Safely Doing Less for Febrile Infants: Reviewing Management in the Setting of the 2021 American Academy of Pediatrics Clinical Practice Guideline.

For more than 4 decades, pediatricians have sought the best practices for effectively managing well-appearing young febrile infants. In 2021, the American Academy of Pediatrics released a clinical practice guideline for the management of well-appearing febrile infants aged 8 to 60 days. The guideline incorporates advancements in testing, such as biomarkers and diagnostic testing in the setting of changing epidemiology, to help risk stratify infants in the newly formed group age 22 to 28 days as well as the group age 29 to 60 days. The new guideline uses inflammatory markers (procalcitonin, C-reactive protein, absolute neutrophil count, and a temperature >38.4°C) to identify infants at low risk for invasive bacterial infection who can potentially avoid the invasive procedures of lumbar puncture, hospitalization, and broad-spectrum antimicrobials. Because of continued ambiguity, incorporating shared decision-making with families in the care of these infants will be important, as will ongoing clinical research to better inform future practice. [Pediatr Ann. 2024;53(6):e202-e207.].

Development and validation of risk prediction model for bacterial infections in acute liver failure patients.

Infections significantly increase mortality in acute liver failure (ALF) patients, and there are no risk prediction models for early diagnosis and treatment of infections in ALF patients. This study aims to develop a risk prediction model for bacterial infections in ALF patients to guide rational antibiotic therapy. The data of ALF patients admitted to the Second Hospital of Hebei Medical University in China from January 2017 to January 2022 were retrospectively analyzed for training and internal validation. Patients were selected according to the updated 2011 American Association for the Study of Liver Diseases position paper on ALF. Serological indicators and model scores were collected within 24 h of admission. New models were developed using the multivariate logistic regression analysis. An optimal model was selected by receiver operating characteristic (ROC) analysis, Hosmer-Lemeshow test, the calibration curve, the Brier score, the bootstrap resampling, and the decision curve analysis. A nomogram was plotted to visualize the results. A total of 125 ALF patients were evaluated and 79 were included in the training set. The neutrophil-to-lymphocyte ratio and sequential organ failure assessment (SOFA) were integrated into the new model as independent predictive factors. The new SOFA-based model outperformed other models with an area under the ROC curve of 0.799 [95% confidence interval (CI): 0.652-0.926], the superior calibration and predictive performance in internal validation. High-risk individuals with a nomogram score ≥26 are recommended for antibiotic therapy. The new SOFA-based model demonstrates high accuracy and clinical utility in guiding antibiotic therapy in ALF patients.

Are we overdiagnosing multisystem inflammatory syndrome in children (MIS-C)? A case series of children with bacterial infection mimicking MIS-C.

INTRODUCTION: Multisystem inflammatory syndrome in children (MIS-C) is a serious hyperinflammatory condition associated with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Usually, the diagnosis of MIS-C is made by criteria defined by international organizations, which include specific clinical features, laboratory findings, and evidence of SARS-CoV-2 infection. We hereby present a case series of three children. The objective of this case series, involving chart review of medical records of children admitted with MIS-C, is to emphasize that the features of MIS-C may overlap with other conditions. CASE PRESENTATION: Three children were presented with MIS-C based on World Health Organization (WHO) criteria and given treatment for the same. However, due to persistent symptoms, they were further worked up and diagnosed to have underlying bacterial infections which included liver abscess, enteric fever, or urinary tract infection. CONCLUSIONS: The criteria for MIS-C may overlap with other conditions, particularly bacterial infection that may lead to overdiagnosis of MIS-C. Therefore, one should be very careful in making an MIS-C diagnosis and other differential diagnoses should be considered when the symptoms persist or worsen.

Machine Learning: A Potential Therapeutic Tool to Facilitate Neonatal Therapeutic Decision Making.

Bacterial infection is one of the major causes of neonatal morbidity and mortality worldwide. Finding rapid and reliable methods for early recognition and diagnosis of bacterial infections and early individualization of antibacterial drug administration are essential to eradicate these infections and prevent serious complications. However, this is often difficult to perform due to non-specific clinical presentations, low accuracy of current diagnostic methods, and limited knowledge of neonatal pharmacokinetics. Although neonatal medicine has been relatively late to embrace the benefits of machine learning (ML), there have been some initial applications of ML for the early prediction of neonatal sepsis and individualization of antibiotics. This article provides a brief introduction to ML and discusses the current state of the art in diagnosing and treating neonatal bacterial infections, gaps, potential uses of ML, and future directions to address the limitations of current studies. Neonatal bacterial infections involve a combination of physiologic development, disease expression, and treatment response outcomes. To address this complex relationship, future models could consider appropriate ML algorithms to capture time series features while integrating influences from the host, microbes, and drugs to optimize antimicrobial drug use in neonates. All models require prospective clinical trials to validate their clinical utility before clinical use.

Recent Advancements in Radiopharmaceuticals for Infection Imaging.

COVID-19 pandemic has heightened the interest toward diagnosis and treatment of infectious diseases. Nuclear medicine, with its powerful scintigraphic, single photon emission computer tomography (SPECT), and positron emission tomography (PET) imaging modalities, has always played an important role in diagnosis of infections and distinguishing them from the sterile inflammation. In addition to the clinically available radiopharmaceuticals, there has been a decades-long effort to develop more specific imaging agents with some examples being radiolabeled antibiotics and antimicrobial peptides for bacterial imaging, radiolabeled antifungals for fungal infections imaging, radiolabeled pathogen-specific antibodies, and molecular engineered constructs. In this chapter, we discuss some examples of the work published in the last decade on developing nuclear imaging agents for bacterial, fungal, and viral infections to generate more interest among nuclear medicine community toward conducting clinical trials of these novel probes, as well as toward developing novel radiotracers for imaging infections.

Multiplexed bacterial pathogen detection and clinical characteristics of orthopedic infection in hospitalized patients.

Introduction: Accurate identification of the etiology of orthopedic infection is very important for correct and timely clinical management, but it has been poorly studied. In the current study we explored the association of multiple bacterial pathogens with orthopedic infection. Methods: Hospitalized orthopedic patients were enrolled in a rural hospital in Qingdao, China. Wound or exudate swab samples were collected and tested for twelve bacterial pathogens with both culture and multiplex real time PCR. Results and discussion: A total of 349 hospitalized orthopedic patients were enrolled including 193 cases presenting infection manifestations upon admission and 156 with no sign of infection. Orthopedic infection patients were mainly male (72.5%) with more lengthy hospital stay (median 15 days). At least one pathogen was detected in 42.5% (82/193) of patients with infection while 7.1% (11/156) in the patients without infection (P < 0.001). S. aureus was the most prevalent causative pathogen (15.5%). Quantity dependent pathogen association with infection was observed, particularly for P. aeruginosa and K. pneumoniae, possibly indicating subclinical infection. Most of the patients with detected pathogens had a previous history of orthopedic surgery (odds ratio 2.8, P = 0.038). Pathogen specific clinical manifestations were characterized. Multiplex qPCR, because of its high sensitivity, superior specificity, and powerful quantification could be utilized in combination with culture to guide antimicrobial therapy and track the progression of orthopedic infection during treatment.

Delayed presentation is associated with serious bacterial infections among febrile infants: A prospective cohort study.

Introduction: Febrile young infants are at risk of serious bacterial infections (SBIs), which are potentially life-threatening. This study aims to investigate the association between delayed presentation and the risk of SBIs among febrile infants. Method: We performed a prospective cohort study on febrile infants ≤90 days old presenting to a Singapore paediatric emergency department (ED) between November 2017 and July 2022. We defined delayed presentation as presentation to the ED >24 hours from fever onset. We compared the proportion of SBIs in infants who had delayed presentation compared to those without, and their clinical outcomes. We also performed a multivariable logistic regression to study if delayed presentation was independently associated with the presence of SBIs. Results: Among 1911 febrile infants analysed, 198 infants (10%) had delayed presentation. Febrile infants with delayed presentation were more likely to have SBIs (28.8% versus [vs] 16.3%, P<0.001). A higher proportion of infants with delayed presentation required intravenous antibiotics (64.1% vs 51.9%, P=0.001). After adjusting for age, sex and severity index score, delayed presentation was independently associated with the presence of SBI (adjusted odds ratio [AOR] 1.78, 95% confidence interval 1.26-2.52, P<0.001). Conclusion: Febrile infants with delayed presentation are at higher risk of SBI. Frontline clinicians should take this into account when assessing febrile infants.

Role of biomarkers in antimicrobial stewardship: physicians' perspectives.

Biomarkers are playing an increasingly important role in antimicrobial stewardship. Their applications have included use in algorithms that evaluate suspected bacterial infections or provide guidance on when to start or stop antibiotic therapy, or when therapy should be repeated over a short period (6-12 h). Diseases in which biomarkers are used as complementary tools to determine the initiation of antibiotics include sepsis, lower respiratory tract infection (LRTI), COVID-19, acute heart failure, infectious endocarditis, acute coronary syndrome, and acute pancreatitis. In addition, cut-off values of biomarkers have been used to inform the decision to discontinue antibiotics for diseases such as sepsis, LRTI, and febrile neutropenia. The biomarkers used in antimicrobial stewardship include procalcitonin (PCT), C-reactive protein (CRP), presepsin, and interleukin (IL)-1ß/IL-8. The cut-off values vary depending on the disease and study, with a range of 0.25-1.0 ng/mL for PCT and 8-50 mg/L for CRP. Biomarkers can complement clinical diagnosis, but further studies of microbiological biomarkers are needed to ensure appropriate antibiotic selection.

Bridging the Translation of ICG-1-Maltotriose: A Multimodal Sensor for Monitoring and Detecting Bacterial Infections.

Bacterial infections lack reliable, specific, and quick detection methods, which incur substantial costs to patients and caretakers. Our team conjugated the FDA-approved fluorescent dye indocyanine green (ICG) with a maltotriose sugar, resulting in two highly specific imaging agents (ICG-DBCO-1-Maltotriose and ICG-Amide-1-Maltotriose) for detecting bacterial infections. We then evaluated the two derivatives using fluorescence imaging (FLI), bioluminescence imaging (BLI), and photoacoustic imaging (PAI) in bacterial infection murine models. Our findings indicate that both imaging agents can correlate with and reliably detect the infection site using FLI and PAI for both Gram-negative and Gram-positive strains, with various bacterial loads. Furthermore, the differences in pharmacokinetic (PK) properties between the two agents allow for one to be used for immediate imaging (2-4 h postinjection), while the other is more effective for longitudinal studies (18-40 h postinjection).

[Optimization of the puncture treatment method for acute bacterial maxillary sinusitis]. / Optimizatsiya metoda punktsionnogo lecheniya ostrogo bakterial'nogo verkhnechelyustnogo sinusita.

OBJECTIVE: Optimization of the method of puncture treatment of acute bacterial maxillary sinusitis (ABMS) through the development of original devices for drainage of the maxillary sinus (MS). MATERIAL AND METHODS: Registration and comparative analysis of the results of puncture methods of treatment of 120 patients with ABMS using developed new original devices for drainage of MS with one channels and with two channels in comparison with the Kulikovsky's needle (KN) was carried out. Based on the results of the analysis, the effectiveness of the original devices was assessed. During the clinical study, patients were divided into two groups: in group I, patients underwent of the MS puncture using KN, in group II, using original devices. Groups I and II, depending on the absence or presence of a block of the natural anastomosis MS, was divided into subgroups A and B, respectively. After puncture of the MS, the pain syndrome was assessed by patients using Visual Analogue Scale (VAS) and by doctors - using Touch Visual Pain (TVP) scale. RESULTS: Our study showed that when puncturing the upper jaw with an original needle with one channels and with two channels, compared with the use of KN, there is a decrease in pain (the average VAS score was 1.5±0.3 and 1.7±0.3 points, respectively; the average TVP scale score was 0.9±0.2 and 1.8±0.3 points, respectively, the difference is significant, p≤0.05). Patients of subgroup IB were manipulated with two KN, patients of subgroup IIB manipulated using the original device with two channels without an additional needle (the average VAS score was 3.0±0.4 and 1.3±0.3 points, respectively; the average TVP scale score was 2.7±0.4 and 1.0±0.2 points, respectively, the difference is significant, p≤0.05). The doctors also assessed the devices used for puncture of the upper jaw. As a result of the study, the high efficiency and safety of using new original devices was established.

Diagnostic accuracy of procalcitonin for bacterial infection in the Emergency Department: a systematic review.

INTRODUCTION AND OBJECTIVE: The care of patients with a suspected infectious process in hospital emergency departments (ED) accounts for 15%-35% of all daily care in these healthcare areas in Spain and Latin America. The early and adequate administration of antibiotic treatment (AB) and the immediate making of other diagnostic-therapeutic decisions have a direct impact on the survival of patients with severe bacterial infection. The main objective of this systematic review is to investigate the diagnostic accuracy of PCT to predict bacterial infection in adult patients treated with clinical suspicion of infection in the ED, as well as to analyze whether the different studies manage to identify a specific value of PCT as the most relevant from the diagnostic point of view of clinical decision that can be recommended for decision making in ED. METHOD: A systematic review is carried out following the PRISMA regulations in the database of PubMed, Web of Science, EMBASE, Lilacs, Cochrane, Epistemonikos, Tripdatabase and ClinicalTrials.gov from January 2005 to May 31, 2023 without language restriction and using a combination of MESH terms: "Procalcitonin", "Infection/Bacterial Infection/Sepsis", "Emergencies/Emergency/Emergency Department", "Adults" and "Diagnostic". Observational cohort studies (diagnostic performance analyses) were included. The Newcastle-Ottawa Scale (NOS) was used to assess the quality of the method used and the risk of bias of the included articles. Observational cohort studies were included. No meta-analysis techniques were performed, but results were compared narratively. RESULTS: A total of 1,323 articles were identified, of which 21 that met the inclusion criteria were finally analyzed. The studies include 10,333 patients with 4,856 bacterial infections (47%). Eight studies were rated as high, 9 as moderate, and 4 as low. The AUC-ROC of all studies ranges from 0.68 (95% CI: 0.61-0.72) to 0.99 (95% CI: 0.98-1). The value of PCT 0.2-0.3 ng/ml is the most used and proposed in up to twelve of the works included in this review whose average estimated performance is an AUC-ROC of 0.79. If only the results of the 5 high-quality studies using a cut-off point of 0.2-0.3 ng/ml PCT are taken into account, the estimated mean AUC-COR result is 0.78 with Se:69 % and Es:76%. CONCLUSIONS: PCT has considerable diagnostic accuracy for bacterial infection in patients treated in ED for different infectious processes. The cut-off point of 0.25 (0.2-0.3) ng/ml has been positioned as the most appropriate to predict the existence of bacterial infection and can be used to help reasonably rule it out.

Expanding antibiotic, vaccine, and diagnostics development and access to tackle antimicrobial resistance.

The increasing number of bacterial infections globally that do not respond to any available antibiotics indicates a need to invest in-and ensure access to-new antibiotics, vaccines, and diagnostics. The traditional model of drug development, which depends on substantial revenues to motivate investment, is no longer economically viable without push and pull incentives. Moreover, drugs developed through these mechanisms are unlikely to be affordable for all patients in need, particularly in low-income and middle-income countries. New, publicly funded models based on public-private partnerships could support investment in antibiotics and novel alternatives, and lower patients' out-of-pocket costs, making drugs more accessible. Cost reductions can be achieved with public goods, such as clinical trial networks and platform-based quality assurance, manufacturing, and product development support. Preserving antibiotic effectiveness relies on accurate and timely diagnosis; however scaling up diagnostics faces technological, economic, and behavioural challenges. New technologies appeared during the COVID-19 pandemic, but there is a need for a deeper understanding of market, physician, and consumer behaviour to improve the use of diagnostics in patient management. Ensuring sustainable access to antibiotics also requires infection prevention. Vaccines offer the potential to prevent infections from drug-resistant pathogens, but funding for vaccine development has been scarce in this context. The High-Level Meeting of the UN General Assembly in 2024 offers an opportunity to rethink how research and development can be reoriented to serve disease management, prevention, patient access, and antibiotic stewardship.

Blood calprotectin as a biomarker for infection and sepsis - the prospective CASCADE trial.

BACKGROUND: Early in the host-response to infection, neutrophils release calprotectin, triggering several immune signalling cascades. In acute infection management, identifying infected patients and stratifying these by risk of deterioration into sepsis, are crucial tasks. Recruiting a heterogenous population of patients with suspected infections from the emergency department, early in the care-path, the CASCADE trial aimed to evaluate the accuracy of blood calprotectin for detecting bacterial infections, estimating disease severity, and predicting clinical deterioration. METHODS: In a prospective, observational trial from February 2021 to August 2022, 395 patients (n = 194 clinically suspected infection; n = 201 controls) were enrolled. Blood samples were collected at enrolment. The accuracy of calprotectin to identify bacterial infections, and to predict and identify sepsis and mortality was analysed. These endpoints were determined by a panel of experts. RESULTS: The Area Under the Receiver Operating Characteristic (AUROC) of calprotectin for detecting bacterial infections was 0.90. For sepsis within 72 h, calprotectin's AUROC was 0.83. For 30-day mortality it was 0.78. In patients with diabetes, calprotectin had an AUROC of 0.94 for identifying bacterial infection. CONCLUSIONS: Calprotectin showed notable accuracy for all endpoints. Using calprotectin in the emergency department could improve diagnosis and management of severe infections, in combination with current biomarkers. CLINICAL TRIAL REGISTRATION NUMBER: DRKS00020521.

Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) for the diagnosis of bacterial periprosthetic joint infection.

BACKGROUND: Periprosthetic joint infection (PJI) is one of the most serious and debilitating complications that can occur after total joint arthroplasty. Therefore, early diagnosis and appropriate treatment are important for a good prognosis. Recently, molecular diagnostic methods have been widely used to detect the causative microorganisms of PJI sensitively and rapidly. The Multiplex Loop-Mediated Isothermal Amplification (LAMP) method eliminates the complex temperature cycling and delays caused by temperature transitions seen in polymerase chain reaction (PCR) methods, making it faster and easier to perform compared to PCR-based assays. Therefore, this study developed a multiplex LAMP assay for diagnosing bacterial PJI using LAMP technology and evaluated its analytical and clinical performance. METHODS: We developed a multiplex LAMP assay for the detection of five bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa, and Escherichia coli, frequently observed to be the causative agents of PJI. The method of analytical sensitivity and cross-reactivity were determined by spiking standard strains into the joint synovial fluid. The analytical sensitivity of the multiplex LAMP assay was compared with that of a quantitative real-time PCR (qPCR) assay. Clinical performance was evaluated using 20 joint synovial fluid samples collected from patients suspected of having bacterial PJI. RESULTS: The analytical sensitivity of the gram-positive bacterial multiplex LAMP assay and qPCR were 105/104 CFU/mL, 103/103 CFU/mL, and 105/104 CFU/mL against S. agalactiae, S. epidermidis, and S. aureus, respectively. For P. aeruginosa and E. coli, the analytical sensitivity of the multiplex LAMP and qPCR assays were 105/104 and 106/104 CFU/mL, respectively. The multiplex LAMP assay detects target bacteria without cross-reacting with other bacteria, and exhibited 100% sensitivity and specificity in clinical performance evaluation. CONCLUSIONS: This multiplex LAMP assay can rapidly detect five high-prevalence bacterial species causing bacterial PJI, with excellent sensitivity and specificity, in less than 1 h, and it may be useful for the early diagnosis of PJI.

Application value of metagenomic next-generation sequencing in hematological patients with high-risk febrile neutropenia.

Background: Metagenomic next-generation sequencing (mNGS) is a novel non-invasive and comprehensive technique for etiological diagnosis of infectious diseases. However, its practical significance has been seldom reported in the context of hematological patients with high-risk febrile neutropenia, a unique patient group characterized by neutropenia and compromised immune responses. Methods: This retrospective study evaluated the results of plasma cfDNA sequencing in 164 hematological patients with high-risk febrile neutropenia. We assessed the diagnostic efficacy and clinical impact of mNGS, comparing it with conventional microbiological tests. Results: mNGS identified 68 different pathogens in 111 patients, whereas conventional methods detected only 17 pathogen types in 36 patients. mNGS exhibited a significantly higher positive detection rate than conventional methods (67.7% vs. 22.0%, P < 0.001). This improvement was consistent across bacterial (30.5% vs. 9.1%), fungal (19.5% vs. 4.3%), and viral (37.2% vs. 9.1%) infections (P < 0.001 for all comparisons). The anti-infective treatment strategies were adjusted for 51.2% (84/164) of the patients based on the mNGS results. Conclusions: mNGS of plasma cfDNA offers substantial promise for the early detection of pathogens and the timely optimization of anti-infective therapies in hematological patients with high-risk febrile neutropenia.

Application of metagenomic next-generation sequencing in optimizing the diagnosis of ascitic infection in patients with liver cirrhosis.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) is an emerging technique for the clinical diagnosis of infectious disease that has rarely been used for the diagnosis of ascites infection in patients with cirrhosis. This study compared mNGS detection with conventional culture methods for the on etiological diagnosis of cirrhotic ascites and evaluated the clinical effect of mNGS. METHODS: A total of 109 patients with ascites due to cirrhosis were included in the study. We compared mNGS with conventional culture detection by analyzing the diagnostic results, pathogen species and clinical effects. The influence of mNGS on the diagnosis and management of ascites infection in patients with cirrhosis was also evaluated. RESULTS: Ascites cases were classified into three types: spontaneous bacterial peritonitis (SBP) (16/109, 14.7%), bacterascites (21/109, 19.3%) and sterile ascites (72/109, 66.1%). In addition, 109 patients were assigned to the ascites mNGS-positive group (80/109, 73.4%) or ascites mNGS-negative group (29/109, 26.6%). The percentage of positive mNGS results was significantly greater than that of traditional methods (73.4% vs. 28.4%, P < 0.001). mNGS detected 43 strains of bacteria, 9 strains of fungi and 8 strains of viruses. Fourteen bacterial strains and 3 fungal strains were detected via culture methods. Mycobacteria, viruses, and pneumocystis were detected only by the mNGS method. The mNGS assay produced a greater polymicrobial infection rate than the culture method (55% vs. 16%). Considering the polymorphonuclear neutrophil (PMN) counts, the overall percentage of pathogens detected by the two methods was comparable, with 87.5% (14/16) in the PMN ≥ 250/mm3 group and 72.0% (67/93) in the PMN < 250/mm3 group (P > 0.05). Based on the ascites PMN counts combined with the mNGS assay, 72 patients (66.1%) were diagnosed with ascitic fluid infection (AFI) (including SBP and bacterascites), whereas based on the ascites PMN counts combined with the culture assay, 37 patients (33.9%) were diagnosed with AFI (P < 0.05). In 60 (55.0%) patients, the mNGS assay produced positive clinical effects; 40 (85.7%) patients had their treatment regimen adjusted, and 48 patients were improved. The coincidence rate of the mNGS results and clinical findings was 75.0% (60/80). CONCLUSIONS: Compared with conventional culture methods, mNGS can improve the detection rate of ascites pathogens, including bacteria, viruses, and fungi, and has significant advantages in the diagnosis of rare pathogens and pathogens that are difficult to culture; moreover, mNGS may be an effective method for improving the diagnosis of ascites infection in patients with cirrhosis, guiding early antibiotic therapy, and for reducing complications related to abdominal infection. In addition, explaining mNGS results will be challenging, especially for guiding the treatment of infectious diseases.

Serious bacterial infection risk in recently immunized febrile infants in the emergency department.

STUDY OBJECTIVES: Fever following immunizations is a common presenting chiefcomplaint among infants. The 2021 American Academy of Pediatrics (AAP) febrile infant clinical practice guidelines exclude recently immunized (RI) infants. This is a challenge for clinicians in the management of the febrile RI young infant. The objective of this study was to assess the prevalence of SBI in RI febrile young infants between 6 and 12 weeks of age. METHODS: This was a retrospective chart review of infants 6-12 weeks who presented with a fever ≥38 °C to two U.S. military academic Emergency Departments over a four-year period. Infants were considered recently immunized (RI) if they had received immunizations in the preceding 72 h prior to evaluation and not recently immunized (NRI) if they had not received immunizations during this time period. The primary outcome was prevalence of serious bacterial infection (SBI) further delineated into invasive-bacterial infection (IBI) and non-invasive bacterial infection (non-IBI) based on culture and/or radiograph reports. RESULTS: Of the 508 febrile infants identified, 114 had received recent immunizations in the preceding 72 h. The overall prevalence of SBI was 11.4% (95% CI = 8.9-14.6) in our study population. The prevalence of SBI in NRI infants was 13.7% (95% CI = 10.6-17.6) compared to 3.5% (95% CI = 1.1-9.3) in RI infants. The relative risk of SBI in the setting of recent immunizations was 0.3 (95% CI = 0.1-0.7). There were no cases of invasive-bacterial infections (IBI) in the RI group with all but one of the SBI being urinary tract infections (UTI). The single non-UTI was a case of pneumonia in an infant who presented with respiratory symptoms within 24 h of immunizations. CONCLUSION: The risk of IBI (meningitis or bacteremia) in RI infants aged 6 to 12 weeks is low. Non-IBI within the first 24 h following immunization was significantly lower than in febrile NRI infants. UTIs remain a risk in the RI population and investigation with urinalysis and urine culture should be encouraged. Shared decision making with families guide a less invasive approach to the care of these children. Future research utilizing a large prospective multi-center data registry would aid in further defining the risk of both IBI and non-IBI among RI infants.

Microbiological diagnosis of pleural infections: a comparative evaluation of a novel syndromic real-time PCR panel.

Current microbial diagnostics for pleural infections are insufficient. Studies using 16S targeted next-generation sequencing report that only 10%-16% of bacteria present are cultured and that 50%-78% of pleural fluids containing relevant microbial DNA remain culture negative. As a rapid diagnostic alternative suitable for clinical laboratories, we wanted to explore a PCR-based approach. Based on the identification of key pathogens, we developed a syndromic PCR panel for community-acquired pleural infections (CAPIs). This was a pragmatic PCR panel, meaning that it was not designed for detecting all possibly involved bacterial species but for confirming the diagnosis of CAPI, and for detecting bacteria that might influence choice of antimicrobial treatment. We evaluated the PCR panel on 109 confirmed CAPIs previously characterized using culture and 16S targeted next-generation sequencing. The PCR secured the diagnosis of CAPI in 107/109 (98.2%) and detected all present pathogens in 69/109 (63.3%). Culture secured the diagnosis in 54/109 (49.5%) and detected all pathogens in 31/109 (28.4%). Corresponding results for 16S targeted next-generation sequencing were 109/109 (100%) and 98/109 (89.9%). For bacterial species included in the PCR panel, PCR had a sensitivity of 99.5% (184/185), culture of 21.6% (40/185), and 16S targeted next-generation sequencing of 92.4% (171/185). None of the bacterial species present not covered by the PCR panel were judged to impact antimicrobial therapy. A syndromic PCR panel represents a rapid and sensitive alternative to current diagnostic approaches for the microbiological diagnosis of CAPI.IMPORTANCEPleural empyema is a severe infection with high mortality and increasing incidence. Long hospital admissions and long courses of antimicrobial treatment drive healthcare and ecological costs. Current methods for microbiological diagnostics of pleural infections are inadequate. Recent studies using 16S targeted next-generation sequencing as a reference standard find culture to recover only 10%-16% of bacteria present and that 50%-78% of samples containing relevant bacterial DNA remain culture negative. To confirm the diagnosis of pleural infection and define optimal antimicrobial therapy while limiting unnecessary use of broad-spectrum antibiotics, there is a need for rapid and sensitive diagnostic approaches. PCR is a rapid method well suited for clinical laboratories. In this paper we show that a novel syndromic PCR panel can secure the diagnosis of pleural infection and detect all bacteria relevant for choice of antimicrobial treatment with a high sensitivity.

Development of an automated amplicon-based next-generation sequencing pipeline for rapid detection of bacteria and fungi directly from clinical specimens.

The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory. However, mNGS is resource, time, and labor-intensive-requiring extensive laborious preliminary benchwork, followed by complex bioinformatic analysis. We aimed to address these shortcomings by developing a largely Automated targeted Metagenomic next-generation sequencing (tmNGS) PipeLine for rapId inFectIous disEase Diagnosis (AMPLIFIED) to detect bacteria and fungi directly from clinical specimens. Therefore, AMPLIFIED may serve as an adjunctive approach to complement SMT. This tmNGS pipeline requires less than 1 hour of hands-on time before sequencing and less than 2 hours of total processing time, including bioinformatic analysis. We performed tmNGS on 50 clinical specimens with concomitant cultures to assess feasibility and performance in the hospital laboratory. Of the 50 specimens, 34 (68%) were from true clinical infections. Specimens from cases of true infection were more often tmNGS positive compared to those from the non-infected group (82.4% vs 43.8%, respectively, P = 0.0087). Overall, the clinical sensitivity of AMPLIFIED was 54.6% with 85.7% specificity, equating to 70.6% and 75% negative and positive predictive values, respectively. AMPLIFIED represents a rapid supplementary approach to SMT; the typical time from specimen receipt to identification of potential pathogens by AMPLIFIED is roughly 24 hours which is markedly faster than the days, weeks, and months required to recover bacterial, fungal, and mycobacterial pathogens by culture, respectively. IMPORTANCE: To our knowledge, this represents the first application of an automated sequencing and bioinformatics pipeline in an exclusively pediatric population. Next-generation sequencing is time-consuming, labor-intensive, and requires experienced personnel; perhaps contributing to hesitancy among clinical laboratories to adopt such a test. Here, we report a strong case for use by removing these barriers through near-total automation of our sequencing pipeline.