What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue?

Infective endocarditis (IE) is a severe and life-threatening disease. Identification of infectious etiology is essential for establishing the appropriate antimicrobial treatment and decreasing mortality. The aim of this study was to explore the potential utility of metataxonomics for improving microbiological diagnosis of IE. Here, next-generation sequencing (NGS) of the V3–V4 region of the 16S rRNA gene was performed in 27 heart valve tissues (18 natives, 5 intravascular devices, and 4 prosthetics) from 27 patients diagnosed with IE (4 of them with negative blood cultures). Metataxonomics matched with conventional diagnostic techniques in 24/27 cases (88.9%). The same bacterial family was assigned to 24 cases; the same genus, to 23 cases; and the same species, to 13 cases. In 22 of them, the etiological agent was represented by percentages > 99% of the reads and in two cases, by ~70%. Staphylococcus aureus was detected in a previously microbiological undiagnosed patient. Thus, microbiological diagnosis with 16S rRNA gene targeted-NGS was possible in one more sample than using traditional techniques. The remaining two patients showed no coincidence between traditional and 16S rRNA gene-targeted NGS microbiological diagnoses. In addition, 16S rRNA gene-targeted NGS allowed us to suggest coinfections that were supported by clinical data in one patient, and minority records also verified mixed infections in three cases. In our series, metataxonomics was valid for the identification of the causative agents, although more studies are needed before implementation of 16S rRNA gene-targeted NGS for the diagnosis of IE.

The Case for Early Antibiotic Commencement and Source Control in Paediatric Subdural Empyema: A Single-Centre Retrospective Case Series

<b><i>Background:</i></b> Subdural empyema is a neurosurgical emergency requiring prompt diagnosis and treatment. There is a debate between the benefits and risks of starting early antibiotics prior to surgical drainage as this is purported to reduce the rate of microbiological diagnosis. Here, we describe our experience of treating this potentially life-threatening condition, advocating for the early commencement of antibiotics and importance of source control in its treatment. <b><i>Methods:</i></b> Retrospective review of a prospectively collected electronic departmental database included all patients who were admitted to our unit with a diagnosis of subdural empyema over an 11-year period (2008–2018). Basic demographic data were collected. Further data pertaining to mode of presentation, surgical approach, causative organism, post-operative antibiotic regime, anti-seizure medications, length of hospital stay, further surgery, and neurological outcomes were extracted. <b><i>Results:</i></b> Thirty-six children underwent 44 operations for subdural empyema at our institution during the study period. Median age was 11.0 (range 0.2–15.8); 47.2% (17/36) were female. Over time, there was decreasing use of burr holes and increasing use of craniectomy as the index surgery. Using a combination of extended culture and polymerase chain reaction, a microbiological diagnosis was achieved in all 36 cases; the commonest causative microorganism was of the <i>Streptococcus anginosus</i> group of bacteria. Seven patients underwent repeat surgery, and 4 patients underwent a concurrent ENT procedure. No risk factors were significant in predicting the likelihood of re-operation (location of subdural empyema, age, index surgery type, inflammatory markers, concurrent ENT procedure, and microorganism) although it was notable that none of the patients undergoing a concurrent ENT procedure underwent repeat surgery (<i>p</i> = 0.29). Median length of stay was 12 days (range 3–74), and there were no inpatient or procedure-related mortalities. Clinical outcomes were good with 94.4% (34/36) categorized as modified Rankin Scale 0–3 at discharge and there were 2 cranioplasty-related complications. <b><i>Conclusions:</i></b> We observed an evolution of practice from limited surgical approaches towards more extensive index surgery over the study period. Given that a microorganism was isolated in all cases using a comprehensive approach, initiation of antibiotic therapy should not be delayed on presentation. Concurrent ENT surgery may be an important factor in providing aggressive source control thereby reducing the need for repeat surgery.

Tuberculosis in the Elderly

The tuberculosis (TB) epidemic is most prevalent in the elderly, and there is a progressive increase in the notification rate with age. Most cases of TB in the elderly are linked to the reactivation of lesions that have remained dormant. The awakening of these lesions is attributable to changes in the immune system related to senescence. The mortality rate from tuberculosis remains higher in elderly patients. Symptoms of active TB are nonspecific and less pronounced in the elderly. Diagnostic difficulties in the elderly are common in many diseases but it is important to use all possible techniques to make a microbiological diagnosis. Recognising frailty to prevent loss of independence is a major challenge in dealing with the therapeutic aspects of elderly patients. Several studies report contrasting data about poorer tolerance of TB drugs in this population. Adherence to antituberculosis treatment is a fundamental issue for the outcome of treatment. Decreased completeness of treatment was shown in older people as well as a higher risk of treatment failure.

Sonication of Kirschner Wire as a Tool for the Microbiological Diagnosis of Diabetic Foot Infection

We hypothesized that biofilm production occurs on stainless steel when incubated with tissue specimens in thioglycolate broth media (TBM). In a diabetic foot infection (DFI) cohort, applying the Kirschner wire and conventional methods were more sensitive than applying only the latter (CI 90%; 0.167 versus 0.375).

Diagnostic Stewardship Aiming at Expectorated or Induced Sputum Promotes Microbial Diagnosis in Community-Acquired Pneumonia

PurposeStudies on aetiology of community-acquired pneumonia (CAP) vary in terms of microbial sampling methods, anatomical locations, and laboratory analyses, since no gold standard exists. In this large, multicentre, retrospective, regional study from Norway, our primary objective was to report the results of a strategic diagnostic stewardship intervention, targeting diagnostic yield from lower respiratory tract sampling. The secondary objective was to report in-hospital CAP aetiology and the diagnostic yield of various anatomical sampling locations.MethodsMedical records from cases diagnosed with in-hospital CAP were collected retrospectively from March throughout May for three consecutive years at six hospitals. Between year one and two, we launched a diagnostic stewardship intervention at the emergency room level for the university teaching hospital only. The intervention was multifaceted aiming at upscaling specimen collection and enhancing collection techniques. Year one at the interventional hospital and every year at the five other emergency hospitals were used for comparison.ResultsOf the 1280 included cases of in-hospital CAP, a microbiological diagnosis was established for 29.1 % among 1128 blood cultures and 1444 respiratory tract specimens. Blood cultures were positive for a pathogenic respiratory tract microbe in 4.9 % of samples, whereas upper and lower respiratory tract samples overall provided a probable microbiological diagnosis in 21.3 % and 47.5 %, respectively. Expectorated or induced sputum overall provided aetiology in 51.7 % of the samples. At the interventional hospital, diagnostic yield from expectorated or induced sputum was significantly enhanced from 41.2 % to 62.0 % after the intervention. Non-typeable Haemophilus influenza and Streptococcus pneumoniae accounted for 25.3 % and 24.7 % of microbiologically confirmed cases, respectively.ConclusionExpectorated or induced sputum outperformed other sampling methods in providing a reliable microbiological diagnosis for in-hospital CAP. A diagnostic stewardship intervention significantly improved diagnostic yield of lower respiratory tract sampling.

Evaluating the Clinical Utility of Staged Bronchoalveolar Lavage (BAL) Fluid Analysis in Adolescent and Adult Acute Leukemia and Lymphoma Patients with Febrile Neutropenia and Lung Infiltrates

Introduction: Lung infiltrates(LI) are seen in 30-45% of the patients with Febrile neutropenia(FN) in haematological malignancies.In FN patients with LI, accurate/probable microbiological diagnosis is possible in only 30 % with conventional blood culture and serological tests. BAL increases the microbiological diagnostic yield. The battery of microbiological tests done in BAL fluid analysis is not uniform.We evaluated a staged approach while analysing the BAL fluid sample, in the first stage we performed the routine cultures and antigen-based tests and in patients with negative results, we performed Polymerase Chain Reaction(PCR) tests guided by the radiological findings. Methods: This was a prospective observational study initiated after Institutional ethics committee approval and conducted at Tata memorial centre,Mumbai between November 2018 and June 2020.BAL testing was done as per a standard protocol(Sampsonas et al.) in hemodynamically stable patients with Spo2 more than 90% and platelet count above 300x10 9/L.Samples were sent for gram stain & bacterial cultures, ziehl neelsen stain and cultures, fungal stain and cultures and Galactomannan (ELISA) and an extra sample was preserved in an EDTA vacutainer at 10-20 degree C. If none of the initial reports were positive, then stored BAL sample was sent for PCR testing guided by the radiology and clinical picture i.e., with nodular infiltrates(Bacteria,Nocardia,Aspergillus,Mucor,P.Jiroveci,Mycobacterium TB,Atypical Mycobacterium TB) and with diffuse micronodular infiltrates Viruses,Legionella,mycoplasma PCR tests were sent. The causal association of the isolated organism was defined as per AGIHO guidelines(G. Maschmeyer et al.) The Primary objective is proportion of patients with a confirmed microbiological diagnosis using staged BAL analysis. The Secondary Objectives are proportion of patients who had a change in antimicrobial therapy,Feasibility of doing a Bronchoscopy and Proportion of Patients who develop Major or Minor complications during procedure and the 4 and 12weeks Clinical and Radiological Outcomes. A sample size of 130 patients was required for incidence of 50%(40-60%) positivity with 10% variation at 95% confidence interval. Results: A total of 172 patients were eligible of which 50 patients are not enrolled due to physician discretion in 37 patients,9 lost for followup and 2 refused consent and one patient expired and one palliated and 122 patients are enrolled and of these BAL couldn't be done in 20 patients due to hypoxia,low platelets,poor GCS at the time of performing BAL and finally BAL is feasible in 83.6%(n=102/122) patients. Baseline characteristics of patients are mentioned in Table 1.Median age of the patients was 30 (15-65) years with 69.6% males (n=85/122). A confirmed microbiological diagnosis (G. Maschmeyer et al.) was established in 71.3%( 81 /122 ) of cases.Microbiological results are depicted in Table2. A change of antimicrobial based on BAL (addition and removal of antimicrobial) was done for 78 patients(63.8%) of which 42 had removal of antibacterial and 11 patients had removal of antifungals. Among 42 patients who had removal of antibiotics, by the end of 4weeks, 36(85.7%) had clinical response and 34 had radiological response,(4 died and 2 lost for followup). By the end of 12weeks, 31 patients had sustained clinical and radiological response (2 died, 2 lost to follow-up and 1 progressive disease). Among 11 patients with removal of antifungals 9 had clinical and radiological response by the end of 4weeks (1 died and 1 non responder) which was sustained at week 12. Complications of BAL One patient had a major complication (persistent hypoxia), while minor complications were recorded in 27/122 (22%) (Hypoxia-16, hypertension-8,tachycardia-3) during procedure and in 21/122 (17%) (Fever-8, bleeds-6, tachycardia-5,hypertension-2)upto 24 hours post procedure. Clinical and Radiological responses as per criteria( Figure 1) Conclusion: BAL fluid analysis improves the diagnostic yield in febrile neutropenia with lung infiltrates. This leads to a change in antimicrobials in a significant number of patients. It contributes to improved outcomes in this patient population. The test is feasible in a large majority, is safe and the staged approach helps in optimisation of resources. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Spondylodiscitis due to Salmonella Typhi: a series of four cases

ABSTRACT Salmonella Typhi is very rarely associated with focal bone and joint complications. Classically, they are described in patients with risk factors such as haemoglobinopathies. We report four cases of spondylodiscitis, where the aetiology was found to be Salmonella Typhi. All four cases were treated successfully with variable duration of ceftriaxone followed by cotrimoxazole. We report these cases to highlight the importance of obtaining a microbiological diagnosis and the possibility of a rare infection in endemic settings.

648. Rapid, Non-invasive Detection of Cryptic Tularemia Using a Plasma-Based Microbial Cell-Free DNA Next-Generation Sequencing Test

Background Francisella tularensis causes severe infections and is a Category A potential bioterrorism agent but its microbiological diagnosis can be challenging. The diagnosis of tularemia is limited by non-specific protean clinical symptoms that overlap with other infectious syndromes, the fastidious nature of Francisella tularensis, along with insensitivity and poor specificity of serology. Rapid, non-invasive diagnosis of tularemia by microbial cell-free DNA (mcfDNA) next-generation sequencing (NGS) of plasma offers a means to overcome these limitations. Methods The Karius Test™ (KT) detects and quantifies mcfDNA in molecules/µL (MPM) from &gt;1400 organisms in plasma (performed at the CLIA certified/CAP accredited Karius laboratory). KT detections of Francisella tularensis were compiled from three medical centers with clinical review by healthcare providers. Results KT detected Francisella tularensis in four patients. All were adult males in whom the diagnosis was unexpected. The most common symptoms were fever and chest pain. White blood cell count neutrophilic predominance and abnormal chest X-ray findings were found in three cases. A broad infectious disease work-up was performed with the initiation of broad spectrum empiric antibiotics in all cases. KT was the first test to identify Francisella tularensis as the microbiological diagnosis with a time to diagnosis of 2.25 days (avg) +/- 0.5 from sample collection (one day from sample receipt in all cases), enabling narrowed, targeted antibiotic treatment. Francisella tularensis mcfDNA concentration was 1772 MPM (avg) +/- 1914. KT was the only test to establish the diagnosis in two cases; tularemia serologies were confirmatory in two cases, one of which had confirmatory culture from lymph node biopsy. Three patients were diagnosed with the pneumonic form of the illness and one with a visceral glandular form. Possible epidemiological exposures were identified in all cases. Conclusion KT enabled rapid, non-invasive, plasma-based diagnosis of diverse clinical manifestations of invasive tularemia against a competing broad infectious and non-infectious differential diagnosis. Timely diagnosis enabled targeted narrowing of antibiotic therapy and successful treatment of pneumonic and glandular forms of the infection. Disclosures All Authors: No reported disclosures