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Development of a multiplexed microsphere PCR for rapid, culturefree detection and Gram-typing of bacteria in human blood samples Fang Liang, Daniel J. Browne, Megan J. Gray, Kate H. Gartlan, David D. Smith, Ross Barnard, Geoff R. Hill, Simon Robert Corrie, and Kate Markey ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00277 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 20, 2018
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ACS Infectious Diseases
Development of a multiplexed microsphere PCR for rapid, culture-free detection and Gram-typing of bacteria in human blood samples Fang Liang,1,2# Daniel J Browne,1# Megan J Gray,1,2 Kate H Gartlan,1,3 David D Smith,1 Ross T Barnard,2,5 Geoff R Hill,1,4 Simon R Corrie6# and Kate A Markey1,3,4,5*# 1
Division of Immunology, QIMR Berghofer Medical Research Institute, 300 Herston Road, Brisbane, Queensland, 4006, Australia 2
School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia campus, Brisbane, Queensland, 4072, Australia
3
School of Medicine, The University of Queensland, St Lucia campus, Brisbane, Queensland, 4072, Australia
4
Department of Haematology and Bone Marrow Transplantation, The Royal Brisbane and Women’s Hospital, Bowen Bridge Road & Butterfield Street, Brisbane, Queensland, 4029, Australia 5
Australian Infectious Disease Research Centre, The University of Queensland, St Lucia campus, Brisbane, Queensland, 4029, Australia
6
Department of Chemical Engineering, ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash and QLD nodes, Monash University, 22 Alliance Lane, Clayton, Victoria, 3800, Australia # Contributed equally to this work. * To whom correspondence should be addressed. Tel: +617 3362 0290; Fax: +617 3845 3509; Email:
[email protected] Present Address: Kate A Markey, Division of Immunology, QIMR Berghofer Medical Research Institute, Brisbane, QLD, 4006, Australia
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Blood stream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected blood stream infection remain centered around blood culture (highly variable timing, hours to days), and empiric use of broad-spectrum antibiotics is often employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g. combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives), however current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Grampositive from Gram-negative bacterial DNA in a 3.5-hour time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labelled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labelled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.
Keywords: Bloodstream infection, PCR, multiplex, Luminex, bacteria, Gram-positive, Gram-negative
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ACS Infectious Diseases
Blood stream infection (BSI), and the clinical syndrome of septic shock which can result, represents an important clinical problem, impacting up to 1.5 million people per year in the USA, and carrying an in-hospital mortality rate of up to 30%.1 In addition to the population at large, sepsis and BSI represent serious complications for patients with malignancy, with many common chemotherapy regimens inducing significant bone marrow suppression.2 Febrile neutropenia is a medical emergency, and the standard of care involves investigation of the underlying microbial cause with blood cultures, empiric broad spectrum antibiotics, and the measurement of serum lactate to guide fluid resuscitation.3-4 The lack of normal immune function in this patient group means that fevers may be the only warning of BSI and subsequent sepsis, justifying their treatment as potential oncological emergencies, even though it is likely that most fevers are unrelated to serious infection. Prolonged neutropenia remains the best-defined risk factor for severe infection, and sophisticated predictive tools that could guide pre-emptive treatment of bacterial infection are lacking. However, these tools are in clinical use for early diagnosis of invasive aspergillosis and pre-emptive treatment of cytomegalovirus infection.5-8 Blood culture remains the standard method to investigate blood stream infection in neutropenic patients, even though the proportion of positive blood cultures remains at only 10-33%,9 and is highly dependent on the health-care setting (e.g. inpatient vs presentation to the emergency department).9 The average time to determination of Gram-status and organism identification is highly dependent on the time to blood culture positivity, which can be as short as 5 hours for fast-growing organisms present at high levels in the blood, and as long as 5 days, when most laboratories discontinue incubation of blood culture bottles. Antimicrobial susceptibility testing typically requires a further 24-48 hours. As BSI-related mortality increases with each hour of delayed treatment, broad-spectrum empiric antibiotic treatment is standard.4 This highlights the urgent need for both predictive assays and rapid diagnostic assays with the capacity to both define and rule-out BSI. Advanced diagnostic techniques including sequencing, multiplexed PCR, mass spectrometry,10 and combinations thereof, are not currently standard clinical practice, despite their promise to deliver faster, more accurate results.11 Many of the emerging techniques are still reliant on blood culture, however those that report detection/identification of bacteria directly from blood usually involve multiplexed PCR (e.g. SeptiFast, SepsiTest, VYOO, Magic-Plex, T2Candida), designed to detect subsets of common sepsis-causing pathogens. However, the majority of multiplexed PCR assays are limited to 30 HPV genotypes in a single tube.16 The advantage of targeting Gram-status is that relatively low multiplexing is required, and a robust test could rule out bacterial infections, or guide patient treatment towards Gram-positive (e.g containing vancomycin or teicoplanin) or Gram-negative regimens (e.g. a broad-spectrum beta-lactam like piperacillintazobactam or a carbapenem). In this study, we implemented a multiplexed microsphere-PCR approach to distinguish between DNA derived from Gram-negative and Gram-positive bacteria, with a Luminex-based flow cytometry readout. Instead of relying on the unpredictable nature of hybridization between PCR amplicons and short probes,17-21 we instead designed the assay to generate Gram-type specific, fluoro-labelled oligonucleotides, in proportion to amplicon concentration, for efficient hybridization to complementary probes bound to Luminex beads. We have validated our assay using organisms that commonly cause bloodstream infections in febrile neutropenic patient groups.9 We achieved comparable detection limits in the assay in comparison to singleplex SYBRgreen qPCR (
