A Real-Time PCR Antibiogram for Drug-Resistant Sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔCt<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 Gram-negative and 2 Gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours

Efficacy of bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR for detecting neonatal sepsis: a case control study

<p>Abstract</p> <p>Background</p> <p>Neonatal sepsis is difficult to diagnose and pathogens cannot be detected from blood cultures in many cases. Development of a rapid and accurate method for detecting pathogens is thus essential. The main purpose of this study was to identify etiological agents in clinically diagnosed neonatal sepsis using bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR (BrRNA-RT-qPCR) and to conduct comparisons with the results of conventional blood culture. Since BrRNA-RT-qPCR targets bacterial ribosomal RNA, detection rates using this approach may exceed those using conventional PCR.</p> <p>Methods</p> <p>Subjects comprised 36 patients with 39 episodes of suspected neonatal sepsis who underwent BrRNA-RT-qPCR and conventional blood culture to diagnose sepsis. Blood samples were collected aseptically for BrRNA-RT-qPCR and blood culture at the time of initial sepsis evaluation by arterial puncture. BrRNA-RT-qPCR and blood culture were undertaken using identical blood samples, and BrRNA-RT-qPCR was performed using 12 primer sets.</p> <p>Results</p> <p>Positive rate was significantly higher for BrRNA-RT-qPCR (15/39, 38.5%) than for blood culture (6/39, 15.4%; p = 0.0039). BrRNA-RT-qPCR was able to identify all pathogens detected by blood culture. Furthermore, this method detected pathogens from neonates with clinical sepsis in whom pathogens was not detected by culture methods.</p> <p>Conclusions</p> <p>This RT-PCR technique is useful for sensitive detection of pathogens causing neonatal sepsis, even in cases with negative results by blood culture.</p

Diagnostic approach to lower airway dysfunction in athletes: a systematic review and meta-analysis by a subgroup of the IOC consensus on ‘acute respiratory illness in the athlete’

Objectives To compare the performance of various diagnostic bronchoprovocation tests (BPT) in the assessment of lower airway dysfunction (LAD) in athletes and inform best clinical practice. Design Systematic review with sensitivity and specificity meta-analyses. Data sources PubMed, EBSCOhost and Web of Science (1 January 1990–31 December 2021). Eligibility criteria Original full-text studies, including athletes/physically active individuals (15–65 years) who underwent assessment for LAD by symptom-based questionnaires/history and/or direct and/or indirect BPTs. Results In 26 studies containing data for quantitative meta-analyses on BPT diagnostic performance (n=2624 participants; 33% female); 22% had physician diagnosed asthma and 51% reported LAD symptoms. In athletes with symptoms of LAD, eucapnic voluntary hyperpnoea (EVH) and exercise challenge tests (ECTs) confirmed the diagnosis with a 46% sensitivity and 74% specificity, and 51% sensitivity and 84% specificity, respectively, while methacholine BPTs were 55% sensitive and 56% specific. If EVH was the reference standard, the presence of LAD symptoms was 78% sensitive and 45% specific for a positive EVH, while ECTs were 42% sensitive and 82% specific. If ECTs were the reference standard, the presence of LAD symptoms was 80% sensitive and 56% specific for a positive ECT, while EVH demonstrated 65% sensitivity and 65% specificity for a positive ECT. Conclusion In the assessment of LAD in athletes, EVH and field-based ECTs offer similar and moderate diagnostic test performance. In contrast, methacholine BPTs have lower overall test performance

Prevalence of lower airway dysfunction in athletes: a systematic review and meta-analysis by a subgroup of the IOC consensus group on ‘acute respiratory illness in the athlete’

Objective To report the prevalence of lower airway dysfunction in athletes and highlight risk factors and susceptible groups. Design Systematic review and meta-analysis. Data sources PubMed, EBSCOhost and Web of Science (1 January 1990 to 31 July 2020). Eligibility criteria Original full-text studies, including male or female athletes/physically active individuals/military personnel (aged 15–65 years) who had a prior asthma diagnosis and/or underwent screening for lower airway dysfunction via self-report (ie, patient recall or questionnaires) or objective testing (ie, direct or indirect bronchial provocation challenge). Results In total, 1284 studies were identified. Of these, 64 studies (n=37 643 athletes) from over 21 countries (81.3% European and North America) were included. The prevalence of lower airway dysfunction was 21.8% (95% CI 18.8% to 25.0%) and has remained stable over the past 30 years. The highest prevalence was observed in elite endurance athletes at 25.1% (95% CI 20.0% to 30.5%) (Q=293, I2=91%), those participating in aquatic (39.9%) (95% CI 23.4% to 57.1%) and winter-based sports (29.5%) (95% CI 22.5% to 36.8%). In studies that employed objective testing, the highest prevalence was observed in studies using direct bronchial provocation (32.8%) (95% CI 19.3% to 47.2%). A high degree of heterogeneity was observed between studies (I2=98%). Conclusion Lower airway dysfunction affects approximately one in five athletes, with the highest prevalence observed in those participating in elite endurance, aquatic and winter-based sporting disciplines. Further longitudinal, multicentre studies addressing causality (ie, training status/dose–response relationship) and evaluating preventative strategies to mitigate against the development of lower airway dysfunction remain an important priority for future research