Evaluation of the Bio-Rad Dx CT/NG/MG® assay for simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in urine.

We evaluated the performance of the Bio-Rad real-time Dx CT/NG/MG® assay for detection of C. trachomatis, N. gonorrhoeae and M. genitalium on a collection of 441 urine samples from sexually transmitted infections, or travellers consultations and from anonymous sperm donors that were previously analysed with the Abbott RealTime CT/NG assay. Samples positive for C. trachomatis or N. gonorrhoeae with the Abbott assay had all previously been confirmed with an in-house real-time PCR assay. Samples positive for M. genitalium with the Bio-Rad assay were subsequently analysed by an in-house real-time PCR. On a total of 441 urines, 104 samples were positive for C. trachomatis, 12 were positive for N. gonorrhoeae and seven were positive for M. genitalium. After retesting of discrepant results, the test results were completely concordant, resulting in a calculated sensitivity and specificity of the Bio-Rad assay of 98.1 % and 100 % for C. trachomatis and of 91.7 % and 100 % for N. gonorrhoeae. Results for M. genitalium with the Bio-Rad assay were also concordant with the results of an in house PCR. We also evaluated the performance of automated nucleic acid extractions of urine samples with the NucliSENS easyMAG (bioMérieux) compared to the manual DNA extraction prescribed by the insert of the kit. The easyMAG extraction gave lower Ct values, relieved inhibition and had a lower hands-on time.

Optimization of HPV DNA detection in urine by improving collection, storage, and extraction.

The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates.

Cost analysis in favor of a combined approach for cytomegalovirus after kidney transplantation: a single-center experience.

BACKGROUND: In kidney transplant recipients, cytomegalovirus (CMV) can cause significant morbidity, mortality, and costs, which can be prevented by universal antiviral prophylaxis or preemptive therapy. METHODS: With the aim to improve our understanding of the advantages and disadvantages of these interventions, we documented resource use for 101 consecutive kidney transplant recipients in our center receiving preemptive therapy and estimated resource use for 2 alternative scenarios. RESULTS: At 100 days after transplantation, the mean total costs of our preemptive strategy including monitoring and treatment with intravenous ganciclovir was €2545 per patient. At €4853 per patient, these costs were highest for the CMV-positive donor/CMV-negative recipient (D+/R-) patient subgroup (n = 28), who frequently require recurrent treatment. A treatment scenario with valganciclovir prophylaxis for D+/R- and R+ patients, in which we ignored late-onset disease after discontinuation of prophylaxis, resulted in an estimated cost of €1892 per patient. A combined approach using valganciclovir prophylaxis in the D+/R- group and a preemptive strategy in the R+ groups would result in the lowest mean and median costs per patient (€1701). CONCLUSION: Our study suggests that a combined approach, using valganciclovir prophylaxis in D+/R- patients and preemptive treatment in R+ patients, may result in the lowest cost. This approach seems reasonable as it restricts expensive prophylactic drug therapy to those who would benefit the most, whereas it limits the risk for drug toxicity and late-onset disease in those at lower risk for CMV.

Performance of different mono- and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel.

An external quality assessment (EQA) panel consisting of a total of 48 samples in bronchoalveolar lavage (BAL) fluid or transport medium was prepared in collaboration with Quality Control for Molecular Diagnostics (QCMD) (www.qcmd.org). The panel was used to assess the proficiency of the three laboratories that would be responsible for examining the 6,000 samples to be collected in the GRACE Network of Excellence (www.grace-lrti.org). The main objective was to decide on the best-performing testing approach for the detection of influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus (RSV), human metapneumovirus, coronavirus, rhinovirus, adenovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila by nucleic acid amplification techniques (NAATs). Two approaches were chosen: (i) laboratories testing samples using their in-house procedures for extraction and amplification and (ii) laboratories using their in-house amplification procedures on centrally extracted samples. Furthermore, three commercially available multiplex NAAT tests-the ResPlex (Qiagen GmbH, Hilden, Germany), RespiFinder plus (PathoFinder, Maastricht, The Netherlands), and RespiFinder Smart 21 (PathoFinder) tests-were evaluated by examination of the same EQA panel by the manufacturer. No large differences among the 3 laboratories were noticed when the performances of the assays developed in-house in combination with the in-house extraction procedures were compared. Also, the extraction procedure (central versus local) had little effect on performance. However, large differences in amplification efficacy were found between the commercially available tests; acceptable results were obtained by using the PathoFinder assays.

Validation of the MicroScan-96 for the species identification and methicillin susceptibility testing of clinical significant coagulase-negative staphylococci.

An automated system, MicroScan WalkAway-96, in conjugation with Combo Pos® 28 panels, was validated for the identification and methicillin susceptibility of coagulase-negative staphylococci (CNS). The performance of this system was evaluated on 428 CNS. Identification results were compared using a validated in-house method. Methicillin susceptibility was compared with oxacillin MIC testing and the presence of the mecA gene by PCR (in-house real-time method). The MicroScan system correctly identified 94.6% of the staphylococci (405 out of 428). 3.5% of the strains (15 out of 428) were not correctly identified. 1.9% of the isolates (8 out of 428) were correctly identified with a low probability. Identification of Staphylococcus warneri and Staphylococcus lugdunensis was determined with the least accuracy. Microscan combines both oxacillin and cefoxitin for determination of the methicillin susceptibility result. Correlation between this result and the mecA method was 97.6%. Correlation with the oxacillin MIC method was also 97.6%. Fourteen isolates showed a discrepant result, 8 were reported to be resistant in mecA-negative strains, 2 were reported false-susceptible in mecA positive strains and 4 strains showed a discrepant result with oxacillin MIC, but not with mecA determination. The automated system can be considered a simple and reliable method for identification and methicillin susceptibility of CNS.

Evaluation of the ESPLINE® Influenza A & B-N assay for the detection of influenza A and B in nasopharyngeal aspirates.

Several direct antigen tests for the detection of influenza often lack sensitivity compared to immunofluorescence (IF) on the specimens and viral culture (VC). We evaluated the performance of a rapid test, the ESPLINE® Influenza A & B-N assay. A total of 302 respiratory specimens were collected at the University Hospital of Antwerp. A first group of 60 samples taken during the H1N1 outbreak (2009-2010) and a second group of 242 samples stored during the seasonal influenza epidemics (2000-2009) were analyzed with the ESPLINE® test. A subset of samples were also evaluated with the BinaxNOW Influenza and the Clearview Exact Influenza. The results were compared to IF on the specimens, VC with IF, and the combination of both, which was considered as the gold standard. The ESPLINE® test's overall sensitivity and specificity were 91% and 97%, during the H1N1 season 80% and 93%, and for the detection of seasonal influenza 93% and 97%, respectively. In comparison to the BinaxNOW Influenza and the Clearview Exact Influenza, all tests demonstrated a similar specificity of 92.0-100% but a significantly different sensitivity of 44.4-86.0%, with the ESPLINE® test being significantly more sensitive. Due to its very good performance and simplicity, the ESPLINE® test facilitates urgent testing. The test seems less sensitive to detect H1N1 compared to seasonal influenza, although the difference is borderline not significant (p = 0.067).

Detection of human papillomavirus DNA in urine. A review of the literature.

The detection of human papillomavirus (HPV) DNA in urine, a specimen easily obtained by a non-invasive self-sampling method, has been the subject of a considerable number of studies. This review provides an overview of 41 published studies; assesses how different methods and settings may contribute to the sometimes contradictory outcomes; and discusses the potential relevance of using urine samples in vaccine trials, disease surveillance, epidemiological studies, and specific settings of cervical cancer screening. Urine sampling, storage conditions, sample preparation, DNA extraction, and DNA amplification may all have an important impact on HPV DNA detection and the form of viral DNA that is detected. Possible trends in HPV DNA prevalence in urine could be inferred from the presence of risk factors or the diagnosis of cervical lesions. HPV DNA detection in urine is feasible and may become a useful tool but necessitates further improvement and standardization.

Culture-based detection of methicillin-resistant Staphylococcus aureus by a network of European laboratories: an external quality assessment study.

Twenty-three hospital laboratories from Europe and Israel participated in an external quality assessment (EQA) of the culture-based detection of methicillin-resistant Staphylococcus aureus (MRSA). Participants also reported the MRSA prevalence in clinical cultures and patient screening specimens, as well as the MRSA screening practices employed at their hospitals. An EQA panel of 18 samples consisting of two MRSA harbouring SCCmec IV and I, and one strain each of methicillin-resistant coagulase-negative S. epidermidis, methicillin-sensitive S. aureus and Escherichia coli as pure strains or in mixtures at 10(7)-1 cfu absolute loads was analysed by the 23 participants. Seventeen (74%) participants identified 17 or more samples correctly. Of these, 15 (88%) utilised a chromogenic medium alone (ChromID, bioMérieux; BBL CHROMagar, BD Diagnostics; MRSA Select, Bio-Rad Laboratories) or combined with a conventional medium and up to three confirmatory tests. Proportions of MRSA among S. aureus isolated from clinical cultures varied widely, even among hospitals within countries, ranging from 11-20% to 61-70%. MRSA carriage rates were less variable (0-20%) between countries. Almost all participants (n=22, 96%) screened patients for MRSA carriage during 2009-2010, of which 15 (68%) screened intensive care unit (ICU) patients alone or combined with other targeted high-risk groups, and 10 (45%) combined nasal screening with another body site.

Lower respiratory tract infection in the community: associations between viral aetiology and illness course.

OBJECTIVES: This study determined associations between respiratory viruses and subsequent illness course in primary care adult patients presenting with acute cough and/or suspected lower respiratory tract infection. METHODS: A prospective European primary care study recruited adults with symptoms of lower respiratory tract infection between November 2007 and April 2010. Real-time in-house polymerase chain reaction (PCR) was performed to test for six common respiratory viruses. In this secondary analysis, symptom severity (scored 1 = no problem, 2 = mild, 3 = moderate, 4 = severe) and symptom duration were compared between groups with different viral aetiologies using regression and Cox proportional hazard models, respectively. Additionally, associations between baseline viral load (cycle threshold (Ct) value) and illness course were assessed. RESULTS: The PCR tested positive for a common respiratory virus in 1354 of the 2957 (45.8%) included patients. The overall mean symptom score at presentation was 2.09 (95% confidence interval (CI) 2.07-2.11) and the median duration until resolution of moderately bad or severe symptoms was 8.70 days (interquartile range 4.50-11.00). Patients with influenza virus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), coronavirus (CoV) or rhinovirus had a significantly higher symptom score than patients with no virus isolated (0.07-0.25 points or 2.3-8.3% higher symptom score). Time to symptom resolution was longer in RSV infections (adjusted hazard ratio (AHR) 0.80, 95% CI 0.65-0.96) and hMPV infections (AHR 0.77, 95% CI 0.62-0.94) than in infections with no virus isolated. Overall, baseline viral load was associated with symptom severity (difference 0.11, 95% CI 0.06-0.16 per 10 cycles decrease in Ct value), but not with symptom duration. CONCLUSIONS: In healthy, working adults from the general community presenting at the general practitioner with acute cough and/or suspected lower respiratory tract infection other than influenza impose an illness burden comparable to influenza. Hence, the public health focus for viral respiratory tract infections should be broadened.

Acute respiratory infection due to Mycoplasma pneumoniae: current status of diagnostic methods.

Because of the absence of well-standardized both in-house and FDA-approved commercially available diagnostic tests, the reliable diagnosis of respiratory infection due to Mycoplasma pneumoniae remains difficult. In addition, no formal external quality assessment schemes which would allow to conclude about the performance of M. pneumoniae diagnostic tests exist. In this review, the current state of knowledge of M. pneumoniae-associated respiratory infections in the context of epidemiological studies published during the past 5 years is discussed, with particular emphasis on the diagnostic strategies used and their impact on results. The role of M. pneumoniae as a cause of respiratory tract infections (RTIs) differs from study to study due to geographical and epidemiological differences, as well as to the application of different diagnostic techniques and criteria used.

Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens.

Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.

Evaluation of the NucliSens miniMAG RNA extraction and real-time NASBA applications for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in throat swabs.

Nucleic acids were extracted from 215 throat swabs from patients with community-acquired pneumonia by the manual Boom extraction, the NucliSens miniMAG and the Qiagen DNA blood kit and amplified respectively by in-house real-time NASBA, NucliSens EasyQ reagents, and real-time PCR for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae. Five out of 215 throat swabs were found to be C. pneumoniae positive by all techniques used. A total of 11 out of 215 throat swabs were positive for M. pneumoniae; 10/215 by Qiagen extraction and PCR amplification and 9/215 by NucliSens miniMAG and NucliSens EasyQ amplification. The NucliSens miniMAG and NucliSens EasyQ applications were successfully coupled to detect both organisms in throat swabs.

Evaluation of different nucleic acid amplification techniques for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from patients with community-acquired pneumonia.

The number of pathogens involved in community-acquired pneumonia, with varying susceptibilities to antimicrobials, is numerous constituting an enormous challenge for diagnostic microbiology. Differentiation of infections due to Streptococcus pneumoniae and those due to Mycoplasma pneumoniae, Chlamydophila pneumoniae, or L. pneumophila as well as those due to viruses is essential to allow correct decisions concerning the antibiotics to be administered. The sensitivity and specificity of real-time simplex and multiplex nucleic acid sequence-based amplification (NASBA), and simplex PCR were compared for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from hospitalized and outpatients with community-acquired pneumonia (CAP). Two hundred fifty one respiratory specimens were collected from 147 patients with CAP. NASBA was done using the NucliSens Basic Kit (bioMérieux). PCR for M. pneumoniae and C. pneumoniae was done as described earlier [Ieven, M., Ursi, D., Van Bever, H., Quint, W., Niesters, H. G. M., and Goossens, H. 1996. Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J. Infect. Dis. 173, 1445-14452.; Ursi, D., Ieven, M., Van Bever, H. P., and Goossens, H. 1998. Construction of an internal control for the detection of Chlamydia pneumoniae by PCR. Mol. Cellul. Probes. 12, 235-238.]. A real-time PCR was developed to detect L. pneumophila whereas a real-time NASBA was designed to detect Legionella spp. All samples with discordant results were re-analysed. Compared to an expanded gold standard the sensitivities of the different techniques, were 77.8%, 100%, and 100% for detection of M. pneumoniae; and 50%, 100%, and 50% for detection of L. pneumophila by PCR, real-time simplex NASBA, and real-time multiplex NASBA, respectively. C. pneumoniae was detected in two samples only. Simplex real-time NASBA proved to be more sensitive than simplex PCR and was also more sensitive than real-time multiplex NASBA, as previously found with spiked clinical specimens. It's practical attractiveness pleads for further optimalisation of the multiplex approach.

Laboratory preparedness and response with a focus on arboviruses in Europe.

BACKGROUND: The global health burden of arboviruses is continuously rising, which results in increasing pressure on local and (inter)national laboratory infrastructures. Timely and accurate diagnosis of cases is one of the main pillars for public health and clinical responses to an arbovirus emergence. AIMS AND SOURCES: This narrative review aims to summarize recent advances and to identify needs in laboratory preparedness and response activities, with a focus on viruses transmitted by arthropods in Europe. The review is based on evidence extracted from PubMed searches, Public Health and clinical laboratory experiences from the authors and the authors' opinions substantiated by peer-reviewed scientific literature. CONTENT: We illustrate the importance of inter-epidemic laboratory preparedness activities to ensure adequate Public Health and clinical responses. We describe the status of arbovirus endemicity and emergence in Europe thereby highlighting the need for preparedness for these viruses. We discuss the components and pitfalls of an adequate laboratory preparedness and response and the broader context of the current landscape of international research, clinical and laboratory preparedness networks. The complexity of arbovirus laboratory preparedness and response is described. IMPLICATIONS: Outbreak preparedness plans need to look beyond national reference laboratories, to include first-line responding onsite hospital laboratories and plans for strengthening of such local capacity and capability as required depending on the nature of the outbreak. In particular, the diagnosis of arbovirus infections is complicated by the existence of geographic overlap of circulation of numerous arboviruses, the overlap in clinical manifestation between many arboviruses and other aetiologies and the existence of cross-reactivity between related arboviruses in serology testing. Inter-epidemic preparedness activities need strong national and international networks addressing these issues. However, the current mushrooming of European preparedness networks requires governance to bring the European preparedness and response to a next level.

Amoxicillin for acute lower respiratory tract infection in primary care: subgroup analysis by bacterial and viral aetiology.

OBJECTIVE: We aimed to assess the effects of amoxicillin treatment in adult patients presenting to primary care with a lower respiratory tract infection (LRTI) who were infected with a potential bacterial, viral, or mixed bacterial/viral infection. METHODS: This multicentre randomized controlled trial focused on adults with LRTI not suspected for pneumonia. Patients were randomized to receive either antibiotic (amoxicillin 1 g) or placebo three times daily for 7 consecutive days using computer-generated random numbers (follow-up 28 days). In this secondary analysis of the trial, symptom duration (primary outcome), symptom severity (scored 0-6), and illness deterioration (reconsultation with new or worsening symptoms, or hospital admission) were analysed in pre-specified subgroups using regression models. Subgroups of interest were patients with a (strictly) bacterial, (strictly) viral, or combined infection, and patients with elevated values of procalcitonin, C-reactive protein, or blood urea nitrogen. RESULTS: 2058 patients (amoxicillin n = 1036; placebo n = 1022) were randomized. Treatment did not affect symptom duration (n = 1793). Patients from whom a bacterial pathogen only was isolated (n = 207) benefited from amoxicillin in that symptom severity (n = 804) was reduced by 0.26 points (95% CI -0.48 to -0.03). The odds of illness deterioration (n = 2024) was 0.24 (95% CI 0.11 to 0.53) times lower from treatment with amoxicillin when both a bacterial and a viral pathogen were isolated (combined infection; n = 198). CONCLUSIONS: Amoxicillin may reduce the risk of illness deterioration in patients with a combined bacterial and viral infection. We found no clinically meaningful benefit from amoxicillin treatment in other subgroups.

Procarboxypeptidase U (proCPU, TAFI, proCPB2) in cerebrospinal fluid during ischemic stroke is associated with stroke progression, outcome and blood-brain barrier dysfunction.

Essentials Little is known of procarboxypeptidase U (proCPU) in cerebrospinal fluid (CSF) of stroke patients. ProCPU levels were studied in CSF of controls and non-thrombolyzed acute ischemic stroke patients. ProCPU is elevated in CSF of stroke patients compared with controls. ProCPU in CSF correlates with stroke progression, outcome, and blood-brain barrier dysfunction. SUMMARY: Background Procarboxypeptidase U (proCPU, TAFI, proCPB2), the zymogen of CPU, which is a potent antifibrinolytic enzyme and a modulator of inflammation, has previously been investigated in plasma of stroke patients, but so far, no information on the proCPU levels in cerebrospinal fluid (CSF) during acute ischemic stroke (AIS) is available. Objectives This case-control observational study investigates proCPU in CSF of AIS patients compared with controls with an intact blood-brain barrier (BBB) and evaluates the relationship of CSF/plasma proCPU ratios with stroke parameters. Methods A sensitive HPLC-based enzymatic assay was used to determine proCPU levels in CSF of non-thrombolyzed patients in the hyperacute phase (< 24 h after onset) of AIS (n = 72). Individuals (n = 32) without stroke, an intact BBB and no apparent abnormalities in biochemical and microbiological tests, served as controls. Relations between the CSF/plasma proCPU ratio and (i) stroke severity, (ii) stroke progression/recurrence, (iii) stroke outcome and (iv) BBB dysfunction (CSF/serum albumin ratio) were assessed. Results Mean (SEM) proCPU levels were elevated in the CSF of stroke patients compared with controls (4.36 (0.23) U L-1 vs. 3.50 (0.23) U L-1 ). Higher median [IQR] CSF/plasma proCPU ratios were found in patients with stroke progression ((6.0 [4.2-6.9]) × 10-3 ) and poor outcome ((6.4 [3.9-7.0]) × 10-3 ) after 3 months (modified Rankin Scale; mRS > 3) compared with patients without progression ((3.9 [2.7-5.4]) × 10-3 ) or better outcome ((4.0 [2.8-5.0]) × 10-3 ). In stroke patients with a disrupted BBB, proCPU ratios were higher compared with stroke patients with an intact BBB ((6.4 [5.8-9.0]) × 10-3 vs. (3.7 [2.8-5.0]) × 10-3 ). Conclusions ProCPU is increased in CSF during hyperacute ischemic stroke and is associated with stroke progression and outcome after 3 months, most likely due to BBB dysfunction in the hyperacute phase of ischemic stroke.

Aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 11 European countries.

OBJECTIVES: To describe the role of bacteria (including bacterial resistance), viruses (including those recently described) and mixed bacterial-viral infections in adults presenting to primary care with lower respiratory tract infection (LRTI). METHODS: In all, 3104 adults with LRTI were enrolled, of whom 141 (4.5%) had community-acquired pneumonia (CAP), and 2985 matched controls in a prospective study in 16 primary care networks in Europe, and followed patients up at 28-35 days. We detected Streptococcus pneumoniae and Haemophilus influenzae and assessed susceptibility, atypical bacteria and viruses. RESULTS: A potential pathogen was detected in 1844 (59%) (in 350 (11%) bacterial pathogens only, in 1190 (38%) viral pathogens only, and in 304 (10%) both bacterial and viral pathogens). The most common bacterial pathogens isolated were S. pneumoniae (5.5% overall, 9.2% in CAP patients) and H. influenzae (5.4% overall, 14.2% in CAP patients). Less than 1% of S. pneumoniae were highly resistant to penicillin and 12.6% of H. influenzae were ß-lactamase positive. The most common viral pathogens detected were human rhinovirus (20.1%), influenza viruses (9.9%), and human coronavirus (7.4%). Influenza virus, human parainfluenza viruses and human respiratory syncytial virus as well as human rhinovirus, human coronavirus and human metapneumovirus were detected significantly more frequently in LRTI patients than in controls. CONCLUSIONS: A bacterial pathogen is identified in approximately one in five adult patients with LRTI in primary care, and a viral pathogen in just under half, with mixed infections in one in ten. Penicillin-resistant pneumococci and ß-lactamase-producing H. influenzae are uncommon. These new findings support a restrictive approach to antibiotic prescribing for LRTI and the use of first-line, narrow-spectrum agents in primary care.

High-dose cefepime as an alternative treatment for infections caused by TEM-24 ESBL-producing Enterobacter aerogenes in severely-ill patients.

This study evaluated retrospectively the efficacy of treatment with cefepime vs. a carbapenem, in combination with amikacin or ciprofloxacin, for seriously-ill patients infected with ESBL-producing Enterobacter aerogenes who were admitted to an intensive care unit. Forty-four episodes of infection were investigated in 43 patients: 21 treated with cefepime; 23 with a carbapenem. The two treatment groups did not differ statistically in terms of age, APACHE II scores, and infection sites, but the average duration of antibiotic exposure was significantly shorter in the cefepime group (8.5 days vs. 11.4 days; p 0.04). Clinical improvement was seen in 62% of patients receiving cefepime vs. 70% of patients receiving a carbapenem (p 0.59). Bacteriological eradication was achieved in 14% of patients receiving cefepime vs. 22% of patients receiving a carbapenem (p 0.76). The 30-day mortality rates related to infection were 33% in the cefepime group and 26% in the carbapenem group (p 0.44). Thus, outcome parameters did not differ significantly between the two groups. Nevertheless, a statistically significant increase in failure to eradicate ESBL-producing E. aerogenes was observed as the MICs of cefepime rose (p 0.017). Pulsed-field gel electrophoresis revealed three distinct clones, but one predominant clone harbouring the bla(TEM-24) gene was associated with most (42/44) of the episodes of infection. It was concluded that cefepime may be an alternative agent for therapy of severe infections caused by TEM-24 ESBL-producing E. aerogenes, although further studies are required to confirm these observations.

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.

Encephalomyelitis-associated antimyelin autoreactivity induced by streptococcal exotoxins.

OBJECTIVE: After implicating Streptococcus pyogenes as causing acute disseminated encephalomyelitis (ADEM) in a child, we wanted to prove that in vivo activation of autoreactive T lymphocytes by superantigens of this Streptococcus contributed to the dramatic demyelination. BACKGROUND: ADEM is a demyelinating disorder of the CNS sharing many similarities with MS. Demyelination in MS is considered to be the result of an autoimmune process mediated by autoreactive T lymphocytes with specificity for myelin antigens. METHODS: Phenotypic analysis and proliferation assays on blood monocytes, as well as isolation of myelin basic protein (MBP)-reactive T-cell lines/clones; and TCR repertorium analysis by PCR-ELISA and cytokine production. RESULTS: 1) The blood T-cell receptor (TCR) repertoire was compatible with in vivo expansion induced by S. pyogenes exotoxins. 2) TCR expression analysis indicated clonal expansion of CD8+ MBP-reactive T cells, suggesting in vivo activation. MBP-reactive T cells showed crossreactivity to S. pyogenes supernatant and exotoxins. 3) Cytokine mRNA quantification of the mononuclear cells revealed a Th2-biased profile. CONCLUSION: In vivo exposure to S. pyogenes may have induced activation of pathogenic myelin reactive T cells, contributing to the dramatic inflammatory demyelination.