Molecular Dx in Bacterial Infections

1. Under IAP President’s Action Plan 2023
Dr Upendra Kinjawadekar – President IAP 2023
Dr Vineet Saxena – Hon Secretary General
Dr Bhaskar Shenoy – National Convenor
Dr Maninder S Dhaliwal – Joint National Convenor
Dr Vasant M Khalatkar – National Co-ordinator

2. Lecture 5
PCR Based
Molecular tests

3. Culture
Broad Spectrum Targeted Treatment
Sepsis
Empiric Treatment
Gram stain
Day 1
Traditional Methods
Day 3-4
Day 2
Multiplex PCR
Allows identification of GP
and GN organisms and
resistance determinants In
1-3 hours
Rapid Methods
Empiric Treatment Targeted Treatment
Rapid tests identify the organisms 24-48 hours earlier than traditional methods
Palavecino E. Rapid Methods for identification of MRSA.
MRSA protocols 2nd Edition. 2013
Susceptibility

4. Future is Now

5. Examples of Molecular Tests by Complexity Level
COMPLEXITY
Sequencing
Genotyping
Quantitative PCR: Viral Loads
Multiplex PCR
Respiratory, Blood Cultures ,Meningo
Encephalitis panel
Two-Three Targets: FluA and B, RSV ,Adeno
One Target: Group B streptococci, MRSA, C difficile

6. Advantages
Faster Results
Shorter Time to Optimal Therapy
Improve Treatment Decisions
Avoid Unnecessary Antibiotics
Support Antimicrobial Stewardship Efforts
Reduce Unnecessary Testing
Reduce Healthcare Costs

7. Fully automated: Extraction, amplification and detection
Automated amplification and detection. Requires separate NAextraction
Examples of Platforms/Instruments
GeneXpert
BD Max
3M Integrated
Cycler
LightCycler eSensor Illumigene
Panther

8. For identifying the pathogen in an infectious disease
For identifying the mechanism of resistance
For monitoring treatment with viral loads , ex:CMV, HIV etc
For infection control , example MRSA screening
For outbreak investigation
For knowing variant. Ex delta/omicron
Role of PCR

9. Conventional PCR
Real time PCR monitors DNA amplification in real time, not at its end as in conventional
PCR. Ex-Genexpert
Reverse Transcriptase (RT) PCR-creates complementary DNA (cDNA) by reverse
transcribing RNA to DNA using reverse transcriptase . Ex- RT-PCR COVID test
Multiplex PCR- simultaneous amplification of different target genes by using different
primers in same PCR reaction. Ex: Multiplex panel Biofire
Types of PCR

10. FLU A
Seasonal A/H1 or H3
2009 H1N1
FLU B
RSV
ParaFlu 1, 2,
3 and 4
hMPV
Adenovirus
Rhino/
Enterovirus
Multiplex PCR: Detection and differentiation of respiratory viruses
Coronavirus
4 subtypes
1. B. pertussis
2. Mycoplasma
pneumoniae
3.Chlamydophila
pneumoniae
Film Array Respiratory Panel (BioFire) Detects 20 respiratory pathogens.
NP swab

11. Case 1
4 year old
High fever and cough since 2 days
O/E congested eyes, tachypnea. Saturation 88%, Bilateral wheeze
CBC Hb 11.8 g%, WBC 5460. N45 L46, platelets 2 lakhs. CRP 116
Started on oseltamivir

12. Would you like to discontinue Oseltamivir?
Yes, here oseltamivir has no action on adenovirus

13. This NP panel has detected both Adenovirus and
Streptococcus Pneumonia. Is this a coinfection?
May not be true as about 30% healthy children
have streptococcal pneumonia carriage in
upper respiratory tract.
Lower respiratory tract sample more reliable
for bacteria.

14. Upper respiratory panels
– Specimen -Throat swab/ Nasopharyngeal swab
Lower respiratory panel
– BAL, sputum expectoration, ET aspirate
Respiratory panels

15. • Can only detect targets for which there exist PCR primers.
For example –cannot detect stenotrophomonas maltophilia (can cause VAP
in 1-5%)
• Resistance gene detected may not belong to organism detected
• Cannot give comprehensive antibiogram unlike cultures
• Colonisers versus pathogen challenging especially for oganisms which
can be both – strep pneumonia, Hib, Moraxella, etc)
• Can only detect the NA, can not differentiate between living and dead
organisms, and hence can not be used to determine the end of therapy.
Limitations

16. Collection of Nasopharyngeal aspirate
Improper sample collection will only lead to detection of multiple organisms creating more confusion.

17. Collection Technique - NPA

18. • Samples can be refrigerated at 2 to 8 deg C b
efore transport to laboratory
• The extract can be stored below -70 deg C for
long duration
Storage of NP sample

19. 6 year old
Fever and cough since 5 days. Sick looking , requiring oxygen support.
Hb 12.2, TLC 5760 N76L21, platelets 1.56. CRP 115
Case 2

20. Started on IV Co-amoxiclav, Vancomycin and azithromycin

22. • Staphylococcal and Streptococcus not detected.
• Vancomycin is to be discontinued?
• No, as NP not a good specimen. Clinical co-relation important
•Oseltamivir added. Is that right?
•Yes, because of detected of influenza coinfection
•How significant is pseudomonas on NP PCR?
•NP is not a reliable specimen for LRTI.
•Pseudomonas unlikely to be significant for healthy child.
• Significant if underlying cystic fibrosis or immune compromised.

23. Monsoon setting. 8 year old, high fever since 3 days with chills, red eyes, bodyache.
Dengue NS1 negative, SGOT 456, SGPT 312
Leptospirosis suspected.
How to confirm?
Case 4
Tropical Infections like Leptospirosis and Ricketssia are
best diagosed within one week of onset using PCR

24. Child with ALL on relapse
Fever since 6 days
No focus of infection
Was treated 10 days back with IV ciprofloxacin for blood culture which grew Ralstonia
mannitolytica as per sensitivity report. Port was removed during the episode.
Blood culture had become sterile, again developed fever
Sepsis PCR panel sent
Case 5

25. Blood culture is sterile
Is there a way to find out causative organism?

28. H/o recurrent boils in 4 year old.
MRSA screening done
Case 7

29. Utility of PCR for screening of MRSA colonisation
Child can be prescribed Mupirocin local application BD for 5 days

30. Case 8
• 8 days premature baby, birth weight 1.2 kg, develops fever and episode of apnea on
Day 6 of cefotaxime plus amikacin.
• Lab reports TLC 20870,PCT 35.96. antibiotics stepped up to meropenem + vanco
• Blood culture flags gram negative growth.
• Identification will happen only next day. Vancomycin stopped
• Is there need for stepping up further gram negative cover?

31. IMP not detected
VIM Not detected
NDM Not detected
KPC Not detected
OXA48 Not detected
Since Carbepenemase was not detected, meropenem can be continued.
Later blood culture grew Burkholderia cepacia sensitive to piperacillin-tazobactam.
This PCR guides timely antibiotic stewardship

32. Conclusions
• Early and accurate diagnosis of infections and appropriate antimicrobial therapy
correlate with positive clinical outcomes
• Several molecular fully automated platforms are available for rapid diagnosis of
infectious diseases and are becoming a useful tool in diagnostic armamentarium
• Curbs unnecessary antibiotic use
– Limit unnecessary/increased appropriate antiviral use
– Limit other laboratory testing/radiology – sepsis workup children
– Manage high-risk patients
- Reduce hospital stay or time in the ER
– Rapid outbreak identification of influenza
– Prevent or limit community spread
– Characterize epidemiology of virus infections

33. End of lecture 5
PCR tests can
recognize
target genes

34. End of lecture 5
Slides prepared by : Dr Maninder
U.L.T.R.A.
Module