Trans-renal DNA for infectious disease diagnosis

Transcription

1 Trans-renal DNA for infectious disease diagnosis Clare Green, Jim Huggett, Alimuddin Zumla Centre for Infectious Diseases and International Health University College London

2 Outline 1. Trans-renal DNA: a brief history and current knowledge 2. Using qpcr to define urinary DNA 3. Putting these lessons into practice: TB trdna 4. Summary

3 What is trans-renal DNA? A B No treatment DNAse RNAse DNAse & RNAse A: Botezatu, I et al. (2000) Clinical Chemistry, 46:8; B: Umansky, S., Xenomics Ltd, USA Cell free nucleic acids Small molecular weight molecules

4 The advantages of a trdna approach for infectious disease diagnosis Non-invasive sample Abundant sample Ease of collection, outside health care facility by the patient Diagnosis irrespective of infection site

5 TrDNA timeline CNAPs Routine foetal discovered RHD genotyping 2000 TrDNA defined Lo (1997) Lancet : 350: Botezatu (2000),Clinical Chemistry :46:8; Finning (2002), Transfusion :42: Melkonyan, (2008), Ann. N.Y. Acad. Sci. :137: Infectious disease diagnosis 2008 Ultrashort fragments

6 TrDNA for infectious disease diagnosis Nucleic acids in urine used to diagnose infections outside the genito-urinary tract 1996 Legionella spp? 1998 Wucheria bancrofti? 2006 Plasmodium falciparum 2007 Leishmania infantum 1999 Dengue virus Mycobacterium tuberculosis

7 The advantages of a trdna approach for tuberculosis diagnosis 9 million people develop TB annually 1.7 million people die from TB per year Up to 70 % of TB patients are HIV positive in sub- Saharan Africa Current diagnostics insensitive & case detection rates low especially in sub-saharan Africa Smear microscopy based on method developed in 1882

8 TB trdna EU framework 6 project involving partners in Europe and Africa Centre for Infectious Diseases and International Health (CIDIH), UCL, UK Ludwig Maximilan University, (LMU) Munich, Germany Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland Instituto Nazionale per le Malattie Infettive (INMI), Rome, Italy University of Zambia (UNZA) & University Teaching Hospital (UTH), Lusaka, Zambia Mbeya Medical Research Programme (MMRP), Mbeya, Tanzania

9 TrDNA for TB diagnosis Detection of Mycobacterium tuberculosis nucleic acids in urine in patients with pulmonary tuberculosis 2002 Kafwabulula (n=63) 2005 Torrea (n=247) 2008 Cannas (n=43) 1999 Aceti (n=13) 2006 Rebollo (n=27)

10 TrDNA for TB diagnosis Detection of Mycobacterium tuberculosis nucleic acids in urine in patients with pulmonary tuberculosis 100 % Detection sensitivity 50 % 600 bp Amplicon size 300 bp Aceti 99 Kafwabaluala 02 Torrea 05 Rebollo 06 Cannas 08

11 Inconsistencies and unknowns 1. Extraction method Sample 2. Storage of urine prior to extraction Extract DNA Measure DNA by PCR DNA 3. Target amplified Result

12 Addressing the inconsistencies Using qpcr 1. Extraction method 2. Storage of urine prior to extraction Human DNA assay targeting ALU J SINEs Inhibition measured by SPUD assay 3. Target amplified Mycobacterium tuberculosis specific targets - IS IS1081

13 Performance of the qpcr assays Assay Use Size (bp) ALU J IS6110 IS1081 Human DNA quantification MTB DNA quantification MTB DNA quantification Efficiency R 2 Linear dynamic range Cq NTC 94 ~1.00 ~ x 10 6 to 5 x ~0.96 ~ x 10 6 to 8 >40 39 ~0.98 ~ x 10 5 to 25 >40 PCR efficiency = 10-1/slope -1 ALU J IS6110 IS1081

14 Extraction techniques Extraction technique optimised for DNA recovery & inhibition DNA recovery

15 Extraction techniques Assessed on patient urines Undiluted extract Country Extracts diluted 1:5 Sex Nolan (2006) Analytical Biochemistry 351: Huggett (2008) BMC Research Notes 1:70

16 Summary of urine extraction and detection Q sepharose method chosen for extraction of DNA from urine In extractions from larger volumes of urine, a 1:5 dilution is required to remove inhibition The amount of human DNA in urine from Italy, Zambia and Tanzania are comparable at baseline

17 Stability of DNA in stored urine Italy

18 Stability of DNA in stored urine Zambia

19 Summary of stability of DNA in stored urine Different stability of DNA in the urine of healthy individuals in Italy and Zambia To avoid any potential degradation, extract DNA from urine as soon as possible after collection

20 MTB specific assays: target choice Multi-copy targets 1. IS6110 Present in 0 to 25 copies in the MTB complex Regions of the insertion sequence have homology with other Mycobacterium species 1. IS1081 Present at 6 copies in M. tuberculosis, M. bovis, M. avium & M. xenopi Van Embden (1993) Journal of Clinical Microbiology; 31:2: 406-9

21 Target choice Ultra-short amplification Q sepharose extraction methods enabled detection of ultrashort fragments Shorter targets improved MTB detection sensitivity in one individual Our assays employ SYBR green and rely on good primer design for MTB specificity Meklonyan (2008) Ann. N.Y. Acad. Sci. 1:137: 83-81

22 Putting these lessons into practice Optimising TB trdna detection IS1081 IS6110 IS6110 Q sepharose Wizard

23 Summary Urine is a very variable sample ALU J can be used to determine human DNA content of sample prior to disease specific analysis Wizard extraction allows a larger volume of extracted DNA to be analysed in one qpcr assay (no qpcr inhibition by the extract)

24 The future Characterise the trdna phenomenon with respect to disease progression Using the qpcr assays outlined, examine MTB trdna in well defined patient groups and DNA in urine further work in progress

25 Thank you Centre for Infectious Diseases & International Health (CIDIH), UCL Jim Huggett Alimuddin Zumla Gillian Rodger Tanya Novak Nina Witt Mike Taylor University of Zambia (UNZA) Glendah Kalunga Peter Mwaba Patrick Katemangwe Ludwig Maximilan University & Mbeya Medical Research Programme (LMU) (MMRP) Michael Hoelscher Klaus Reither Gasper Nyika Jutta Jung Instituto Nazionale per le Malattie Infettive (INMI) Angela Cannas Enrico Giradi Ludovica Calvo Foundation for Innovative New Diagnostics (FIND) Elizabeth Talbot Mark Perkins