The microbiological outcomes of culture-negative blood specimens using 16S rRNA broad-range PCR sequencing: a retrospective study in a Canadian province from 2018 to 2022.

Broad-range 16S rRNA PCR and sequencing of 1,183 blood specimens from 853 unique patients yielded an interpretable sequence and bacterial identification in 29%, 16S rRNA amplification with uninterpretable sequences in 53%, and no amplification in 18%. This study highlights the potential utility of this technique in identifying fastidious gram-negative and anaerobic bacteria but the frequent recovery of environmental and contaminant organisms argues for its judicious use. IMPORTANCE: The existing literature focuses on its performance compared to blood cultures in patients with sepsis, leaving a gap in the literature regarding other blood specimens in suspected infectious syndrome across the severity spectrum. We aimed to characterize its microbiological outcomes and provide insight into its potential clinical utility.

Spinal epidural abscess as predicting factor for the necessity of early surgical intervention in patients with pyogenic spondylitis.

BACKGROUND: Pyogenic spondylitis is a condition with low incidence that can lead to neurological sequelae and even life-threatening conditions. While conservative methods, including antibiotics and bracing, are considered the first-line treatment option for pyogenic spondylitis, it is important to identify patients who require early surgical intervention to prevent progressive neurologic deficits or deterioration of the systemic condition. Surgical treatment should be considered in patients with progressive neurologic deficits or deteriorating systemic condition. However, currently, there is a lack of treatment guidelines, particularly with respect to whether surgical treatment is necessary for pyogenic spondylitis. This study aims to analyze the radiological epidural abscess on MRI and clinical factors to predict the need for early surgical intervention in patients with pyogenic spondylitis and provide comprehensive insight into the necessity of early surgical intervention in these patients. METHODS: This study retrospectively reviewed 47 patients with pyogenic spondylitis including spondylodiscitis, vertebral osteomyelitis, epidural abscess, and/or psoas abscess. All patients received plain radiographs, and a gadolinium-enhanced magnetic resonance imaging (MRI) scan. All patients have either tissue biopsies and/or blood cultures for the diagnosis of a pathogen. Demographic data, laboratory tests, and clinical predisposing factors including comorbidities and concurrent other infections were analyzed. RESULTS: We analyzed 47 patients, 25 of whom were female, with a mean age of 70,7 years. MRI revealed that 26 of 47 patients had epidural abscesses. The surgical group had a significantly higher incidence of epidural abscess than the non-surgical group (p = 0.001). In addition, both CRP and initial body temperature (BT) were substantially higher in the surgical group compared to the non-surgical group. There was no significant difference between the surgical group and the non-surgical group in terms of age, gender, comorbidities, and concurrent infectious disorders, as well as the number of affected segments and affected spine levels. However, the surgical group had lengthier hospital stays and received more antibiotics. CONCLUSION: The presence of an epidural abscess on MRI should be regarded crucial in the decision-making process for early surgical treatment in patients with pyogenic spondylitis in order to improve clinical outcomes.

Diagnosing Fracture-Related Infections: Where Are We Now?

Accurate diagnosis of fracture-related infection (FRI) is critical for preventing poor outcomes such as loss of function or amputation. Due to the multiple variables associated with FRI, however, accurate diagnosis is challenging and complicated by a lack of standardized diagnostic criteria. Limitations with the current gold standard for diagnosis, which is routine microbiology culture, further complicate the diagnostic and management process. Efforts to optimize the process rely on a foundation of data derived from prosthetic joint infections (PJI), but differences in PJI and FRI make it clear that unique approaches for these distinct infections are required. A more concerted effort focusing on FRI has dominated more recent investigations and publications leading to a consensus definition by the American Orthopedics (AO) Foundation and the European Bone and Joint Infection Society (EBJIS). This has the potential to better standardize the diagnostic process, which will not only improve patient care but also facilitate more robust and reproducible research related to the diagnosis and management of FRI. The purpose of this minireview is to explore the consensus definition, describe the foundation of data supporting current FRI diagnostic techniques, and identify pathways for optimization of clinical microbiology-based strategies and data.

Mycobacterium chelonae Infection Identified by Metagenomic Next-Generation Sequencing as the Probable Cause of Acute Contained Rupture of a Biological Composite Graft-A Case Report.

We present the case of a 72-year-old female patient with acute contained rupture of a biological composite graft, 21 months after replacement of the aortic valve and the ascending aorta due to an aortic dissection. Auramine-rhodamine staining of intraoperative biopsies showed acid-fast bacilli, but classical culture and molecular methods failed to identify any organism. Metagenomic analysis indicated infection with Mycobacterium chelonae, which was confirmed by target-specific qPCR. The complexity of the sample required a customized bioinformatics pipeline, including cleaning steps to remove sequences of human, bovine ad pig origin. Our study underlines the importance of multiple testing to increase the likelihood of pathogen identification in highly complex samples.

Impact of Early-Onset Sepsis and Antibiotic Use on Death or Survival with Neurodevelopmental Impairment at 2 Years of Age among Extremely Preterm Infants.

OBJECTIVE: To evaluate the hypothesis that early-onset sepsis increases risk of death or neurodevelopmental impairment (NDI) among preterm infants; and that among infants without early-onset sepsis, prolonged early antibiotics alters risk of death/NDI. STUDY DESIGN: Retrospective cohort study of infants born at the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network centers (2006-2014) at 22-26 weeks of gestation and birth weight 401-1000 g. Early-onset sepsis defined as growth of a pathogen from blood or cerebrospinal fluid culture ≤72 hours after birth. Prolonged early antibiotics was defined as antibiotics initiated ≤72 hours and continued ≥5 days without culture-confirmed infection, necrotizing enterocolitis, or spontaneous perforation. Primary outcome was death before follow-up or NDI assessed at 18-26 months corrected age. Poisson regression was used to estimate adjusted relative risk (aRR) and CI for early-onset sepsis outcomes. A propensity score for receiving prolonged antibiotics was derived from early clinical factors and used to match infants (1:1) with and without prolonged antibiotic exposure. Log binomial models were used to estimate aRR for outcomes in matched infants. RESULTS: Among 6565 infants, those with early-onset sepsis had higher aRR (95% CI) for death/NDI compared with infants managed with prolonged antibiotics (1.18 [1.06-1.32]) and to infants without prolonged antibiotics (1.23 [1.10-1.37]). Propensity score matching was achieved for 4362 infants. No significant difference in death/NDI (1.04 [0.98-1.11]) was observed with or without prolonged antibiotics among the matched cohort. CONCLUSIONS: Early-onset sepsis was associated with increased risk of death/NDI among extremely preterm infants. Among matched infants without culture-confirmed infection, prolonged early antibiotic administration was not associated with death/NDI.

Culture negative empyema in a critically ill child: an opportunity for rapid molecular diagnostics.

BACKGROUND: Nucleic acid amplification technologies (NAAT) are advancing our ability to make rapid molecular diagnoses in patients with serious culture negative infections. This is the first report of PCR coupled to electrospray ionization mass spectrometry use in the evaluation of complicated community acquired pneumonia in a pediatric patient. CASE PRESENTATION: We present a case of culture negative empyema in a critically ill, Caucasian, 2-year-old girl who was treated with broad-spectrum empiric antibiotics, in which the length of stay was prolonged by adverse effects of the empiric antibiotic treatment. PCR coupled to electrospray ionization mass spectrometry was applied to culture negative fluid and tissue samples from the patient in order to determine the etiology of the empyema. CONCLUSIONS: Using this method, Streptococcus mitis/viridans was identified as the pathogen. A retrospective review of cases of empyema in children at our institution found that 87.5% of cases were negative for identification of a pathogen and antibiotics were administered to 100% of cases prior to collecting pleural fluid for culture. Understanding the role of Streptococcus mitis/viridans group in the etiology of empyema using an advanced NAAT coupled with mass spectrometry can enlighten clinicians as to the impact of this pathogen in community acquired pneumonia and help assist with antibiotic stewardship.

Candida parapsilosis in Late Prosthetic Joint Infections Among Immunocompetent Patients: A Case Series.

Introduction: Fungal prosthetic joint infections (PJIs) are very rare in immunocompetent patients. PJI can present either early, delayed, or as late chronic infections. Diagnosis of fungal PJI presenting late is challenging due to the difficulty in isolation as well as the clinical presentation very similar to an aseptic loosening. There are no clear guidelines regarding the management of these patients. Case Report: We present five cases of Candida parapsilosis PJI presenting as late chronic infections. All five patients were culture-negative preoperatively, immunocompetent, and with good soft-tissue condition. There were three infected knee prostheses and two infected hip hemiarthroplasty. All of them were treated with extensive debridement, meticulous sampling, and extended culture. We treated all of them with long-term antifungals without any disease reactivation or recurrence till the last follow-up. Conclusion: Fungal infection should be suspected in immunocompetent patients presenting early with features of aseptic loosening but without typical signs of periprosthetic infection. Revision with long-term suppressive therapy can give good results in these patients.

Dogs can detect an odor profile associated with Staphylococcus aureus biofilms in cultures and biological samples.

Introduction: The study investigated the utilization of odor detection dogs to identify the odor profile of Staphylococcus aureus (S. aureus) biofilms in pure in vitro samples and in in vivo biosamples from animals and humans with S. aureus periprosthetic joint infection (PJI). Biofilms form when bacterial communities aggregate on orthopedic implants leading to recalcitrant infections that are difficult to treat. Identifying PJI biofilm infections is challenging, and traditional microbiological cultures may yield negative results even in the presence of clinical signs. Methods: Dogs were trained on pure in vitro S. aureus biofilms and tested on lacrimal fluid samples from an in vivo animal model (rabbits) and human patients with confirmed S. aureus PJI. Results: The results demonstrated that dogs achieved a high degree of sensitivity and specificity in detecting the odor profile associated with S. aureus biofilms in rabbit samples. Preliminary results suggest that dogs can recognize S. aureus volatile organic compounds (VOCs) in human lacrimal fluid samples. Discussion: Training odor detection dogs on in vitro S. aureus, may provide an alternative to obtaining clinical samples for training and mitigates biosecurity hazards. The findings hold promise for culture-independent diagnostics, enabling early disease detection, and improved antimicrobial stewardship. In conclusion, this research demonstrates that dogs trained on in vitro S. aureus samples can identify the consistent VOC profile of PJI S. aureus biofilm infections. The study opens avenues for further investigations into a retained VOC profile of S. aureus biofilm infection. These advancements could revolutionize infectious disease diagnosis and treatment, leading to better patient outcomes and addressing the global challenge of antimicrobial resistance.

Determining the Clinical Utility of 16S rRNA Sequencing in the Management of Culture-Negative Pediatric Infections.

The use of 16S rRNA sequencing in culture-negative infections has improved identification of bacterial pathogens in select scenarios, but its clinical impact requires further elucidation, especially in the pediatric population. This retrospective study aims to determine the clinical utility of 16S rRNA sequencing on the clinical management of pediatric culture-negative infections in our institution. Significant clinical utility was identified in 30 (40.5%) of 74 clinical samples (p < 0.0001). Of all specimens, pulmonary samples yielded the most clinical utility (n = 9, 30%), followed equally by joint fluid (n = 6, 20%) and bone (n = 6, 20%), with no difference between fluid and fresh tissue specimens (p = 0.346). Although the difference was not statistically significant (p = 0.4111), the overall use of broad-spectrum coverage was decreased. The median number of antibiotics was decreased from two to one (p < 0.0001) based on 16S rRNA sequencing results. The results suggest that 16S rRNA sequencing has a significant impact on decreasing the number of antibiotics used in the treatment of pediatric culture-negative infections. 16S rRNA sequencing performed on pulmonary specimens has the highest likelihood of identifying a pathogen compared to other specimen types. Additional cost-benefit analysis needs to be completed to further determine clinical benefit.

Early outcome of culture-negative infection in open fractures of the lower limb: A prospective study.

Background: Culture-negative infections in open long bone fractures are frequently encountered in clinical practice. We aimed to identify the rate and outcome of culture-negative infections in open long bone fractures of lower limb. Methodology: A prospective cohort study was conducted from November 2015 to May 2017 on Gustilo and Anderson Grade III open long bone fractures of the lower limb. Demographic data, injury details, time from injury to receiving antibiotics and index surgical procedure were noted. Length of hospital stay, number of additional surgeries and occurrence of complications were also noted. Patients with infected open fractures were grouped as culture positive or culture negative depending on the isolation of infecting microorganisms in deep intraoperative specimen. The clinical outcome of these two groups was statistically analysed. Results: A total of 231 patients with 275 open fractures involving the femur, tibia or fibula were studied. There was clinical signs of infection in 84 patients (36.4%) with 99 fractures (36%). Forty-three patients (51.2%) had positive cultures and remaining 41 patients had negative cultures (48.8%). The rate of culture-negative infection in open type III long bone fractures in our study was 17.7%. There was no statistical difference in the clinical outcome between culture-negative and culture-positive infections. Conclusion: Failure to identify an infective microorganism in the presence of clinical signs of infection is routinely seen in open fractures and needs to be treated aggressively.

Rare Case of Community-Acquired Endocarditis Caused by Neisseria meningitidis Assessed by Clinical Metagenomics.

The most common causes of infective endocarditis (IE) are Staphylococcus, Streptococcus, Enterococcus, and HACEK-related organisms. In 15-30% of the IE cases, standard blood cultures remain sterile. We aimed at identifying the causative agent of a blood-culture-negative IE by whole metagenome shotgun sequencing (WMGS). A 54-year old woman diagnosed with community-onset pneumonia by a general practitioner, was admitted with dyspnea, cough and fever. The patient's blood cultures were repeatedly negative. The transesophageal echocardiography and transthoracic echocardiography showed an echo density on the left coronary leaflet of the aortic valve and signs suggestive of a ruptured abscess of the mitro-aortic junction. The patient underwent a semi-urgent aortic valve replacement by a mechanical prosthetic valve. We extracted DNA from the surgically-removed fresh valve tissue. The extraction procedure included bacterial/fungal DNA enrichment procedure. Nextera XT library prepared from the valve DNA extract was sequenced (2 × 250) on an Illumina MiSeq instrument. Sequence reads were mapped against bacterial genomic sequences, 16S rRNA genes and clade-specific taxonomic markers. Most of the 103,136 sequencing reads classified as bacterial were assigned to Neisseria meningitidis. In line with these data, mapping of reads against clade-specific and 16S rRNA gene markers revealed N. meningitidis as the most represented species. Assembled metagenomic fragments had the best average nucleotide identity (ANI) with N. meningitidis. Comparison of assembled contigs to reference alleles showed that this strain belongs to the ST-41/44 complex. N. meningitidis is commonly associated with meningitis and/or septicemia but should not be neglected as a causative agent of IE, which became exceedingly rare with the introduction of antibiotics. Our data show that WMGS may be used as a diagnostic procedure to strengthen the diagnosis of IE and to obtain draft genomic sequence of the pathogen and typing information.

Two-stage revision for the culture-negative infected total hip arthroplasty : A comparative study.

AIMS: Periprosthetic joint infection (PJI) remains a challenging complication following total hip arthroplasty (THA). It is associated with high levels of morbidity, mortality and expense. Guidelines and protocols exist for the management of culture-positive patients. Managing culture-negative patients with a PJI poses a greater challenge to surgeons and the wider multidisciplinary team as clear guidance is lacking. PATIENTS AND METHODS: We aimed to compare the outcomes of treatment for 50 consecutive culture-negative and 50 consecutive culture-positive patients who underwent two-stage revision THA for chronic infection with a minimum follow-up of five years. RESULTS: There was no significant difference in the outcomes between the two groups of patients, with a similar rate of re-infection of 6%, five years post-operatively. Culture-negative PJIs were associated with older age, smoking, referral from elsewhere and pre-operative antibiotic treatment. The samples in the culture-negative patients were negative before the first stage (aspiration), during the first-stage (implant removal) and second-stage procedures (re-implantation). CONCLUSION: Adherence to strict protocols for selecting and treating culture-negative patients with a PJI using the same two-stage revision approach that we employ for complex culture-positive PJIs is important in order to achieve control of the infection in this difficult group of patients. Cite this article: Bone Joint J 2018;(1 Supple A)100-B:3-8.