Search

Investigate the performance of real-time 16S PCR and third-generation 16S sequencing in the diagnosis of external ventricular drain related infections (EVDRI). 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. 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. 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. [ABSTRACT FROM AUTHOR]

Abstract 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

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract 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.

An experiment was conducted to investigate the efficacy of a cultured Synergistes jonesii inoculum in degrading the Leucaena leucocephala (leucaena) toxins: 3-hydroxy-4(1 H)-pyridone and 3-hydroxy-2(1 H)-pyridone (3,4- and 2,3-DHP). Sixteen stall-housed Bos indicus steers naïve to leucaena were fed varying combinations of forage-harvested leucaena and Chloris gayana (Rhodes grass). Dietary treatments, offered at 25 g dry matter/kg LW.day, were: 25% leucaena; 50% leucaena; 100% leucaena; and 50% leucaena, switched to 50% Medicago sativa (lucerne) after 6 weeks at time of inoculation. The experiment was 10 weeks in duration, consisting of a 6-week pre-inoculation period, followed by inoculation with cultured S. jonesii , and a 4-week post-inoculation period. Mean daily dry matter intake was recorded. Twenty-four-hour urine collections and rumen fluid samples were obtained weekly for estimation of total urinary DHP, and detection of S. jonesii using nested polymerase chain reaction analysis including presence of single nucleotide polymorphisms (SNP), respectively. In the pre-inoculation period, total urinary DHP increased quickly to high levels, then gradually declined after Week 3 with 2,3-DHP the dominant isomer through to Week 6. Indigenous strains of S. jonesii were sporadically detected by PCR analysis, indicating S. jonesii was present before inoculation but at the lower limits of detection. After inoculation there was no change in the rate of total DHP degradation or the frequency of detection of S. jonesii , although there was increased rate of degradation of 2,3-DHP. SNP indicated the presence of different strains of S. jonesii in both indigenous and cultured S. jonesii. DMI was low, especially in the 100% treatment, due in part to the high stem content of the forage-harvested leucaena and probable DHP toxicosis. It was concluded that the cultured S. jonesii inoculum did not fully protect animals against leucaena toxicity. Leucaena is a highly valuable tree legume, which produces high quality feed for cattle in northern Australia and in many tropical countries. However, it contains toxic secondary compounds that have deleterious effects on animal production and health. Since 1984, toxicity has been controlled with a bacterium inoculum supplied as rumen fluid, and more recently as a fermenter cultured inoculum. Our findings indicate that the artificially grown inoculum does not fully protect cattle consuming leucaena from toxicity. Addressing this issue, both with improved inoculum and/or with new alternate solutions for managing toxicity, will have significant implications for improving cattle production and health in tropical regions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Copyright of Tropical Grasslands / Forrajes Tropicales is the property of International Centre for Tropical Agriculture - CIAT and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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.