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Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction.

ABSTRACT: Development and implementation of rapid antimicrobial susceptibility testing is critical for guiding patient care and improving clinical outcomes, especially in cases of sepsis. One approach to reduce the time-to-answer for antimicrobial susceptibility is monitoring the inhibition of DNA production, as differences in DNA concentrations are more quickly impacted compared to optical density changes in traditional antimicrobial susceptibility testing. Here, we use real-time PCR to rapidly determine antimicrobial susceptibility after short incubations with antibiotic. Application of this assay to a collection of 144 isolates in mock blood culture, covering medically relevant pathogens displaying high rates of resistance, provided susceptibility data in under 4 hours. This assay provided categorical agreement with a reference method in 96.3% of cases across all species. Sequencing of a subset of PCR amplicons showed accurate genus level identification. Overall, implementation of this method could provide accurate susceptibility results with a reduced time-to-answer for a number of medically relevant bacteria commonly isolated from blood culture.

SUBMITTER: Maxson T

PROVIDER: S-EPMC6292663 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction.

Maxson Tucker T Blancett Candace D CD Graham Amanda S AS Stefan Christopher P CP Minogue Timothy D TD

PloS one 20181213 12

Development and implementation of rapid antimicrobial susceptibility testing is critical for guiding patient care and improving clinical outcomes, especially in cases of sepsis. One approach to reduce the time-to-answer for antimicrobial susceptibility is monitoring the inhibition of DNA production, as differences in DNA concentrations are more quickly impacted compared to optical density changes in traditional antimicrobial susceptibility testing. Here, we use real-time PCR to rapidly determine ...[more]

PMID: 30543695

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Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Project description:BackgroundInvasive meningococcal disease (IMD) is a highly lethal disease. Diagnosis is commonly performed by culture or Realtime-PCR (qPCR).AimsOur aim was to evaluate, retrospectively, whether culture positivity correlates with higher bacterial load and fatal outcome. Our secondary aim was to compare culture and qPCR sensitivity.MethodsThe National Register for Molecular Surveillance was used as data source. Cycle threshold (CT), known to be inversely correlated with bacterial load, was used to compare bacterial load in different samples.ResultsThree-hundred-thirteen patients were found positive for Neisseria meningitidis by qPCR, or culture, or both; 41 died (case fatality rate 13.1%); 128/143 (89.5%) blood samples and 138/144 (95.8%) CSF were positive by qPCR, 37/143 (25.9%) blood samples and 45/144 (31.2%) CSF were also positive in culture. qPCR was 3.5 times (blood) or 3.1 times (CSF) more sensitive than culture in achieving a laboratory diagnosis of IMD (OR 24.4; 95% CI 12.2-49.8; p < .10-4; Cohen's κ 0.08 for blood and OR 49.0; 95% CI 19.1-133.4; p<10-4; Cohen's κ 0.02; for CSF). Positivity of culture did not correlate with higher bacterial loads in blood (mean CT 27.7±5.71, and CT 28.1±6.03, p = 0.739 respectively in culture positive or negative samples) or in CSF (mean CT 23.1±4.9 and 24.7±5.4 respectively in positive or negative CSF samples, p = 0.11).CT values in blood from patients who died were significantly lower than in patients who survived (respectively mean 18.0, range 14-23 and mean 29.6, range 16-39; p<10-17). No deaths occurred in patients with CT in blood over 23. Positive blood cultures were found in 10/25 (40%) patients who died and in 32/163 (19.6%) patients who survived, p = 0.036, OR 2.73; 95% CL 1.025-7.215), however 60% of deaths would have remained undiagnosed with the use of culture only.ConclusionsIn conclusion our study demonstrated that qPCR is significantly (at least 3 times) more sensitive than culture in the laboratory confirmation of IMD. The study also demonstrated that culture negativity is not associated with lower bacterial loads and with less severe cases. On the other side, in patients with sepsis, qPCR can predict fatal outcome since higher bacterial load, evaluated by qPCR, appears strictly associated with most severe cases and fatal outcome. The study also showed that molecular techniques such as qPCR can provide a valuable addition to the proportion of diagnosed and serotyped cases of IMD.

Dataset Information

Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction.

Publications

Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction.

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