CURRICULUM VITAE, MATS NILSSON Name: Mats Nilsson Date and place of birth: August 22, 1969, Björnekulla, Sweden Office: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Box 1031, Se-171 21 Solna, Sweden http://www.su.se/profiles/matsn-1.191373 Professional preparation: 1993 M.Sc., Biology, Uppsala University, Sweden. Graduate studies - 1998 PhD, Medical Genetics, Uppsala University. Title: Padlock probes: Circularizing oligonucleotides for localized detection of DNA sequence variants. Supervisor: Prof. Ulf Landegren. Postdoc studies - 1998-1999 Beijer Fellow, Uppsala University, Medical Genetics. 1999-2000 EMBO Fellow at Leiden University, Molecular Cell Biology, Molecular Cytometry. Docent (Associate Professor) 2003 Uppsala University, Molecular Medicine. Present Appointments 2014-04-01--Site Director, Science for Life Laboratory in Stockholm. 2012-07-01—Professor of Biochemistry (Molecular Diagnostics) at Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University. 2013-07-01—Visiting Professor of Molecular Diagnostics, Department of Immunology, Genetics and Pathology, Uppsala University, Sweden. Previous Appointments 2013-01-01- 2015-06-31 Scientific Director, Science for Life Laboratory. 2009-01-01—2014-12-31 VR Research Fellow (Rådsforskartjänst; Swedish Research Council). 2009-05-01—2013-02-28 Professor of Molecular Diagnostics, Department of Immunology, Genetics and Pathology, Uppsala University, Sweden. 2008-01-01—2008-12-31 Researcher (Göran Gustafsson research fellow) at the Department of Genetics and Pathology, Uppsala University, Sweden. 2006-01-01—2007-12-31 Research assistant (FoAss; MedFak) at the Department of Genetics and Pathology, Uppsala University, Sweden. 2002-01-01—2005-12-31 Research assistant (FoAss; Swedish Reaserch Council) at the Department of Genetics and Pathology, Uppsala University, Sweden. 2001-01-01—2001-12-31 Beijer research fellow, Genetics and Pathology, Uppsala University. PROFILE My research is focused on technology development in the field of molecular analysis. I have pioneered the padlock and selector probe technologies and rolling circle amplification (RCA). The major application areas are genomics, cancer, and infectious diagnostics. The research is translational and cross-disciplinary, involving collaborations with clinicians and researchers in biomedical disciplines as well as researchers in engineering and physics. I have participated in many Framework 6 and 7 consortia (MolTools, COMICS, EUROGENESCAN, TWOBIAS, READNA, and DIATOOLS), two IMI projects (RAPP-ID and ONCOTRACK), one IMI2 project (EBOLA-MoDRAD), as well as in nationally funded projects in Sweden (COMDIA, BioNanoLab, Molecular Nano Diagnostics, FLU-ID, and more). I have co-founded 5 biotech startups (ParAllele Inc, South San Francisco, Olink Bioscience AB, Halo Genomics AB, Q-linea AB, and EMPE Diagnostics). ParAllele has been acquired by Affymetrix and Halo Genomics by Agilent. The Swedish companies employ more than 70 people. Many of the PhD students graduating in my group have gone to successful postdocs at Stanford, and many are now pursuing industrial careers (2 CEO and 3 CSO). TUTORED STUDENTS Graduated students (PhD): Malte Kuhnemund, 2016; Anja Mezger, 2015; Camilla Russell, 2015; Marco Mignardi 2015; Lucy Mathot 2014 (co-supervisor); Anna Engström 2013. (co-supervisor); Rongqin Ke, 2012; Ida Grundberg, 2011.; Sara Henriksson, 2010; Magnus Isaksson, 2010; Henrik Johansson, 2010; Chatarina Larsson, 2009; Jenny Göransson, 2008; Jonas Melin, 2006. (co-supervisor); Jonas Jarvius, 2006. (co-supervisor); Johan Stenberg, 2006; Fredrik Dahl, 2005; Johan Banér, 2003 (co-supervisor); Dan-Oscar Antson, 2001 (co-supervisor)

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Current PhD students: Elin Lundin, Tomasz Krzywkowski, Iván Hernández, Sibel Ciftci, Xiaoyan Qian, Felix Neumann. SELECTED PUBLICATIONS 2012-2016 (107 peer-reviewed articles, 34 reviews and book chapters that have been cited over 6700 times. H-factor = 38) 1. Gómez de la Torre, T.Z., Ke, R., Mezger, A., Svedlindh, P., Strømme, M. & Nilsson, M. Sensitive detection of spores using volume-amplified magnetic nanobeads. Small 8, 2174- 2177 (2012). 2. Engström, A., Zardán Gómez de la Torre, T., Stromme, M. & Nilsson, M. Detection of rifampicin resistance in Mycobacterium tuberculosis by padlock probes and magnetic nanobead-based readout. PLoS ONE 8, e62015 (2013). 3. Ke, R., Mignardi, M., Pacureanu, A., Svedlund, J., Botling, J., Wahlby, C. & Nilsson, M. In situ sequencing for RNA analysis in preserved tissue and cells Nat. Methods 10, 857-860 (2013). 4. Russell, C., Welch, K., Jarvius, J., Cai, Y., Brucas, R., Nikolajeff, F., Svedlindh, P. & Nilsson, M. Gold nanowire based electrical DNA detection using rolling circle amplification. ACS Nano 8, 1147–1153 (2014). 5. Kuhnemund, M. & Nilsson, M. Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore. Biosens Bioelectron 67, 11-17 (2015). 6. Mezger, A., Gullberg, E., Göransson, J., Zorzet, A., Herthnek, D., Tano, E., Nilsson, M. & Andersson, D.I. A General Method to Rapidly Determine Antibiotic Susceptibility and Species in Bacterial Infections. J. Clin. Microbiol. 53, 425-432 (2015). 7. Mignardi, M., Mezger, A., Qian, X., La Fleur, L., Botling, J., Larsson, C. & Nilsson, M. Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ Nucleic Acids Res. 43, e151 (2015). 8. Pavankumar, A.R., Engström, A., Liu, J., Herthnek, D. & Nilsson, M. Proficient detection of multidrug-resistant Mycobacterium tuberculosis by padlock probes and lateral flow nucleic acid biosensors. Anal. Chem. 88, 4277−4284 (2016). 9. Kühnemund, M., Wei, Q., Darai, E., Wang, Y., Hernandez-Neuta, I., Yang, Z., Tseng, D., Ahlford, A., Mathot, L., Sjoblom, T., Ozcan, A. & Nilsson, M. Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy. Nature Communications in press (2017). 10. Kuhnemund, M., Hernandez-Neuta, I., Sharif, M.I., Cornaglia, M., Gijs, M.A. & Nilsson, M. Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules. Nucleic Acids Res (2017). PATENTS AND APPLICATIONS (last 5 years) one per case, there are several continuations and divisions) 1. Ahlford, A., Nilsson, M. & El-Heliebi, A. In vivo collection and localized quantification and profiling of

circulating cells, proteins and nucleic acids. in US provisional 62253907 (2015). 2. Asalapurum, P. & Nilsson, M. Diagnostic device and related method. in SE-1650197-5 (2016).

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CURRICULUM VITAE, RAVI KUMAR V BANDA

Name: Venkata Ravi Kumar, Banda Date of Birth: 11March 1954, Bhimavaram, A.P. Present Address: XCyton Diagnostics Limited, 449, 10th cross, IV Phase, Peenya Industrial Estate, Bangalore 560058, INDIA QUALIFICATIONS Degree Subject Institute University Year M.B.B.S JIPMER Pondicherry 1978 Ph.D Neuro Chemistry IISC, Bangalore 1985

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WORK EXPERIENCE Chairman and Managing Director, XCyton Diagnostics Limited, December 1994 to the present Scientist and Group Leader Astra Research Centre India, (ARCI) January 1986 to December 1993 Guided Students for Ph.D. at The Indian Institute of Science during 1989-1993 and Currently Guiding PhD Students at NIMHANS Registrar, Department of Psychiatry, Christian Medical College, Vellore, March 1978 to June 1979 CURRENT AREA OF RESEARCH Development of Molecular and Immunno-diagnostic kits for Infectious syndromes. ABSTRACTS AND PUBLICATIONS 10 research papers in High Impact International Journals. In last two years: Prasad P, Vishnu Bhat B, Ravi Kumar BV*, ….Shruthi B Syndrome Evaluation System (SES) versus blood culture (BACTEC) in the diagnosis and management of neonatal sepsis - a Randomized Controlled Trial Ind. J. Paediatrics 2016 DOI 10.1007/s12098-015-1956-3 Joseph L…B V Ravi Kumar, Anna Nilsson Aetiology of Pneumonia in Children in India – Data from Community Acquired Pneumonia Etiology Study (CAPES) Cohort: J.Global Health Vol 5 201-210 2015 PATENTS Indian Patents (Two granted, Four applications pending), PCT Patent (Two granted in 33 countries and USA; Four applications pending), US Patent: One Granted PRODUCTS DEVELOPED: SES Syndrome Evaluation System: SES is a molecular Diagnostic platform for detection of pathogens in critical infections. SES reduces death by 82% because it is ten times faster four times more sensitive, prompts early targeted therapy reduces antibiotic usage by 60%, reduces hospital stay by 40% and reduces overall health care costs. HIV-CheX: In the market since 1997. The kit is a third generation synthetic peptide based ELISA for detection of HIV 1& 2. Developed in collaboration with Prof. P. Balaram, Molecular Biophysics Unit, I.I.Sc and Prof. V Ravi, Neurovirology, NIMHANS. HEP-CheX C: In the market since 2001. Third generation ELISA using synthetic peptides and developed in collaboration with Dr. Navin Khanna of ICGEB and Pro. Subrat Panda of AIIMS, NewDelhi CYSTI-CheX; In the market since 2001. ELISA kit that detects antibodies to cysticercus ES antigens in CSF of patients. It is a patented product of AstraZeneca Research Foundation India and developed in collaboration with Prof. V Ravi, Neurovirology, NIMHANS, Bangalore JEV-CheX: In the market since September, 2004. IgM capture ELISA developed in collaboration with NIMHANS under the aegis of DBT. XCYTO-SCREEN: A series of DNA macro array chips for the diagnosis of:eye infections such as Viral Retinitis, Kerato Conjunctivitis, Uveities and Infectious Endophtalmitis, HPV Infections of cervix by high risk carcinogenic genotypes, Meningitis and Encephalitis, Developed in Collaboration with Sankarnetralaya, LV Prasad Eye Institute, Centre for Cellular &Molecular Biology and All India Institute of Medical Sciences. Launched in September 2007. AWARDS Lockheed Martin DST award – India Innovation Growth Programme - 2013 Bio-Singaporeawardforthe“MostImportantTechnologyinnovationCompanyofAsia–2009”.Biospectrum Award for the Best Bio-Product of the year 2008 for XCyto-Screen range of Products BioSpectrum Award for the best bio product of the Year 2004 for JEV-CheX Rabo Bank award for Bio-Products of the year 2003 for HIV-CheX and HEP-CheX C Prof. Giri Memorial Gold Medal for Best Thesis (1986), Indian Institute of Science, Bangalore, India The Best House-Surgeon award (1978), JIPMER, Pondicherry, India.

Proposal Form

STRATEGIC INDO-SWEDISH COOPERATIVE

PROGRAMME ON “HEALTH AND DISEASE PREVENTION”

A. PROJECT

1. Title of Project

Point-of-care multiplex confirmatory diagnostic test for multi/extensively drug-resistant tuberculosis

2. Specific research field

Field Sub-field

Antimicrobial resistance - Innovative treatment, diagnosis and preventive strategies - Tuberculosis

Addressing the need for point-of-care testing (POCT) to improve treatment outcomes and efficient utilization of health resources. Development and evaluation of antimicrobial resistance control strategies to reduce the risk of acquisition, transmission and infection by antimicrobial-resistant pathogens in inpatient and community settings.

※ please be sure to be specific.

3. Project Duration

01/07/2017 - 31/05/2020 (3 years)

※ Project duration should be 3 to 4 years all together.

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4. Summary of Project

Keywords

Mycobacterium tuberculosis

Antibiotic resistance

Multi and extensively drug-resistant tuberculosis

Inexpensive point-of-care test

Confirmatory multiplex Test kit

Padlock probes

Rolling circle amplification

Lateral flow biosensors Resource clinics-RNTCP microscopy centres

Objectives

India became the largest hub for tuberculosis (TB), mainly due to the lack of confirmatory diagnostic tests at primary TB diagnostic clinics like RNTCP and sputum microscopy centres and non-availability of quick drug susceptibility tests. We have developed a proof-of-concept method by combining DNA amplification and lateral flow for detection of causative agent of TB, Mycobacterium tuberculosis, and its drug susceptibility pattern against antibiotics used to treat MDR-TB, in 60 min. Our aim is to develop an inexpensive and self-sustainable ‘XDR-TB’ diagnostic test requiring NO electricity-NO refrigeration-NO pipetting-NO skilled labor and completely suitable for Indian TB care. Therefore, our objectives are (1) to optimize the proof-of-concept test to meet clinical sensitivity, (2) to provide type confirmatory answer (YES/NO) for the presence of MDR/XDR-TB, in 60 min by developing visual ‘red signals’. (3) After clinical evaluation at different geographic locations of Indian RNTCP and sputum microscopy centres, the LF-MDx assay will be transformed into LF-MDx test kit format, suitable for the use of semi-skilled personnel at resource-limited clinics.

Approach

A proof-of-concept method was obtained for a test by combining isothermal amplification method with later flow technology was established by Prof. Nilsson’s group (Sweden) for the detection of most common mutations of MDR/XDR-TB. The test was well scrutinized for correct identification of MDR/XDR-TB genotypes by wisely integrating the stringent molecular method (padlock probe-dependent rolling circle amplification, PLP-RCA) and very sensitive lateral flow biosensors (LFNAB) to produce ‘red color’ visual signals. After complete optimization, the method will be initially validated on clinical samples obtained from the Swedish Institute for Communicable Disease Control (Sweden). Later its clinical sensitivity and specificity will be evaluated on sputum samples obtained from TB patients in association with National Institute for Research in Tuberculosis (NIRT-Chennai) and Christian Medical College (CMC Hospital-Vellore). Thereafter, the test will be optimized for the use at primary TB diagnostic centres via research & development-association with the commercial partners (EMPE Diagnostics-Sweden and XCyton Diagnostics-India), who will also transform the test into self-sustainable kit format. Finally, the MDR/XDR-TB test kit will be checked for its operational feasibility and geographical variability at primary TB diagnostic centres associated with CMC-Vellore and Kalinga Institute of Technology (KIIT University & Hospital, Bhubaneswar-Orissa).

Expected

Outcome

• Development and commercialization of the inexpensive, rapid and multiplex confirmatory point-of-care test kit for MDR/XDR-TB, which is not based on PCR.

• A test can precisely indicate ‘which mutation is present’ and ‘presence or absence’ of wild-type genes by developing the ‘red’ colored visual signals in 60 min, while the patient can wait in the clinic.

• Establishment of the world’s first test kit that combines isothermal isothermal amplification with lateral flow technology to can provide YES/NO answer that helps the clinician to choose appropriate antibiotic, from the day one.

• This inexpensive LF-MDx test can bring the diagnosis time from 6 months (3-6 months) to 60 min, which eventually reduces the treatment cost by at least 6 times.

• First self-sustainable product of its kind. Usage of battery operable thermocycler and usage of dry reagents avoids the requirement of electricity and cold storage.

※ Attention - font: Times New Roman, size: 11 points / Do not exceed space provided.

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5. Personal Data of Principal Investigators

INDIA

Name of PI Ravi Kumar, B.V.

Title/ Designation Managing Director

Department Research & Development

Organization XCyton Diagnostics Ltd.

Address

No.449, 10th Cross, 4th Phase, Peenya Industrial Area Bangalore 560 058, India

Office phone # +91 (0)80-2836 7581-85

Cell phone # +9198440-98051

Fax # +91 (0)80-2836 7582

e-mail ravikumar@xcyton.com

Date of Birth March 11, 1954

SWEDEN

Name of PI Mats Nilsson

Title/ Designation Professor

Department Department of Biochemistry and Biophysics

Organization Stockholm University, Stockholm, Sweden

Address

Science for Life Laboratory, Karolinska Institutet Science Park Tomtebodavägen 23A 171 65 Solna, Sweden

Office phone # +46 (0)762 756 161

Cell phone # +46 (0)730 537 876

Fax #

e-mail mats.nilsson@scilifelab.se

Date of Birth August 22, 1969

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6. Project partners

PartnersinIndianTeam

Name Organization, Division Title Degree Specialty

Joy Sarojini Michael

Vice Principal, Head of Microbiology, Christian Medical College Vellore - 632004

Prof.

MBBS MD

Hospital Infection Control, Mycobacterium tuberculosis infections, Laboratory validation of diagnostic methods, Antibiotic resistance,

Ravi Kumar V. Banda

XCyton Diagnostics Ltd., Bangalore – 560058

CEO

MBBS Ph. D.

Clinical diagnostics, Commercial partner for product development

Avinash Sonawane

Dean, School of Biotechnology Head of Immunology, KIIT University & Hospital, Bhubaneswar, Orissa-751024

Prof. Ph. D.

Antimicrobial factors of Mycobacterium tuberculosis, vaccine development

Sridharan Koppula

Dy. Chief of Lab. Services, Professor of Microbiology, Sri Ramachandra Medical College and Research Institute and Hospital (SRMC & H) Porur, Chennai – 600 116

Prof. MBBS MD

Antimicrobial drug susceptibility testing, Laboratory validation of diagnostic methods, Patient care.

PartnersinSwedishTeam

Name Organization,Division Title Degree Specialty

Mats Nilsson

Department of Biochemistry and Biophysics, Stockholm University, Science for Life Laboratory, 17165 Stockholm

Prof. Ph. D.

Molecular diagnostics, Infectious diseases, Interdisciplinary research, Tuberculosis, cancer

Pavankumar Asalapuram

EMPE Diagnostics AB Nobles väg 3, Karolinska Institute Science Park, 17165 Stockholm

CEO Ph.D.

Point-of-care lateral flow technology, Molecular diagnostic tools, Tuberculosis, Enteric infections.

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B. TECHNICAL INFORMATION

1. Objectives of Project (up to 200 words)

India is a high Tuberculosis (TB) burden countries in the world, where ~40% its population is at risk of developing TB infections. We have established a proof-of-concept multiplex molecular method called LF-MDx, using padlock probe-dependent isothermal rolling circle amplification for robust discrimination of single-nucleotide variants. Like a point-of-care test, ‘red color’ visual signals are developed on specially designed lateral flow strip cassettes showing the presence of most common mutations causing MDR/XDR-TB and their respective wild type genes, in approximately 60 min. Therefore, the aim of this study is to develop an inexpensive, point-of-care, multiplex molecular diagnostic test for resource-limited TB clinics, including RNTCP and sputum microscopy centres, based on our proof-of-concept assay. Objectives of this study include: (1) Elaboration of the combinatorial proof-of-concept method into a fully developed LF-MDx test kit. (2) Test-kit optimization by conducting large-scale evaluation at reference and resource-limited TB laboratories, across India to meet clinical sensitivity. (3) Provision of confirmatory answer (YES/NO) for the presence of MDR/XDR-TB in 60 min, during the first patient visit to PTBDC. (4) Development of inexpensive and self-sustainable diagnostic test (NO electricity-NO refrigeration-NO pipetting- NO skilled labor) suitable for Indian primary health care set-up, in a kit format.

2. Justification for collaboration, including background to the proposed project, each partner’s expertise and specific contribution to the project, and the added value of the proposed collaboration (up to 400 words)

Tuberculosis (TB) is a major infectious disease causing 9 million new cases/year (3800 deaths/day) and remains medical-socio-economical challenge. The pathogen, Mycobacterium tuberculosis often develops desistance by acquiring chromosomal mutations. Today’s TB diagnostic tests can provide rapid results but they all are PCR-based, a method that is inherently difficult for large-scale multiplexing and good infrastructure. As an alternative, mutations linked to drug resistance can be detected by padlock probes (PLP) with unimolecular precision enabling highly multiplexed assays for multi-drug/extensively drug resistance MDR/XDR-TB are possible in a one-step reaction. Prof. Nilsson is global expert in developing cutting-edge molecular diagnostics for the disease surveillance and diagnosis. His PLP technology is being used in clinical research laboratories worldwide. Nilsson contributes the proof-of-concept molecular method for this proposal, enabling detection of MDR/XDR-TB. Initial testing using clinical TB isolates, in association with Prof. Sven Hoffner (Karolinska Institute-Sweden) has been established but needs optimization for global use. Dr. Pavankumar, pharmaceutical-biotechnologist, co-founded EMPE Diagnostics that develops combinatorial and confirmatory point-of-care diagnostics. His 10 years of translational research, including PLP-lateral flow concept became foundation for this project. EMPE’s clinical consortium with eight WHO Supernational reference laboratories in Europe and India for clinical evaluation can help to develop the LF-MDx test to comply international diagnostic requirements. Dr. Ravi, physician-scientist, founded XCyton Diagnostics developing molecular assays for critical infections as Sepsis Encephalitis, Meningitis, Febrile Neutropeniaetc and antibiotic resistance. He has won Lockheed Martin DST awad and associated with several Govt. organisations. XCyton is a producer-supplier of molecular diagnostic services to all intensive care units across a wide-range of hospitals across India and provide clinical service. Prof. Joy Michael has been extensively evaluated drug-resistance TB diagnostics, including Microscopically Observed Drug susceptibility (MODS) assay, LED fluorescent microscope, GeneXpert, and Line probe assays. She has received the Biovision Lilly Award for her research in developing/evaluating new diagnostics. Dr. Michael will contribute her extensive experience piloting LF-MDx test and leverage her collaborations with Indian RNTCP to develop sustainable strategies.

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Prof. Avinash is experienced in biomarker research, developing antimicrobial targets and vaccines for tuberculosis. Being coordinated national and international research projects, Avinash will help to evaluate LF-MDx test in hospital-associated resource-limited clinics. His will coordinate the evaluation of LF-MDx test in the village-based medical campls. Prof. Sridhar, a clinicican-cum-microbiologist and coordinates interdisciplinary laboratory-driven projects in the hospital. He is responsible of laboratory management and evaluates the suitability to used medical device in the clinical laboratory.

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3. Descriptions of the Cooperative research project – maximum 6 pages (font size 12 pt)

Background and clinical implications of TB: TB is a common communicable disease but in many cases, it becomes lethal infection, if not properly treated. TB in humans is most commonly caused by the bacterium Mycobacterium tuberculosis, which is the second most common cause of mortality due to infectious diseases. About 9.6 million new TB cases and 1.5 million deaths (3,800/day) in 2014 (WHO Global Report 2014) but the mortality rate is expected to increase up to 70%, among immunocompetent individuals. Though TB is largely curable, the Bacillus Calmette-Guérin (BCG) is the only vaccine available against TB since 1921, which protects severe cases of TB in children, however, the protection elicited by the vaccine against pulmonary TB in adults, representing much of the disease burden and the transmissible form, is very poor. The lowest levels of protection have been observed in countries with the high TB prevalent settings, like India, which contribute to 26% of the global TB burden. Thus, the control of TB heavily relies on effective antibiotic treatment, which if not implemented and used properly, leads to drug-resistance. MDR-TB treatment is very complicated and involves the use of more toxic drugs with prolonged treatment duration from months to years. Such complications in MDR-TB treatment easily transforms it to the dangerous extensively drug-resistant (XDR) TB, where the patients often become carriers of spreading MDR-TB. Recently, the TBNET and RESIST-TB networks suggested that the identification of MTB mutations in clinical isolates coding for katG, inhA, rpoB, embB, rrs, rpsL, and gyrA genes has implications for the management of TB patients, pending the results of in vitro DST. Though the improved infection control measures and better diagnostic methods are needed to reduce the spread of MDR/XDR-TB strains, it remains a challenge for resource-limited clinical laboratories due to high cost of sample processing. Global health challenges of TB: Prevalence of drug-resistant TB has increased steadily since 1990s, jeopardizing the global TB control programmes. The MDR-TB strains [resistant to at least the two main first-line anti-TB drugs rifampicin (RIF) and isoniazid (INH)] represent a major challenge for the control of the disease. Treatment of MDR-TB is cumbersome as it requires the use of toxic second-line anti-TB drugs for at least 18-24 months (Balaji et al 2010; Ahuja et al. 2012; WHO 2014). As per the WHO Global Tuberculosis Report 2014, 3.5% of new and 20.5% of previously treated cases are infected with MDR-TB strains, and the prevalence is expected to increase. Only the countries, India, China and the Russian Federation contribute to 60% of the estimated global burden of MDR-TB. However, the XDR-TB is defined as an MDR-TB strain that is also resistant to any second-line antibiotics like fluoroquinolone (FQ) and at least one of the three injectable drugs amikacin (AMK), kanamycin (KAN), and capreomycin (CAP). Adding the fuel to the existing drug resistance problem, XDR-TB may in fact be practically impossible to treat with currently available drugs, potentially bringing us back to the pre-antibiotic era, leaving the patients in isolation. The XDR-TB has been reported in 84 countries, where almost 10% of MDR-TB cases are reported to have XDR-TB. Drug-resistant TB is serious global health problem. Inadequately treated patients become chronic carriers and can spread drug-resistant TB strains in-and-across the communities due to globalization and exchange of people. Financial and societal burden of TB: Public health measures to control or reverse the problem, where dissemination has already occurred, can be extremely expensive and are often beyond the means of countries with high prevalence of drug-resistant strains. The treatment for one drug-susceptible TB patient costs is up to $ 500; while it increases to US$ 9 235 in middle-income countries and US$ 48 553 in developing countries for the treatment (WHO 2014). Therefore, an average of ~$ 28 894 costs for the treatment of one MDR-TB patient. India the second-high TB burden countries in the world with up to 3 million cases/year. An average patient in India spends up to INR 5000 for one first-line DST, but the treatment costs about INR 2 lakhs for MDR-TB treatment and INR 5 lakhs. The patients usually undergo empirical first-line antibiotics treatment (Isonaizid+Rifampicin+Ethambutol+Pirazynamide) for 2 months, followed by

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(Isonaizid+ Rifampicin) for 4 months, as a continuation phase. It is important to mention that the MDR/XDR-TB patients should take 8-12 second or third generation antibiotics for 2 to 3 years, along with bi-weekly injections, without any guarantee of complete cure. Since, continuous transmission of drug-resistant strains by even a small number of individuals may develop into a serious problem across the world and if ignored, the treatment costs become unmanageable and could lead to socio-economic collapse. Problems in current TB diagnostics: TB patients form low- and middle-income countries do not have access to advanced health care facilities and equipped laboratories. In fact, the TB cases are detected by microscopic examination of sputum samples, which is, despite being a relatively cheap method is not a very sensitive method. It is reported that ~40-50% of sputum microscopic results are either error prone or require re-testing, and of course, the problem with drug resistance cannot be addressed with this technique. Only a few patients are referred to district or national reference laboratories where phenotypic culture DST can be performed, which takes up to 6 months to obtain results. In contrast, polymerase chain reaction (PCR)-based nucleic acid amplification tests (NAAT) provide rapid results for the detection of drug-resistant MTB strains through various automated and semi-automated techniques. For example, the GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA, U.S.A.) provides rapid indicative identification of MDR-TB directly from sputum samples in less than 2 h. However, it is restricted to only specific detection of RIF resistance. The probe-hybridization assay, GenoType MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) can detect MDR-TB but requires multiple manual steps and specific instruments. Although the solid-phase reverse hybridization line-probe assay (LPA) can rapidly detect RIF and INH resistance, it is not recommended by WHO for the detection of second-line drug-resistance attributes. Conclusively, the capacity and requirement for instrumentation and skilled personnel of currently available NAATs hinder prompt detection of MDR-TB in resource-limited settings. Therefore, a proficient method that can address the molecular detection challenges of MDR-TB and be implemented in peripheral laboratory settings is required to provide easily interpreted first-hand information about MDR-TB isolates. In M. tuberculosis, drug resistance is not caused by horizontal gene transfer like in many other bacterial species, but instead drug resistance arises only via the acquisition of spontaneous chromosomal mutations. Resistance to RIF is caused by mutations within an 81-bp region of the rpoB gene, called the RIF resistance-determining region. Most clinical isolates resistant to RIF have a mutation in codon 516, 526 or 531. INH resistance is most commonly mediated by mutations at codon 315 in the katG gene, and at nucleotide position - 15 in the promoter region of inhA. FQ resistance is linked to mutations in the A-subunit of the DNA gyrase, encoded by gyrA, where mutations are found in the quinolone resistance determining region, codons 88-94, while the mutations at codons 90 and 94 are commonly observed. Resistance to the second-line injectable drugs AMK, KAN and CAP is associated with mutations in the 16S rRNA gene rrs, specifically at position 1401 (Engström et al. 2012). Therefore, it is important to identify all the resistance markers to enable appropriate antibiotic treatment. An alternative to performing lengthy phenotypic-based methods is to utilize molecular-based methods for the detection of mutations associated with drug resistance. These methods can yield an answer in matter of a few hours, instead of weeks or months in the case of phenotypic-based DST methods. There are several commercially available tests developed to detect drug-resistant TB; however, all are based on PCR, a method that is inherently difficult to perform in a multiplex manner. PCR-based methods also require extensive assay re-optimization in the case of assay design alterations. Principle and preliminary results as a proof-of-concept: We have developed a combinatorial proof-of-concept molecular method for the detection of XDR-TB in 60 min, by joining the stringent PLP-dependent RCA and sensitive lateral flow biosensor (LFNAB) chemistry (Pavankumar et al. 2016). The PLPs are linear oligonucleotides comprising target-specific sequences at the 5’ and 3’ ends with a linker segment containing sequences for amplification,

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identification and detection in between (Nilsson et al. 1994). As the probe ends hybridize in juxtaposition on the target specific site, a perfect match at the 3′ end is required for ligation, efficiently discriminating point mutations. The linker segment in the middle contains sequences with functions for amplification, identification, and detection. The circularized padlock can undergo RCA to produce a single-stranded concatemer, containing multiple complementary repeats of the PLP sequence (Baner et al. 1998; Baner et al. 2003). To improve the sensitivity, the concatemer is restriction-digested, re-ligated into new circles, and subjected to an additional round of RCA, known as circle-to-circle amplification (C2CA; Dahl et al. 2004). The PLPs were designed to target wild type and their corresponding mutated loci in the genes rpoB, katG, gyrA, and rrs, and in the inhA promoter region. The signals are developed by addition of the monomerized amplicons and gold nanoparticle-oligonucleotide conjugates to the Lateral flow cassette, which is hybridized to already capture-impregnated probes enabling visualization by the naked eye (Pavankumar et al. 2016). Using this technique, we have analysed XDR-TB samples with a set of 18 PLPs in one reaction, in one tube. Visualization of signal on the low-density array was possible with as little as 300pg, which corresponds to 600 target molecules. These preliminary experiments showed satisfactory efficiency and robust discrimination of mismatching target, which is proposed to improve further for the applications in general clinical labs and hospitals. The figure below illustrates the strategy:

Figure 1. Proof-of-principle of LF-MDx test: Linker segment (gray) of the PLP contains detection and restriction sites, while the 5′ and 3′ arms (black) are designed to hybridize to the target sequence (orange; A). Upon matched hybridization, PLPs are ligated and the circularized probes can undergo RCA. The amplicons are digested using a restriction oligonucleotide. The products are again amplified by RCA (C2CA), monomerized and hybridized in a sandwich fashion to their common tags of AuNP oligonucleotides and to the respective WT or MUT oligonucleotide tags immobilized on the LF strips to produce visual signals (B). Blue are the restriction oligonucleotides, which will serve as templates to produce C2CA concatemers, which are further restriction digested to apply on lateral flow strips. Visual signals are developed when the immobilized streptavidin-

bound oligonucleotides are hybridized to its respective C2CA monomers and thiolated oligonucleotides conjugated with gold nanoparticles.

Figure 2. Self-sustainable LF-MDx test: About 2-3 ml of sputum sample is collected in a sample collection bottle and concentrated and lysed in tube 1, containing MTB-lysis solution (mixture of two enzymes, diazonium salts and detergents). Lysate is transferred to tube 2 containing hybridization-ligation mixture. After the incubation, the contents are transferred to tube 3 for Isothermal amplification (RCA). RCA products are then digested in tube 4, and transferred to tube 5 for C2CA. Apmlicons are further digested in tube 6 containing digestion mix that monomerizes the C2CA amplicons. The monomers are mixed with 10 µl of AUNP-conjugates in the same tube and applied on sample holder (step 7) to see the visual signals. Specific amplicons of katG 315 wt, katG 315 ACC, rpoB 531 wt, rpoB 531 TTG, rpoB 526 wt, rpoB 526 TAC, rrs 1401 wt, rrs 1401 G, gyrA 94 wt, gyrA 94 GGC, gyrA 90 wt, gyrA 90 GTG, inhA -15 wt, and inhA -15T. The red signals are

developed at their respective places where the specific LF strip oligonucleotides are immobilized. References: WHO Global TB report 2014; Balaji et al. PLos One. 2010, 5:e9527; Ahuja et al. PLoS Medicine. 2012, 9: e1001300; Engström et al. JCM. 2012. 50:2026-33; Nilsson et al. Science. 1994, 30:2085-8; Baner et

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al. Nucleic Acids Res. 1998, 26:5073-8.; Baner et al. NAR. 2003, 31:e103; Dahl et al. PNAS. 2004, 101:4548-53; Pavankumar et al. Anal. Chem. 2016, 88, 4277-84. Impact Drug-resistant TB is a major global health problem. To decrease morbidity and mortality, appropriate actions must be undertaken. Ideally, antibiotic treatment should always be guided by DST and full characterization of drug-resistant M. tuberculosis. Despite being generally robust, sensitive and specific, a major drawback with phenotypic DST methods is that they are inherently lengthy procedures. An alternative to examining the phenotype is to examine the genotype responsible for that phenotype, i.e. drug resistance. An advantage with molecular-based methods aiming at detecting mutations associated with drug resistance in M. tuberculosis is that their turn-around time is very short. Time may be crucial as patients with active disease may die or transmit drug-resistant bacteria to others before appropriate treatment has been implemented. The proposed collaboration between Sweden and India will be benefited in the similar way, where the developed technology at Prof. Nilsson’s laboratory (Sweden) can be evaluated and improved according to actual clinical conditions in India, to develop a novel, inexpensive, self- sustainable, TB drug-resistance-specific, easily operable test for the provision of confirmatory results, even in Indian peripheral labs. This collaborative initiative is constituted to a through clinical experts like CMC-Vellore (clinical evaluation through chain of their highly infrastructure and peripheral clinics) and SRMC-Chennai (University hospital mostly treats rural patients) and KIIT-Bhubaneshwar (that operates primary health centres at 3 villages), and a business partner, XCyton Diagnostics Ltd. for product development and commercial evaluation. At the end, the project would produce an inexpensive test kit for rapid detection of XDR-TB in 60 min, without the requirement of electricity. The Swedish company, EMPE Diagnostics develops dry reagents, battery operable thermocycler for this purpose and produces the lateral flow cassettes. Essentially the test would bring the current XDR-TB diagnostic time to 60 min. Such type of collaborations connects the bench to the bedside with clinical evaluation of technology from the lab in a high prevalence clinical setting translating science for society and global health. Implementation and Actor constellations: This collaborative project will be operated between Prof. Mats Nilsson, Stockholm University/Science for Life Laboratory, Stockholm, Sweden and EMPE Diagnostics, a Swedish commercial partner with Indian commercial partner Dr. Ravi Kumar, of XCyton Diagnostics Ltd., and Indian clinical partners CMC-Vellore, SRMC-Chennai and KIIT-Bhubaneshwar. Their expertise and contributions to the project as follows: Prof. Mats Nilsson: A well-known expert in molecular diagnostics, especially developing cutting-edge technologies for the disease surveillance and diagnosis. He is a developer of padlock probe technology that is being used in several research and clinical laboratories worldwide. His experience with cancer and infectious diseases including TB even at single molecule precision can be evidenced by several publications in premier scientific journals like Science, Nature Genetics, Nature Biotechnology, Nature Methods, Nano Letters, etc. and is being applicable to develop novel solutions to the existing health care problems. Having this experience, Nilsson contributes the proof-of-concept for this proposal, which needs the clinical evaluation and further optimization to make it into a user-friendly format. Nilsson is also a co-inventor of EU patent for LF-MDx concept and the current format of lateral flow cassette design. Dr. Pavankumar Asalapuram: A pharmacist by education but a molecular biologist by training. His interest is to develop affordable diagnostics that can be used in the first contact point between the doctor and the patient, especially suitable for resource-limited clinical setups. Pavankumar has by now developed 5 diagnostic platforms including PLP-RCA and lateral flow MDR-TB test for Tuberculosis. He has published bout 25 peer-reviewed scientific articles and holds 3 international patents around biomedical and molecular diagnostics. Pavankumar has developed successful collaborations between

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researchers and clinicians in Europe and Asia. He is the co-founder EMPE Diagnostics AB, which is a facility to develop lateral flow nucleic acid biosensor solutions that holds the patent on developing lateral flow cassettes. Prof. Sven Hoffner: Director of the WHO Supranational TB Reference Laboratory at the Swedish Institute for Communicable Disease Control (SMI), Solna, Sweden and Assoc. Prof at the Karolinska Institute, Stockholm, Sweden, will collaborate with Prof. Nilsson in the optimizion and evaluation of the molecular method. SMI has a large culture collection consisting of both drug-resistant and drug-susceptible M. tuberculosis clinical isolates and other mycobacterial species. Dr. Ravi Kumar Banda: The physician-scientist turned entrepreneur founded XCyton Diagnostics Ltd. of Bangalore, India. Dr. Ravi Kumar and team had developed a Molecular Diagnostic tool called Syndrome Evaluation System (SES) a multiplex format for simultaneous detection of all pathogens which can cause a syndrome. SES can diagnose bacteria, fungi, RNA viruses, DNA viruses and parasites in a single sample in a single test in seven hours’ time. SES for eye infections, brain infections, sepsis and antibiotic resistance. SES was developed by XCyton Diagnostics Ltd. in collaboration with many institutes under NMITLI programme of CSIR, Government of India. XCyton Diagnostics has very good and approved facilities to handle Mycobacterium samples. He also takes care of the commercial aspects of the offered product according to the governmental regulations. Dr. Joy Sarojini Michael: Faculty In-charge of the Mycobacteriology division in the Department of Microbiology at Christian Medical College, Vellore. Since 2007, Dr. Michael has been extensively involved in the research of new tuberculosis diagnostics, including Microscopically Observed Drug susceptibility (MODS) assay, LED fluorescent microscope, and GeneXpert assays for diagnosis of extra pulmonary TB and childhood TB, and Line probe assays for MTBDR and MTBDR SI assays for diagnosis of MDR and XDR TB. She has received the Biovision Lilly Award for her contributions for research in this field of new diagnostics and policy making. Her laboratory is a NABL & RNTCP accredited to perform culture & DST and CB-NAAT for Programmatic Management of drug resistant TB (PMDT) program of RNTCP. Dr. Michael will contribute her extensive experience and leverage her close collaborations to influence sustainable strategies. Prof. Avinash Sonawane: has huge experience in biomarker research, especially antimicrobial targets and developing vaccines for tuberculosis. Several of his coordinated national and international research projects evaluating biomedical solutions is proposed for the hospital-associated resource-limited primary care clinics in that geographical area. His involvement in the project is very useful since the test can be tested in the village-based clinics. Prof. Sridhar Sathyamurthy, A clinician holds MD in Microbiology, is an expert in clinical microbiology and coordinates interdisciplinary projects in the hospital. He is responsible of laboratory management and evaluates the suitability to used medical device in the clinical laboratory. SRMC Hospital is one of the largest hospitals in Southern India attracts patients from all parts of the country, majorly southern India. They have a very huge laboratory patient sample collection and clinical validation system that could help to evaluate LF-MDx test. Plan for contract between the parties: The parties will set up a contract that regulates aspects that will be important to agree on among the partners to be able to execute the project in an efficient way, and to avoid future conflicts, and if conflicts would arise, how to settle them. Any Intellectual Property that may arise within the project will be shared among the parties between Sweden and India. Work plan (schedule, milestones and deliverables): Based on the proposal, our expertise and the need for improved TB diagnostics, the following aims were made, which will be clearly explained under section 4 below: 1. Full optimization and standardization of the developed proof-of-principle molecular method based on padlock probes, rolling circle amplification in combination with lateral flow biosensors for the detection of MDR/XDR-TB.

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2. Protocol optimization and evaluation including optimization of analytical sensitivity and specificity enabling detection of samples contain scanty pathogens. 3. Validation of the development of the product with clinical samples in a large-scale fashion. 4. Field trials in regional laboratories, primary health clinics and village medical camps. 5. Development of dry reagents, battery operable thermocycler and LF-MDx test kit format.

0-12 months

13-24 months

25-36 months

Project Milestones

Aim I

• Standardization of the developed molecular method at Nilsson’s lab.

• Optimization of the multiplex-lateral flow readout at EMPE Diagnostics.

• Clinical sensitivity and specificity compared with DST gold standards at Xcyto Diagnostics.

• Optimization of dry reagents at EMPE Diagnostics. Aim II

• In-lab validation of LF-MDx test with reference clinical samples in Sweden and India

• Enhancement of efficiency (specific & sensitivity) with clinical samples at CMC-Vellore.

• Optimization of the test using battery operable thermocycler and dry reagents

• Design for commercial prototype at EMPE Diagnostics and XCyton Diagnostics Ltd

Aim III

• Validation of the developed product in the laboratories of CMC-Vellore, SRMC-Chennai and XCyton Diagnostics Ltd. • Evaluation of prototype in the labs of CMC-Vellore, EMPE Diagnostics and XCyton Diagnostics Ltd.

Aim IV

• Clinical evaluation at CMC chain of hospitals, SRMCH and KIIT-Bhubaneswar village medical camps • Optimize the testing and storage conditions.

Dissemination

Literature survey, scientific and technical discussions, transfer of knowledge, lab and field evaluations, visits between countries and focus on further development of the proposal

Deliverables:

1. Development and optimization of the developed molecular method for detection LF-MDx – Patent & Publication

2. Evaluation of efficiency of LF-MDx test in terms of cost, clinical sensitivity, social-economic and commercial interest – Public health & commercial initiative

3. Development of self-sustainable diagnostic test for LF-MDx using dry reagents, battery operable thermocycler with devoid of pipetting - Patent & commercial initiative

4. Clinical validation of the developed prototype in India – Publication & commercialization 5. Field trials in peripheral labs and site-visits – Transfer of knowledge and human relationships

between countries 6. Commercialization of LF-MDx test kit – Commercial interest between the countries

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4. Plan for the execution of the Cooperative Project-withcleardescriptionofresearchactivitiesonbothsidesaswellasoftheelementsofcooperationandexchange,3pagesmaximum

1. Standardization of the developed the molecular method: Based on the preliminary results, the developed molecular method was tested with the DNA of a reference strain, and three resistance clinical isolates of M. tuberculosis. The analytical sensitivity of the assay was determined to be as little as 300 pg target DNA, equivalent to 600 target molecules. However, the following assessments are to be made for real-time analysis:

• Optimization of analytical sensitivity and specificity: The analytical sensitivity of the molecular method will be optimized for performance of both smear-negative and smear-positive sputum samples. Other genes or genomic regions will be included for further improving the clinical sensitivity and specificity of the method. The analytical specificity of the probes will be evaluated and optimized.

• Developing the dry reagents: EMPE Diagnostics in association with Nillson’s lab, all the reagents of PLP-RCA molecular method will be lyophilised to develop dry reagents that can withstand the environmental conditions of India.

• Standardization of visual readout format: Design and printing of the oligo-array on lateral flow strips will be optimized for LF-MDx format for optimal detection by the naked eye. Protocols, handling and reagents will initially be tested and the full assay (including PLP-RCA molecular method and lateral flow readout) will be evaluated on clinical samples obtained from SMI.

• Scaling-up to a kit format: With the help of the industrial collaborator, EMPE Diagnostics, a ready-made LF-MDx kit format will be developed. Further optimizations focusing on preparation of reagents in a large-scale fashion enabling cost-effective production, robust handling and long-term shelf-life will be developed in association with XCyton.

2. Assessment of Limit of Detection (LOD) the molecular method: The developed protocol for the molecular method will be assessed to identify LOD, as follows:

• Standardization of the assay to detect resistant organisms in proportions >1% in a sample: As the developed method, can detect both wild type (susceptible) as well as mutant (resistant) bacteria, the system will be tuned to detect resistance phenotypes only, when the proportion of resistant bacteria crosses 1% of the total bacterial load in a sample. Hence the standardization requires samples known amount of resistant phenotypes and that will be obtained from CMC-Vellore.

• These samples can be of two types: (i) liquid cultures, containing resistant bacteria, anywhere between 0.2% - 1.5%, and is determined by phenotypic proportion method at NIRT, Chennai. These samples contain the resistant bacterial population between <1% - 80%, and these are already available in the strain repository at CMC-Vellore, SMI, and SRMC-Chennai. (ii) Artificially constituted samples, where in to a standard drug susceptible culture different proportions of resistant bacteria are added.

• Phenotypic proportion method: The standard MDR-TB and XDR-TB clinical samples will be obtained from CMC-Vellore, SMI, and SRMC-Chennai and number of organisms will be determined as a part of phenotypic proportion method. The known colony forming units of the organisms will be added to sputum obtained from TB patients and the minimum number of resistant bacilli will be detected by molecular method.

• Sensitivity and specificity: The sensitivity of molecular method based on the minimum number of organisms that can be detected, will be estimated. In addition, NIRT, Chennai will help us with the various resistant and susceptible strains collected from across the nation and deposited at the institute. The XDPL will establish the standard DNA or the cultures and quantitative

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assays in-house for further development based on the variations between batch to batch quality control during and after the prototype development.

• Enhancement of sensitivity of the assay: In case, if the analytical sensitivity of the molecular method is not sufficient to detect sputum smear-positive samples qualified as “scanty”, or detect sputum smear-negative culture positive samples work to enhance the sensitivity of the method will be carried out to detect smear negative/culture positive cases.

• Precision study: The minimum number of bacteria determined in LOD will be added to sputum samples obtained from TB patients and the consistency of detection in the hands of two different technicians in 10 days will be determined. This will be done for the twelve different MDR mutations accounting for 90% of all resistant cases. Similar exercise for all XDR mutations will be done.

• Stabilization of the dry reagents and lateral flow strips: The dry formulations of enzymes, oligonucleotide-conjugated gold nanoparticles, lateral flow test cassettes will be thoroughly tested at respective time intervals for their quality and efficiency. Based on the variations, if any, all the materials will be optimized according to the clinical evaluation of bacterial samples done by EMPE Diagnostics and XCyton Diagnostics.

• Accelerated stability studies: The stability studies will be conducted as per the ICH guidelines in order to determine the shelf life of the product. Further, to improve the stabilization based on the required clinical conditions as well as the geographical/climatic conditions to explore wide-range opportunities.

• Development of a manufacturing process: The EMPE Diagnostics will produce dry reagent formulations and latera flow cassettes, while XCyton Diagnostics Ltd. will develop prototype kits.

3. Validation during the developmental stage by CMC-Vellore: The validation include: (a) Bacterial cultures with known mutations will be used during the standardization of the molecular method protocols. (b) In a double blind study, a set of 200 resistant samples and 100 susceptible culture samples will be tested for determining the analytical/laboratory accuracy of the method. (c) A set of sputum samples from cases suspected of drug resistance or known cases of drug resistance will be tested for sensitivity and specificity. 4. Clinical or field evaluation of the test: The prospective clinical evaluation I: About 600 smear-positive sputum samples obtained from Category II patients at CMC-Vellore and XCyton Diagnostics, in association with NIRT-Chennai, would be used. The results obtained by in-house developed kit would be compared with those obtained using the Hain Genotype MTBDRplus and Genotype MTBDRsl and phenotypic DST methods. These patients are referred to NIRT with suspicion of drug resistance from various parts of India. 5. Clinical evaluation of LF-MDx test kits: A demonstration project, initially in six district TB centres, will be installed. The personnel will be trained to use the test and they will be monitored to evolve a national testing protocol for all centres and a training module can be prepared for RNTCP. After the first-phase of evaluation, the test kits are evaluated in CMC-Vellore, SRMC and KIIT hospital set-ups, primary health care centers and village based medical camps to assess its performance and usage.

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5. Facilities related to project activity available at the institutions where the project will be carried out:

At the Collaborating Indian Institutions At the Collaborating Swedish Institutions

• Clinically evaluate the performance of the test at CMC-Vellore

• Evaluate the commercial feasibility at XCyton Diagnostics Ltd.

• Development of test kits at XCyton Diagnostics Ltd.

• Organization of clinical evaluation in peripheral TB diagnostic laboratories of CMC-chain of hospitals, SRMC University hospital set-up and KIIT’s village medical camps.

• Finalization of commercial kit format according to the Indian diagnostic needs

• Complete optimization of the molecular method at Nilsson’s lab

• Evaluate the test on clinical samples obtained from SMI or EMPE Diagnostics.

• EMPE diagnostics will dry reagents, lateral flow test cassettes.

• EMPE Diagnostics will undertake the stability analysis of reagents of LF-MDx test kit.

• EMPE will also select a battery operable thermocycler and prepare full-kit format.

• The commercial format will be finalized and the technology will be transferred to India.

6. Information of visiting persons for technical mission (per annum).

Estimation of the expected plan of visits; any further modification, according to the needs of joint project has to be communicated and approved by the participants institutions.

A. India to Sweden

Name Organization Duration Purpose

1st Year Joy Michael

& Ravi kumar Banda

CMC-Vellore 25 days Tech transfer and trouble shooting

2nd Year

Sridhar Sathyamurthy & Avinash Sonawane

SRMC & KIIT 15 days Trouble shooting

3rd Year Ravi kumar Banda XCyton 10 days

Discussions and finalization of kit-format

B. Sweden to India

Name Organization Duration Purpose

1st Year Pavankumar Asalapuram EMPE 25 days Tech transfer and

discussions

2nd Year Pavankumar Asalapuram EMPE 15 days Trouble shooting

3rd Year Mats Nilsson

Stockholm University 10 days

Discussions and finalization of kit-format

※ Attention: Please add lines if necessary.

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7. Expected results of Cooperation (e.g. joint publications, patents etc.)

Are any of the expected results likely to have commercial value? (up to 100 words)

1. Development and optimization of the developed molecular method for detection LF-MDx – Patent & Publication

2. Evaluation of efficiency of LF-MDx test in terms of cost, clinical sensitivity, social-economic and commercial interest – Public health & commercial initiative

3. Development of self-sustainable diagnostic test for LF-MDx using dry reagents, battery operable thermocycler with devoid of pipetting - Patent & commercial initiative

4. Clinical validation of the developed prototype in India – Publication & commercialization

5. Field trials in peripheral labs and site-visits – Transfer of knowledge and human relationships between countries

6. Commercialization of LF-MDx test kit – Commercial interest between the countries

8. Personal and Professional Data (CV) of Indian and Swedish PIs must be attached.

(CV must describe the expertise of the PIs in the proposed field of work by citing relevant scientific publications or patent applications during the last 5 years. Do not exceed two pages A4 size for each PI (Font: Times New Roman, size: 11 points).

9. Complementary resources.

Mats Nilsson participates in two projects that aims to develop point of care tests for Ebola and Influenza virus respectively: -The IMI2 project EbolaMoDRAD, coordinated by Ali Mirazimi, Swedish Centre for Disease Control. -The SSF funded project FLU-ID. Main applicant Dag Winkler, Chalmers.

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C. FINANCIAL INFORMATION

Budget or resources for the project; one for the Swedish side and one for the Indian side

Indianside- 1styear

Equipment +consumables +Sr. Scientist+ Research Associate 1+ Research Associate 2+ travel and housing expenses in Sweden. Salary including taxes : 1080000 Equipment : 2400000 Consumables + testing at CMC : 5000000 Travel : 100000 Housing expenses : 470000 Sum : 9050000

- 2ndyearEquipment +consumables +Sr. Scientist+ Research Associate 1+ Research Associate 2+ travel and housing expenses in Sweden. Salary including taxes : 1296000 Consumables + Product development : 2438000 Testing cost at CMC : 1050000 Testing cost at SRMC : 1050000 Testing cost at KIIT : 1050000 Travel : 200000 Housing expenses : 470000 Sum : 7554000 - 3rdyearEquipment +consumables +Sr. Scientist+ Research Associate 1+ Research Associate 2+ travel and housing expenses in Sweden. Salary including taxes : 1555200 Product development : 4088000 Testing cost at CMC : 500000 Testing cost at SRMC : 500000 Testing cost at KIIT : 500000 Media & Chemical : 4500000 Clinical evaluation : 3600000 Travel : 100000 Housing expenses : 70000 Sum : 15413200

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Swedishside

- 1styearStockholm University: 1 postdoc + 0.25 technician + reagents + travel and housing expenses in India. Salary including LKP : 850 Reagents : 386 Travel : 30 Housing expenses : 30 OH (35%) : 454 Sum :1750 kSEK

EMPE Diagnostics:

5% of PI´s salary (0,6 manmonths) and 4 manmonths for work on stabilization of reagents and prototype development.

Total cost Requested funding Salary : 440 220 Reagents : 30 15 Travel : 30 15 Sum : 500 kSEK 250 kSEK

Total Requested Budget: 1750 kSEK (SU) + 250 kSEK (EMPE)= 2 000 kSEK

- 2ndyearTotal Requested Budget: 1750 kSEK (SU) + 250 kSEK (EMPE)= 2 000 kSEK

- 3rdyear

Total Requested Budget: 1750 kSEK (SU) + 250 kSEK (EMPE)= 2 000 kSEK

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2

Nomination (not exceeding 1 A4 page). Motivate why your candidate should be nominated by the University; describe how the nominee will contribute to the department’s research, and how the department will provide support to the candidate.

Sverige satsar f.n. historiskt stora summor i framtida infrastrukturer användbara för strukturstudier med kristallografi, t.ex. MAX IV och ESS. Frielektronlaserteknologier (FEL) börjar visa potential och det finns anledning att tro att detta kommer kunna bli mycket viktiga verktyg inom livsvetenskaperna. Dessutom finansierar VR SWEDSTRUCT, som nyligen blivit en svensk nod i EU programmet för integrerad strukturbiologi INSTRUCT.

Sverige har en internationellt stark ställning inom strukturbiologi och kristallografi, men med ett pågående generationsskifte. Institutionen för Biokemi och Biofysik har sedan länge en stark verksamhet inom EPR och NMR (Gräslund/Mäler), metoder med en klar synergi med kristallografi. DBB ser dessa förutsättningar som en möjlighet att bygga upp en nationellt ledande miljö för strukturbiokemi och har med detta mål sedan 2007 aktivt rekryterat unga lovande gruppledare inom kompletterande vetenskapliga områden med ett fokus på strukturstudier. Vi har nu etablerade starka grupper med unga tillsvidareanställda gruppledare inom strukturell enzymologi (Högbom) samt membrantransport (Drew). Pål Stenmark representerar en tredje viktig inriktning inom strukturbiokemi: studier av medicinskt relevanta proteiner och inhibitorstudier/läkemedelsdesign.

Samtliga dessa inriktningar är väl integrerade med övriga institutionen, på DBB finns ett 10-tal forskargrupper som använder strukturstudier som kompletterande metod. Pål samarbetar även med ämnesmässigt närliggande institutioner vid Stockholms universitet, till exempel institutionen för molekylär biovetenskap (MBW) och institutionen för organisk kemi.

Pål har etablerat en framgångsrik forskargrupp på DBB. Han har tre postdoktorer och en doktorand i sin grupp, och gruppen utvecklas snabbt. Pål har en internationellt framstående roll inom strukturella studier av Botulinum neurotoxinerna och deras receptorer (Structure 2013, Nature communications 2013). Pål har även flera framgångsrika samarbeten med forskargrupper nationellt och internationellt, t.ex. med KI och Harvard. Han har även flera framgångsrika samarbeten inom SU och DBB (PNAS 2013, NAR 2010, NAR 2013). Totalt har Pål redan långt mer än 30 mycket högkvalitativa publikationer. Pål är även en uppskattad lärare som undervisar på flera kurser samt är kursansvarig. Han driver en omtyckt grundkurs samt en bred kemi-seminarieserie. Tillsammans med övriga meriter gör det han skulle vara ett mycket välkommet tillskott till DBBs permanenta lärarkollegium.

Om Pål skulle bli en "Wallenberg Academy Fellow" är institutionen beredd att täcka minst 50% av lönekostnaderna efter det att hans forskarassistentjänst har löpt ut. Vi avser också att sträva efter att han kan anställas permanent som t.ex. lektor vid DBB. Institutionen tillhandahåller lokaler i form av kontor och labb, och delfinansierar lönekostnader för doktorander. Dessutom tillhandahåller institutionen biokemisk basutrustning.

Professor Lena Mäler, Prefekt

D. Signatures of the Principal Investigators & Institutions

▶ Swedish PI

Mats Nilsson 2017-01-16 Name Signature Date____

▶ Indian PI

Ravikumar Banda 2017-01-16

Name Signature Date _

B. Declaration from the Heads of the Collaborating Institutions

It is certified that

i) the Institutions agree to participate in this Joint Research Project

ii) the Institutions shall provide necessary facilities for implementing the Joint Research Project

iii) the Institutions assume financial & other management responsibilities for the duration of the project to be carried out at their institution and

iv) the back-up funding for manpower, consumable etc. is available for this Joint Research Project.

Signature of the Heads of the Institutions (through scanned exchanges)

▶ Head of the Swedish Institution

Lena Mäler 2017-01-16 Name Signature Date

▶ Head of the Indian Institution

Ravikumar Banda 2017-01-16 Name Signature Date