1. A newly developed integrated Lab-on-Chip versatile platform for fast detection of MDR tuberculosis and malaria
Daniela M. Cirillo
On behalf of the FP7 TMREST Consortium
Emerging Bacterial Pathogens Unit
Div. of Immunology, Transplantation and Infectious Diseases
San Raffaele Scientific Institute
Milano, ITALY

2. Diagnostic tools for poverty related diseases: Malaria
Early diagnosis of malaria and its effective and timely treatment is a life saving intervention
Microscopy remains the gold standard
Needs well trained microscopist
Labour intensive and time consuming
Chance of misdiagnosis is high
May not consistently detect all infecting parasites accurately (mixed infection cases) i.e. P. knowlesi has been falsely diagnosed as P. malariae
Low level of parasitaemia especially problematic
Rapid diagnostic tests – RDTs
Rapid and easier to use than microscopy
Poor diagnosis for non-falciparum malaria i.e. P. knowlesi cross reacts with P. falciparum
Do not work well with low parasitaemia

3. Molecular Diagnosis Tools
Standard PCR is not routine (takes 1-2 days) but is carried out in cases of discrepancy between microscopy and RDT
Currently drug resistance of the infecting parasites is not assessed during diagnosis

4. The TM-REST Project Tuberculosis Malaria
hSR, ITALY - FZB, GERMANY - UNISI, ITALY - NCIPD-NRL, BULGARIA - ST srl, ITALY - UHLD, ALBANIA - UNIG, UK - FIND, SWITZERLAND - HPA-MRU-QM, UK
Tuberculosis Malaria
Rapid identification of MTC
Rapid diagnosis of MDR cases
Spoligotyping
Rapid identification of Plasmodium pathogenic species
Rapid diagnosis of malaria drug resistant cases
The integrated PCR and Microarray lab-on-chip tool is an innovation over the conventional molecular diagnostics for its robustness, simplicity of use and price

5. TM-REST CONSORTIUM
San Raffaele Scientific Institute (Italy): P Miotto, AM Cabibbe, D Zallocco, DM Cirillo
Univ. of Siena (Italy): E Lazzeri, F Santoro, G Pozzi
Univ. of Glasgow (United Kingdom): J Mugasa, L Ranford-Cartwright
Queen Mary and Westfield College, Univ. of London (United Kindom): V Nikolayevskyy, YBalabanova, F Drobniewski and Samara Oblast TB service (Russian Federation): I Kontsevay, O Ignatyeva , S Mironova ,A Kritsky, A Kovalyov, J Chinkova, A Rybkina
National Reference Center for Mycobacteria, Forschungszentrum Borstel (Germany): S Niemann
Univ. Hospital of Lung Diseases “Prof. Dr. Shefqet Ndroqi” (Albania): S Tafaj
National Centre for Infectious and Parasitic Diseases (Bulgaria): E Bachiiska, S Panaiotov
Foundation for Innovative New Diagnostics (Switzerland): M Perkins
ST Microelectronics (Italy): G Ventimiglia, D Spinella, T Barbuzzi

6. TM-REST organization Development of the Chip (ST/Veredus)
TB Spoligo (Borstel)
TB ID MDR
Probe design and protocol optimization (Milano /Siena)
(London Samara)
“field testing”
Malaria (Glagow)
Samples collction (Tirana, Sofia,Samara)

7. Lab-on-Chip for molecular diagnostics
PCR:
Ultra-Fast PCR
Asymmetric Cy-5 PCR strategy
Microarray:
Orientation probes
Hybridization Control probes
Hybridization Negative Controls probes
Lab-on-chip architecture
2 PCR reactors of 12.5 uL volume each (Total 25 ul)
1 Hybridization chamber of 30 uL
A 126 spots DNA microarray
2 in-let ports compatible with standard
micro-pipettor tips
Integrated Heaters and Sensors
All the reaction modules are fluidically integrated

8. The TCS platform Highly-trained personnel not required
Easy to use microfluidic interface
PCR time reduced (by approximately 70%)
Fully customizable temperatures and reaction times
Suitable for diagnostic applications

9. In-Check platform workflow
Sample preparation (DNA/RNA)
PCR Mix loading
Amplification (on TCS)
Hybridization (on TCS)
Washing & Drying
Microarray acquisition and analysis (OR)
15 min - 1h 30 min
30 min
4 min

10. Automatic generation of final report
software
Chip information
Automatic generation of final report

11. Probe design in M. tuberculosis assay
Identification of MTB
- based on IS 6110 transposon element
- 16S RNA
First-line drug resistance detection
- RIF-R: rpoB (RRDR; cod )
- INH-R: katG (cod. 315)
- INH-R: inhA prom. region (nt -8, -15)

12. Data analysis for each probe replicate
Probe selection WT
MUT
Data analysis for each probe replicate
Best probe selection
Threshold settings

13. Tuberculosis – Test evaluation for RIF/INH-R detection

14. Patients recruitment and testing isolates
Partner
Samara
Sofia
Tirana
Patients
459 67
300
MTB cultures
200
MTB DNA
(crude extracts from cultures)
180
(crude extracts from sputum) - 89
Proportion of MDR strains in the collection
30.3%
100%
2.0%
Genotyping results available
(24 loci VNTR + spoligotyping) 37
(VNTR + spoligotyping)
100
(24 loci VNTR and spoligotyping)
Phenotypic DST results available
Molecular DST results available 59 10

15. Results from Samara P99 by V.Nikolayesky, Irina Kontsevaya
Two versions of chips tested in London:
TB1.0 (RIF+INH) - initial validation, optimization of protocols and probes selection
TB4.0 (RIF+INH+ID) – further validation
No of tests performed:
TB1.0
TB4.0
DNA from cultures
160
118
DNA from sputum 18 10

16. Statistical Analysis

17. spoligochip reference collection key species genotype 9679/00
M. tuberculosis
H37Rv ATCC
9564/00
M.bovis
ATCC
2336/02
Haarlem
4850/03
EAI
1797/03
2333/99
Uganda I
2169/99
2191/99
Uganda II
2176/99
10493/01
Ghana
2570/02
5390/02
Cameroon
7968/03
LAM
2151/03
S-type
2318/06
4412/04
X-type
9953/04
7936/01
Delhi/CAS
2637/02
4445/02
Beijing
1934/03
10462//01
M. africanum
Afri 2
5434/02
M.africanum
Afri 1a
1449/02
10473/01
Afri 1b
10494/01
7011/02
M.pinnedpedii
Seal
4258/00
Bovis
9577/99
M.caprae
Caprae
416/01
M. microti
llama

18. comparision of chip-spoligo and membrane-spoligo

19. Probe analysis for the spoligochip
some probes give weaker signals than others
(very often: 2; often: 8; casually: 14,15,17,27,33)
only one false positive signal (probe 26, Afri I)
Sig Mean/ Back Mean > 10 → 1 (positive signal)
< 10 → 0 (negative signal)
> 90% specificity and sensitivity
however, cut off (10) needs to be verified in further experiments
individual cut offs may be reasonable (e.g. for spacer 2)
almost 100% concordance between results obtained with membrane based and chip based spoligotyping system

20. Malaria chip Conclusion
Detection of human plasmodia based on SSUrRNA gene amplification and detection with 5 species specific probes
Detection of mixed infection
Detection of parasite species is 100% specific and sensitive to 1-5 parasites/ µL blood
Detection of parasites resistant to common antimalarial drugs:
Amplification condition and probes have been included on the LoC for detection of resistance to four commonly used antimalarials
Chloroquine
Pyrimethamine
Proguanil
Atovaquone
Very fast – less than an hour from blood sample to result

21. Conclusion Output of the project:
Prototype and preliminary evaluation on strains and clinical samples of:
Rapid test for TB/NTMs ID and MDR TB
Spoligochip for TB
Rapid test for Malaria diagnosis and identification of DR in F. falciparum
All tests will be further validated on field clinical specimens

22. Thanks for your attention!
Way forward:
Further testing on clinical samples
Company strategy on investment in diagnostics
Thanks to all the partners of the consortium
Thanks for your attention!