Molecular Diagnostics in ID: Boon or Bane
Dr Ramasubramanian
The story so far…
• Rapid
• Highly sensitive & specific
• Detection limit
• Decreased TAT
• Noncultivable or slow growing pathogens
• Defining transmission
• Determining drug resistance
• Monitoring treatment efficacy
Peer Pressure...
Ram
Gopalakrishnan
Molecular Diagnostics
• Diagnostic Sensitivity – ability to detect a disease
• Diagnostic Specificity – degree to which an assay is negative when the disease is absent
• Analytical Sensitivity – lowest detection limit
• Analytical Specificity – check cross reactivity with other organisms
• Reproducibility – Intra & Inter batch variations
Courtesy Dr Camilla Rodrigues
Clin Chem Lab Med 2019; aop
Bane of Rapid Testing (PCR)
• Requires infrastructure
• Experienced staff
• Expensive
• Multiple genes involved
• Dual infections & dead organisms
• Silent mutations
• Validation
Dtsch Arztebl Int 2018; 115: 822–32
Dtsch Arztebl Int 2018; 115: 822–32
Sample Shipping & Storage
• Characterize specimen
• Optimal collection
• Storage
• Handling & shipping
FEMS Immunol Med Microbiol 49 (2007) 184-91
False Positives & Negatives
• Contaminated lab reagents & equipment
• Environmental contamination
• Mixed infections
• Timing of collection
• Inhibitory substances
False negatives in PCR
• Specimen level
▪ Inhibitory substances e.g. haem, storage
▪ Specimen
▪ Paucibacillary samples
▪ Unequal distribution in
samples
• Extraction level
▪ Loss of DNA
▪ Insufficient lysis
▪ Reagents - SDS, ethanol
▪ Improper vortexing
• PCR level
▪ Less Mg 2+ ions
▪ Incomplete denaturation
▪ Incorrect primer sequence.
▪ Poor thermal conductivity
• Others
▪ Glove powder
Courtesy Dr Camilla Rodrigues
Biological Relevance
J Clin Microbiol June 2013:51;6: 1841-49
Now What ???
Parkinson’s Sixth Law – The progress of Science varies
inversely with the number of journals published
Proficiency Testing & Quality Management
• Standards must be equivalent
• Signal to noise ratio monitoring
• External proficiency testing
• Rigorous quality control
• Specimen stability & integrity (storage times, transport times, anticoagulants used)
• QA of equipments
IT Issues
• Understanding character & coverage of database
• ‘Polluted database’
• Laboratory cooperation
• Integration of LIS & interpretation of tests
“ Diagnostic resistance ”
• Adaptive evolution
• Gene deletions / new mutations
• Resistance genes vs. expressed resistance & vice versa
Cost
• Instruments
• Probes
• Type of assay
Open Forum Infectious Diseases.4,4. 1-3. (2017)
Health Economics and Evidence:
EDITORIAL COMMENTARY • CID 2018:66 (1 June) • 1687Am J Clin Pathol 1974:61;867-72
Lets take a step back…
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