PPT MOLECULAR DIG gram +ve bacteria

1. Molecular diagnostic methods for detection of drug
resistance in gram positive bacteria
Dr. Ajit Kumar singh
MD(Laboratory Medicine) 2nd year PGT
Chittaranjan National Cancer Institute
Newtown Kolkata 700160
Moderator
Dr Subhranshu Mandal
MD microbiology
Associate Professor
Department of Laboratory Medicine
Chittaranjan National Cancer Institute
Kolkata 700160

2. Introduction
• Antimicrobial resistance (AMR) among bacteria is an escalating public
health emergency
• 1.27 million deaths were attributed to bacterial AMR in 2019
• If left unchecked, AMR may lead to an estimated 10 million deaths
each year by 2050
• Global increases in AMR during the COVID-19 pandemic, largely
driven by antibiotic overuse and breakdowns in infection control
Ref-JAC Antimicrob Resist
https://doi.org/10.1093/jacamr/dlad018

3. Introduction
• Patients with infections caused by antibiotic-susceptible organism
• Narrow-Spectrum agents can decrease antibiotic pressure
• Reduce selection for increase antibiotic-resistant species
• Decrease cost and minimize microbiome disturbances

4. Currently used methods for antimicrobial
susceptibility testing

5. • Microorganism has been recovered in pure culture
• Require a large inoculum
• Take several days for final results
• Miss pathogens present at low levels
• Less expensive than Genotypic diagnostics, provide clear information
about
• Both resistance and susceptibility
• MIC value
Phenotypic identification methods

6. Genotypic AST methods
• Detect the presence of genes or mutations that predict AMR
• Provide results within a few hours (sometimes a few minutes)
• Performed on isolated bacteria
• Performed directly on patient specimens without culturing it
• Genotypic AST methods detect one or a small number of resistance
factors
• Syndromic panels detect multiple microorganisms and resistance
genes/mutations from a single specimen
Ref-https://doi.org/10.1093/jacamr

7. Phenotypic methods vs Genotypic AST methods

8. Important Gram-Positive

9. Resistance Mechanisms in Clinically
Important Gram-positive Pathogens

10. Resistance Mechanisms in Clinically
Important Gram-positive Pathogens

11. Resistance Mechanisms in Clinically
Important Gram-positive Pathogens

12. Antibiogram for Staphylococcus spp.
CLSI M02 and CLSI M07

13. Antibiogram for Enterococci
CLSI M02 and CLSI M07

14. Antibiogram for Pneumococci
CLSI M02 and CLSI M07

15. Contaminated Surfaces
and Shared Items
Frequent Contact
Cleanliness
Crowding
Compromised Skin
Factors that Facilitate Transmission
Antimicrobial
Use

16. Susceptible groups
Diabetics
Immunocompromised (HIV, Cancer, Transplant, lupus)
Extended hospital stay
Indwelling catheters/ prosthetic devices
Elderly
Dialysis patients
IDUs
H/o MRSA

17. Detection of drug resistance in gram positive bacteria
Traditional Culture-Based Methods
Molecular Techniques
Innovative Approaches
Mass Spectrometry-Based Methods
Bioinformatics Approach for Detection of AMR Genes and Databases
Microfluidics, Biosensors and Nanotechnology
Future Perspectives and Emerging Technologies
Nanopore Sequencing.
Digital PCR
The Integration of CRISPR-Cas Systems with Aptamers
Machine Learning and Predictive Analytics

18. Molecular Techniques
• Nucleic Acid Amplification Technology (NAAT) in AST
• Polymerase Chain Reaction (PCR) and Multiplex PCR
• Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
• PCR Combined with Restriction Fragment Length Polymorphism (PCR–RFLP)
• Real-Time Polymerase Chain Reaction (qPCR)
• Isothermal Amplification Methods
• Next-Generation Sequencing (NGS)
• Whole-Genome Sequencing (WGS)
• Metagenomics for Antimicrobial Surveillance
• DNA Microarray
• Fluorescence In Situ Hybridization (FISH)

19. Conventional Polymerase Chain Reaction
• Conventional PCR comprises three steps:
• (i) Denaturing of the double stranded DNA at 95°C
• (ii) Annealing of the PCR primers at 50 to 60°C
• (Iii) Extension of the DNA at 72°C
• The PCR-amplified gene product can be visualized by running agarose
gels and staining, DNA with ethidium bromide or other fluorescent
DNA chelating dyes.
• The whole process, including amplification and visualization, can take
between 4 and 5 h.

20. Conventional Polymerase Chain Reaction
Source: Chittranjan national cancer institute , Department of molecular pathology

21. Reverse Transcriptase Polymerase Chain Reaction
• The process of RT-PCR involves transcribing an RNA molecule into a
complementary DNA molecule (cDNA) and amplifying it using PCR
• Compared to DNA molecules, cDNA molecules generated from the original
RNA have a higher degree of purity, as they lack contaminants such as
proteins that may affect the accuracy of the test
• As a result, cDNA is more specific and can be more easily detected by
primer
• This technique is
• Specificity
• Sensitivity
• Reliability

22. Real-Time Polymerase Chain Reaction (qPCR)
• Based on PCR technology
• Amplify and detect or quantify a target DNA
• Real time basis
• Advantages
• Quantitative
• Take less time
• Contamination rate
• Sensitivity and Specificity

23. Real-Time Polymerase Chain Reaction (qPCR)
• Detection of amplification products of real time PCR by
• Nonspecific method use SYBR green dye
• Specific method use fluorescent labeled oligonucleotide prob
• Two type of hybridization probes are commonly used
• TaqMan
• Molecular beacon
• Post amplification melting curve analysis is used for quantification of the
nucleic acid load

24. Real-Time Polymerase Chain Reaction (qPCR)
Source: Chittranjan national cancer institute , Department of molecular pathology

25. What is Syndromic Testing?

26. Traditional vs. Syndromic Testing
1. Rogers BB et al. Arch Pathol Lab Med. 2015;139:636-641.
2. Brendish NJ et al. Lancet Respir Med. 2017:401-411.

27. Traditional Diagnostic Testing Makes the
Clinician Choose Among Speed, Accuracy, and
Comprehensiveness
PCR=polymerase chain reaction; MDx=molecular diagnostic
1. Blaschke AJ. Diagn Microbiol Infect Dis. 2012;74(4):349-355.
2. Mardis ER. Annu Rev Genomics Hum Genet. 2008;9:387-402.

28. Other Potential Cost Savings Benefits
↓ ED wait times ↓LOS
↓ TAT ↓ isolation times
↓ Hospital-acquired
infections
↓ chest radiographs
↓ morbidity
↓ ABX use/duration ↓ ancillary testing ↓ admissions ↑ patient satisfaction
↓ contamination risk

29. The BioFire FilmArray System
All-in-one integration of
Sample
preparation
Amplification Detection

30. The BioFire FilmArray System
A simple workflow that can be performed
Any
time
Any
tech
Any
size institution
Any
shift

31. Pouch Preparation
5. The lid of the sample injection vial is closed and
the vial is inverted 3 times to mix the sample
6. The sample/buffer mixture is injected into the
pouch through the red inlet port
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2. Hydration solution is injected into the
pouch through the blue inlet port
3. Sample buffer is added to the sample
injection vial
1. The pouch is inserted into the
loading block
The BioFire® FilmArray® Instrument is
now ready to set-up
2 minutes of
hands-on
time
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4. The sample is added to the sample injection vial
using the transfer pipette
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32. Sample Extraction, Amplification, and
Detection: It’s All in the Pouch
The Pouch is loaded into the
BioFire® FilmArray® Instrument

33. What Happens During the Run?
8. The BioFire® FilmArray® Instrument performs a melt to
confirm the presence or absence of assay-specific
temperature signatures of the second-stage PCR
product for each well in the array.
7. Each well is pre-spotted with a single pair of second-stage PCR
primers, resulting in specific amplification of target DNA only. A
fluorescent double-stranded DNA binding dye monitors each
reaction.
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run-time
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6. Products from the first-stage PCR are diluted to remove any
remaining PCR primers, combined with fresh master mix, and
prepared to be aliquoted into each well of the array.
5. Nucleic acid moves to the first-stage PCR chamber.
Reverse transcriptase converts target RNA to DNA,
followed by a high-order multiplex PCR.
4. Elution buffer allows the nucleic acids to be released from
the beads. Beads and other waste products are pushed
back to previous blisters.
3. Nucleic acid is captured by adsorption to magnetic beads. A
series of wash steps remove proteins, cellular debris, and
other potential PCR inhibitors.
2. Nucleic acid moves from the lysis chamber to the first
purification chamber.
1. Sample moves into lysis chamber where bead beating
occurs. Ceramic beads break up cells and viruses and
release the nucleic acid.

34. Pathogen Identification
In order for an organism to be identified as positive:1
• Two out of three wells must be positive with a melt peak
• Melting peaks must fall within their assay-specific temperature range
• Melting peaks must be significantly similar to each other
• Both internal controls must pass (RNA/DNA process and PCR 2)
1. FilmArray GI [Instruction Booklet]. Salt Lake City, UT: BioFire Diagnostics, LLC.
2. Ririe KM. Analytic Biochem. 1997;245:154-60.
Melting curve analysis2

35. Detection Summary

36. Thank you…

37. Easy
• No sample pre-processing
• Sample buffer added to FAIV
• Process is the same for sputum and BAL
sample types:
1. With flocked swab, stir sample for
10 seconds
2. Swab breaks off into sample FAIV
• Swab comes in the pneumonia panel kit
FAIV = FilmArray Injection Vials

38. Reporting Timeline for Species Identification
and Susceptibilities
Time from Positive Blood Culture Result Available to Clinical Team
1-2 hours Gram stain
2-8 hours Gram-positive organism identification
and resistance mechanism, inpatient only
Verigene results of Gram-positive
organisms only
12- 15 hours Gram-negative preliminary
susceptibilities reported
Disk diffusion results for Gram Negative
only
24-48 hours Definitive organism identification MALDI-TOF results confirming
Verigene results for Gram-positive
organisms or new identification for
Gram-negative organisms
48-72 hours Definitive susceptibility results Traditional susceptibility testing to
confirm Verigene resistance
mechanisms for Gram-positive
organisms and disk diffusion results
for Gram-negative organisms

39. Timeline of major molecular techniques for the
diagnosis of bacterial infections

40. [2002]

41. Staph aureus today!!
Most common cause of skin and soft tissue infections
MC cause of cellulitis, osteomyelitis, septic arthritis, surgical wounds
MC cause of nosocomial infections
MC cause of health care associated endocarditis (52%) and in IDUs
(57%)
Common cause of bacteremia, foodborne disease, implant infection,
abscess etc

42. The ambler classification of β-lactamases, which is
based on each enzyme’s primary protein structure

43. Mechanism of Methicillin Resistance
 Horizontal transfer of
genomic island
SCCmec
 Contains gene mecA
 Produces PBP2a
protein responsible
for resistance