Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping.

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.

Presence of microorganisms in children with pharyngotonsillitis and healthy controls: a prospective study in primary healthcare.

PURPOSE: Most 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. METHODS: This observational prospective study in primary healthcare includes 77 children aged < 15 with a sore throat and 34 asymptomatic children, all of whom were sampled from the tonsils with an E-swab® 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. RESULTS: A 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. CONCLUSION: Bacteria were more common than viruses in both study groups. The high rate of pathogens in asymptomatic children interferes with diagnoses based on aetiology.

Invasive pneumococcal disease in persons with predisposing factors is dominated by non-vaccine serotypes in Southwest Sweden.

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 < 0.001) while non-PCV13 serotypes increased, mainly among patients ≥65 years and in patients with predisposing factors, including cardiovascular disease, pulmonary disease and malignancy (p < 0.001 for all). Patients with predisposing factors, including those with malignancy, immune deficiency or renal disease, were more likely to have IPD caused by a serotype not included in PCV13 rather than a vaccine-included serotype. Serotype 3 was associated with intensive care unit admissions while serotype 1 and 7F caused IPD among healthier and younger patients. PCV13 serotypes were associated with invasive pneumonia, and non-PCV13 serotypes were associated with bacteremia with unknown focus and with manifestations other than pneumonia or meningitis. 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.

Bacteria and viruses in the upper respiratory tract of Congolese children with radiologically confirmed pneumonia.

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.

High rate of antibiotic resistance among pneumococci carried by healthy children in the eastern part of the Democratic Republic of the Congo.

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 < 0.05). The predominant serotypes were 19F, 11, 6A/B/C/D and 10A. More than 50% of the pneumococcal isolates belonged to a serotype/serogroup not included in PCV13. Eighty per cent of the isolates were not susceptible to benzylpenicillin and non-susceptibility to ampicillin and ceftriaxone was also high (42 and 37% respectively). Almost all the isolates (94%) were resistant to trimethoprim-sulphamethoxazole, while 43% of the strains were resistant to ≥3 antibiotics. CONCLUSIONS: Our study shows alarmingly high levels of reduced susceptibility to commonly used antibiotics in pneumococci carried by healthy Congolese children. This highlights the importance of local antibiotic resistance surveillance and indicates the needs for the more appropriate use of antibiotics in the area. The results further indicate that improved living conditions are needed to reduce the pneumococcal burden, in addition to PCV13 vaccination.

Intact Pneumococci Trigger Transcription of Interferon-Related Genes in Human Monocytes, while Fragmented, Autolyzed Bacteria Subvert This Response.

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.

Household air pollution and pneumococcal density related to nasopharyngeal inflammation in mothers and children in Ethiopia: A cross-sectional study.

BACKGROUND: Three billion people in low- and middle-income countries are exposed to household air pollution as they use biomass fuel for cooking. We investigated the associations between solid fuel use and nasopharyngeal (NP) inflammation, as well as the associations between high pneumococcal density and NP inflammation, in mothers and children in rural and urban Ethiopia. MATERIALS AND METHODS: Sixty pairs of mothers (median age, 30 years; range, 19-45 years) with a child (median age, 9 months; range, 1-24 months) were included from rural Butajira (n = 30) and urban Addis Ababa (n = 30) in Ethiopia. The cohort was randomly selected from a previous study of 545 mother/child pairs included 2016. Questionnaire-based data were collected which included fuel type used (solid: wood, charcoal, dung or crop waste; cleaner: electricity, liquefied petroleum gas). Nasopharyngeal (NP) samples were collected from all mothers and children and analyzed for the levels of 18 cytokines using a Luminex immunoassay. Pneumococcal DNA densities were measured by a real-time multiplex PCR and a high pneumococcal density was defined as a cyclic threshold (Ct) value ≤ 30. RESULTS: Mothers from rural areas had higher median CXCL8 levels in NP secretions than those from urban areas (8000 versus 1900 pg/mL; p < 0.01), while rural children had slightly higher IL-10 levels than those from the urban area (26 vs 13 pg/mL; p = 0.04). No associations between fuel type and cytokine levels were found. However, a high pneumococcal density was associated with higher levels of cytokines in both mothers (CCL4, CXCL8, IL-1ß, IL-6 and VEGF-A) and children (CCL4, CXCL8, IL-1ß, IL-6 and IL-18). CONCLUSIONS: No significant associations were found between solid fuel use and NP inflammation in Ethiopian mothers and children, but the inflammatory activity was higher in individuals living in the rural compared to the urban area. In addition, high cytokine levels were associated with high pneumococcal density in both mothers and children, indicating a significant impact of NP pathogens on inflammatory mediator levels in upper airways.

Co-occurrence of bacteria and viruses and serotype distribution of Streptococcus pneumoniae in the nasopharynx of Tanzanian children below 2 years of age following introduction of the PCV13.

Introduction: Pneumococcal conjugate vaccines have reduced severe disease attributed to vaccine-type pneumococci in children. However, the effect is dependent on serotype distribution in the population and disease development may be influenced by co-occurrence of viral and bacterial pathogens in the nasopharynx. Methods: Following introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in Tanzania we performed repeated cross-sectional surveys, including 775 children below 2 years of age attending primary healthcare centers. All children were sampled from nasopharynx and pneumococci were detected by single-target PCR. Pneumococcal serotypes/groups and presence of viruses and other bacteria were determined by two multiplex PCR assays. Results: The prevalence of PCV13 vaccine-type pneumococci decreased by 50%, but residual vaccine-types were still detected in 21% of the children 2 years after PCV13 introduction. An increase in the non-vaccine-type 15 BC was observed. Pneumococci were often co-occurring with Haemophilus influenzae, and detection of rhino/enterovirus was associated with higher pneumococcal load. Discussion: We conclude that presence of residual vaccine-type and emerging non-vaccine-type pneumococci in Tanzanian children demand continued pneumococcal surveillance. High co-occurrence of viral and bacterial pathogens may contribute to the disease burden and indicate the need of multiple public health interventions to improve child health in Tanzania.

Surface-Shaving of Staphylococcus aureus Strains and Quantitative Proteomic Analysis Reveal Differences in Protein Abundance of the Surfaceome.

Staphylococcus aureus is a pathogen known to cause a wide range of infections. To find new targets for identification and to understand host-pathogen interactions, many studies have focused on surface proteins. We performed bacterial-cell surface-shaving, followed by tandem mass tag for quantitative mass spectrometry proteomics, to examine the surfaceome of S. aureus. Two steps were performed, the first step including surface protein-deficient mutants of S. aureus Newman strain lacking important virulence genes (clfA and spa, important for adhesion and immune evasion and srtAsrtB, linking surface-associated virulence factors to the surface) and the second step including isolates of different clinical origin. All strains were compared to the Newman strain. In Step 1, altogether, 7880 peptides were identified, corresponding to 1290 proteins. In Step 2, 4949 peptides were identified, corresponding to 919 proteins and for each strain, approximately 20 proteins showed differential expression compared to the Newman strain. The identified surface proteins were related to host-cell-adherence and immune-system-evasion, biofilm formation, and survival under harsh conditions. The results indicate that surface-shaving of intact S. aureus bacterial strains in combination with quantitative proteomics is a useful tool to distinguish differences in protein abundance of the surfaceome, including the expression of virulence factors.

Low rate of pneumococci non-susceptible to penicillin in healthy Swedish toddlers.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (p < 0.001). 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; p < 0.05). 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. CONCLUSIONS: 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.

Antibiotic use in children under 5 years of age in Northern Tanzania: a qualitative study exploring the experiences of the caring mothers.

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, have remained largely unknown. Therefore, the aim of this study was 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 into 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, posed barriers for healthcare 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 healthcare, thus resembling the function of the community health worker. CONCLUSIONS: To improve antibiotic use in children under 5 years of age in Northern Tanzania, the precarious situation that women often find themselves in as they access treatment for their sick children needs to be taken into consideration. It is necessary to improve structures, including the healthcare system, socioeconomic inequalities and promoting gender equality both in the household and in the public arena to reduce misuse of antibiotics. Meanwhile, equipping community health workers to support Tanzanian women in appropriate healthcare seeking for their children, may be a feasible target for intervention.

Treatment of periprosthetic joint infections guided by minimum biofilm eradication concentration (MBEC) in addition to minimum inhibitory concentration (MIC): protocol for a prospective randomised clinical trial.

INTRODUCTION: Prosthetic 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 ANALYSIS: This 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 DISSEMINATION: Approval is received from the Swedish Ethical Review Authority, no 2020-01471 and the Swedish Medical Products Agency, EudraCT, no 2020-003444-80. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov ID: NCT04488458.

Household fuel use and its association with potential respiratory pathogens among healthy mothers and children in Ethiopia.

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.

High Levels of Fine Particulate Matter (PM2.5) Concentrations from Burning Solid Fuels in Rural Households of Butajira, Ethiopia.

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.

Tanzanian primary healthcare workers' experiences of antibiotic prescription and understanding of antibiotic resistance in common childhood infections: a qualitative phenomenographic study.

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.

Prevalence and patient related factors associated with Extended-Spectrum Beta-Lactamase producing Escherichia coli and Klebsiella pneumoniae carriage and infection among pediatric patients in Tanzania.

Extended-Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae (EPE) is increasing worldwide, though less documented in low-income settings. Here we determined the prevalence of EPE infection and carriage, and patient factors associated with EPE-carriage among pediatric patients in three health care levels in Tanzania. Between January and April 2016, 350 febrile children (median age 21 months) seeking care at a university or a regional referral hospital, or a health centre in Moshi municipality, Tanzania, were included. Socio-demographic characteristics were collected using a questionnaire. Rectal swabs and blood cultures were collected from all children (n = 350) and urinary samples from 259 children at admission. ESBL-phenotype and antimicrobial susceptibility were determined for Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) isolates. Only one EPE case (E. coli) in blood and four in urine (one E. coli and three K. pneumoniae) were found, whereas (n = 90, 26%) of the children were colonized in feces (ESBL-E. coli; n = 76, ESBL-K. pneumoniae, n = 14). High resistance rates were seen in fecal ESBL-E. coli (n = 76) against trimethoprim-sulfamethoxazole (n = 69, 91%), gentamicin (n = 51, 67%), ciprofloxacin (n = 39, 51%) and chloramphenicol (n = 27, 35%) whereas most isolates were sensitive to amikacin (n = 71, 93%). Similar rates were seen for fecal ESBL-K. pneumoniae. Resistance to first line antibiotics were also very high in fecal E. coli not producing ESBL. No sociodemographic factor was associated with EPE-carriage. Children colonized with EPE were younger than 12 months (n = 43, 48%) and often treated with antibiotics (n = 40, 44%) in the previous two months. After adjustment for age children admitted to the intensive care unit had higher odds of EPE fecal carriage compared with those in the general wards (OR = 3.9, 95%CI = 1.4-10.4). Despite comparatively high rates of fecal EPE-carriage and previous antibiotic treatment, clinical EPE cases were rare in the febrile children. The very high resistant rates for the EPE and the non-ESBL producing E. coli to commonly used antibiotics are worrying and demand implementation of antibiotic stewardship programs in all levels of health care in Tanzania.

Does extraction of cardiac implantable electronic devices improve outcome in patients with Staphylococcus aureus bacteraemia?

BACKGROUND: 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 electronic devices (CIED) is recommended in SAB patients but studies evaluating long-term prognosis are scarce. MATERIALS AND METHODS: 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. RESULTS: SAB patients with CIED (n = 33) compared to non-CIED patients (n = 593) were older (83 versus 70 years, p = .0001), had a higher 30-day mortality (12/33, 36% versus 119/593, 20%, p = .044) and higher incidence of IE (9/33, 27% versus 41/593, 7%, p = .0006). One-year mortality was 19/33 (58%) among the SAB CIED patients. Echocardiography was performed in all nine patients with CIED-IE but only in 14/24 (58%) of the 24 SAB CIED patients that were considered not having IE. However, if patients with very early mortality were excluded, echocardiography was performed in 14/17 (82%) of SAB CIED-non-IE patients. CIED extraction/explantation during intravenous antibiotic treatment was only performed in three patients with SAB CIED-IE and in one non-IE patient. One year post treatment initiation, 14 out of 33 SAB CIED patients were alive of whom only one had CIED extraction/explantation performed as part of treatment. CONCLUSION: Staphylococcus aureus bacteraemia in CIED patients is associated with poor prognosis but in a subgroup of patients survival beyond one year was seen despite retainment of the electronic device.

Decreased number of hospitalized children with severe acute lower respiratory infection after introduction of the pneumococcal conjugate vaccine in the Eastern Democratic Republic of the Congo.

INTRODUCTION: acute lower respiratory infections (ALRI) are a leading killer of children under five worldwide including the Democratic Republic of the Congo (DR Congo). We aimed to determine the morbidity and case fatality rate due to ALRI before and after introduction of the 13-valent pneumococcal conjugate vaccine (PVC13) in DR Congo 2013. METHODS: data were collected from medical records of children with a diagnosis of ALRI, aged from 2 to 59 months, treated at four hospitals in the Eastern DR Congo. Two study periods were defined; from 2010 to 2012 (before introduction of PCV13) and from 2014 to 2015 (after PCV13 introduction). RESULTS: out of 21,478 children admitted to the hospitals during 2010-2015, 2,007 were treated for ALRI. The case fatality rate among these children was 4.9%. Death was significantly and independently associated with malnutrition, severe ALRI, congenital disease and symptoms of fatigue. Among the ALRI hospitalised children severe ALRI decreased from 31% per year to 18% per year after vaccine introduction (p = 0.0002) while the fatality rate remained unchanged between the two study periods. Following introduction of PCV13, 63% of the children diagnosed with ALRI were treated with ampicillin combined with gentamicin while 33% received ceftriaxone and gentamicin. CONCLUSION: three years after PCV13 introduction in the Eastern part of the DR Congo, we found a reduced risk of severe ALRI among children below five years. Broad-spectrum antibiotics were frequently used for the treatment of ALRI in the absence of any microbiological diagnostic support.

Inflammatory Mediator Profiles in Secretory Otitis Media in Relationship to Viable Bacterial Pathogens and Bacterial and Viral Nucleic Acids.

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-ß (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-1ß, 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.

A Pangenome Approach for Discerning Species-Unique Gene Markers for Identifications of Streptococcus pneumoniae and Streptococcus pseudopneumoniae.

Correct identifications of isolates and strains of the Mitis-Group of the genus Streptococcus are particularly difficult, due to high genetic similarity, resulting from horizontal gene transfer and homologous recombination, and unreliable phenotypic and genotypic biomarkers for differentiating the species. Streptococcus pneumoniae and Streptococcus pseudopneumoniae are the most closely related species of the clade. In this study, publicly-available genome sequences for Streptococcus pneumoniae and S. pseudopneumoniae were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for S. pneumoniae and nine for S. pseudopneumoniae were identified. These species-unique gene marker candidates were verified by PCR assays for identifying S. pneumoniae and S. pseudopneumoniae strains isolated from clinical samples. All determined species-level unique gene markers for S. pneumoniae were detected in all S. pneumoniae clinical isolates, whereas fewer of the unique S. pseudopneumoniae gene markers were present in more than 95% of the clinical isolates. In parallel, taxonomic identifications of the clinical isolates were confirmed, using conventional optochin sensitivity testing, targeted PCR-detection for the "Xisco" gene, as well as genomic ANIb similarity analyses for the genome sequences of selected strains. Using mass spectrometry-proteomics, species-specific peptide matches were observed for four of the S. pneumoniae gene markers and for three of the S. pseudopneumoniae gene markers. Application of multiple species-level unique biomarkers of S. pneumoniae and S. pseudopneumoniae, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.