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IntroductionProsthetic joint infections (PJIs) are disastrous complications for patients and costly for healthcare organisations. They may promote bacterial resistance due to the extensive antibiotic use necessary in the PJI treatment. The PJI incidence is estimated to be 1%–3%, but the absolute numbers worldwide are high and increasing as large joint arthroplasties are performed by the millions each year. Current treatment algorithms, based on implant preserving surgery or full revision followed by a semitailored antibiotic regimen for no less than 2–3 months, lead to infection resolution in approximately 60% and 90%, respectively. Antibiotic choice is currently guided by minimum inhibitory concentrations (MICs) of free-living bacteria and not of bacteria in biofilm growth mode. Biofilm assays with relatively rapid output for the determination of minimum biofilm eradication concentrations (MBECs) have previously been developed but their clinical usefulness have not been established.Methods and analysisThis single-blinded, two-arm randomised study of hip or knee staphylococcal PJI will evaluate 6-week standard of care (MIC guided), or an alternative antibiotic regimen according to an MBEC-guided-based decision algorithm. Sixty-four patients with a first-time PJI treated according to the debridement, antibiotics, and implant retention principle will be enrolled at a single tertiary orthopaedic centre (Sahlgrenska University Hospital). Patients will receive 14 days of standard parenteral antibiotics before entering the comparative study arms. The primary outcome measurement is the proportion of changes in antimicrobial regimen from first-line treatment dependent on randomisation arm. Secondary endpoints are unresolved infection, how microbial properties including biofilm abilities and emerging antimicrobial resistance correlate to infection outcomes, patient reported outcomes and costs with a 12-month follow-up.Ethics and disseminationApproval is received from the Swedish Ethical Review Authority, no 2020-01471 and the Swedish Medical Products Agency, EudraCT, no 2020-003444-80.Trial registration numberClinicalTrials.gov ID:NCT04488458.

Background Antibiotic resistance is a threat to global child health. Primary healthcare workers play a key role in antibiotic stewardship in the community, but few studies in low-income countries have described their experiences of initiating antibiotic treatment in children. Thus, the present study aimed to describe primary healthcare workers’ experiences of antibiotic prescription for children under 5 years of age and their conceptions of antibiotic resistance in Northern Tanzania. Methods A qualitative study involving individual in-depth interviews with 20 prescribing primary healthcare workers in Moshi urban and rural districts, Northern Tanzania, was performed in 2019. Interviews were transcribed verbatim, translated from Kiswahili into English and analysed according to the phenomenographic approach. Findings Four conceptual themes emerged during the analysis; conceptions in relation to the prescriber, the mother and child, other healthcare actors and in relation to outcome. The healthcare workers relied mainly on clinical examination and medical history provided by the mother to determine the need for antibiotics. Confidence in giving advice concerning non-antibiotic treatment varied among the participants and expectations of antibiotic treatment were perceived to be common among the mothers. Antibiotic resistance was mainly perceived as a problem for the individual patient who was misusing the antibiotics. Conclusions To increase rational antibiotic prescription, an awareness needs to be raised among Tanzanian primary healthcare workers of the threat of antibiotic resistance, not only to a few individuals, but to public health. Guidelines on childhood illnesses should be updated with advice concerning symptomatic treatment when antibiotics are not necessary, to support rational prescribing practices and promote trust in the clinician and mother relationship.

Background: Antimicrobial resistance is a serious threat to the global achievements in child health thus far. Previous studies have found high use of antibiotics in children in Northern Tanzania, but the experiences of the primary care-givers, who play a key role in accessing and administering antibiotics for the sick child, has remained largely unknown. We aimed to understand mothers’ conceptions of antibiotic use in their children, which is of importance when forming strategies to improve antibiotic use in the community. Method: A qualitative study including eight focus group discussions with mothers of under-five children in Moshi urban and rural districts, Northern Tanzania, was performed during 2019. The discussions were recorded, transcribed verbatim, translated to English and analysed according to the phenomenographic approach. Findings: Three conceptual themes emerged during analysis; (1) conceptions of disease and antibiotics, (2) accessing treatment and (3) administering antibiotics. Antibiotics were often perceived as a universal treatment for common symptoms or diseases in children with few side-effects. Although mothers preferred to attend a healthcare facility, unforeseen costs, long waits and lack of financial support from their husbands, pose barriers for care-seeking. However, pharmacies were perceived as a cheap and convenient option to access previously used or prescribed antibiotics. Some mothers sought advice from a trusted neighbour regarding when to seek care, thus resembling the function of the community health worker (CHW). Conclusions: To improve antibiotic use in children under five years of age in Northern Tanzania, we suggest targeting behaviour of individuals alone will not be sufficient. Improving structures, including the healthcare system and socio-economic inequality, are needed for sustained results. In the meantime, equipping CHWs to support women in appropriate healthcare seeking for their children, may be a feasible target for intervention.

Secretory otitis media (SOM) is characterized by persistence of fluid in the middle ear, often following an episode of acute otitis media. Our hypothesis is that failure to eliminate bacterial or viral pathogens may result in persistent low-grade inflammation. In this study, we analyzed inflammatory mediators in middle ear fluids from 67 children with SOM. This was combined with determinations of viable bacteria by culture along with detection of bacterial and viral genetic material by real-time polymerase chain reaction (PCR). The inflammatory mediators found at the highest concentrations (>30 ng/mL) were stem cell growth factor-β (median 110 ng/mL), CXCL1, IL-16, IL-8, migration inhibitory factor, CXCL10, and CXCL9. Among bacterial pathogens, Moraxella catarrhalis and Haemophilus influenzae dominated, regardless of detection methods, while rhinovirus dominated among viral pathogens. Middle ear fluid levels of interleukin (IL)-1α, IL-17, IL-1β, fibroblast growth factor basic, and tumor necrosis factor correlated strongly with presence of bacteria detected either by culture or PCR, while IL-1RA, IL-3, IL-6, IL-8, CCL3, CCL4, and granulocyte-colony stimulating factor correlated significantly with real-time PCR values. CXCL10, CXCL9, CCL2, and TRAIL correlated significantly with viral nucleic acid levels. To conclude, persistence of viral and bacterial pathogens may fuel persistent inflammation in SOM. Bacteria caused a broad inflammatory response, while viruses chiefly elicited the interferon-induced chemokines CXCL9 and CXCL10.

Staphylococcus aureus bacteraemia (SAB) is recognized as an infection that is difficult to treat and with high risk of device related infection. Extraction/explantation of cardiac implantable elect...

Peptide biomarker candidates for the respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus are presented. First, bacterial cultures representing the genetic variability in each of the four species, were analyzed. The peptide biomarker candidates were then experimentally verified to be present in a clinical situation by analyzing true positive clinical samples. The most promising peptide biomarkers were used in a targeted MS mode, demonstrating their use for future clinical implementation., Graphical Abstract Highlights Discovery of peptide biomarker candidates of respiratory tract pathogens S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus as target pathogens. Peptide biomarker candidates were experimentally verified in clinical samples. Targeted MS using promising peptide biomarker candidates shown as proof-of-concept., Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Background Over 90% of Ethiopians still rely on solid fuels for cooking food. The pollution from the burning process causes adverse respiratory outcomes including respiratory infections. This study aimed to assess the association of the pollution with nasopharyngeal occurrence of potential pathogens. Methods We conducted a comparative cross-sectional study in urban and rural settings in Ethiopia in 2016. Questionnaire-based data were collected from 168 mothers and 175 children aged below two years. Multiplex real-time PCR assays were performed on nasopharyngeal secretions for detection of bacteria and viruses and for the identification of pneumococcal serotypes/groups. Results High rates of bacteria and viruses in the nasopharynx were detected by PCR among both the children and the mothers. Among the detected viruses, enterovirus was more commonly detected among rural children than among children from urban areas. Streptococcus pneumoniae and Haemophilus influenzae were both more prevalent among children and mothers from rural areas compared with urban groups and among those using solid fuels compared with cleaner fuel users. Children from rural households using solid fuels and children whose mothers had educational status below high school had four times higher odds for detection of S. pneumoniae compared with those households using cleaner energy or those children having mothers with a higher educational status, respectively. One or more serotype/serogroup was identified in about 40% of the samples that were positive for pneumococci. Out of all identified serotypes/serogroups, 43% in the children and 45% in the mothers belonged to PCV13, indicating the larger majority of detected pneumococci being non-PCV13 serotypes. Conclusion This study presented a high carriage rate of S. pneumoniae and H. influenzae among both children and their mothers, especially in rural areas and among solid fuel users. Thus, interventions should target cleaner energy sources to the public and promote maternal education.

Background Acute pneumonia remains a leading cause of death among children below 5 years of age in the Democratic Republic of the Congo (DR Congo), despite introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in 2013. Potential pathogens in the nasopharynx of hospitalised children with pneumonia have not been studied previously in DR Congo. Here we compare clinical characteristics, risk factors and nasopharyngeal occurrence of bacteria and viruses between children with severe and non-severe pneumonia. Methods Between June 2015 and June 2017, 116 children aged from 2 to 59 months hospitalised due to radiologically confirmed pneumonia at Panzi referral university hospital, Bukavu, Eastern DR Congo were included in the study and sampled from nasopharynx. A multiplex real-time PCR assay for detection of 15 different viruses and 5 bacterial species was performed and another multiplex PCR assay was used for pneumococcal serotype/serogroup determination. Results During the study period 85 (73%) of the children with radiologically confirmed pneumonia met the WHO classification criteria of severe pneumonia and 31 (27%) had non-severe pneumonia. The fatality rate was 9.5%. Almost all (87%) children were treated with antibiotics before they were hospitalised, in most cases with amoxicillin (58%) or trimethoprim-sulfamethoxazole (20%). The frequency of potential pathogens in the nasopharynx of the children was high, and any viral or bacterial nucleic acids present at high levels, irrespective of species or type, were significantly associated with severe pneumonia as compared with non-severe cases (52% versus 29%, p = 0.032). White blood cell count > 20,000/μL and C-Reactive Protein > 75 mg/dL were associated with severe pneumonia at admission. Fatal outcome was in the multivariable analysis associated with having a congenital disease as an underlying condition. One or more pneumococcal serotypes/serogroups could be identified in 61 patients, and out of all identified serotypes 31/83 (37%) were non-PCV13 serotypes. Conclusions The occurrence of any bacteria or any viruses at high levels was associated with severe pneumonia at admission. Children with congenital disorders might need a higher attention when having symptoms of acute respiratory infection, as developed pneumonia could lead to fatal outcome.

Purpose. Correct serotype identification of Streptococcus pneumoniae (pneumococcus) is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. Furthermore, correct identification and differentiation of the pathogenic S. pneumoniae from closely related commensal species of the mitis group of the genus Streptococcus are essential for correct serotype identification. Methodology. A new protocol for determining the existing 98 serotypes of pneumococcus was developed, applying two PCR amplifications and amplicon sequencing, using newly designed internal primers. The new protocol was validated using S. pneumoniae genome sequences, reference strains with confirmed serotypes and clinical isolates, and comparing the results with those from the traditional Quellung reaction or antiserum panel gel precipitation, in addition to real-time PCR analysis. The taxonomic identifications of 422 publicly available (GenBank) genome sequences of S. pneumoniae , Streptococcus pseudopneumoniae and Streptococcus mitis were assessed by whole-genome sequence average nucleotide identity based on blast (ANIb) analysis. Results. The proposed sequetyping protocol generates a 1017 bp whole cpsB region sequence, increasing resolution for serotype identification in pneumococcus isolates. The identifications of all GenBank genome sequences of S. pneumoniae were confirmed, whereas most of the S. pseudopneumoniae and almost all of the S. mitis genome sequences did not fulfil the ANIb thresholds for species-level identification. The housekeeping biomarker gene, groEL, correctly identified S. pneumoniae but often misclassified S. pseudopneumoniae and S. mitis as S. pneumoniae . Conclusions. These studies affirm the importance of applying reliable identification protocols for S. pneumoniae before serotyping; our protocols provide reliable diagnostic tools, as well as an improved workflow, for serotype identification of pneumococcus and differentiation of serogroup 6 types.

Objectives: To determine the antibiotic susceptibility and serotype distribution of colonizing Streptococcus pneumoniae in Tanzanian children. Serial cross-sectional surveys were performed following the national introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in December 2012. Methods: A total of 775 children less than 2 years of age were recruited at primary health centres in Moshi, Tanzania between 2013 and 2015, and samples were obtained from the nasopharynx. S. pneumoniae were isolated by culture and tested for antibiotic susceptibility by disc diffusion and E-test methods; molecular testing was used to determine serotype/group. Results: Penicillin non-susceptibility in the isolated pneumococci increased significantly from 31% (36/116) in 2013, to 47% (30/64) in 2014 and 53% (32/60) in 2015. Non-susceptibility to amoxicillin/ampicillin and ceftriaxone was low (n = 8 and n = 9, respectively), while 97% (236/244) of the isolates were non-susceptible to trimethoprim–sulfamethoxazole. The majority of the children (54%, n = 418) had been treated with antibiotics in the past 3 months, and amoxicillin/ampicillin were overall the most commonly used antibiotics. Carriage of penicillin-non-susceptible pneumococci was more common in children with many siblings. The prevalence of PCV13 serotypes among the detected serotypes/groups decreased from 56% (40/71) in 2013 to 23% (13/56) in 2015. Conclusions: Penicillin non-susceptibility in S. pneumoniae colonizing Tanzanian children increased during an observation period shortly after the introduction of PCV13. Measures to ensure rational use of antibiotics and more effective systems for surveillance of antibiotic resistance and serotype distribution are needed to assure continued effective treatment of pneumococcal disease. Keywords: Streptococcus pneumoniae, Drug resistance, Bacterial, Air pollution, Pneumococcal vaccines, Nasopharyngeal colonization

The use of solid fuel, known to emit pollutants which cause damage to human health, is the primary energy option in Ethiopia. Thus, the aim of this study was to measure the level of household air pollution by using the 24-h mean concentration of fine particulate matter (PM2.5) in 150 randomly recruited households in rural Butajira, Ethiopia. Data relating to household and cooking practices were obtained by conducting face-to-face interviews with the mothers. The 24-h mean (standard deviation) and median PM2.5 concentrations were 410 (220) and 340 µg/m3, respectively. Households using only traditional stoves and those who did not open the door or a window during cooking had a significantly higher mean concentration compared with their counterparts. There is a statistically significant correlation between the mean concentration of PM2.5 and the self-reported cooking duration. The pollution level was up to 16 times higher than the WHO 24-h guideline limit of 25 μg/m3, thus leaving the mothers and children who spend the most time at the domestic hearth at risk of the adverse health effects from solid fuel use in Ethiopia. Thus, effective short- and long-term interventions are urgently needed.

PurposeMost studies on paediatric pharyngotonsillitis focus on group A streptococci. This study, however, analyses a broad spectrum of bacteria and viruses related to paediatric pharyngotonsillitis and evaluates their associated clinical symptoms and courses.MethodsThis observational prospective study in primary healthcare includes 77 children aged ®for analysis with culture and PCR for 14 bacteria and 15 viruses. Patients were evaluated clinically, and their symptoms recorded in diaries for 10 days. Participants were followed up for 3 months by reviewing medical records.ResultsA pathogen was detected in 86% of patients and in 71% of controls (P = 0.06). Bacteria were found in 69% of patients and 59% of controls (P = 0.3), and viruses in 36% and 26%, respectively (P = 0.3). Group A streptococci was the most common finding, with a prevalence of 49% and 32%, respectively (P = 0.1). Clinical signs were not useful for distinguishing pathogens. None of the controls and 16% of the patients reconsulted for a sore throat within 3 months.ConclusionBacteria were more common than viruses in both study groups. The high rate of pathogens in asymptomatic children interferes with diagnoses based on aetiology.

Background The pneumococcal conjugate vaccine PCV7 was introduced in Southwest Sweden in the child vaccination program in 2009, followed by PCV13 in 2010 and PCV10 in 2015. In this retrospective cohort study we assessed the pneumococcal serotype distribution in relation to predisposing factors, clinical manifestations and outcome during seven years after PCV introduction. Methods Clinical data from 1278 patients with 1304 episodes of invasive pneumococcal disease (IPD) between January 2009 and December 2015 in Region Västra Götaland, Sweden, were retrospectively collected from medical records. Pneumococcal isolates were serotyped by gel diffusion and/or Quellung reactions performed at the Public Health Agency in Sweden. Associations between serotypes and clinical characteristics were statistically evaluated by use of Fisher’s exact test, Mann-Whitney U test and Logistic regression analysis, whereas IPD episodes caused by serotypes over time were analyzed by Mantel-Haenszel chi-square test. Results With the exception of serotype 3, the prevalence of PCV13 serotypes decreased during the study period, from 76% (n = 157) of all IPD episodes in 2009 to 25% (n = 42) in 2015 (p p Conclusions Non-PCV13 serotypes caused the majority of IPD cases in Southwest Sweden, especially in patients ≥65 years and in patients with predisposing factors. Serotype 3, included in PCV13, was prevalent and often caused severe disease.

BackgroundRespiratory pathogens including Streptococcus pneumoniae and Haemophilus influenzae, are implicated in the pathogenicity of acute lower respiratory infection (ALRI). These are also commonly found in both healthy and sick children. In this study, we describe the first data on the most frequent bacteria and viruses detected in the nasopharynx of children from the general population in the Eastern DR Congo.MethodsFrom January 2014 to June 2015, nasopharyngeal samples from 375 children aged from 2 to 60 months attending health centres for immunisation or growth monitoring were included in the study. Multiplex real-time PCR assays were used for detection of 15 different viruses and 5 bacterial species and for determination of pneumococcal serotypes/serogroups in the nasopharyngeal secretions.ResultsHigh levels of S. pneumoniae were detected in 77% of cases, and H. influenzae in 51%. Rhinovirus and enterovirus were the most commonly found viruses, while respiratory syncytial virus (RSV) was rare (1%). Co-occurrence of both bacteria and viruses at high levels was detected in 33% of the children. The pneumococcal load was higher in those children who lived in a dwelling with an indoor kitchen area with an open fire, i.e. a kitchen with an open fire for cooking located inside the dwelling with the resultant smoke passing to the living room and/or bedrooms; this was also higher in children from rural areas as compared to children from urban areas or children not living in a dwelling with an indoor kitchen area with an open fire/not living in this type of dwelling. Immunization with 2-3 doses of PCV13 was associated with lower rates of pneumococcal detection. Half of the identified serotypes were non-PCV13 serotypes. The most common non-PCV13 serotypes/serogroups were 15BC, 10A, and 12F, while 5, 6, and 19F were the most prevalent PCV13 serotypes/serogroups.ConclusionsThe burden of respiratory pathogens including S. pneumoniae in Congolese children was high but relatively few children had RSV. Non-PCV13 serotypes/serogroups became predominant soon after PCV13 was introduced in DR Congo.

In this retrospective cohort study, 626 consecutive SAB patients were identified in routine diagnostics (November 2014-October 2016). Patient characteristic, infective endocarditis (IE) incidence and mortality were compared for patients with and without CIED.SAB patients with CIED (

Background Acute pneumonia remains a leading cause of death among children below 5 years of age in the Democratic Republic of the Congo (DR Congo), despite introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in 2013. Pathogens in the nasopharynx of hospitalised children with pneumonia have not been studied previously in DR Congo. Here we compare clinical characteristics, risk factors and nasopharyngeal occurrence of bacteria and viruses between children with severe and non-severe pneumonia Methods Between June 2015 and June 2017, 116 children aged from 2 to 59 months hospitalised due to radiologically confirmed pneumonia at Panzi referral university hospital, Bukavu, eastern DR Congo were included in the study and sampled from nasopharynx. A multiplex real-time PCR assay for detection of 15 different viruses and 5 bacterial species was performed and another multiplex PCR assay was used for pneumococcal serotype/serogroup determination. Results During the study period 85 (73%) of the children with radiologically confirmed pneumonia met the WHO classification criteria of severe pneumonia and 31 (27%) had non-severe pneumonia. The fatality rate was 9.5%. Almost all (87%) children were treated with antibiotics before they were hospitalised, in most cases with amoxicillin (58%) or trimethoprim-sulfamethoxazole (20%). Children with severe pneumonia were more commonly treated with trimethoprim-sulfamethoxazole before hospitalisation than children with non-severe pneumonia (OR 4.75; 95%CI 1.04–21.65, p = 0.043), as were children who died at the hospital as compared to children who recovered (OR 3.98; 95%CI 1.1–14.4, p = 0.035). Any viral or bacterial nucleic acids present at high levels in nasopharynx were significantly associated with severe pneumonia as compared with non-severe cases (52% versus 29%, p = 0.032). High levels of pneumococci or respiratory syncytial virus (RSV) were associated with fatal outcome. One or more serotypes/serogroups could be identified in 61 patients, and out of all identified pneumococcal serotypes 31/83 (37%) were non-PCV13 serotypes. Conclusions The occurrence of any bacteria or any viruses at high levels, irrespective of species, was associated with severe pneumonia. High levels of RSV or pneumococci were associated with higher risk of fatality. Pre-hospitalisation treatments with broad spectrum antibiotics were common but were associated with a more severe outcome.

Background Pneumococcal conjugate vaccines have been introduced in the infant immunisation programmes in many countries to reduce the rate of fatal pneumococcal infections. In the Democratic Republic of the Congo (DR Congo) a 13-valent vaccine (PCV13) was introduced in 2013. Data on the burden of circulating pneumococci among children after this introduction are lacking. In this study, we aimed to determine the risk factors related to pneumococcal carriage in healthy Congolese children after the vaccine introduction and to assess the antibiotic resistance rates and serotype distribution among the isolated pneumococci. Methods In 2014 and 2015, 794 healthy children aged one to 60 months attending health centres in the eastern part of DR Congo for immunisation or growth monitoring were included in the study. Data on socio-demographic and medical factors were collected by interviews with the children’s caregivers. Nasopharyngeal swabs were obtained from all the children for bacterial culture, and isolated pneumococci were further tested for antimicrobial resistance using disc diffusion tests and, when indicated, minimal inhibitory concentration (MIC) determination, and for serotype/serogroup by molecular testing. Results The pneumococcal detection rate was 21%, being higher among children who had not received PCV13 vaccination, lived in rural areas, had an enclosed kitchen, were malnourished or presented with fever (p value

The use of solid fuel, known to emit pollutants which cause damage to human health, is the primary energy option in Ethiopia. Thus, the aim of this study was to measure the level of household air pollution by using the 24-h mean concentration of fine particulate matter (PM2.5) in 150 randomly recruited households in rural Butajira, Ethiopia. Data relating to household and cooking practices were obtained by conducting face-to-face interviews with the mothers. The 24-h mean (standard deviation) and median PM2.5 concentrations were 410 (220) and 340 µg/m3, respectively. Households using only traditional stoves and those who did not open the door or a window during cooking had a significantly higher mean concentration compared with their counterparts. There is a statistically significant correlation between the mean concentration of PM2.5 and the self-reported cooking duration. The pollution level was up to 16 times higher than the WHO 24-h guideline limit of 25 μg/m3, thus leaving the mothers and children who spend the most time at the domestic hearth at risk of the adverse health effects from solid fuel use in Ethiopia. Thus, effective short- and long-term interventions are urgently needed.

Exposure to household air pollution has been linked to chronic obstructive pulmonary disease, respiratory symptoms and reduced lung function. This study aims to assess respiratory symptoms and lung function among Ethiopian women in relation to exposure to HAP. We conducted a cross-sectional study among non-smoking women responsible for household cooking. Data was collected on socio-demographic characteristics, respiratory symptoms and risk factors using a validated questionnaire. Spirometry with reversibility testing was performed according to American Thoracic Society/European Respiratory Society guidelines. We used independent t-test and multivariable logistic regression to compare the means and measure association respectively. A total of 545 women participated in the study out of which 231 (42.3%) performed spirometry with at least three acceptable manoeuvres. Everyone in the rural group and 43% of the urban group were exposed to HAP from solid fuels during cooking. The odds of developing at least one respiratory symptom when compared with those using cleaner fuels are twice as high for women cooking within the living house. We also found significantly lower forced expiratory volume in the first second (FEV1) (L) among solid fuels users compared with cleaner energy users. Given the larger population settlement in the rural areas and the use of solid fuel as the only energy source, there is a higher risk of developing chronic respiratory health problems for those women in Ethiopia.

Clinical and environmental-associated strains (n=17), genotypically related to Corynebacterium spp., yet distinct from any species of the genus Corynebacterium with validly published names, have been isolated during the last 20 years and tentatively identified as Corynebacterium sanguinis, although the combination, "Corynebacterium sanguinis" was never validly published. The comprehensive genotypic and phenotypic characterisations and genomic analyses in this study support the proposal for recognizing the species within the genus Corynebacterium, for which the name, Corynebacterium sanguinis sp. nov., is reaffirmed and proposed. Strains of Corynebacterium sanguinis are Gram-positive, non-motile, non-spore-forming, short, pleomorphic and coryneform bacilli, growing aerobically, with CO

Correct identification ofStreptococcus pneumoniae(pneumococcus) and differentiation from the closely related species of the Mitis group of the genusStreptococcus, as well as serotype identification, is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. In this study, we assessed the taxonomic identifications of 422 publicly available genome sequences ofS. pneumoniae, S. pseudopneumoniaeandS. mitis, using different methods. Identification ofS. pneumoniae, by comparative analysis of thegroELpartial sequence, was possible and accurate, whereasS. pseudopneumoniaeandS. mitiscould be misclassified asS. pneumoniae, suggesting thatgroELis unreliable as a biomarker for differentiatingS. pneumoniaefrom its closest related species. The genome sequences ofS. pneumoniaeandS. pseudopneumoniaefulfilled the suggested thresholds of average nucleotide identity (ANI), i.e., > 95% genome sequence similarity to the sequence of respective type strains for identification of species, whereas none of theS. mitisgenome sequences fulfilled this criterion. However, ANI analyses of all sequencesversusall sequences allowed discrimination of the different species by clustering, with respect to species type strains. Thein silicoDNA-DNA distance method was also inconclusive for identification ofS. mitisgenome sequences, whereas presence of the “Xisco” gene proved to be a reliable biomarker forS. pneumoniaeidentification. Furthermore, we present an improved sequetyping protocol including two newly-designed internal sequencing primers with two PCRs, as well as an improved workflow for differentiation of serogroup 6 types. The proposed sequetyping protocol generates a more specific product by generating the whole gene PCR-product for sequencing, which increases the resolution for identification of serotypes. Validations of both protocols were performed with publicly availableS. pneumoniaegenome sequences, reference strains at the Culture Collection University of Gothenburg (CCUG), as well as with clinical isolates. The results were compared with serotype identifications, using real-time Q-PCR analysis, as well as the Quellung reaction or antiserum panel gel-precipitation. Our protocols provide a reliable diagnostic tool for taxonomic identification as well as serotype identification ofS. pneumoniae.

Acute pharyngotonsillitis denotes tonsillar inflammation caused by bacteria or viruses. Here, we investigated if beta-hemolytic streptococci (β-HS) tonsillitis would differ in inflammatory mediator response from tonsillitis of other causes. Tonsillar secretions were obtained from 36 acute pharyngotonsillitis patients and 16 controls. Bacteria were cultured quantitatively and 18 different viruses were quantified by real-time polymerase chain reaction. Cytokine and prostaglandin E2 (PGE2) levels were determined by enzyme-linked immunosorbent assays. Almost half of the patients' tonsillar secretions yielded high counts of β-HS, and most samples contained viruses, irrespective of whether β-HS were present or not. The Epstein-Barr virus (EBV) was the most common virus (patients 62% and controls 13%). Compared to controls, patients' secretions had higher levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF), and PGE2, while few samples contained IL-12, IL-10, or interferon-gamma (IFN-γ). The presence of β-HS in tonsillitis secretions could not be distinguished by any of the measured mediators, while the presence of EBV DNA tended to be associated with enhanced levels of IL-1β and IL-8. The results suggest a common inflammatory response in acute pharyngotonsillitis, regardless of causative agent. The suggested correlation between intense inflammation and the presence of EBV DNA in tonsillitis secretions may be due to reactivation of the virus and/or the EBV-containing B cells.

Modern microbial systematics requires a range of methodologies for the comprehensive characterization, classification and identification of microorganisms. While whole-genome sequences provide the ultimate reference for defining microbial phylogeny and taxonomy, selected biomarker-based strategies continue to provide the means for the bulk of microbial systematic studies. Proteomics, the study of the expression of genes, as well as the structure and function of the resulting proteins, offers indirect measures of genome sequence data. Recent developments in applications of proteomics for analyzing microorganisms have paralleled the growing microbial genome sequence database, as well as the evolution of mass spectrometry (MS) instrumentation and bioinformatics. MALDI-TOF MS, which generates proteomic mass patterns for 'fingerprint'-based characterizations, has provided a marked breakthrough for microbial identification. However, MALDI-TOF MS is limited in the number of targets that can be detected for strain characterization. Advanced methods of tandem mass spectrometry, in which proteins and peptides generated from proteins, are characterized and identified, using LC-MS/MS, provide the ability to detect hundreds or thousands of expressed microbial strain markers for high-resolution characterizations and identifications. Model studies demonstrate the application of proteomics-based analyses for bacterial species- and strain-level detection and identification and for characterization of environmentally relevant, metabolically diverse bacteria. Proteomics-based approaches represent an emerging complement to traditional methods of characterizing microorganisms, enabling the elucidation of the expressed biomarkers of genome sequence information, which can be applied to 'proteotyping' applications of microorganisms at all taxonomic levels.

A peculiar trait of pneumococci ( Streptococcus pneumoniae ) is their propensity to undergo spontaneous lysis during stationary growth due to activation of the enzyme autolysin (LytA), which fragments the peptidoglycan cell wall. The fragments that are generated upon autolysis impair phagocytosis and reduce production of interleukin-12 (IL-12) and gamma interferon (IFN-γ) by human leukocytes in response to intact pneumococci, thereby impeding crucial host defenses. The objective was to identify additional monocyte genes whose transcription is induced by intact pneumococci and subverted by autolyzed bacteria. Monocytes were isolated from healthy blood donors and stimulated for 3 h with UV-inactivated S. pneumoniae (Rx1PLY − LytA + strain), which is capable of autolyzing, its LytA − isogenic autolysin-deficient mutant, or a mixture of the two (containing twice the initial bacterial concentration). Gene expression was assessed by Illumina microarray, and selected findings were confirmed by reverse transcription-quantitative real-time PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and flow cytometry. In all, we identified 121 genes that were upregulated to a significantly higher degree by intact than autolyzed pneumococci. These included IFNB1 and a large set of interferon-induced genes, such as IFIT3, RSAD2, CFCL1, and CXCL10 genes, as well as IL12B and CD40 genes. RT-qPCR revealed that transcription of these genes in response to intact pneumococci diminished when autolyzed pneumococci were admixed and that this pattern was independent of pneumolysin. Thus, transcription of interferon-related genes is triggered by intact pneumococci and subverted by fragments generated by spontaneous bacterial autolysis. We suggest that interferon-related pathways are important for elimination of pneumococci and that autolysis contributes to virulence by extinguishing these pathways.

Acute otitis media (AOM) is an inflammatory response to microbes in the middle ear, sometimes associated with rupture of the tympanic membrane. Human leukocytes produce different patterns of inflammatory mediators in vitro when stimulated with Gram-positive and Gram-negative bacteria, respectively. Here, we investigated the cytokine and prostaglandin E2 (PGE2) responses in middle ear fluids (MEFs) from children with spontaneously perforated AOM, and related the mediator levels to the presence of pathogens detected by culture (live) or PCR (live or dead). Furthermore, the in vivo cytokine pattern was compared with that induced in leukocytes stimulated by dead bacteria in vitro. MEFs with culturable pathogenic bacteria contained more interleukin (IL)-1β (median: 110 μg/L vs.

Streptococcus pneumoniae is a major pathogen in humans. The pathogenicity of this organism is related to its many virulence factors, the most important of which is the thick pneumococcal capsule that minimizes phagocytosis. Another virulence-associated trait is the tendency of this bacterium to undergo autolysis in stationary phase through activation of the cell wall-bound amidase LytA, which breaks down peptidoglycan. The exact function of autolysis in pneumococcal pathogenesis is, however, unclear. Here, we show the selective and specific inefficiency of wild-type S. pneumoniae for inducing production of phagocyte-activating cytokines in human peripheral blood mononuclear cells (PBMC). Indeed, clinical pneumococcal strains induced production of 30-fold less tumor necrosis factor (TNF), 15-fold less gamma interferon (IFN-γ), and only negligible amounts of interleukin-12 (IL-12) compared with other closely related Streptococcus species, whereas the levels of induction of IL-6, IL-8, and IL-10 production were similar. If pneumococcal LytA was inactivated by mutation or by culture in a medium containing excess choline, the pneumococci induced production of significantly more TNF, IFN-γ, and IL-12 in PBMC, whereas the production of IL-6, IL-8, and IL-10 was unaffected. Further, adding autolyzed pneumococci to intact bacteria inhibited production of TNF, IFN-γ, and IL-12 in a dose-dependent manner but did not inhibit production of IL-6, IL-8, and IL-10 in response to the intact bacteria. Fragments from autolyzed bacteria inhibited phagocytosis of intact bacteria and reduced the in vitro elimination of pneumococci from human blood. Our results suggest that fragments generated by autolysis of bacteria with reduced viability interfere with phagocyte-mediated elimination of live pneumococci.

The present study aimed to improve the rate of detection of blood-borne microbes by using PCRs with pan-bacterial and Candida specificity. Seventeen per cent of the blood samples (n = 178) collected from 107 febrile patients with haematological malignancies were positive using standard culture (BacT/Alert system). Candida PCR was positive in 12 patients, only one of whom scored culture-positive. Bacterial PCR using fresh blood samples was often negative, but the detection rate increased when the blood was pre-incubated for 2 days. These data indicate that PCR assays might be a complement for the detection of blood-borne opportunists in immunocompromised haematology patients.

Secretory otitis media (SOM) is characterised by persistent fluid in the middle ear cavity, but the cause is unknown. We investigated the clinical, bacteriological and immunological effects of treatment with probiotic bacteria on SOM.In this double-blind pilot/preliminary study, 60 children with long-standing SOM (median 6 months) who were scheduled for insertion of tympanostomy tubes were randomised to nasal spray treatment with Streptococcus sanguinis, Lactobacillus rhamnosus or placebo for 10 days before surgery. Clinical evaluation was carried out after 10 days of treatment. Middle ear fluid (MEF) was collected during surgery for quantification of cytokines and detection of bacteria by culture and polymerase chain reaction (PCR). Nasopharyngeal swabs were obtained before treatment and at surgery.Complete or significant clinical recovery occurred in 7/19 patients treated with S sanguinis compared to 1/17 patients in the placebo group (p0.05). In the L rhamnosus treatment group, 3/18 patients were cured or much better (p = 0.60 compared with placebo). Spray treatment did not alter the composition of the nasopharyngeal flora or the cytokine pattern observed in the nasopharynx or MEF, except for a higher level of IL-8 found in the nasopharynx of L rhamnosus treated children.This study shows that spray treatment with S sanguinis may be effective against SOM. The mechanism for the effect remains to be investigated.

The mechanisms that determine the duration of fecal shedding of norovirus in humans have not been described in detail.We investigated serum inflammatory mediator levels in relation to the duration of viral shedding in norovirus infection.A prospective cohort study of patients hospitalized with acute norovirus genogroup II infection. Rectal swab samples were obtained at inclusion and day 7, 14, 21 and 28. Serum levels of 42 inflammatory mediators were determined with a Luminex-based cytokine assay. Sera from 20 healthy blood donors served as controls.Altogether, 28 patients (54% women, median age 83 years, median duration of symptoms 3 days) were included. Twelve subjects cleared the virus within 14 days and 16 were norovirus-RNA positive for21 days, constituting the two study groups ("rapid" vs. "slow" clearance). Individuals with norovirus infection had higher levels of IL-18, CXCL9, CXCL10, soluble IL-2 receptor and macrophage migration inhibitory factor (MIF), compared to controls (p0.05), with the highest median concentrations in the slow clearance group. In contrast, CCL5 levels were lower in the slow compared to the rapid clearance group (median 54 vs. 134 ng/mL, p0.05), and lower in norovirus-infected patients than in controls.Low levels of CCL5 were associated with longer duration of viral shedding, suggesting that CCL5 may influence the clearance of norovirus.

Infection caused by Streptococcus pneumoniae is the leading cause of mortality in children worldwide. The aim of this study was to determine if a noted increase in non-susceptibility to penicillin among pneumococcal clinical isolates from young children reflected a similar increase in healthy children.During 2004-2005, before the conjugate pneumococcal vaccine was introduced in Sweden, 663 healthy children (13-24 months of age) attending 17 child health centres in Gothenburg, Sweden, were cultured for bacteria in the nasopharynx. Social factors were identified through a parental questionnaire. Pneumococcal serotypes and antibiotic resistance rates were determined. Antibiotic resistance was also monitored in 162 simultaneously obtained nasopharyngeal pneumococci isolated from clinical samples.The healthy children frequently carried pneumococci (45%), Moraxella catarrhalis (54%), and Haemophilus influenzae (22%). The carriage rates for all these pathogens were higher in children attending day care centres compared to children staying at home (p0.001). The dominating pneumococcal serotypes were 6B, 19F, 23F, and 6A. Non-susceptibility to penicillin was low (4.0%) and only exceeded by that to trimethoprim-sulfamethoxazole (9.8%). Both rates were higher in the clinical isolates (9.3% and 16.7%, respectively; p0.05). No relationships to geographic area, day care attendance, recent antibiotic use, or travel abroad were shown for any specific serotype or for the presence of penicillin-non-susceptible pneumococci in the healthy children.Pneumococcal resistance rates in the healthy child population were low and did not reflect the higher rates noted at the laboratory in clinical samples obtained before and during the study.

Upon bacterial stimulation, tissue macrophages produce a variety of cytokines that orchestrate the immune response that clears the infection. We have shown that Gram-positives induce higher levels of interleukin-12 (IL-12), interferon-gamma (IFN-gamma), and tumor necrosis factor (TNF) from human peripheral blood mononuclear cells (PBMCs) than do Gram-negatives, which instead induce more of IL-6, IL-8, and IL-10. Here, we study whether these patterns follows or crosses taxonomic borders. PBMCs from blood donors were incubated with UV-inactivated bacteria representing 37 species from five phyla. IL-12, TNF, IL-1beta, IL-6, IL-8, and IL-10 were measured in the supernatants after 24 h and IFN-gamma after 5 days. Irrespective of phylogenetic position, Gram-positive bacteria induced much more IL-12 (nine times more on average) and IFN-gamma (seven times), more TNF (three times), and slightly more IL-1beta (1.5 times) than did Gram-negatives, which instead induced more IL-6 (1.5 times), IL-8 (1.9 times), and IL-10 (3.3 times) than did Gram-positives. A notable exception was the Gram-positive Listeria monocytogenes, which induced very little IL-12, IFN-gamma, and TNF. The results confirm the fundamental difference in innate immune responses to Gram-positive and Gram-negative bacteria, which crosses taxonomic borders and probably reflects differences in cell wall structure.