Background. The frequent lack of a microbiological diagnosis in community-acquired pneumonia (CAP) impairs pathogen-directed antimicrobial therapy. This study assessed the use of comprehensive ...multibacterial, multiviral molecular testing, including quantification, in adults hospitalized with CAP. Methods. Clinical and laboratory data were collected for 323 adults with radiologically-confirmed CAP admitted to 2 UK tertiary care hospitals. Sputum (96%) or endotracheal aspirate (4%) specimens were cultured as per routine practice and also tested with fast multiplex real-time polymerase-chain reaction (PCR) assays for 26 respiratory bacteria and viruses. Bacterial loads were also calculated for 8 bacterial pathogens. Appropriate pathogen-directed therapy was retrospectively assessed using national guidelines adapted for local antimicrobial susceptibility patterns. Results. Comprehensive molecular testing of single lower respiratory tract (LRT) soecunebs achieved pathogen detection in 87% of CAP patients compared with 39% with culture-based methods. Haemophilus influenzae and Streptococcus pneumoniae were the main agents detected, along with a wide variety of typical and atypical pathogens. Viruses were present in 30% of cases; 82% of these were codetections with bacteria. Most (85%) patients had received antimicrobials in the 72 hours before admission. Of these, 78% had a bacterial pathogen detected by PCR but only 32% were culture-positive (P < .0001). Molecular testing had the potential to enable de-escalation in number and/or spectrum of antimicrobials in 77% of patients. Conclusions. Comprehensive molecular testing significantly improves pathogen detection in CAP, particularly in antimicrobial-exposed patients, and requires only a single LRT specimen. It also has the potential to enable early de-escalation from broad-spectrum empirical antimicrobials to pathogen-directed therapy.
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BFBNIB, NUK, PNG, UL, UM, UPUK
Faster respiratory pathogen detection and antibiotic resistance identification are important in critical care due to the severity of illness, significant prior antibiotic exposure and infection ...control implications. Our objective was to compare the performance of the commercial Unyvero P55 Pneumonia Cartridge (Curetis AG) with routine bacterial culture methods and in-house bacterial multiplex real-time PCR assays. Seventy-four bronchoalveolar lavage specimens from patients admitted to a Scottish intensive care unit (ICU) over a 33-month period were tested prospectively by routine culture and viral PCR and retrospectively by Unyvero P55 and in-house bacterial PCR. Sensitivity/specificity was 56.9%/58.5% and 63.2%/54.8% for the Unyvero P55 and in-house bacterial PCR panels respectively; sensitivity for in-panel targets was 63.5 and 83.7% respectively. Additional organisms were detected by Unyvero P55 and in-house bacterial PCR panels in 16.2% specimens. Antibiotics were changed on the basis of routine test results in 48.3% cases; of these, true-positive or true-negative results would have been obtained earlier by Unyvero P55 or in-house bacterial PCR panel in 15 (53.6%) and 17 (60.7%) cases respectively. However, a false-negative molecular test result may have been acted upon in six (21.4%) cases with either assay. Sensitivity/specificity of Unyvero P55 antibiotic resistance detection was 18.8%/94.9% respectively. Molecular testing identified a number of respiratory pathogens in this patient cohort that were not grown in culture, but resistance detection was not a reliable tool for faster antibiotic modification. In their current set-up, molecular tests may only have benefit as additional tests in the ICU pneumonia setting.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
•ResistancePlus GC assay was compared to in house methods for 206 clinical samples.•Sensitivity for N. gonorrhoeae and gyrA detection was 98.5% and 97.1%.•Sensitivity/Specificity for ciprofloxacin ...resistance detection was 98.8%/98.9%.•A minority resistant variant of 27 % was detected in a mixed population.
There is growing concern due to the emergence of multidrug resistance in Neisseria gonorrhoeae. A rapid molecular test which guides and provides antimicrobial susceptibility knowledge prior to start of treatment is needed. This study evaluated the clinical performance of the ResistancePlus GC assay compared to in-house PCR and antimicrobial susceptibility results for ciprofloxacin resistance. Samples were selected from a range of sites with corresponding cultures isolated from the same patient episode. The ResistancePlus GC assay displayed high sensitivity for N. gonorrhoeae detection (98.5%) and gyrA detection (97.1%). There was high agreement (98.9%) between the ResistancePlus GC assay and culture phenotype. Mixed population testing showed that the assay was able to detect resistance in a sample containing a minority variant of 27% resistant. The ResistancePlus GC assay performed well and could be used to provide a clinically relevant indication of ciprofloxacin susceptibility for the treatment of gonorrhoea.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Background
WGS is increasingly being applied to healthcare-associated vancomycin-resistant Enterococcus faecium (VREfm) outbreaks. Within-patient diversity could complicate transmission ...resolution if single colonies are sequenced from identified cases.
Objectives
Determine the impact of within-patient diversity on transmission resolution of VREfm.
Materials and methods
Fourteen colonies were collected from VREfm positive rectal screens, single colonies were collected from clinical samples and Illumina WGS was performed. Two isolates were selected for Oxford Nanopore sequencing and hybrid genome assembly to generate lineage-specific reference genomes. Mapping to closely related references was used to identify genetic variations and closely related genomes. A transmission network was inferred for the entire genome set using Phyloscanner.
Results and discussion
In total, 229 isolates from 11 patients were sequenced. Carriage of two or three sequence types was detected in 27% of patients. Presence of antimicrobial resistance genes and plasmids was variable within genomes from the same patient and sequence type. We identified two dominant sequence types (ST80 and ST1424), with two putative transmission clusters of two patients within ST80, and a single cluster of six patients within ST1424. We found transmission resolution was impaired using fewer than 14 colonies.
Conclusions
Patients can carry multiple sequence types of VREfm, and even within related lineages the presence of mobile genetic elements and antimicrobial resistance genes can vary. VREfm within-patient diversity could be considered in future to aid accurate resolution of transmission networks.
•Molecular assays reliably detect norovirus from stool and vomit.•Fast turnaround and simplicity of Xpert Norovirus suit it to near patient testing.•The BD-MAX System is useful for high-throughput ...norovirus testing.•xTAG GPP had lower norovirus sensitivity/specificity compared to other platforms.
Norovirus is a leading cause of infectious gastroenteritis, characterized by outbreaks of diarrhoea and vomiting in closed settings. Nucleic acid amplification tests allow rapid and sensitive laboratory diagnosis of norovirus, with a number of commercial platforms now available.
Evaluate the performance of the Becton Dickinson BD-MAX™System, Cepheid Xpert® Norovirus Assay, and Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) for norovirus detection in stool. Assess the performance of the Xpert® Norovirus Assay and BD-MAX™ in vomit samples.
163 diarrhoeal stool samples were tested on four diagnostic systems (laboratory-defined real time RT-PCR (assigned as gold standard), BD MAX™, Xpert® Norovirus Assay, and xTAG® GPP). A further 70 vomit samples were tested on the Xpert and BD MAX platforms.
In stool, sensitivity and specificity of the BD-MAX™ was 96.8% and 100%, for Xpert® Norovirus Assay was 91.9% and 100%, and for xTAG® GPP was 79.0% and 87.1%. In vomit samples positive and negative percent agreement was 95.6% and 92.0%, between the BD-MAX™ and Xpert® Norovirus.
The BD-MAX™ System with user defined settings and the Xpert® Norovirus Assay showed acceptable sensitivity and specificity for detection of norovirus from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a number of other clinically useful enteropathogens. Clinical laboratories must consider skill mix, budget, and sample throughput to determine the best fit for their service.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Co-infections with bacterial or fungal pathogens could be associated with severity and outcome of disease in COVID-19 patients. We, therefore, used a 16S and ITS-based sequencing approach to assess ...the biomass and composition of the bacterial and fungal communities in endotracheal aspirates of intubated COVID-19 patients. Our method combines information on bacterial and fungal biomass with community profiling, anticipating the likelihood of a co-infection is higher with (1) a high bacterial and/or fungal biomass combined with (2) predominance of potentially pathogenic microorganisms. We tested our methods on 42 samples from 30 patients. We observed a clear association between microbial outgrowth (high biomass) and predominance of individual microbial species. Outgrowth of pathogens was in line with the selective pressure of antibiotics received by the patient. We conclude that our approach may help to monitor the presence and predominance of pathogens and therefore the likelihood of co-infections in ventilated patients, which ultimately, may help to guide treatment.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
QIAstat-Dx Respiratory Panel V2 (RP) is a novel molecular-method-based syndromic test for the simultaneous and rapid (∼70-min) detection of 18 viral and 3 bacterial pathogens causing respiratory ...infections. This report describes the first multicenter retrospective comparison of the performance of the QIAstat-Dx RP assay to the established ePlex Respiratory Pathogen Panel (RPP) assay, for which we used 287 respiratory samples from patients suspected with respiratory infections. The QIAstat-Dx RP assay detected 312 (92%) of the 338 respiratory targets that were detected by the ePlex RPP assay. Most of the discrepant results have been observed in the low-pathogen-load samples. In addition, the QIAstat-Dx RP assay detected 19 additional targets in 19 respiratory samples that were not detected by the ePlex RPP assay. Nine of these discordant targets were considered to represent true positives after discrepancy testing by a third method. The main advantage of the QIAstat-Dx system compared to other syndromic testing systems, including the ePlex RPP assay, is the ability to generate cycle threshold (
) values, which could help with the interpretation of results. Taking the data together, this study showed good performance of the QIAstat-Dx RP assay in comparison to the ePlex RPP assay for the detection of respiratory pathogens. The QIAstat-Dx RP assay offers a new, rapid, and accurate sample-to-answer multiplex panel for the detection of the most common viral and bacterial respiratory pathogens and therefore has the potential to direct appropriate therapy and infection control precautions.
Transferable linezolid resistance due to
,
,
and
-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of
...in linezolid-resistant
isolates from Scotland. Six linezolid-resistant
isolated from urogenital samples were confirmed to carry the
gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The
gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to
dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to
-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn
transposon in pWE0254-1 carrying linezolid (
), macrolide (
) and spectinomycin ANT(9)-Ia resistance genes. OptrA amino acid sequences differed by 0-20 residues. We report multiple variants of
on distinct plasmids in diverse strains of
. It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.
Digital video has become a dominant form of student learning in and beyond the classroom, and thus its pervasive nature in contemporary learning environments commands scholarly inquiry. In this paper ...we explore a participatory design-based research approach to the integration of video hook technology in the post-primary science classroom (students aged 12–15). Video hooks were designed with the intention of engaging students and augmenting their interest in science. Teachers across ten schools voluntarily agreed to implement the video hooks, and with their students (N = 128) engage in a qualitative, observational methodology to ascertain their effect. Triangulated data was collected through teacher interviews (N = 10), structured lesson observation and researcher journal documentation. Results reveal that student reaction was instant and impactful with evidence of both triggered and maintained student interest.
Abstract
Organic chemistry often represents a key impasse for students during their third level science education. Both intrinsic and extrinsic factors contribute to perceived difficulties in ...learning the subject. Moreover, the teaching of
multi-step organic synthesis
at third level has well-documented challenges. At University College Cork (UCC), we have adopted a strategy to engage students at the mid to late degree stage using an innovative teaching hook, to inspire interest and engagement. The approach was taken to outline chronologically some of the seminal breakthroughs in synthesis, and organic chemistry more generally, from the 1950 s to a few of the current leaders of today. Importantly the focus is on the people, the circumstance and the stories surrounding them. As a relevance-based hooking strategy grounded in a storytelling pedagogy, the goal was to inform students of the potential in organic chemistry and thus spark their interest for the subsequent lectures. Over multiple interventions, feedback from students has been highly positive. We posit that the design framework behind the ‘The People and Personalities’ could be adapted to many disciplines in a similarly successful manner. Overall, this approach proved inspirational for students, and was a most timely intervention in their degree program.
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