Practice of blood culture: Can we do better? Dr Purva Mathur

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1 Practice of blood culture: Can we do better? Dr Purva Mathur

2 Importance Role of Microbiological Cultures in ASP IDSA Guideline; April 13, 2016 ASP with a culture-guided de-escalation of antibiotics successfully reduces Length of stay Mortality Cost of antimicrobials, DDDs Antimicrobial resistance rate Highly recommended for hospitals.

3 BSIs Morbidity & mortality > 30,000 deaths (U.S. hospitals) Mortality: 34 to 52% Septicemia affected nearly 1 out of every 23 hospitalized patients (4.2%) (2009) Aggregate cost: $15.4 billion or 4.3% of all hospital costs.

4 Rapid identification: Multiple positive impacts on patient outcomes Reductions in mortality Morbidity LOS Antibiotic use Cost of treatment AMR

5 Blood collection Empirical & often broad-spectrum antimicrobial therapy Continued till etiological agent is identified & AST results are available Target (tailor) therapy

6 Delay in microbial identification Empirical & often broad-spectrum antimicrobial therapy Target (tailor) therapy 50% BSIs Up to 70% fungemia Receive incorrect therapy during the empirical treatment period before culture results are available

7 Efficient communication Gram staining Microorganism identification ASTs Timely switches from empirical therapy to targeted therapy Essential for providing safe, effective, and efficient care of patients with BSIs Updated Bundles in Response to New Evidence The leadership of the Surviving Sepsis Campaign (SSC) has believed since its inception that both the SSC Guidelines and the SSC performance improvement indicators (1) will evolve as new evidence that improves our understanding of how best to care for patients with severe sepsis and septic shock becomes available. With publication of 3 trials (2,3,4) that do not demonstrate superiority of required use of a central venous catheter (CVC) to monitor central venous pressure (CVP) and central venous oxygen saturation (ScvO2) in all patients with septic shock who have received timely antibiotics and fluid resuscitation compared with controls or in all patients with lactate >4 mmol/l, the SSC Executive Committee has revised the improvement bundles as follows: TO BE COMPLETED WITHIN 3 HOURS OF TIME OF PRESENTATION*: 1. Measure lactate level 2. Obtain blood cultures prior to administration of antibiotics r 3. Administer broad spectrum antibiotics 4. Administer 30ml/kg crystalloid for hypotension ao lcta te 4mmol/L * Time of presentation is defined as the time of triage in the emergency department or, if presenting from another care venue, from the earliest chart annotation consistent with all elements of severe sepsis or septic shock ascertained through chart review. TO BE COMPLETED WITHIN 6 HOURS OF TIME OF PRESENTATION: 5. Apply vasopressors (for hypotension that does not respond to initial fluid resuscitation) to maintain a mean Marterial pressure ( AP) 65mmHg 6. In the event of persistent hypotension after initial fluid administration (MAP < 65 f mm Hg) or i a4 in tial lcta te was m m ol/l, re-assess volume status and tissue perfusion and document findings according to Table Re-measure lactate if initial lactate elevated.

8 Detection of microorganisms by conventional method Limitations Blood-cultures Gold standard Provides causal organism for further AST & optimization of antimicrobial therapy 1 to 3 days Non-cultivable/fastidious microorganisms Culture neg. if treatment begun prior to blood sampling Timing of blood-culture collection Volume of blood cultures

9 An Ideal Platform??? Should identify a broad spectrum of pathogens (bacteria, fungi, viruses, and protozoa) Determine susceptibility to a battery of antibiotics Allow analysis of specimens in high or low throughput Low cost per sample Minimum hands-on time User friendly Generate timely results (6 hours or less).

10 Continuous monitoring systems BACTEC 9000 series (BD) BacT/Alert 3D system (bio-merieux) Colorimetric or fluorimetric detection of increased CO2 levels due to growth VersaTREK (TREK) Measures change in gas pressure (production of CO2) in the headspace CO2: system signals positive; removed and sub-cultured

11 Identification of pathogens by advanced culture-based methods Gram staining after positive signal Pathogen ID & AST from blood is usually performed using microdilution broth identification with automated continuously-monitoring systems: Vitek 2 (biome rieux), Phoenix (BD) Micro Scan Walk Away (Siemens) Or semi-automated systems Micronaut (Merlin)

12 Continuously monitoring expert-based systems have decreased turnaround time Mean of 6.75 h for complete ID of bacteria ~18 h for fungi Including antibiotic susceptibility reporting (Vitek 2 data)

13 Rapid Assays Based on 2 methodological groupings Nucleic acid-based detection tests Proteomic-based methods using mass spectrometry (MS)

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15 Rapid pathogen identification by Mass spectrometry Notion of using mass spectrometry for identification of bacteria was proposed in 1975 Not possible to analyze intact proteins (they would have fragmented in the process)

16 Mass spectrometry Technology for analysis of intact macromolecules: invented in the 1980s In 1985, Koichi Tanaka described a soft desorption ionization method using ultrafine metal powder & glycerol that enabled mass spectrometric analysis of biological macromolecules Awarded the Nobel Prize in Chemistry

17 Hillenkamp & Karas reported Soft desorption ionization using an organic compound matrix Term matrix-assisted laser desorption ionization (MALDI) was coined. Advances in IT Computer science Development of validated comprehensive databases of mass spectra representing diverse types of bacteria and fungi Enabled automation of MALDI-TOF MS and associated data analysis An important tool for the Clinical Microbiology labs

18 MALDI-TOF MS Most common application of MALDI-TOF MS: ID of pure microbial isolates grown by culture. Protein pattern matching by MALDI-TOF MS More accurate than conventional biochemical phenotypic testing Faster and less expensive than 16S DNA sequencing

19 MALDI-TOF Two MALDI-TOF instruments currently FDA approved Microflex Biotyper (Bruker) Vitek Mass Spectrometry System (biomerieux)

20 Particle sizes (mass spectrum) is unique to each organism; identified by comparison with a library of standard reference spectra. Allocation of a numerical score to each identification log score 2.0: acceptable identification species >1.7 but <2.0: identification to the genus level Results: minutes

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22 Can be applied to broth media from a positive BC bottle (not FDA approved) Diagnostic yield ~ 80%; turnaround time of minutes ID of yeast in positive BC broth: more challenging than bacteria Polymicrobial BSIs: detects at least 1 of the organisms, but it rarely (<10% of cases) detects all organisms

23 Relies on culture (median hour delay) They cannot directly detect AMR In order to accelerate diagnosis, it is desirable to detect & identity pathogens directly from the patient s blood, avoiding the culture step.

24 Direct detection from blood: Nucleic Acid Technology Molecular techniques applied directly to whole blood samples Same-day identification of pathogens Early pathogen-specific targeting of antimicrobial therapy.

25 Identification using NAT Two categories (1) Methods applied after cultivation steps & using positive BCs/ single colonies (2) assays that can be applied directly to drawn blood or other primarily sterile specimens, like CSF.

26 Identification using NAT Three procedures for applying NAT assays in ID (1) Pathogen-specific methods (2) multiplex assays covering several different pathogens typical for a certain infection type (3) Universal broad-range assays involving conserved target sequences, eg. eubacterial 16S/ 23S rdna, & pan fungal 8S or 18S rdna These rdnas are usually in multiple copies. More sensitive detection

27 Initial assays: designed for detection of single pathogen of interest Not useful Current assays are based on two main strategies: Identification of a selected group of pathogens using specific targets (i.e., SeptiFast, VYOO, Magicplex) Detection of a broad range of pathogens using universal/conserved targets (i.e., SepsiTest, PCR/ESI- MS).

28 Commercially Available Molecular Assays for Diagnosis of BSIs from Whole Blood 1. LightCycler SeptiFast (Roche) Multiplex real-time PCR assay that detects 25 pathogens including five Candida species and Aspergillus fumigatus. meca gene may be detected with a separate test Initial volume of blood: 3 ml Pathogen detection: specific fluorescent probes. The time-to-result: hours. Widely evaluated Recent meta-analysis on 6,012 patients from 35 selected studies. Sensitivity 75.0% (95% confidence interval, %) Specificity 92.0% (95% confidence interval,

29 2. SepsiTest (Molzym, Germany) Based on broad-range PCR amplification followed by sequencing. Two 1 ml aliquots of blood are processed in duplicate Human DNA is selectively degraded Largest study (N = 342) sensitivity 87% specificity 85.8% Slow turnaround time of 8 12 hours

30 Multiplexed PCR analysis Detects 34 pathogens 6 species of Candida & A. fumigatus Several resistance genes meca, vana & vanb, blashv & blactx-m Result: 8 hours 5 ml of blood Total DNA: applied to an affinity chromatographic column that specifically binds the microbial DNA Human DNA is depleted during the extraction step Sensitivity 38.0% %. VYOO (SIRS-Lab)

31 Magicplex Sepsis Real-Time Test (Seegene) Three PCR reactions: to achieve ID at species level Conventional PCR amplification Primers for 91 microorganisms 85 bacteria, 5 Candida & A. fumigatus 3 resistance genes (meca, vana and vanb) Real-time PCR for identification of group or genera level of pathogens present. Human DNA is removed prior to the lysis of microorganisms. Result: 6 hours. Only one study published. Sensitivity: 65.0% Specificity: and 92.0% Second real-time PCR is performed to achieve the identification at species level

32 T2 magnetic resonance (MR; T2 Biosysystems) Automated nanoparticle-based PCR assay; can detect ~1 CFU /ml Candida sp. in blood in ~ 3 hours. Initial pilot study using spiked blood samples: 98% PPV 100% negative predictive value between T2 MR results & BC for Candida sp. First FDA-approved assay for direct detection Five most common Candida sp.

33 Biofire Film- Array system (biomerieux) Uses nested multiplex PCR Identify 19 bacterial pathogens (staphylococci, streptococci, Enterococcus, Listeria, Acinetobacter, N.meningitidis, P. aeruginosa & members of Enterobacteriaceae 5 yeast 3 resistance markers (meca, vana/b, & KPC) Directly from positive blood cultures. Sensitivities for pathogen ID >90%; resistance markers is 100%. The turnaround time: 1 hour.

34 PCR electrospray ionization mass spectrometry Combines PCR & mass spectrometry in an automated, high-throughput platform Not FDA approved Can identify nearly all human pathogens & selected ABR markers (cultured material/directly from blood samples) Actionable information: 8 hours Only one study High concordance between conventional ID from blood culture & this method Costly Robust

35 PCR/ESI-MS technology The base compositions of multiple amplicons from different regions of the genome are compared to an extensive database: Pathogen ID is achieved Not as informative as sequencing Broad bacteria and Candida detection assay (BAC assay; Ibis): identifies > 600 bacteria & Candida species. BAC assay also detects resistance genes (meca), (vana and vanb), and blakpc.

36 Especially relevant when the different microorganisms are present in different abundances Using several non-overlapping primer pairs may allow amplification of the less abundant species

37 New Version of PCR/ESI-MS Principal changes: larger volume of blood (5mL) 1-6 specimens can be analyzed at a time. Mass spectrometer: Bench-top instrument Better sensitivity for detection in direct clinical specimens. Further evaluations are currently underway.

38 Peptide Nucleic Acid Fluorescent In Situ Hybridization Molecular Stains PNA FISH stains (AdvanDx) Commercially available for direct identification of selected pathogens from + BC Targets species-specific rrna, (abundant in growing bacteria and yeast Kits are currently available to differentiate between S. aureus & CoNS; E.faecalis & Enterococcus species; E. coli, K.pneumoniae & P. aeruginosa; & Candida species. Turnaround time: ~ 90 minutes. ST & SP: 96%- 100%.

39 In 2013, a faster and less labor-intensive QuickFISH (AdvanDx) assay was introduced with a turnaround time of 20 minutes. Excellent sensitivity and specificity results, similar to those seen with the original PNA- FISH assays

40 Yeast Traffic Light PNA-FISH assay PNA-FISH assay with clear antimicrobial therapy implications 3-probe system that stains C. albicans & C. parapsilosis green, C. tropicalis yellow, C. glabrata & C. krusei red Color scheme designed to provide input on whether fluconazole therapy is likely to be effective. When associated with concomitant antimicrobial stewardship: reduced use of echinocandins and saved $1729 per patient

41 Assays for S. aureus and resistance determinants S. aureus: 20% of nosocomial BSI Empiric therapy with vancomycin β-lactams are superior to vanco for T/t of MSSA 2 FDA-approved real-time PCR assays that detect S aureus & MRSA from positive blood cultures: GeneOhm StaphSR (BD, Sparks, MD) Xpert MRSA/SA (Cepheid, Sunnyvale, CA) assays. Both have clinical accuracy >97% for detection of S. aureus and differentiation of MR

42 The Xpert MRSA/SA (Cepheid) Rapid, automated PCR for SA & MR in +BC. Novel multiplex real-time assay for 2 genes: staphylococcal protein A (spa) gene (specific for S. aureus) meca gene 98.3% 100% sensitivity 98.6% 99.4% specific < 1 hour

43 The Verigene assay (Nanosphere) Automates NA extraction from positive BC broth Has GP microarray panel for meca,vana/vanb & GN panel for MDR Labs can select which panel to test based on the Gram stain morphology. Polymicrobial BSI poses a challenge.

44 Challenges of NAT Use of whole blood is challenging. Excess of human DNA Hemoglobin Use of small volume of blood (1 to 5mL) Conventional culture methods use ml Bacterial load in BSIs can be as low as 1 10 CFU/mL; limitation for detection of pathogen DNA. Range of detection Antimicrobial susceptibility Technical complexity Effort Overall costs Differentiation of viable from nonviable microorganisms microbial DNA aemia? Higher sensitivity/ only DNA (degraded pathogens)

45 Economic Evaluation Decreased time of reporting Cost savings are derived from targeted deescalation of empiric broad-spectrum antimicrobial therapy Reduced time to appropriate targeted therapy Patient s length of hospital stay No economic evaluation studies that comply with guidelines for full economic evaluation done till date for rapid testing techniques

46 Clinical and economic impacts of rapid pathogen identification in positive blood cultures

47 Impact of GeneXpert MRSA/SA (Cepheid) on clinical outcomes Reduced mean time to initiation of appropriate therapy (49.8 hours to 5.2 hours) Significantly shorter LOS (6.2 days) cost saving of $ per patient Trend toward lower mortality rates (18% vs 26%).

48 Clinical and economic impact of MALDI-TOF for direct identification of organisms in positive blood cultures Using near real time antimicrobial stewardship based on MALDI-TOF results, time to adjustment of antimicrobial therapy was shortened by 46 hours in BSI. Significant decrease in ICU LOS by 1.2 days hospital LOS by 1.8 days Cost saving: $ per patient

49 Feasibility of Implementation Considerable capital investment Cost-per-test of the NATs: higher Implementation may be affected specific hospital environments Laboratory settings staff competencies specimen volume budget considerations Ability to provide active notification of test results to clinicians or pharmacists: Crucial

50 Implementing any new test in a hospital setting often encounters resistance efforts to control budgets related to reagents human resources other factors Selection of an appropriate laboratory technique that best suits an institution often depends on making a business case, demonstrating potential quality outcomes or cost- effectiveness metrics

51 Which Practices are best to improve patients outcome? Recent Meta-analysis

52 Interventions Rapid molecular technique, with additional direct communication Rapid molecular technique, with no additional direct communication Rapid phenotypic technique, with additional direct communication Rapid phenotypic technique, with no additional direct communication Rapid Gram stain Outcomes Time to targeted therapy is the primary outcome of interest

53 Results Rapid Gram stain reporting decreases time to targeted antimicrobial therapy decrease morbidity/mortality length of a hospital stay associated hospital costs

54 Rapid Molecular Techniques without Additional Direct Communication Treatment is inconsistent but often substantially faster than after standard testing

55 Rapid Molecular Techniques with Additional Direct Communication PNA-FISH method/ GeneXpert real-time PCR platform - MRSA. Timeliness of treatment in hospital settings is improved Significant and homogeneous reduction in mortality

56 Rapid Phenotypic Techniques with Additional Direct Communication Additional direct communication interventions were initiated by: ID fellow making recommendations directly to the physician in charge By laboratory staff immediately phoning ID & AST results directly to physicians By a clinical microbiologist phoning clinically relevant information and treatment advice

57 Generally substantial improvement over standard testing practices. Rapid phenotypic techniques with direct communication likely improves the timeliness of targeted therapy.

58 Impact on mortality? whether the implementation of a rapid technique and communication practice(s) reduces mortality. Strong correspondence between the timeliness of targeted therapy & mortality can be observed The relationship fails to reach significance.

59 IDSA Should ASPs Advocate for Rapid Diagnostic Testing on Blood Specimens to Optimize Antibiotic Therapy and Improve Clinical Outcomes? Comment: Availability of rapid diagnostic tests is expected to increase; thus, ASPs must develop processes and interventions to assist clinicians in interpreting and responding appropriately to results. Evidence Summary The use of rapid molecular assays and mass spectrometry is associated with improvements in time to initiation of appropriate antibiotic therapy, rates of recurrent infection, mortality, length of stay, and hospital costs

60 Should ASPs Work With the Microbiology Laboratory to Perform Selective or Cascade Reporting of Antibiotic Susceptibility Test Results? Comment: some form of selective or cascaded reporting is reasonable. After implementation, ASPs should review prescribing to ensure there are no unintended consequences.

61 Potential harm of rapid identification techniques Lack of timely and accurate detection of a BSI agent, despite rapid testing of positive blood culture bottles. Inaccurate identification of the microorganism might lead to inappropriate and ineffective changes in antimicrobial therapy. Polymicrobial If rapid methods are unable to detect them, outcome would be similar to that of the falsenegative result

62 BSI-causing microbes identified by the rapid technique may not behave according to the institutional antibiogram.

63 IDSA ASP Guidelines April 13, 2016 Should ASPs Work With the Microbiology Laboratory to Develop Stratified Antibiograms, Compared With Non-stratified Antibiograms? Stratification can expose important differences in susceptibility, which can help ASPs develop optimized treatment recommendations A single institutional, or hospital-wide antibiogram may mask important susceptibility differences across units within the institution

64 Potential harm False-positive (contaminated cultures) False-negative (insufficient growth in the blood culture) Rapid reporting of results may give physicians a false sense of accuracy, causing them to overlook the basic limitations inherent in the blood culture process itself.

65 Routine methods are still the definitive reference standard, and any discrepancies between a rapid method and the definitive culture result should be closely monitored

66 Future research needs Limited number of good-quality studies evaluating the impact of rapid testing on reducing the time to targeted therapy for hospitalized patients. Both immediate and longer-term outcome data necessary Impact in varied hospital settings, such as small or nonacademic institutions A better understanding of how batching and other such routines for testing samples

67 BIOMARKERS OF SEPSIS

68 C-reactive protein If CRP fails to reduce after Treatment: Signifies inappropriate treatment Increased Morbidity & Mortality Prognostic importance

69 Prohormone of calcitonin Normal Serum PCT levels: <0.1 ng/ml During sepsis, PCT is constitutively released from tissues Levels of PCT increase in 4 12 h after inflammation Circulating levels declines with treatment: Better kinetic profile than CRP and cytokines.

70 IDSA In Adults in ICUS With Suspected Infection, Should ASPs Advocate PCT Testing as an Intervention to Decrease Antibiotic Use? Recommendation: Suggest the use of serial PCT measurements as an ASP intervention to decrease antibiotic use

71 Lipopolysaccharide-binding protein Acute-phase protein stimulated by IL-6 & IL-1 Baseline levels low (1 15 g/ml) Greatly increase during infection Limitations: it cannot differentiate between recent and old infection Recently: Soluble form of CD14 used as a biomarker of sepsis because its levels are equivalent to PCT.

72 Inflammatory cytokines TNF-α, IL-1β, IL-8, and IL-6: released in infections In GN sepsis, TNF and IL-1β levels elevated. IL-6 can be measured more reliably in plasma; long half-life Elevated levels of IL-6 in septic patients are associated with an increased mortality

73 Chemokines Many inflammatory chemokines: considered as potential biomarkers of sepsis Chemokine IL-8 for diagnosis of sepsis Monocyte chemoattractant protein-1 (MCP) for prediction of sepsis mortality

74 Pro-adrenomedullin and pro-vasopressin Serum levels rise during the early phase of septic shock; decrease during later phases Pro-ADM: excellent prognostic biomarker for the severity and outcome of sepsis

75 Pentraxin Belong to the superfamily of proteins involved in acute immunological responses which act as pattern recognition receptors. Increased levels of PTX3 are associated with the severity of sepsis.

76 Macrophage migration inhibitory factor Distinguishes among survivors and nonsurvivors Fails to discriminate infectious from noninfectious causes of inflammation.

77 New biomarkers Triggering receptor expressed on myeloid cells-1 (TREM-1) bacterial or fungal infections induces expression of TREM-1. Sensitivity and specificity: more than CRP and PCT.

78 CD163 Trans-membrane molecule Until now only revealed on the membrane of mononuclear phagocytes. Blood levels of serum scd163 can be used as a biomarker of inflammatory diseases

79 micrornas Type of endogenous noncoding small RNAs with approximately 22 nucleotides in length. Circulating mirnas have been recently identified as promising biomarkers for sepsis. mir-150 might be in related to immune system dysfunctions in sepsis;

80 SUMMARY Septicemia remains a major cause of hospital mortality. Rapid pathogen ID in BSI can lead to improved clinical outcomes, shorter hospital stays, lower health- care costs. Rapid pathogen ID, with/ without ABR genes enables targeted treatment Several rapid pathogen identification methods, such as PNA-FISH, MALDI-TOF, PCR, multiplex microarrays, are being applied to positive blood cultures.

81 PNA-FISH: Well-validated method; new QuickFISH system has reduced turnaround time to 20 minutes, enabling species- identification results to be reported in the same time frame as Gram staining. Application of MALDI-TOF directly to positive blood cultures: in experimental phase; has the potential to identify a much broader range of organisms than PNA-FISH.

82 PCR-based methods, including GeneXpert (1 hour), FilmArray (1 hour), and Verigene (2.5 hours), are somewhat slower than QuickFISH and MALDI-TOF but have little or no sample processing and include selected antibiotic resistance genes.

83 It is now technically feasible to amplify microbial pathogen NA targets directly from blood samples Large body of literature, evaluating the accuracy of systems such as SeptiFast, SepsiTest, & most recently, T2 MR.

84 Molecular approaches provide information that is clinically relevant and complementary but not equivalent to that provided by conventional blood culture methods. Before these methods can be adopted and gain wide- spread acceptance, clinicians must learn how to use the information in managing BSIs.

85 None of the methods will replace subculturing positive blood culture isolates to agar plates for definitive identification and antimicrobial susceptibility testing. Adoption of such technologies involves additive clinical laboratory costs, expertise.

86 Clinical and economic benefits only when such approaches are combined with a robust AMSP to help translate the results from the laboratory to the end users (ie, clinicians) and help them make informed patient care decisions. ACTIVE LIASONING, COMMUNICATION

87 QUALITY PROGRAM For hospital patients with bloodstream infections, lack of timely identification of the microorganism and targeted therapy results in preventable adverse patient outcomes (e.g., mortality) PREVENTABILITY/IMPROVEMENT Time to report organism identification commonly ranges from hrs PRACTICES/ INTERVENTION Rapid Gram stain Rapid molecular technique Rapid molecular technique, with rapid/direct communication Other rapid phenotypic technique Other rapid phenotypic technique, with rapid/direct communication IMMEDIATE OUTCOMES Time to target therapy Time to report organism identification HEALTH CARE/ HEALTH OUTCOMES Mortality Broad-spectrum antibiotic use Associated hospital costs Hospital length of stay ASSOCIATED HARMS Lack of timely detection of BSI agent Inaccurate identification of agent and target antimicrobial Atypical microorganism susceptibility not defined by rapid technique Reporting results for false positive blood cultures False negative results LMBP QI analytic framework: BSI evidence review question. For hospital inpatients who are admitted for, or are found to have BSIs, what practices are effective at increasing the timeliness of providing targeted therapy to improve clinical outcomes?