Empiric Antibiotic Therapy in 1402 Children With Parapneumonic Effusion/Pleural Empyema in Germany: A Long-term Surveillance Study.

OBJECTIVE: This study investigated empiric antibiotic treatment (EAT), guideline adherence, antibiotic streamlining and clinical outcomes in 1402 hospitalized children with pediatric parapneumonic effusion/pleural empyema (PPE/PE). METHODS: A nationwide surveillance study collected data on EAT, clinical course/outcome, pathogens, susceptibility testing and antibiotic streamlining of children with PPE/PE in Germany between 2010 and 2018. Subgroups were compared using χ2 test/Fisher exact test, Mann-Whitney U test and linear regression analysis adjusting for patient age where appropriate. RESULTS: Complete data on EAT were available for 1402 children. In children with monotherapy (n = 567) and in children with combination therapy of 2 antibiotics (n = 589), the most commonly used antibiotics were aminopenicillin/beta-lactamase inhibitor [138/567 (24.3%) and 102/589 (17.3%)] and cefuroxime [291/567 (51.3%) and 294/589 (49.9%)]. The most common combinations with these beta-lactams were macrolides, aminoglycosides and clindamycin. We observed no difference in clinical severity/outcome between EAT with aminopenicillin/beta-lactamase inhibitor and cefuroxime, neither when used in monotherapy nor when used in combination therapy of 2 antibiotics. Species diagnosis of Streptococcus pneumoniae (n = 192), Streptococcus pyogenes (n = 111) or Staphylococcus aureus (n = 38) in polymerase chain reaction or culture from pleural fluid or blood resulted in a switch to an appropriate narrow-spectrum beta-lactam therapy in 9.4%, 18.9 % and 5.2% of children. In a subset of children with reported bacterial susceptibility testing, penicillin resistance was reported in 3/63 (4.8%) of S. pneumoniae and methicillin resistance in S. aureus was reported in 10/32 (31.3%) of children. CONCLUSION: This study points to antibiotic overtreatment in children with PPE/PE, particularly the frequent use of combinations of antibiotics. Children receiving combinations of antibiotics did not show differences in clinical outcomes. The low rate of children with streamlined antibiotic therapy even upon pathogen detection indicates a necessity for antibiotic stewardship measures in PPE/PE and the need of investigating other potential therapeutic strategies as anti-inflammatory therapy.

Diagnosis and therapy of Mycobacterium marinum: a single-center 21-year retrospective analysis.

BACKGROUND AND OBJECTIVES: In Europe, infections with Mycobacterium (M.) marinum are rare. We conducted a retrospective single-center study to assess the clinical spectrum of M. marinum infection and its diagnosis, treatment and outcome under real-world conditions. PATIENTS AND METHODS: Eighteen patients presenting with M. marinum infections between 1998 and 2018 were identified in the data warehouse of the University Hospital Würzburg and considered for detailed analysis. RESULTS: Twelve patients reported aquatic exposure. In 16/18 cases the upper extremities were affected. No invasive infections were detected. Mean time to diagnosis was 15 weeks. Histology revealed granulomatous inflammation in 14 patients while mycobacterial cultures were positive for M. marinum in 16 cases. Most patients received antibiotic monotherapy (14/18) while combination therapy was administered in four cases. Treatment (with a median duration of 10 weeks) was successful in 13 patients. Five patients were lost to follow-up. CONCLUSIONS: Our retrospective analysis of M. marinum infections at a German tertiary referral center revealed a considerable diagnostic delay and the relevance of microbiological culture, PCR and histology for diagnosis. Monotherapy with clarithromycin (rather than doxycycline) appeared as a reasonable treatment option while immunosuppressed or -compromised patients and those with extended disease received combination therapy.

A streamlined method for the fast and cost-effective detection of bacterial pathogens from positive blood cultures for the BacT/ALERT blood culture system using the Vitek MS mass spectrometer.

BACKGROUND AND OBJECTIVE: Prompt pathogen identification of blood stream infections is essential to provide appropriate antibiotic treatment. Therefore, the objective of this prospective single centre study was to establish an inexpensive, fast and accurate protocol for bacterial species identification with SDS protein-extraction directly from BacT/Alert® blood culture (BC) bottles by VitekMS®. RESULTS: Correct species identification was obtained for 198/266 (74.4%, 95%-CI = [68.8%, 79.6%]) of pathogens. The protocol was more successful in identifying 87/96 (91.4%, 95%-CI = [83.8%, 93.2%]) gram-negative bacteria than 110/167 (65.9%, 95%-CI = [58.1%, 73.0%]) gram-positive bacteria. The hands-on time for sample preparation and measurement was about 15 min for up to five samples. This is shorter than for most other protocols using a similar lysis-centrifugation approach for the combination of BacT/Alert® BC bottles and the Vitek® MS mass spectrometer. The estimated costs per sample were approx. 1.80€ which is much cheaper than for commercial kits. CONCLUSION: This optimized protocol allows for accurate identification of bacteria directly from blood culture bottles for laboratories equipped with BacT/Alert® blood culture bottles and VitekMS® mass spectrometer.

Periprosthetic joint infection: Comparison of automated multiplex-PCR Unyvero i60 ITI cartridge system with bacterial culture and real-time PCR.

BACKGROUND: In the past, various efforts have been made to investigate diagnostic tools for periprosthetic-joint-infection (PJI). It is little-known about the diagnostic utility of polymerase-chain-reaction (PCR) in this context, especially concerning the role of multiplex-PCR assays comparing with conventional tissue culture. OBJECTIVE: Evaluation of an automated-multiplex-PCR cartridge system for patients with suspicion of PJI in comparison with conventional microbiological culture and 16S-rDNA-PCR. METHODS: On suspicion of PJI synovial fluid specimen were taken preoperatively or periprosthetic tissue was collected intraoperatively. Microbiological analysis included conventional culture, 16S-rDNA-PCR and automated-multiplex-PCR (Unyvero-i60-ITI®). The European-Bone-and-Joint-Infection-Society (EBJIS) criteria were used for PJI diagnosis. Positive and negative percent agreement was calculated. Total percentage agreement and Cohen's kappa coefficient were calculated. Sensitivity, specificity and positive predictive value of conventional culture, 16S-rDNA-PCR and multiplex-PCR were calculated. Ten specimens of proved PJI were used as control group. RESULTS: Fifty specimen were suitable for culture. 14 (28%) were classified as PJI, 36 (72%) were aseptic. Coagulase-negative staphylococci was the most frequent detected pathogen. Concordance-rate between mPCR and culture results was 75.6% with a Cohen's kappa of 0.28. Concordance-rate between mPCR and 16S-rDNA was 82.9%, Cohen's kappa was 0.13. Concordance analysis between culture results and 16S-rDNA lead to a concordance-rate of 88.9%. Cohen's kappa was calculated with 0.6. With regard to the microbiological culture as reference, sensitivity of the mPCR was 0.33 and specificity was 0.91. Sensitivity and specificity of the 16S-rDNA-PCR was 0.55 and 0.97. The positive predictive value was 0.57 for the mPCR and 0.83 for the 16S-rDNA-PCR. CONCLUSIONS: Due to fair agreement between mPCR and conventional microbiological culture, the tested multiplex-PCR could be an additional instrument for the detection of PJI but is not superior over the conventional culture.

Farming Practice Influences Antimicrobial Resistance Burden of Non-Aureus Staphylococci in Pig Husbandries.

Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms.

Antimicrobial Resistance Profiles of Coagulase-Negative Staphylococci in Community-Based Healthy Individuals in Germany.

Coagulase-negative staphylococci (CoNS) are common opportunistic pathogens, but also ubiquitous human and animal commensals. Infection-associated CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin- and multidrug-resistant isolates. Less is known about AMR patterns of CoNS colonizing the general population. Here we report on AMR in commensal CoNS recovered from 117 non-hospitalized volunteers in a region of Germany with a high livestock density. Among the 69 individuals colonized with CoNS, 29 had reported contacts to either companion or farm animals. CoNS were selectively cultivated from nasal swabs, followed by species definition by 16S rDNA sequencing and routine antibiotic susceptibility testing. Isolates displaying phenotypic AMR were further tested by PCR for presence of selected AMR genes. A total of 127 CoNS were isolated and Staphylococcus epidermidis (75%) was the most common CoNS species identified. Nine isolates (7%) were methicillin-resistant (MR) and carried the mecA gene, with seven individuals (10%) being colonized with at least one MR-CoNS isolate. While resistance against gentamicin, phenicols and spectinomycin was rare, high resistance rates were found against tetracycline (39%), erythromycin (33%) and fusidic acid (24%). In the majority of isolates, phenotypic resistance could be associated with corresponding AMR gene detection. Multidrug-resistance (MDR) was observed in 23% (29/127) of the isolates, with 33% (23/69) of the individuals being colonized with MDR-CoNS. The combined data suggest that MR- and MDR-CoNS are present in the community, with previous animal contact not significantly influencing the risk of becoming colonized with such isolates.

Performance of Three SARS-CoV-2 Immunoassays, Three Rapid Lateral Flow Tests, and a Novel Bead-Based Affinity Surrogate Test for the Detection of SARS-CoV-2 Antibodies in Human Serum.

For the control of immunity in COVID-19 survivors and vaccinated subjects, there is an urgent need for reliable and rapid serological assays. Based on samples from 63 COVID-19 survivors up to 7 months after symptom onset, and on 50 serum samples taken before the beginning of the pandemic, we compared the performances of three commercial immunoassays for the detection of SARS-CoV-2 IgA and IgG antibodies (Euroimmun SARS-COV-2 IgA/IgG, Mikrogen recomWell SARS-CoV-2 IgA/IgG, and Serion ELISA agile SARS-CoV-2 IgA/IgG) and three rapid lateral flow (immunochromatographic) tests (Abbott PanBio COVID-19 IgG/IgM, Nadal COVID-19 IgG/IgM, and Cleartest Corona 2019-nCOV IgG/IgM) with a 50% plaque-reduction neutralization test (PRNT50) representing the gold standard. Fifty-seven out of 63 PCR-confirmed COVID-19 patients (90%) showed neutralizing antibodies. The sensitivity of the seven assays ranged from 7.0% to 98.3%, and the specificity ranged from 86.0% to 100.0%. Only one commercial immunoassay showed a sensitivity and specificity of greater than 98%.

Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9.

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp614→Gly) variant with single-base resolution in patient samples.

Effect of Prehospital Antibiotic Therapy on Clinical Outcome and Pathogen Detection in Children With Parapneumonic Pleural Effusion/Pleural Empyema.

BACKGROUND: Parapneumonic pleural effusion and pleural empyema (PPE/PE) are complications of community-acquired pneumonia. The objective of this study was to analyze prehospital antibiotic therapy (PH-ABT) of children with PPE/PE and investigate its effects on clinical outcome and pathogen detection. METHODS: Prospective nationwide active surveillance in Germany between October 2010 and June 2018. Children and adolescents <18 years of age with pneumonia-associated PE or PPE requiring drainage or with persistence of PPE/PE >7 days were included. RESULTS: A total of 1724 children with PPE/PE were reported, of whom 556 children (32.3% of 1719 with available data) received PH-ABT. Children with PH-ABT had a shorter median hospital length of stay (15 vs. 18 days, P < 0.001), a longer time from onset of symptoms until hospital discharge (25 vs. 23 days, P = 0.002), a lower rate of intensive care unit admission (58.3% vs. 64.4%, P = 0.015) and fewer infectious complications (5.9% vs. 10.0%; P = 0.005). Bacterial pathogens in blood or pleural fluid culture were detected in 597 (34.5%) of 1513 children. Positive culture results were less frequent in children with than without PH-ABT (81/466 [17.4%] vs. 299/1005 [29.8%]; P < 0.001), whereas detection rates in pleural fluid samples by polymerase chain reaction were similar (91/181 [50.3%] vs. 220/398 [55.3%]; P = 0.263). CONCLUSIONS: In children with PPE/PE, PH-ABT significantly reduced the overall rate of bacterial pathogen detection by culture, but not by polymerase chain reaction. PH-ABT was associated with a lower rate of infectious complications but did not affect the overall duration of disease. We therefore speculate that the duration of PPE/PE is mainly a consequence of an infection-induced inflammatory process, which can only partially be influenced by antibiotic treatment.

The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition.

FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.

Investigating RNA-Protein Interactions in Neisseria meningitidis by RIP-Seq Analysis.

Deep sequencing technology has revolutionized transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and has revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacterial pathogens. Moreover, RNA-seq is nowadays also widely used to comprehensively explore the interaction between RNA-binding proteins and their RNA targets on a genome-wide level in many human-pathogenic bacteria. In particular, immunoprecipitation of an RNA-binding protein (RBP) of interest followed by isolation and analysis of all bound RNAs (RNA immunoprecipitation (RIP)) allows rapid characterization of its RNA regulon. Here, we describe an experimental approach which employs co-immunoprecipitation (coIP) of the RNA-binding chaperone Hfq along with bound RNAs followed by deep-sequencing of co-purified RNAs (RIP-Seq) from a genetically modified strain of Neisseria meningitidis expressing a chromosomally encoded Hfq-3×FLAG protein. This approach allowed us to comprehensively identify both mRNAs and sRNAs as targets of Hfq and served as an excellent starting point for sRNA research in this human pathogenic bacterium.

The CRISPR/Cas system in Neisseria meningitidis affects bacterial adhesion to human nasopharyngeal epithelial cells.

Neisseria meningitidis, a commensal ß-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.

Postoperative Tropheryma whipplei endophthalmitis - a case report highlighting the additive value of molecular testing.

Introduction.Tropheryma whipplei is the causative agent of Whipple's disease. Gastrointestinal and lymphatic tissues are affected in the majority of cases, resulting in diarrhoea, malabsorption and fever. Here, we report a rare case of ocular manifestation in a patient lacking the typical Whipple symptoms. Case presentation. A 74-year-old Caucasian female presented with blurred vision in the right eye over a period of 1-2 months, accompanied by stinging pain and conjunctival hyperaemia for the last 2 days. Upon admission, visual acuity was hand motion in the affected eye. Ophthalmological examination showed typical signs of intraocular inflammation. Diagnostic and therapeutic pars plana vitrectomy including vitreous biopsy and intravitreal instillation of vancomycin and amikacin was performed within hours of initial presentation. Both microscopic analysis and microbial cultures of the vitreous biopsy remained negative for bacteria and fungi. The postoperative antibiotic regime included intravenous administration of ceftriaxone in combination with topical tobramycin and ofloxacin. Due to the empirical therapy the inflammation ceased and the patient was discharged after 5 days with cefpodoxime orally and local antibiotic and steroidal therapy. Meanwhile, the vitreous body had undergone testing by PCR for the eubacterial 16S rRNA gene, which was found to be positive. Analysis of the PCR product revealed a specific sequence of T. whipplei. Conclusion. In our patient, endophthalmitis was the first and only symptom of Morbus Whipple, while most patients with Whipple's disease suffer from severe gastrointestinal symptoms. 16S rDNA PCR should be considered for any intraocular infection when microscopy and standard culture methods remain negative.

Improved performance of the artus Mycobacterium tuberculosis RG PCR kit in a low incidence setting: a retrospective monocentric study.

Tuberculosis (TB) and the spread of Mycobacterium tuberculosis complex (MTBC) strains resistant against rifampin (RIF) and isoniazid (INH) pose a serious threat to global health. However, rapid and reliable MTBC detection along with RIF/INH susceptibility testing are challenging in low prevalence countries due to the higher rate of false positives. Here, we provide the first performance data for the artus MTBC PCR assay in a low prevalence setting. We analyze 1323 respiratory and 311 non-respiratory samples with the artus MTBC PCR assay as well as by mycobacterial culture and microscopy. We propose retesting of specimens in duplicate and consideration of a determined cycle-threshold value cut-off greater than 34, as this significantly increases accuracy, specificity, and negative predictive value without affecting sensitivity. Furthermore, we tested fourteen MTBC positive samples with the GenoType MTBDRplus test and demonstrate that using an identical DNA extraction protocol for both assays does not impair downstream genotypic testing for RIF and INH susceptibility. In conclusion, our procedure optimizes the use of the artus MTB assay with workload efficient methods in a low incidence setting. Combining the modified artus MTB with the GenoType MTBDRplus assays allows rapid and accurate detection of MTBC and RIF/INH resistance.

Neisseria genomics: current status and future perspectives.

High-throughput whole genome sequencing has unlocked a multitude of possibilities enabling members of the Neisseria genus to be examined with unprecedented detail, including the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae. To maximise the potential benefit of this for public health, it is becoming increasingly important to ensure that this plethora of data are adequately stored, disseminated and made readily accessible. Investigations facilitating cross-species comparisons as well as the analysis of global datasets will allow differences among and within species and across geographic locations and different times to be identified, improving our understanding of the distinct phenotypes observed. Recent advances in high-throughput platforms that measure the transcriptome, proteome and/or epigenome are also becoming increasingly employed to explore the complexities of Neisseria biology. An integrated approach to the analysis of these is essential to fully understand the impact these may have in the Neisseria genus. This article reviews the current status of some of the tools available for next generation sequence analysis at the dawn of the 'post-genomic' era.

Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence.

BACKGROUND: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. RESULTS: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. CONCLUSIONS: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.

The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq.

Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of -35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.

Comparative Genome Sequencing Reveals Within-Host Genetic Changes in Neisseria meningitidis during Invasive Disease.

Some members of the physiological human microbiome occasionally cause life-threatening disease even in immunocompetent individuals. A prime example of such a commensal pathogen is Neisseria meningitidis, which normally resides in the human nasopharynx but is also a leading cause of sepsis and epidemic meningitis. Using N. meningitidis as model organism, we tested the hypothesis that virulence of commensal pathogens is a consequence of within host evolution and selection of invasive variants due to mutations at contingency genes, a mechanism called phase variation. In line with the hypothesis that phase variation evolved as an adaptation to colonize diverse hosts, computational comparisons of all 27 to date completely sequenced and annotated meningococcal genomes retrieved from public databases showed that contingency genes are indeed enriched for genes involved in host interactions. To assess within-host genetic changes in meningococci, we further used ultra-deep whole-genome sequencing of throat-blood strain pairs isolated from four patients suffering from invasive meningococcal disease. We detected up to three mutations per strain pair, affecting predominantly contingency genes involved in type IV pilus biogenesis. However, there was not a single (set) of mutation(s) that could invariably be found in all four pairs of strains. Phenotypic assays further showed that these genetic changes were generally not associated with increased serum resistance, higher fitness in human blood ex vivo or differences in the interaction with human epithelial and endothelial cells in vitro. In conclusion, we hypothesize that virulence of meningococci results from accidental emergence of invasive variants during carriage and without within host evolution of invasive phenotypes during disease progression in vivo.

Therapy of 645 children with parapneumonic effusion and empyema-A German nationwide surveillance study.

OBJECTIVE: To evaluate the initial management of pediatric parapneumonic effusion or pleural empyema (PPE/PE) with regard to length of hospital stay (LOS). METHODS: Collection of pediatric PPE/PE cases using a nationwide surveillance system (ESPED) from 10/2010 to 06/2013, in all German pediatric hospitals. Inclusion of PPE/PE patients <18 years of age requiring drainage or with a PPE/PE persistence >7 days. Staging of PPE/PE based on reported pleural sonographic imaging. Comparison of LOS after diagnosis between children treated with different forms of initial invasive procedures performed ≤3 days after PPE/PE diagnosis: pleural puncture, draining catheter, intrapleural fibrinolytic therapy, surgical procedures. RESULTS: Inclusion of 645 children (median age 5 years); median total LOS 17 days. Initial therapy was non-invasive in 282 (45%) cases and invasive in 347 (55%) cases (pleural puncture: 62 [10%], draining catheter: 153 [24%], intrapleural fibrinolytic therapy: 89 [14%], surgical procedures: 43 [7%]). LOS after diagnosis did not differ between children initially treated with different invasive procedures. Results remained unchanged when controlling for sonographic stage, preexisting diseases, and other potential confounders. Repeated use of invasive procedures was observed more often after initial non-invasive treatment or pleural puncture alone than after initial pleural drainage, intrapleural fibrinolytic therapy or surgery. CONCLUSIONS: Initial treatment with intrapleural fibrinolytic therapy or surgical procedures did not result in shorter LOS than initial pleural puncture alone. Larger prospective studies are required to investigate which children benefit significantly from more intensive forms of initial invasive treatment. Pediatr Pulmonol. 2017;52:540-547. © 2016 The Authors. Pediatric Pulmonology Published by Wiley Periodicals, Inc.