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…