndromic management protocols. RESULTS: Among the 587 recruited, the prevalences of infections due to HSV, Treponema pallidum, Chlamydia trachomatis (lymphogranuloma venereum [LGV]), Haemophilus ducreyi, Calymmatobacterium granulomatis, and HIV-1 were 48%, 14%, 11%, 10%, 1%, and 75%, respectively. The prevalence T. pallidum of was higher among men (P = 0.03), and an association was seen among HIV-1-seronegatives on univariate and multivariate analyses (P < 0.001; = 0.01). The prevalence of C trachomatis (LGV) was higher among females (P = 0.004), and an association was seen among HIV-1-seropositives on univariate analysis (P = 0.04). At follow-up, 40/407 (10%) showed a decreased healing tendency, not associated with ulcer etiology or HIV-1 seropositivity. CONCLUSION: Response to syndromic management of GUD was acceptable and not associated with HIV-1 coinfection.

Effects of vasoactive intestinal peptide on prolactin secretion in three species of passerine birds.

Previous work on domesticated species has indicated that vasoactive intestinal peptide (VIP) is an important prolactin-releasing factor in these birds, but no comparative work in passerine birds has been reported. This study showed that iv injections of VIP (50-100 microg/kg body mass) result in a dramatic, but transitory, rise in plasma prolactin in Mexican jays (Aphelocoma ultramarina). Significant increases in prolactin were also observed following VIP injection in blue jays (Cyanocitta cristata) and zebra finches (Poephilla guttata). At the dosage we used, maximum levels of prolactin attained were slightly lower (Mexican jays) or very similar (blue jay and zebra finch) to the maximum prolactin levels observed in other, breeding birds of the same species. In zebra finches that initially had low prolactin, VIP injection resulted in a greater than 10-fold increase in prolactin within 10 min, but those individuals that already had elevated prolactin showed no further increase in response to VIP. Slow-release pellets of VIP implanted subcutaneously in Mexican jays and releasing 10 or 15 microg VIP/day (two or three pellets) produced a significant increase in plasma prolactin (78 and 92% rise, respectively) compared to birds with placebo pellets or with with one pellet releasing only 5 microg/day.