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j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
qPCR analysis of molecular targets for developing world pathogen diagnosis;
a multi-step approach to a multi-step problem
Dr. Jim Huggett
Centre for Infectious DiseasesUniversity College LondonWindeyer Building 46 Cleveland StreetLondon, W1T 4JF
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Tuberculosis, Malaria and HIV/AIDS
Kill > 5 million people per annum (0.1 % world population)
Why infectious diseases affecting the developing world?
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
•Charities currently provide large amounts of antibiotics/anti viral treatment to the developing world
•Recent pledges from Europe and the US to provide large amounts of aid for therapy
•Large pharmaceutical companies agreeing to provide drugs at reduced prices for the developing world
•Hardly any of these consider diagnosis
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
A. Zumla, J.M. Grange, Tuberculosis in: G.C. Cook. A. Zumla (Eds.), Mansons 21 edition, Saunders, 2002, pp. 997-1051
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Study performed at Zambia’s main tertiary hospital
The therapy for many of the diseases was available
The lack of accurate diagnosis let these children down
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Diagnosis
•The first stage in combating an infection
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
1. Modified molecular technologies for affordable, simple diagnosis of infectious disease (Molecular Diagnostics)
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
What is required for a new Diagnostic test to be used?
Cost
Need
Simplicity Local capacity EfficacyEfficacy
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Diagnostic test efficacy
•Sensitivity, Specificity
•New methods established through scientific research
•It is essential that the research is conducted correctly with appropriate controls
•The publication describing the result needs to be realistic in what can be concluded
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Pneumocystis Pneumonia (PCP)
Caused by the fungus P. jirovecii in immunocompromised
Symptoms:
High fever, non-productive cough, shortness of breath (especially on exertion), weight loss and night sweats.
Definitive diagnosis by pathologic identification of the causative organism in induced sputum or bronchoalveolar lavage (BAL)
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Molecular diagnosis of PCP
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Detection of Pneumocystis carinii with DNA amplification
Wakefield et alThe Lancet Volume 336, Issue 8713 1990
Early Molecular diagnosis of PCP
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
PCR designed to amplify part of the Large subunit of the mitochondrial rRNA gene
Primers referred to as H & E
Ideal due mitochondrial multiplicity
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
TM difference 5-15.3 °C
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
95 – 30 seconds56 – 30 seconds72 – 60 seconds
H & EPjHSP70a
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Sweeping statements about the role of molecular diagnosis based on a single assay should be approached with caution.
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Measure DNA by PCR
Sample
DNA
Result
Extract DNA
PCR in a multi-step approach
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Sample
DNA
Result
Extract DNA
Measure DNA by Real time PCR
Measure DNA by Real time PCR
New PCP assay beginning
PjHSP70a
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Assay characteristics PjHSP70a
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
59 %67 %24 %19 %Coefficient of Variation~2~35~439~597,247Average readout (n =24)550500500,000~ Copy Number
Assay characteristics PjHSP70a
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Sample
DNA
Result
Extract DNA
Measure DNA by Real time PCR
Extract DNA
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Patients
Consecutive adult HIV-infected patients undergoing BAL for assessment of respiratory episodes (139 BAL from 132 patients)
n = 62 PCP defined by:Typical clinical/radiological presentationGrocott silver stain (+)Response to anti-Pneumocystis therapy
n = 75 Alternative diagnosis defined by:Atypical clinical/radiological presentationGrocott silver stain (-)No anti-Pneumocystis therapy givenConfirmed alternative path/micro diagnosis
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
DNA extraction
•DNeasy tissue kit vs QIAamp UltraSens (Qiagen)
•Broncho alveolar lavage samples: 200 µl vs 750 µl
•UltraSens specially designed to extract nucleic acids from body fluid.
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
1
10
100
1000
10000
100000
HSP
70 ~
copi
es
DNA extraction comparison PCP patients
DNeasy QIAamp
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
DNeasy QIAamp 0.01
0.1
1
10
100
1000
HSP
70 ~
copi
es
DNA extraction comparison controls
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Implications for a molecular diagnostic test ?
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Sensitivity = 100 %Specificity = 48.6 %
PCP 0.1
1
10
100
1,000
10,000
100,000
1,000,000
p= <0.0001
Patients
HSP
70 ~
copy
num
ber
0.1
1
10
100
1000
10000
100000
1000000
p= <0.0001
Patients
HSP
70 ~
copy
num
ber
PCP Alternative diagnosis
DNeasy QIAamp
Sensitivity = 93 %Specificity = 96.9 %
Sensitivity = 98.4 %Specificity = 95.9 %>10 copy cut-off
QIAamp H & ESensitivity = 96.8 %Specificity = 68 %
Alternative diagnosis
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Sample
DNA
Result
Extract DNA
Measure DNA by Real time PCR
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Sample
DNA
Result
Extract DNA
Measure DNA by Real time PCR
To assess a procedures diagnostic efficacy its individual components must be considered individually
Prof. Richard Tedder:“you are starting to realise that these procedures have a beginning, a middle and an end”
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Last thought
•Molecular diagnostic research using real time PCR may never be applicable in a regional clinic in the developing world
•But real time PCR currently represents the most versatile and accurate quantitative molecular method we have.
•This type of research is essential as newer simpler molecular technologies are developed
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
•Center for Infectious Diseases and International Health, University College London
Alimuddin ZumlaGraham RookRob Miller
Acknowledgements
•Queen Mary's School of Medicine, University of London Stephen Bustin
Tanya NovakNina Witt
•Funding.DFID, EU, AIDCO, BLF, Dr Hadwen Trust
•EMBL, CambridgeMartin Taylor
•University Teaching Hospital, Lusaka, ZambiaPeter Mwaba
•Dep. Infectious Diseases & Tropical Medicine, University of MunichMichael Hoelscher
Andreas Lorenz
•Sigma-GenosysTania NolanNatalie Simpson
•Corbett ResearchGreg NowakThomas KaiserAlec Pinto
j.huggett@ucl.ac.uk Centre for Infectious Diseases & International Health
Thank you