use of molecular methods for diagnostics of infectious diseases

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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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Page 1: Use of Molecular Methods for Diagnostics of Infectious Diseases

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 ?

4The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

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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9The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

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

47

12

85

29

36

1436

12

95

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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

The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

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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

The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

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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