Use of molecular methods for diagnostics of infectious diseases
Tiit Talpsep
The 7th Baltic Congress in Laboratory Medicine
Pärnu, September 9 - 11, 2004
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Diagnostic methods in microbiology
Task of the method – to make the microorganism “visible” and “measureable”
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
•Microscopy•Cultivation•Bio-testing•Immunological methods•Biochemical methods•Molecular methods
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•What is molecular diagnostics ?
•Why to use molecular diagnostics ?
•In-house or commercial PCR ?
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Topics to be discussed:
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Molecular methods are necessary if the traditional methods provide poor
results
• Microscopy gives false positive results - - T.vaginalis, N.gonorrhoeae
• Intracellular pathogens – viruses, M.genitalium • Low sensitivity – Chlamydia
sp.,Neisseria sp.• Seropositivity is common – Chlamydia sp.• Subtyping is mandatory – HSV, HPV, HCV• Microbial growth is slow – M. tuberculosis
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
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Diagnostic methods for detection of Chlamydia trachomatis -
the most common STD in EuropeWHO: - sensitivity > 90% specificity > 99%
- serology – for scientific purpose only
Method Sensitivity Specificity Price(€) Comments
Cell culture 40-85% 100% 6,3 Low sensitivity
Immunofl. 50-90% 85-95% 5,2 Needs expertise
Gen-Probe 70-85% 95-97% 10,9 Low sensitivity
PCR 70-95% 97-100% 10,4 ?Contamination!
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
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Molecular diagnostics – how it works?
• Every organism contains some unique,
species specific DNA sequences
• Molecular diagnostics makes the species specific DNA visible
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
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Molecular diagnostics is a set of methods to study primary structure (sequence) of DNA
•Hybridization with complementary sequences
•Amplification (synthesis) of species specific sequences
PCR – polymerase chain reaction
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
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Key issues
1. place – who & where, what & how
2. price – how much does it cost
3. proficiency - how much high quality information it provides
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PCR laboratory
Sample handling
DNA preparation
Clean room
Stock solutions
Laboratory
Mixing site
Thermocycler
Amplification
Detection
Documentation
QC & QAQuality control & assurance
R & D(Research and development)
Alternatives: - commercial kits - robots + kits
No alternative
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• Quality management• Validation• Regular examination of instruments and reagents• Proficiency testing by interlaboratory
comparisons- Labquality- 90 €- Instand - 150 €- QCMD - 590 €
Quality Control is expensive
Estonia:Molecular diagnostics of infect. dis. – 15 ? labsAccrediated according to ISO 15189 – 1 ? lab
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
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Hard choice - what is the best
• In-house (home-brew) PCR or commercial PCR kits
• Simple thermocycler or fancy robot
• Ordinary PCR or real-time PCRCost & Quality?!
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
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Cost of one analysis (€)(excluding human resources, infrastructure costs, etc.)
Method Consumables Equipment
PCR kit 15-19 + 0,3
PCR kit (RU) 0,7 + 0,1
Real-time 16-21 + 0,6
In-house 8 – 10 (all costs, incl. R&D; QC;QA;etc.)
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
Estonian Health Insurance Fund – 10,4 €
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QCMD proficiency panel- Participants - in-house - commercial - gen-probe
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
N. gonorrhoeaeEST-1; SWE-1; FIN-0; LI-0; LT-0
M. tuberculosisEST-1; SWE-2; FIN-1; LI-
0; LT-0
Herpes simplex virusEST-1; SWE-3; FIN-2; LI-0; LT-0
Hepatitis C virusEST-3; SWE-2; FIN-1; LI-1; LT-0
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85
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1436
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QCMD proficiency panel - % Correct
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
N. gonorrhoeae:In-house* – 90,0Commercial – 81,5
HCV:In-house – 89,5Commercial* – 95,0
M. tuberculosis:In-house* – 86,4 Commercial – 82,0Genprobe – 80,7
HSV:In-house* – 86,1 Commercial – 76,2
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AdvantagesMolecular methods
•High sensitivity and specificity•Detects pathogen, not immune response•Quick results•High transport toleration
In-house (home-brew) PCR methods•Cost effective•High sensitivity•High quality•Fast implementation of scientific discoveries•Customer friendly
R&D is absolutely necessary
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Bottlenecks in Estonia
• QC & QA is not yet everyday practice
• Poor cooperation between laboratories
• Insufficient communication with doctors
• Nonsense investments into fancy equipment
robots give money back if you run50 000 – 200 000 analysis per year
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What can we do better
• Create networking between laboratories• Take second opinion
• Honest dialogue with doctors• Education
• Concentration into specialized laboratories• QC + QC + QC – to build up the trust
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Some people do not believe into the advantages of PCR
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
“I think I may say without contradiction that when the Paris Exhibition closes, electric light will close with it, and no more will be heard of it”
Prof. Erasmus Wilson Oxford University, 1878
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Compliments to:
• Kai Jõers• Raili Randoja• Kärt Denks
• Kadri Raudsep• Avo Karus
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
•Kaido Beljaev •Paul Naaber•Katrin Reimand•Milvi Topman •Rain Lehtme
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Thank you for your attention!