multiplex pcr and early diagnostics for sepsis

1. BIOFIRE: UTILITY IN
CRITICAL CARE
DR ADITYA NATH SHUKLA
REGENCY HEALTHCARE
KANPUR

2. SEPSIS AND MORTALITY
• Key contributor to mortality ( 10-45%)
• Prompt recognition and appropriate management is key to success
• Early initiation of appropriate antimicrobials is key to survival
• Early identification of pathogen is key
• Conventional diagnostic methods have prolonged TAT
• Need for rapid diagnostics

3. A FAST DIAGNOSIS CAN ENSURE TIMELY TREATMENT,
WHICH MAY REDUCE MORTALITY
26%
42%
61%
0
10
20
30
40
50
60
70
Infection/sepsis Severe sepsis Septic shock
Mortality
rate
(%)
Mortality Rate of Sepsis, Severe Sepsis, and Septic
Shock1
Timely treatment is essential to prevent the progression of sepsis to septic
shock and reduce mortality1-3
1. Alberti C et al, for the European Sepsis Study Group. Am J Respir Crit Care Med.
2005;171(5):461-468.
2. Shorr AF et al. Crit Care Med. 2011;39(1):46-51.
3. Moore LJ et al. Surg Clin North Am. 2012;92(6):1425-1443.

4. SEPSIS AND ANTIMICROBIAL RESISTANCE
• Delay in appropriate antimicrobials is directly related to mortality
• Explosion in use of broad spectrum antibiotics
• Hesitancy and resistance to de escalate antibiotics
• Emergence of antibiotic resistance
• Not all sepsis is because of bacteria (high TAT for cultures)
• Need to conserve use of antibiotics to prolong them

5. GOALS OF RAPID DIAGNOSTIC TESTING (RDT)
• Decreases time to identification of pathogen from days to hours
• Can distinguish between viral vs bacterial infections
• Allows streamlining to appropriate therapy
• Facilitate the decision about whether or not to prescribe
antibiotics
• Prevent the use of unnecessary antibiotics
.

6. GOALS OF RAPID DIAGNOSTIC TESTING (RDT)
• Facilitates de-escalation
• Decreased antimicrobial resistance
• Many studies have shown decreased
• Duration of illness
• Length of stays
• Costs
• 30-day all cause mortality
.

7. IMPACTS OF RAPID DIAGNOSTIC TESTING IN
OPTIMIZING ANTIMICROBIAL SELECTION
• Source: adapted from Goff, DA et al. (2012) Using rapid diagnostic tests to optimize antimicrobial selection in antimicrobial stewardship programs. Pharmacother 32(8): 677-687.

8. RAPID DIAGNOSTIC TESTS
GRAM-POSITIVES, GRAM-NEGATIVES, YEAST
• PCR (polymerase chain reaction)
• GeneXpert (Cepheid); FilmArray (Biofire) & others
• Turnaround time @60min
• PNA FISH (peptide nucleic acid fluorescence in situ hybridization)
• GNR Traffic Light PNA FISH (AdvanDx), Yeast Traffic Light PNA FISH (AdvanDx)
• Turnaround time @90min
• Nucleic Acid
• Verigene (Nanosphere)
• Turnaround time @120 – 150min
• MALDI-TOF matrix-assisted laser desorption ionization-time of flight
• MALTI Biotyper (Bruker Daltonics); VITEK MS (bioMerlieux)
• Turnaround time 10-30min; very expensive; no resistance markers

9. BIOFIRE
• Multiplex realtime PCR assay
• Syndromic approach to identification of pathogen
• Tests for a comprehensive group of targets
• Targets updated over period of time with new emerging pathogens of concern
• Identification of mechanism of resistance
• Results are fast and accurate if performed appropriately

10. • Tests for a variety of
pathogens that
cause respiratory,
blood, and
gastrointestinal
infections, as well
antimicrobial
resistance genes
Comprehensive
• Run time of about
1 hour
Fast
• 2 minutes of
hands-on time
Easy
FILMARRAY:
THE FASTEST WAY TO BETTER RESULTS

11. BIOFIRE FILM ARRAY PANELS
• Tests for viruses, bacteria, yeast, parasites and resistance genes
• Panels available:
• Respiratory
• Blood culture identification
• Gastrointestinal
• Meningitis/ encephalitis
• Pneumonia
• Bone and joint infection

12. RESPIRATORY PANEL
• Under EUA by FDA
• 15 viruses and 4 bacteria including (SARS COV 2)
• Sample nasopharyngeal swab
• TAT45 min
• Identified

14. • Comparative study pre and post
implementation of rapid respiratory panel (
Biofire)
• Patiients > 3months of age
• Acute respiratory illness
• Prior to implementation of RRP all patients
were subjected to PCR for RSV, Influenza A & B,
Para influenza 1-3

15. Shorter time to identification of
pathogen
Shorter time duration of antibiotic use
Shorter length of hospital stay
Shorter time of isolation stay

16. MENINGITIS/ ENCEPHALITIS PANEL
• 14 pathogens
• Sample CSF (0.2ml)
• TAT of 1 hour
• CSF culture positive only 75-80%, PCR can be used to rule out bacterial
meningitis and thus reduce antibiotic use
• >10% of all viral encephalitis have normal CSF findings, all viral meningitis
should be initiated with acyclovir and continued till second PCR is negative

17. ME PANEL

18. • 13 studies
• 3764 pts
• 3000+ pooled In analysis
• Senstivity 90% specificity
97%
• 11 % false positive (
streptococcus
pneumonia)
• False negatives HSV1-2,
enterovirus and
cryptococcus
• Index methods ( bacterial
culture, viral PCR,
Cryptococcal Ag of fungal
culture

19. OUR EXPERIENCE
• 59 yrs male
• h/o fever 3 days
• Altered sensorium for 24 hrs
• Corresponding blood sugar
88mg%
• CSF Biofire ME panel:
• Found to be HIV +ve

20. OUR EXPERIENCE
• Treatment modified on D1 as
per CSF ME panel
• ART added as CD4 counts low
D4
• Antibiotic cover modified
• Patient discharged in 10 days
time

21. OUR EXPERIENCE
• 66 yrs old /Female
• Non diabetic
• Left D4-5 dermatomal vesicular
lesions for 4 days
• Altered sensorium and restless 2
days
• MRI: non committal
• CSF: clear, Sugar 53, Proteins84,
cells60 all mononuclear, few RBC

22. OUR EXPERIENCE
• 66 yrs old /Female
• Non diabetic
• Left D4-5 dermatomal vesicular
lesions for 4 days
• Altered sensorium and restless 2
days
• MRI: non committal
• CSF: clear, Sugar 53, Proteins84,
cells60 all mononuclear, few RBC
• Most commonly used panel in our setup
• Able to identify pathogen in less than 6 hrs time
• Treatment titrated accordingly
• Able to reduce antibiotic use

23. PNEUMONIA PANEL
• 33 targets
• Semiquantitative results
• Sample: sputum, BAL, MiniBAL
• TAT 1 hr
• Resistance genes

24. PNEUMONIA PANEL
• Claimed sensitivity of 96.2% specificity of 98.3%
• Found to vary between 75% to 91% against cultures
• Data limited in ICU population ( most on antibiotics and frequent MDRO)

25. OUR EXPERIENCE
• 85 years old female
• k/c COPD , DM2 on inhalers
• Presented with fever for 2 days, cough for 2 days breathlessness for 1 day
• CXR : Diffuse opacities bilateral lower lobes
• ABG: respiratory acidosis with hypoxia, intubated and ventilated
• Routine investigation, ET secretions for stain, C&S, Biofire pneumonia panel
• S Procal 2.5 ng, CRP 28
• Broad spectrum antibiotics, nebulized bronchodilators, steroids other supportive
management

26. OUR EXPERIENCE

27. OUR EXPERIENCE
• Tt Modified
• Antibiotics descalated
• Patient improved with routine management
• Weaned off ventilator

28. • 24 yrs old female
• k/c autoimmune encephalitis, seizure
disorders
• History of recent hospital admission
for refractory status
• Presented with c/o fever 2 days,
depressed consciousness 1 day,
respiratory distress 1 day
• In shock with resp distress
• Intubated ventilated and resuscitated
• TLC 750, S PCT 15ng
• CXR Left lower lobe consolidation
• HRCT < B/l Basal consolidation
• Biofire : BAL

31. • Pneumonia panel only provides semiquantitative results
• Does not distinguish between dead and live bacteria and virus
• Negative report does not totally rule out bacterial/ viral infection
• Concomitant cultures are needed for recovery of isolated and
susceptibility results
• Detection of resistant genes can guide choice of initial antibiotic cover
which has to be modified as per report of culture and clinical response

32. BLOOD CULTURE IDENTIFICATION PANEL
• 43 targets ( Bacteria both Gram +ve and –ve; yeast and
resistance genes
• TAT 1 hr
• Sample is positive blood culture

33. BIOFIRE: ADVANTAGES VIS A VIS CULTURE
• Fast time to pathogen detection and identification of resistant profiles
• detection of virus and atypical pathogens
• Multiple targets at same time
• Detection of pathogens even with ongoing antibiotics
• Better utilization of antibiotics
• Potential positive impact on : nosocomial pneumonia management,
LOS, cost of treatment, antimicrobial stewardship program, better
infection control practices, prevention of spread

34. BIOFIRE: DISADVANTAGES VIS A VIS CULTURE
• Over detection ( problems with clinical decision making)
• Presence of resistant genes may not be due to true
pathogen
• Initial cost of equipment and test panels
• Not widely available
• Needs further studies for validation of results