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

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

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

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

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

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)

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

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

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

Automatic generation of final report E@syCheck software Chip information Automatic generation of final report

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. 516-526-531) - INH-R: katG (cod. 315) - INH-R: inhA prom. region (nt -8, -15)

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

Tuberculosis – Test evaluation for RIF/INH-R detection

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

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

Statistical Analysis

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

comparision of chip-spoligo and membrane-spoligo

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

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

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

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!