Revised version (INFD-D-20-00242): impact of 16S rDNA sequencing on clinical treatment decisions: a single center retrospective study.

BACKGROUND: PCRs targeting 16S ribosomal DNA (16S PCR) followed by Sanger's sequencing can identify bacteria from normally sterile sites and complement standard analyzes, but they are expensive. We conducted a retrospective study in the Strasbourg University Hospital to assess the clinical impact of 16S PCR sequencing on patients' treatments according to different sample types. METHODS: From 2014 to 2018, 806 16S PCR samples were processed, and 191 of those were positive. RESULTS: Overall, the test impacted the treatment of 62 of the 191 patients (32%). The antibiotic treatment was rationalized in 31 patients (50%) and extended in 24 patients (39%), and an invasive procedure was chosen for 7 patients (11%) due to the 16S PCR sequencing results. Positive 16S PCR sequencing results on cerebrospinal fluid (CSF) had a greater impact on patients' management than positive ones on cardiac valves (p = 0.044). The clinical impact of positive 16S PCR sequencing results were significantly higher when blood cultures were negative (p < 0.001), and this difference appeared larger when both blood and sample cultures were negative (p < 0.001). The diagnostic contribution of 16S PCR was higher in patients with previous antibiotic treatment (p < 0.001). CONCLUSION: In all, 16S PCR analysis has a significant clinical impact on patient management, particularly for suspected CSF infections, for patients with culture-negative samples and for those with previous antibiotic treatments.

Added diagnostic value of broad-range 16S PCR on periprosthetic tissue and clinical specimens from other normally sterile body sites.

AIMS: Evaluation of 16S PCR in addition to the standard culture to improve the pathogen detection rate in clinical specimens. METHODS AND RESULTS: Microbiological culture and direct 16S PCR was performed on specimens from suspected prosthetic joint infection patients (cohort-1) and on tissues and fluids from other normally sterile body sites (cohort-2). Based on clinical and microbiological data, the detection rate for both methods was assessed, assuming no superiority of either 16S PCR or culture. In cohort-1, 469 specimens were obtained. Culture was positive in 170 (36·2%) specimens, 16S PCR detected 70 (41·2%) of those pathogens. Additionally, 16S PCR detected pathogens in 13 of 299 (4·3%) culture-negative specimens. In cohort-2, pathogens were cultured in 52 of 430 (12·1%) specimens and 16S PCR revealed those pathogens in 32 (61·5%) specimens. 16S PCR detected pathogens in 31 of 378 (8·2%) culture-negative specimens. CONCLUSIONS: Overall, the yield with 16S PCR was low. For cohort-1 16S PCR detected pathogens in 4·3% of culture-negative specimens, where this was 8·2% for cohort-2. SIGNIFICANCE AND IMPACT OF THE STUDY: Although direct 16S PCR cannot replace culture, it may offer a valuable additional diagnostic option for detection of difficult to culture micro-organisms in culture-negative clinical specimens.

Comparison of two commercial broad-range PCR and sequencing assays for identification of bacteria in culture-negative clinical samples.

BACKGROUND: Culturing has long been the gold standard for detecting aetiologic agents in bacterial infections. In some cases, however, culturing fails to detect the infection. To further investigate culture-negative samples, amplification and subsequent sequencing of the 16S rRNA gene is often applied. The aim of the present study was to compare the current method used at our Department of Clinical Microbiology, based on the MicroSeq ID system (Applied Biosystems, USA) with the Universal Microbe Detection (UMD) SelectNA kit (Molzym, Germany). METHODS: 76 culture-negative samples were first processed with the MicroSeq ID analysis, where total DNA was extracted and the 16S gene amplified and sequenced with the MicroSeq ID system. Samples were subsequently processed with the UMD SelectNA analysis, where human DNA was removed during the DNA extraction procedure and the 16S gene amplified in a real-time PCR and sequenced. RESULTS: 22 of 76 samples (28.9%) were positive for bacteria with the UMD SelectNA, which was significantly more (p = 0.0055) than the MicroSeq ID where 11 of 76 samples (14.5%) were positive. The UMD SelectNA assay identified more relevant bacterial pathogens than the MicroSeq ID analysis (p = 0.0233), but also found a number of species that were considered contaminations. CONCLUSIONS: The UMD SelectNA assay was valuable for the identification of pathogens in culture-negative samples; however, due to the sensitive nature of the assay, extreme care is suggested in order to avoid false positives.

Diagnosis of external ventricular drainage related infections with real-time 16S PCR and third-generation 16S sequencing.

OBJECTIVE: Investigate the performance of real-time 16S PCR and third-generation 16S sequencing in the diagnosis of external ventricular drain related infections (EVDRI). METHODS: Subjects with suspected EVDRI were prospectively included at Uppsala University Hospital. Subjects were included into three groups: subjects with negative CSF culture with and without antibiotic treatment and subjects with positive CSF culture, respectively. CSF was analysed with real-time 16S PCR and third-generation 16S sequencing. Real-time 16S PCR positivity/negativity and number of 16S sequence reads were compared between groups. For culture positive subjects, species identification in third-generation sequencing and routine culture was compared. RESULTS: 84 subjects were included. There were 18, 44 and 22 subjects in the three groups. Real-time PCR was positive in 17 of 22 subjects in the culture positive group and negative in 61 of the 62 subjects in the two culture negative groups. The sensitivity and specificity for real-time 16S PCR compared to culture was estimated to 77% and 98%, respectively. Species identification in 16S sequencing and culture was concordant in 20 of 22 subjects. The number of 16S sequence reads were significantly higher in the culture positive group than in both culture negative groups (p < 0.001). There was no significant difference in number of 16S sequences between the two culture negative groups. CONCLUSIONS: Real-time 16S PCR predict culture results with sufficient reliability. Third-generation 16S sequencing could enhance sensitivity and species identification in diagnostics of EVD-related infections. False negative culture results appear to be uncommon in patients with suspected EVDRI.

Influence of 16S ribosomal RNA gene polymerase chain reaction and sequencing on antibiotic management of bone and joint infections.

INTRODUCTION: Amplification of the 16S ribosomal RNA gene by polymerase chain reaction (PCR) followed by analysis of generated sequences can be an important adjunct to conventional cultures. OBJECTIVE: To determine how the results of this approach influence physicians' decisions regarding the management of bone and joint infections. METHOD: Clinical and laboratory findings of patients seen at the Queen Elizabeth II Health Sciences Centre (Halifax, Nova Scotia) between December 2005 and September 2009 were reviewed. Patients who had negative cultures but likely or possible bone and joint infections were further evaluated using 16S rRNA PCR. The impact of the 16S rRNA PCR result on antibiotic management was evaluated and it was assessed whether untreated patients with negative 16S rRNA PCR subsequently presented with infections, suggesting a false-negative result. RESULT: A total of 36 patients (mean age 62 years) were reviewed. Thirty-two patients were evaluated by infectious disease consultants; of these, 20 were considered likely to have infections. Seventeen patients were admitted with suspected prosthetic joint infections. Twenty-nine patients received antimicrobial treatment before the sample for the 16S rRNA PCR assay was obtained. Of the 36 patients, 26 (72.2%) were treated appropriately with modifications to their antibiotic regimen in response to the 16S rRNA PCR assay results. Antimicrobials were discontinued for 19 patients based on negative PCR assay and, in seven patients, antibiotics were changed based on a positive result. There were no relapses among patients with negative PCR assay in whom antibiotics were discontinued. CONCLUSION: 16S ribosomal RNA gene PCR and sequencing is a valuable tool in the guidance of antimicrobial therapy for bone and joint infections.

HISTORIQUE: L'amplification de la réaction en chaîne de la polymérase (PCR) du gène de l'ARN ribosomique 16S, suivie de l'analyse des séquences générées, peut être un ajout important aux cultures habituelles. OBJECTIF: Déterminer en quoi les résultats de cette approche influent sur les décisions des médecins à l'égard de la prise en charge des infections osseuses et articulaires. MÉTHODOLOGIE: Les chercheurs ont analysé les résultats cliniques et de laboratoire des patients vus au Queen Elizabeth II Health Sciences Centre de Halifax, en Nouvelle-Écosse,entre décembre 2005 et septembre 2009. Les patients dont les cultures étaient négatives, mais qui présentaient une infection osseuse ou articulaire probable ou possible, subissaient une évaluation plus approfondie au moyen de la PCR de l'ARNr 16S. Les chercheurs ont évalué les répercussions du résultat de la PCR de l'ARNr 16S sur la prise en charge des antibiotiques et ont déterminé si les patients non traités dont la PCR de l'ARNr 16S était négative ont ensuite souffert d'infections, laissant ainsi supposer un résultat faux négatif. RÉSULTAT: Au total, les chercheurs ont analysé le dossier de 36 patients (d'un âge moyen de 62 ans). Trente-deux patients ont été évalués par des consultants en infectiologie, et de ce nombre, 20 ont été considérés comme susceptibles d'avoir une infection. Dix-sept patients ont été hospitalisés en raison d'une présomption d'infection articulaire prosthétique. Vingt-neuf patients ont reçu un traitement antimicrobien avant l'obtention de l'échantillon en vue de la PCR de l'ARNr 16S. Des 36 patients, 26 (72,2 %) ont été traités correctement par des modifications au régime antibiotique après l'obtention des résultats de la PCR de l'ARNr 16S. Le traitement aux antimicrobiens a été interrompu chez 19 patients en raison d'une PCR négative et chez sept patients, on l'a remplacé par un autre en raison d'un résultat positif. Les chercheurs n'ont constaté aucune rechute chez les patients dont la PCR était négative et à qui on avait arrêté d'administrer des antibiotiques. CONCLUSION: La PCR et le séquençage du gène de l'ARN ribosomique 16S est un outil précieux pour orienter le traitement antimicrobien des infections osseuses et articulaires.

A case of Capnocytophaga canimorsus endocarditis in a non-immunosuppressed host: the value of 16S PCR for diagnosis.

Capnocytophaga canimorsus is a rare cause of endocarditis and is particularly unusual in non-immunosuppressed hosts. It is associated with animal bites, particularly those from dogs. This case describes a healthy 59-year-old woman, with no identifiable risk factors or dog bite history, who presented with fever of unknown origin. Echocardiography demonstrated an aortic valve mass and root abscess, in keeping with endocarditis, requiring urgent valve replacement surgery. Eight sets of blood cultures were drawn in total; after prolonged incubation, one set grew C. canimorusus. There was initial uncertainty over this being the causative organism, given the lack of immunosuppression or dog bite history, but 16S PCR of the valve identified the same organism, permitting targeted treatment. This case highlights the value of valve 16S PCR as a diagnostic tool in endocarditis.

Granulicatella adiacens: An uncommon diagnosis of prosthetic hip joint infection. A case report with review of the literature.

We report a case of prosthetic hip infection in a 79 year old man caused by Granulicatella adiacens. The diagnosis was achieved using broad range 16S PCR gene analysis at an early stage, after joint aspiration and culture failed to yield a pathogen. Staged revision surgery together with administration of appropriate antibiotics resulted in cure. Granulicatella adiacens is a nutritionally variant streptococcus (NVS). It has been increasingly reported to cause significant morbidities involving various systems. Its insidious growth due to complex growth requirements, has made its diagnosis challenging, and often delays appropriate antibiotic administration.

Bartonella quintana as a cause for prosthetic valve endocarditis and post-sternotomy mediastinitis.

An otherwise healthy patient, with minimal clinical, biochemical and peroperative signs of infection, was diagnosed with Bartonella quintana prosthetic valve endocarditis by 16S PCR. The patient subsequently developed a post-sternotomy mediastinitis and Bartonella quintana was the only detected pathogen. Bartonella quintana can cause severe infections in individuals not classically at risk, and may be missed in the routine diagnostic work-up of endocarditis.

Limited diagnostic possibilities for bloodstream infections with broad-range methods: A promising PCR/electrospray ionization-mass spectrometry platform is no longer available.

Fast and accurate detection of causative agents of bloodstream infections remains a challenge of today's microbiology. We compared the performance of cutting-edge technology based on polymerase chain reaction coupled with electrospray ionization-mass spectrometry (PCR/ESI-MS) with that of conventional broad-range 16S rRNA PCR and blood culture to address the current diagnostic possibilities for bloodstream infections. Of 160 blood samples tested, PCR/ESI-MS revealed clinically meaningful microbiological agents in 47 samples that were missed by conventional diagnostic approaches (29.4% of all analyzed samples). Notably, PCR/ESI-MS shortened the time to positivity of the blood culture-positive samples by an average of 34 hr. PCR/ESI-MS technology substantially improved current diagnostic tools and represented an opportunity to make bloodstream infections diagnostics sensitive, accurate, and timely with a broad spectrum of microorganisms covered.

An unusual case of seronegative, 16S PCR positive Brucella infection.

INTRODUCTION: Brucella is a zoonotic infection commonly diagnosed by isolation of the organism from blood culture or positive serological testing. It is an uncommon cause of a pyrexia of unknown origin in the United Kingdom. CASE PRESENTATION: We describe the case of a 14-year-old girl with no history of travel who presented with pyrexia, weight loss, arthralgia, multiple splenic abscesses and a subsequent pleural effusion, the latter of which isolated a Brucella species on 16S rRNA PCR. The patient responded well to initiation of treatment for brucellosis and on repeat imaging, after 3 months, the splenic abscesses had resolved. CONCLUSION: This unique case demonstrates uncommon complications of brucellosis and the challenges of diagnosing the organism, the latter of which can be alleviated by the utilization of molecularbased technologies. This patient had a negative serology result for brucellosis, which highlights the need to interpret serology results with caution in non-endemic regions for brucellosis.

The impact of PCR in the management of prosthetic joint infections.

An accurate diagnosis of prosthetic joint infection (PJI) remains a challenging clinical problem and is essential for the success of treatment regardless of the treatment option chosen by patients and surgeons. In recent years, PCR for the diagnosis of PJI has received much attention. Here, we review the impact of common PCR-based techniques on identifying causative organisms, antibiotic management and economics of PJI.