Genetic diversity among pandemic 2009 influenza viruses isolated from a transmission chain.

BACKGROUND: Influenza viruses such as swine-origin influenza A(H1N1) virus (A(H1N1)pdm09) generate genetic diversity due to the high error rate of their RNA polymerase, often resulting in mixed genotype populations (intra-host variants) within a single infection. This variation helps influenza to rapidly respond to selection pressures, such as those imposed by the immunological host response and antiviral therapy. We have applied deep sequencing to characterize influenza intra-host variation in a transmission chain consisting of three cases due to oseltamivir-sensitive viruses, and one derived oseltamivir-resistant case. METHODS: Following detection of the A(H1N1)pdm09 infections, we deep-sequenced the complete NA gene from two of the oseltamivir-sensitive virus-infected cases, and all eight gene segments of the viruses causing the remaining two cases. RESULTS: No evidence for the resistance-causing mutation (resulting in NA H275Y substitution) was observed in the oseltamivir-sensitive cases. Furthermore, deep sequencing revealed a subpopulation of oseltamivir-sensitive viruses in the case carrying resistant viruses. We detected higher levels of intra-host variation in the case carrying oseltamivir-resistant viruses than in those infected with oseltamivir-sensitive viruses. CONCLUSIONS: Oseltamivir-resistance was only detected after prophylaxis with oseltamivir, suggesting that the mutation was selected for as a result of antiviral intervention. The persisting oseltamivir-sensitive virus population in the case carrying resistant viruses suggests either that a small proportion survive the treatment, or that the oseltamivir-sensitive virus rapidly re-establishes itself in the virus population after the bottleneck. Moreover, the increased intra-host variation in the oseltamivir-resistant case is consistent with the hypothesis that the population diversity of a RNA virus can increase rapidly following a population bottleneck.

Short- and long-term mortality in patients with community-acquired severe sepsis and septic shock.

BACKGROUND: Severe sepsis and septic shock have a high 30-day mortality (10-50%), but the long-term mortality is not well described. The purpose of this study was to describe long-term mortality among patients with community-acquired severe sepsis or septic shock compared to a population-based reference cohort. METHODS: Two hundred and twelve patients who, within the first 24 h after arrival at the hospital, presented with infection and had failure of at least 1 organ system were included. A population-based reference group of 79,857 patients was identified, and data on comorbidities were extracted from the National Danish Patient Registry. We analyzed the hazard ratio for mortality at predefined intervals. RESULTS: Absolute mortality within the first 30 days was 69/211 (33%, 95% confidence interval (CI) 25-41%), with a cumulative mortality of 121/211 (57%, 95% CI 48-69%) for the entire follow-up. Among septic patients who survived the first 30 days, the mortality hazard ratio was 2.7 (95% CI 1.7-4.3) until day 365, and among septic patients who survived the first year, the 1-4 y mortality hazard ratio was 2.3 (95% CI 1.7-3.3), compared to the community-based reference persons (multivariate Cox regression controlling for age, sex, and Charlson comorbidity index). CONCLUSIONS: Patients with severe sepsis and septic shock who survived the first 30 days had a 2.7 times higher mortality hazard in the first year and a 2.3 times higher mortality hazard in the next 3 y, compared to persons of similar age, sex, and comorbidity level.

Performance of matrix-assisted laser desorption-time of flight mass spectrometry for identification of clinical yeast isolates.

Accurate and fast yeast identification is important when treating patients with invasive fungal disease as susceptibility to antifungal agents is highly species related. Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) provides a powerful tool with a clear potential to improve current diagnostic practice. Two MALDI-TOF-MS-systems (BioTyper/Bruker and Saramis/AXIMA) were evaluated using: (i) A collection of 102 archived, well characterised yeast isolates representing 14 different species and (ii) Prospectively collected isolates obtained from clinical samples at two participating laboratories. Of the 102 archived isolates, 81 (79%) and 92 (90%) were correctly identified by Saramis/AXIMA and BioTyper/Bruker respectively. Saramis/AXIMA was unable to separate Candida albicans, C. africana and C. dubliniensis in 13 of 32 isolates. After manual interpretation of the mass spectra output, all 13 isolates were correctly identified, resulting in an overall identification performance of 92%. No misidentifications occurred with the two systems. Of the routine isolates one laboratory identified 99/99 (100%) and 90/99 (91%) to species level by Saramis/Axima and conventional identification, respectively, whereas the other laboratory identified 83/98 (85%) to species level by both BioTyper/Bruker and conventional identification. Both MALDI-TOF-MS systems are fast, have built-in databases that cover the majority of clinically relevant Candida species, and have an accuracy that outperforms our conventional identification systems.

Treatment of acute otitis media in general practice: quality variations across countries.

BACKGROUND: Recommendations for antibiotic treatment of acute otitis media (AOM) have changed over the years, and today many experts recommend initial observation. However, antibiotic prescribing should be considered in children aged <2 years or if AOM is accompanied by discharging ear. OBJECTIVES: To investigate the quality of treatment of AOM in general practice and to explore the influence of selected GP and patient characteristics on antibiotic prescribing. METHODS: During the winter 2008, a prospective registration of patients diagnosed with AOM was conducted in general practice in Lithuania, Kaliningrad, Spain, Argentina, Sweden and Denmark. Some 1175 patients diagnosed with AOM were registered. Information about age and sex of the patient, duration of symptoms (days), temperature >38.5°C, ear discharge and the antibiotic treatment given was recorded. RESULTS: Danish GPs had the lowest antibiotic prescription rate for AOM [72.7% (95% confidence interval (CI) = 67.0-77.8)] and GPs in Kaliningrad had the highest [97.1% (95% CI = 89.8-99.6)]. Narrow-spectrum penicillin was almost exclusively prescribed in the two Nordic countries, while broad-spectrum penicillins, often in combination with clavulanic acid, were prescribed in the other four countries. Macrolides comprised 5-10% of prescriptions. Antibiotic prescribing was associated with the following characteristics of the patients: symptoms for >3 days, ear discharge and fever. CONCLUSION: The majority of patients with AOM were treated with antibiotics in all six countries, but considerable variations in both prescribing rate and choice of antibiotics were identified.

Species identification of clinical isolates of anaerobic bacteria: a comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems.

We compared two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Shimadzu/SARAMIS and Bruker) on a collection of consecutive clinically important anaerobic bacteria (n = 290). The Bruker system had more correct identifications to the species level (67.2% versus 49.0%), but also more incorrect identifications (7.9% versus 1.4%). The system databases need to be optimized to increase identification levels. However, MALDI-TOF MS in its present version seems to be a fast and inexpensive method for identification of most clinically important anaerobic bacteria.

Health Alliance for prudent antibiotic prescribing in patients with respiratory tract infections (HAPPY AUDIT) -impact of a non-randomised multifaceted intervention programme.

BACKGROUND: Excessive use of antibiotics is worldwide the most important reason for development of antimicrobial resistance. As antibiotic resistance may spread across borders, high prevalence countries may serve as a source of bacterial resistance for countries with a low prevalence. Therefore, bacterial resistance is an important issue with a potential serious impact on all countries. Initiatives have been taken to improve the quality of antibiotic prescribing in primary care, but only few studies have been designed to determine the effectiveness of multifaceted strategies across countries with different practice setting. The aim of this study was to evaluate the impact of a multifaceted intervention targeting general practitioners (GPs) and patients in six countries with different health organization and different prevalence of antibiotic resistance. METHODS: GPs from two Nordic countries, two Baltic Countries and two Hispano-American countries registered patients with respiratory tract infections (RTIs) in 2008 and 2009. After first registration they received individual prescriber feedback and they were offered an intervention programme that included training courses, clinical guidelines, posters for waiting rooms, patient brochures and access to point of care tests (Strep A and C-Reactive Protein). Antibiotic prescribing rates were compared before and after the intervention. RESULTS: A total of 440 GPs registered 47011 consultations; 24436 before the intervention (2008) and 22575 after the intervention (2009). After the intervention, the GPs significantly reduced the percentage of consultations resulting in an antibiotic prescription. In patients with lower RTI the GPs in Lithuania reduced the prescribing rate by 42%, in Russia by 25%, in Spain by 25%, and in Argentina by 9%. In patients with upper RTIs, the corresponding reductions in the antibiotic prescribing rates were in Lithania 20%, in Russia 15%, in Spain 9%, and in Argentina 5%. CONCLUSION: A multifaceted intervention programme targeting GPs and patients and focusing on improving diagnostic procedures in patients with RTIs may lead to a marked reduction in antibiotic prescribing. The pragmatic before-after design used may suffer from some limitations and the reduction in antibiotic prescribing could be influenced by factors not related to the intervention.

Pig-associated methicillin-resistant Staphylococcus aureus: family transmission and severe pneumonia in a newborn.

Carriage of pig-associated methicillin-resistant Staphylococcus aureus (MRSA) is known to occur in pig farmers. Zoonotic lineages of MRSA have been considered of low virulence and with limited capacity for inter-human spread. We present a case of family transmission of pig-associated MRSA ST398, which resulted in a severe infection in a newborn.

Health Alliance for Prudent Prescribing, Yield and Use of Antimicrobial Drugs in the Treatment of Respiratory Tract Infections (HAPPY AUDIT).

BACKGROUND: Excessive and inappropriate use of antibiotics is considered to be the most important reason for development of bacterial resistance to antibiotics. As antibiotic resistance may spread across borders, high prevalence countries may serve as a source of bacterial resistance for countries with a low prevalence. Therefore, bacterial resistance is an important issue with a potential serious impact on all countries. The majority of respiratory tract infections (RTIs) are treated in general practice. Most infections are caused by virus and antibiotics are therefore unlikely to have any clinical benefit. Several intervention initiatives have been taken to reduce the inappropriate use of antibiotics in primary health care, but the effectiveness of these interventions is only modest. Only few studies have been designed to determine the effectiveness of multifaceted strategies in countries with different practice setting. The aim of this study is to evaluate the impact of a multifaceted intervention targeting general practitioners (GPs) and patients in six countries with different prevalence of antibiotic resistance: Two Nordic countries (Denmark and Sweden), two Baltic Countries (Lithuania and Kaliningrad-Russia) and two Hispano-American countries (Spain and Argentina). METHODS/DESIGN: HAPPY AUDIT was initiated in 2008 and the project is still ongoing. The project includes 15 partners from 9 countries. GPs participating in HAPPY AUDIT will be audited by the Audit Project Odense (APO) method. The APO method will be used at a multinational level involving GPs from six countries with different cultural background and different organisation of primary health care. Research on the effect of the intervention will be performed by analysing audit registrations carried out before and after the intervention. The intervention includes training courses on management of RTIs, dissemination of clinical guidelines with recommendations for diagnosis and treatment, posters for the waiting room, brochures to patients and implementation of point of care tests (Strep A and CRP) to be used in the GPs'surgeries. To ensure public awareness of the risk of resistant bacteria, media campaigns targeting both professionals and the public will be developed and the results will be published and widely disseminated at a Working Conference hosted by the World Association of Family Doctors (WONCA-Europe) at the end of the project period. DISCUSSION: HAPPY AUDIT is an EU-financed project with the aim of contributing to the battle against antibiotic resistance through quality improvement of GPs' diagnosis and treatment of RTIs through development of intervention programmes targeting GPs, parents of young children and healthy adults. It is hypothesized that the use of multifaceted strategies combining active intervention by GPs will be effective in reducing prescribing of unnecessary antibiotics for RTIs and improving the use of appropriate antibiotics in suspected bacterial infections.

Quality indicators for diagnosis and treatment of respiratory tract infections in general practice: a modified Delphi study.

OBJECTIVE: To develop a set of quality indicators focusing on the diagnosis and treatment of respiratory tract infections in general practice. DESIGN: A modified 2-round Delphi study. SETTING: General practice. SUBJECTS: A panel of 27 experts (13 countries) comprising mainly general practitioners, clinical microbiologists, and clinical pharmacologists were asked to rate the relevance of 59 quality indicators for diagnosis and treatment of respiratory tract infections with regard to reducing antimicrobial resistance and improving patient health. A thorough literature review was carried out to ensure that all potential quality indicators were considered. OUTCOME: Consensus for a quality indicator was reached if > or =75% of experts scored the item > or =5 on a 7-point Likert scale, ranging from 1 (=completely disagree) through 4 (=uncertain) to 7 (=completely agree). RESULTS: A 96% response rate was achieved in both Delphi rounds. A total of 41 of the proposed 59 quality indicators attained consensus. None of the quality indicators focusing on the diagnostic process achieved consensus. Consensus was attained for 14 quality indicators focusing on the decision regarding antibiotic treatment and for 27 quality indicators focusing on the choice of antibiotics. CONCLUSION: This study resulted in a final set of 41 quality indicators concerning respiratory tract infections in general practice. These quality indicators may be used to strengthen general practitioners' focus on their management of patients with respiratory tract infections and to identify where it is possible to make improvements.

Pivmecillinam versus sulfamethizole for short-term treatment of uncomplicated acute cystitis in general practice: a randomized controlled trial.

OBJECTIVE: To investigate whether short-term treatment with pivmecillinam was more effective than sulfamethizole in patients with acute uncomplicated urinary tract infection (UTI). DESIGN: Randomized controlled trial. SETTING: General practice, Denmark. SUBJECTS: Patients (n = 167) with uncomplicated UTI confirmed by positive urine phase-contrast microscopy. MAIN OUTCOME MEASURES: Drug efficacy based on clinical and bacteriological cure. RESULTS: Urinary symptoms disappeared first in patients treated with pivmecillinam, but after five days there was no significant difference in clinical cure rate between the two antibiotics. At the follow-up visit 7-10 days after initiation of treatment, 95.4% of patients treated with pivmecillinam and 92.6% of patients treated with sulfamethizole had no persistent cystitis symptoms (difference 2.8%, CI -4.5%; 10.0%). Bacteriological cure was observed in 68.8% of patients randomized to pivmecillinam and in 77.9% randomized to sulfamethizole (difference -9.2%, CI -24.7%; 6.3%). Some 26.8% of patients randomized to pivmecillinam experienced a new UTI within 6 months after treatment compared with 18.4% of patients randomized to sulfamethizole (difference 8.4%, CI -4.5%;21.4%). No patients developed septicaemia with urinary pathogens within one year after initial treatment. CONCLUSION: Patients treated with a three-day regime of pivmecillinam experienced faster relief of symptoms compared with patients treated with a three-day regime of sulfamethizole. Five days after initiation of treatment there was no significant difference in clinical and bacteriological cure between the two antibiotic regimes.

Dysgonomonas capnocytophagoides bacteraemia in a neutropenic patient treated for acute myeloid leukaemia.

Dysgonomonas capnocytophagoides, formerly known as CDC group DF-3, is an opportunistic pathogen associated with diarrhoea and very rarely bacteraemia. We report a case of D. capnocytophagoides found in blood cultures from a severely neutropenic patient treated for acute myeloid leukaemia. The isolate was found resistant to penicillin, cephalosporins, meropenem, aminoglycosides and ciprofloxacin, and susceptible to ampicillin, tetracycline, chloramphenicol, clindamycin and trimethoprim-sulphamethoxazole. It was identified using conventional phenotypic testing but remained unidentified by the automated identification system (Vitek-2) as this system did not contain DF-3 or D. capnocytophagoides in its database.

Clonal relationship of recent invasive Haemophilus influenzae serotype f isolates from Denmark and the United States.

Surveillance performed after the introduction of general Haemophilus influenzae serotype b (Hib) vaccination in Denmark identified 13 cases of invasive bacteraemic H. influenzae serotype f (Hif) disease in adults over a period of 7 years. Bacteraemic respiratory tract infections accounted for 61 % of cases, but meningitis, epiglottitis and osteoarthritis were also seen. Recent Danish isolates were compared to recent American isolates, historical Hif strains and non-Hif invasive strains. Results of conventional serotyping were confirmed by PCR detection of the serotype-f-specific cap and bexA gene sequences. Multilocus enzyme electrophoresis typing revealed that recent Danish and American isolates belonged to a single Hif clone, which may be undergoing expansion. The need for accurate serotyping of H. influenzae to enable reliable monitoring for Hib replacement by other capsular types is emphasized.

C-reactive protein measurement in general practice may lead to lower antibiotic prescribing for sinusitis.

BACKGROUND: Symptoms of bacterial sinusitis overlap with viral sinusitis, and it is difficult to distinguish between the two conditions based only on a clinical examination. Uncertain diagnosis results in the significant overuse of antibiotics, which is considered to be one of the most important reasons for development of bacterial resistance to antibiotics. A raised C-reactive protein (CRP) level is an indicator of bacterial infection and the CRP rapid test has been shown to be useful for the diagnosis of bacterial sinusitis in general practice. AIMS: To examine whether general practitioners (GPs) who use the CRP rapid test in their practice have a lower antibiotic prescribing rate for sinusitis than GPs who do not use the test. DESIGN OF STUDY: Observational design. SETTING: General practice in Denmark. METHOD: A group of GPs registered all contacts (n = 17 792) with patients who had respiratory tract infections during a 3-week period between 1 November 2001 and 31 January 2002. GPs who used a CRP rapid test were compared with GPs who did not, and the treatment of their patients (n = 1444) with suspected sinusitis was compared. RESULTS: A CRP rapid test was used by 77% (n = 281) of the GPs. In the group of GPs using a CRP rapid test, the rate of antibiotic prescribing was 59% (95% confidence interval [CI] = 56 to 62) compared with 78% (95% CI = 73 to 82) in the group of GPs who did not use a CRP test. Performing a CRP rapid test was the factor that exerted the greatest influence on whether the patients were prescribed antibiotics, and the level of CRP had a strong influence on the prescribing rate. CONCLUSION: The CRP rapid test has a substantial influence on the treatment of sinusitis, and implementing the test in general practice may lead to a reduction in antibiotic prescribing to patients with sinusitis.

[Malaria in the county of Funen 1987-1999]. / Malaria på Fyn 1987-1999.

INTRODUCTION: We describe epidemiological, prophylactic, and clinical aspects of imported malaria in the county of Funen, 1987-1999. MATERIAL AND METHODS: The medical records of 136 patients were reviewed for age, gender, nationality, place of exposure, chemoprophylaxis, time lag from departure to diagnosis, Plasmodium species, treatment, and complications. Data on prescribed chemoprophylaxis dispensed from the pharmacies in the county of Funen were recorded. RESULTS: Seventy-two per cent of the patients were Danish, 28% foreigners. Sixty per cent of the cases were caused by P. falciparum, of more than 90% which was acquired in sub-Saharan Africa. Cases of benign malaria were most often acquired in SE Asia. In the 49 patients with falciparum malaria, who had taken chemoprophylaxis, only 31 (63%) were fully compliant. Compliance was 76% in patients taking chloroquine phosphate + proguanil and 36% in patients taking only chloroquine phosphate. Six patients had complications, but all recovered. DISCUSSION: Contributory causes in a large number of the reported cases of imported malaria in this study were no chemoprophylaxis or poor compliance. With respect to falciparum malaria, prescription of non-recommended chemoprophylaxis also contributed. Chemoprophylaxis dispensed from the pharmacies on Funen over recent years indicates that general practitioners are aware of altered recommendations.

[Staphylococcus aureus infections--the clinical picture and treatment]. / Infektioner med Staphylococcus aureus--klinik og behandling.

Infections caused by Staphylococcus aureus comprise relatively benign local skin infections, as well as serious generalised conditions. In Denmark, more than 85% of all S. aureus isolates are found resistant to penicillin, whereas resistance to methicillin is rare (< 1%) and therefore one of the penicillinase-stable penicillins is still the drug of choice. Dicloxacillin is usually chosen, because of its superior penicillinase stability. Infections caused by methicillin resistant strains may be treated with different combinations of macrolides, fusidic acid, aminoglycosides, fluoroquinolones, and rifampicin, but vancomycin is generally used. Newer drugs like the oxazolidinones and streptogramins are effective against methicillin-resistant strains and will be available in Denmark within a short time. Antibiotic treatment, however, is most often only a supplement to surgical intervention. This paper deals with the clinical picture and treatment of some common S. aureus infections.

[Quick simultaneous analyses are important when MERS-coronavirus infection is suspected].

In September 2012 a novel coronavirus (CoV) caused severe respiratory tract infections in patients from The Arabian Peninsula. It was named Middle East respiratory syndrome (MERS)-CoV. Here, a small series of case stories illustrates how simultaneous analyses for MERS-CoV and other agents of infections at the loca l laboratory resulted in rapid rejection of suspicion of MERS and establishment of the true cause of disease. Relevant treatment was initiated and the patients were discharged from hospital.

Quality indicators for treatment of respiratory tract infections? An assessment by Danish general practitioners.

BACKGROUND: In 2008, a set of 41 quality indicators for antibiotic treatment of respiratory tract infections (RTIs) in general practice were developed in an international setting as part of the European project HAPPY AUDIT. OBJECTIVES: To investigate Danish general practitioners' (GPs') assessment of a set of internationally developed quality indicators and to explore if there is an association between the GPs' assessment of the indicators and their practice characteristics as well as their antibiotic prescription pattern. METHODS: A total of 102 Danish GPs were invited to assess the 41 quality indicators. The GPs were categorized into two groups according to their assessment of indicators. Data concerning practice characteristics and antibiotic treatment were obtained during a three-week registration of patients with RTIs and were linked to the GPs' assessments of the indicators. RESULTS: A total of 62 (61%) responded. Quality indicators focusing on the frequency of prescribing of narrow-spectrum penicillin were rated as suitable by more than 80% of the Danish GPs, while quality indicators concerning cephalosporins or quinolones were rated suitable by less than half of the GPs. The antibiotic prescribing pattern differed significantly and the GPs who disagreed on most indicators prescribed more macrolides and less narrow-spectrum penicillin than the GPs who agreed on most indicators. CONCLUSION: Even though an international expert panel agreed on a set of quality indicators for antibiotic treatment of RTIs, only a few of them were rated suitable by the GPs, who are supposed to use them.

[Rational antibiotic treatment can be achieved through audits and teaching]. / Rationel antibiotikabehandling kan opnås ved audit og undervisning.

A prerequisite for rational antibiotic treatment is the suspicion that the disease is caused by bacteria and that treatment will reduce symptoms and complications. In primary health care most infections are caused by bacteria. The European project HAPPY AUDIT found that an intervention targeting doctors and patients in primary care led to a considerable decrease in antibiotic prescribing. Teaching of hospital doctors, combined with a restriction in the choice of antibiotics, resulted in a decrease in multidrug-resistant strains. A concerted effort is needed to curb the growing resistance problem.

Molecular screening for Candida orthopsilosis and Candida metapsilosis among Danish Candida parapsilosis group blood culture isolates: proposal of a new RFLP profile for differentiation.

Candida orthopsilosis and Candida metapsilosis are recently described species phenotypically indistinguishable from Candida parapsilosis . We evaluated phenotyping and molecular methods for the detection of these species among 79 unique blood culture isolates of the C. parapsilosis group obtained during the years 2004-2008. The isolates were screened by PCR amplification of the secondary alcohol dehydrogenase-encoding gene ( SADH) followed by digestion with the restriction enzyme Ban I, using C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96139 and C. metapsilosis ATCC 96144 as controls. Isolates with RFLP patterns distinct from C. parapsilosis were characterized by sequence analysis of the ITS1-ITS2, 26S rRNA (D1/D2) and SADH regions. Restriction patterns for the 3 species with each of 610 restriction enzymes were predicted in silico using 12 available sequences. By PCR-RFLP of the SADH gene alone, four isolates (5.1 %) had a pattern identical to the C. orthopsilosis reference strain. Sequence analysis of SADH and ITS (internal transcribed spacer) regions identified two of these isolates as C. metapsilosis. These results were confirmed by creating a phylogenetic tree based on concatenated sequences of SADH, ITS and 26S rRNA gene sequence regions. Optimal differentiation between C. parapsilosis, C. metapsilosis and C. orthopsilosis was predicted using digestion with NlaIII, producing discriminatory band sizes of: 131 and 505 bp; 74, 288 and 348 bp; and 131, 217 and 288 bp, respectively. This was confirmed using the reference strains and 79 clinical isolates. In conclusion, reliable discrimination was obtained by PCR-RFLP profile analysis of the SADH gene after digestion with NlaIII but not with BanI. C. metapsilosis and C. orthopsilosis are involved in a small but significant number of invasive infections in Denmark.