Background Accurate microbiologic diagnosis is important for appropriate management of infectious diseases. Sequencing-based molecular diagnostics are increasingly used for precision diagnosis of infections. However, their clinical utility is unclear. Methods We conducted a retrospective analysis of specimens that underwent 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) followed by Sanger sequencing at our institution from April 2017 through March 2019. Results A total of 566 specimens obtained from 460 patients were studied. Patients were considered clinically infected or noninfected based on final diagnosis and management. In 17% of patients, 16S rRNA PCR/sequencing was positive and in 5% of patients, this test led to an impact on clinical care. In comparison, bacterial cultures were positive in 21% of patients. Specimens with a positive Gram stain had 12 times greater odds of having a positive molecular result than those with a negative Gram stain (95% confidence interval for odds ratio, 5.2–31.4). Overall, PCR positivity was higher in cardiovascular specimens (37%) obtained from clinically infected patients, with bacterial cultures being more likely to be positive for musculoskeletal specimens (P < .001). 16S rRNA PCR/sequencing identified a probable pathogen in 10% culture-negative specimens. Conclusion 16S rRNA PCR/sequencing can play a role in the diagnostic evaluation of patients with culture-negative infections, especially those with cardiovascular infections.

Objective: To investigate the molecular epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) in infants in a neonatal intensive care unit (NICU) using whole-genome sequencing. Design: Investigation of MSSA epidemiology in a NICU. Setting: Single-center, level IV NICU. Methods: Universal S. aureus screening was done using a single swab obtained from the anterior nares, axilla, and groin area of infants in the NICU on a weekly basis. Core genome multilocus sequence type (cgMLST) analysis was performed on MSSA isolates detected over 1 year (2018–2019). Results: In total, 68 MSSA-colonized infants were identified, and cgMLSTs of 67 MSSA isolates were analyzed. Overall, we identified 11 cgMLST isolate groups comprising 39 isolates (58%), with group sizes ranging from 2 to 10 isolates, and 28 isolates (42%) were unrelated to each other or any of the isolate groups. Cases of infants colonized by MSSA were scattered throughout the 1-year study period, and isolates belonging to the same cgMLST group were typically detected contemporaneously, over a few weeks or a few months. Overall, 13 infants (19.7%) developed MSSA infections: bacteremia (n = 3), wound infection (n = 5), conjunctivitis (n = 4), and cellulitis (n = 1). We detected no association between these clinically manifest infections and specific cgMLST groups. Conclusions: Although MSSA isolates in infants in a NICU showed high diversity, most were related to other isolates, albeit within small groups. cgMLST facilitates an understanding of the complex transmission dynamics of MSSA in NICUs, and these data can be used to inform better control strategies.

Introduction Post-operative meningitis (POM) is a life-threatening complication of neurosurgery. Diagnosis is often difficult due to pre-existing inflammation and antecedent antimicrobial use. Bacterial cerebrospinal fluid (CSF) cultures may reveal no growth, but empiric antibiotics are typically given due to the high morbidity and mortality associated with POM. 16S rRNA gene PCR/sequencing is a molecular methodology that can identify the presence of bacteria regardless of viability for culture. Case Presentation A patient presented with a rapid onset of fever associated with headache, neck pain, nausea and altered mental status 11 days after undergoing laser interstitial thermal therapy for treatment of recurrent astrocytoma at another hospital. Based on clinical presentation and imaging, POM was suspected, and empiric antibacterial therapy was started. Microbiological stains and cultures of CSF were negative. Due to persistent fevers, 16S rRNA gene PCR/sequencing was done on CSF; it detected a member of the order Enterobacteriales most closely resembling Serratia species. All antimicrobials were stopped except for cefepime, which was given for 2 weeks. The patient’s mental status fully recovered. Conclusion The application of 16S rRNA gene PCR/sequencing in the setting of POM is of value by improving the quality of patient care and decreasing costs by antimicrobial de-escalation. Further studies regarding the positive and negative predictive values of this test are required.

BackgroundPolymerase chain reaction (PCR)/sequencing targeting the 16S ribosomal RNA (rRNA) gene to detect bacteria in normally sterile tissues and fluids has become increasingly popular in clinical medicine. This culture-independent technique can detect bacteria that are nonviable or difficult to cultivate using conventional methods. The clinical value of this type of testing is not well defined. We aimed to assess the diagnostic value of 16S rRNA PCR/Sanger sequencing as a clinical diagnostic assay at Mayo Clinic.MethodsThis is an interim analysis of the first 173 of 478 patients who had 16S rRNA PCR/Sanger sequencing done on sterile tissues or fluids at our institution from April, 2017 to November, 2018 as part of routine clinical practice. Medical records are being retrospectively reviewed, with results compared with those of culture.ResultsWe reviewed 207 specimens from 173 patients (musculoskeletal 79%, cardiovascular 7%, central nervous system 4%, other 9%) that underwent 16S rRNA PCR/Sanger sequencing by clinical request (Table 1). In 90% of these specimens, the test was pre-planned rather than added-on. Nine specimens were excluded from analysis, as cultures were not performed. Overall concordance of culture with PCR/sequencing was 81% (160/197; P < 0.0001). Of 44 culture-positive specimens, PCR detected the same bacterium in 21 (48%) (Table 2). 45% (20/44) of those with positive cultures and 46% of those with positive PCR/sequencing results had received prior antimicrobial therapy (Table 3). PCR was negative in 139/144 specimens that were culture-negative (97%). PCR/sequencing was helpful in detecting a putative bacterial pathogen in 4 patients with negative cultures (Table 4).ConclusionOverall, 16S rRNA PCR/Sanger sequencing improved diagnostic yield compared with culture in a minority of cases. The described assay is limited by its inability to detect polymicrobial infections, a technical limitation that could possibly be addressed using massive parallel sequencing. Careful selection of cases and a save and add-on approach may be more cost-effective than upfront testing, although this was requested in a minority of cases. DisclosuresRobin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.