in

November 10, 2016 | National University of Singapore

TUBERCULOSIS Research

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Organised by

CONTENTS

Event Info

Programme

Speakers & Talks

Posters

Contact

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SPRINT-TB (Singapore Programme of Research Investigating New Approaches to Treat-

ment of Tuberculosis) is a translational bench-to-bedside research programme bringing

together clinicians and scientists working in tuberculosis (TB) research, with the aim of im-

proving medicines and approaches to treat and eradicate this high-priority infectious dis-

ease. SPRINT-TB started in 2014 at National University of Singapore and is supported by

the National Research Foundation Singapore under its Translational and Clinical Research

(TCR) Flagship Programme administered by the Singapore Ministry of Health’s National

Medical Research Council.

We are pleased to welcome you at the SPRINT-TB 2nd Annual Symposium—continuing

the series of events that SPRINT-TB hosts annually, each year featuring renowned scientists,

clinicians and key opinion leaders in TB.

EVENT INFO

SPRINT-TB 2nd Annual Symposium

in Tuberculosis Research

Date

November 10, 2016

Venue

CeLS Auditorium, Level 1, Centre for Life Sciences,

National University of Singapore, 28 Medical Drive,

Singapore 117456

Website

www.tuberculosis.sg

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PROGRAMME

November 10, 2016

8.30 am Arrival & Registration

9.00 am

Welcome Address

Prof. Nicholas Paton, SPRINT-TB Programme Director, NUS,

Singapore

Session 1: Mycobacterial Biology & Drug Discovery

Chair: Dr. Martin Gengenbacher, Department of Microbiology &

Immunology, NUS, Singapore

9.10 am

TB Systems Biology – Tools to Take On a Killer

Prof. David Sherman

Center for Infectious Diseases Research, USA

Keynote Speaker

10.05 am

Stress Resistance, Cell Division and In Vivo Persistence

Prof. Sabine Ehrt

Weill Cornell Medical College, USA

Keynote Speaker

10.45 am Tea Break

11.00 am

Flipping threhalose monomycolate across the inner

membrane in mycobacteria

Asst. Prof. Chng Shu Sin, Department of Chemistry, NUS,

Singapore

11.30 am

Membrane Targeting Antimycobacterials – Really?

A/Prof. Mei Lin Go, Department of Pharmacy, NUS,

Singapore

12.00 pm

New ClpP1P2 Inhibitors with Reduced Activity Against the

Human Proteasome: the Next Generation of TB drugs?

A/Prof. Brian Dymock, Department of Pharmacy, NUS,

Singapore

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Session 2: Tuberculosis in Clinic

Chair: A/Prof. Raymond Lin, Department of Laboratory Medicine,

NUH, Singapore

1.50 pm

Human Challenge Studies in Enteric Fever

Prof. Brian Angus

University of Oxford, UK

Keynote Speaker

2.30 pm

Clinical Comparison Study of QFT and T-SPOT in the Detection

of LTBI and Active Pulmonary TB

Dr. Kiyoyasu Fukushima

Nagasaki Genbaku Isahaya Hospital, Japan

Keynote Speaker

3.10 pm Targeting Tuberculosis and Tissue Destruction

Dr. Catherine Ong, Department of Medicine, NUS, Singapore

3.40 pm Tea Break

3.55 pm TB Treatment in the Age of Smartphones

Dr. James Molton, Department of Medicine, NUS, Singapore

4.25 pm

Developing Animal Model Relapse Platforms for Testing Sterilizing Activity of Drug Combinations for Tuberculosis Dr. Rupangi Verma, Department of Medicine, NUS, Singapore

SPRINT-TB Young Investigator

4.50 pm End of Symposium

12.30 pm

Pyrazinamid: Old Drug, New Mechanisms of Resistance

Pooja Gopal, Department of Microbiology & Immunology,

NUS, Singapore

SPRINT-TB Young Investigator

12.55 pm Lunch & Poster Session

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David earned his PhD in Biochemistry from

Vanderbilt University and performed

post‑graduate work at the Rockefeller

University and at Washington University in St. Louis. He began work on M.

tuberculosis while at PathoGenesis Corp., where he played a lead role in the

discovery and early development of the anti‑TB agent PA‑824. Prof. Sherman is

the Tuberculosis BL-3 Director and full Professor at the Center for Infectious

Disease Research, USA. He is an established expert in TB molecular research, with

interests iin virulence and latency of M. tuberculosis and TB drug discovery.

Professor Center for Infectious Diseases Research

Seattle, WA, USA

TB Systems Biology—Tools to Take On a Killer

The M. tuberculosis (MTB) response to drugs is controlled at the systems-level

through gene networks. To design more effective TB therapies, we must

understand the systems-scale consequences of drug perturbation under different

conditions. To develop this insight, we are probing the MTB regulatory network in

a variety of ways. We are using a library of transcription factor (TF) overexpression

mutants, and screening for TFs that influence drug susceptibility when

overexpressed. In combination with transcriptomics studies and new in silico tools

to explore the growing TB gene regulatory network, we have identified several key

TFs and novel drug effector mechanisms.

SPEAKERS & TALKS

KEYNOTE SPEAKER

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Sabine received her Ph.D. in Microbiology from

the Friedrich-Alexander University of Erlangen-

Nürnberg, Germany and performed

postdoctoral research at the Department of International Medicine and

Infectious Diseases at Cornell University Medical College and at the School of

Public Health at the University of California at Berkeley. In 1999, Dr. Ehrt joined

the faculty of the Department of Microbiology and Immunology at Weill Cornell

Medical College. Prof. Ehrt’s research interests are centered on host-pathogen

interactions in the context of tuberculosis. She is a recipient of an Irma T. Hirschl

Career Scientist Award and an Excellence in Mentoring Award from the WMC

Postdoctoral Association.

Stress Resistance, Cell Division and In Vivo Persistence

Among the factors that contribute to the success of M. tuberculosis as a

pathogen is its ability to withstand potentially bactericidal host defenses and to

resist elimination by an activated immune system. Acidification of the

macrophage phagosome represents one such antimycobacterial defense

mechanism. We identified acid hyper-susceptible M. tuberculosis mutants and

report their phenotypic and mechanistic characterization that highlights a link

between stress resistance, cell division and pathogenesis.

Professor Weill Cornell Medical College

New York, NY, USA

KEYNOTE SPEAKER

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Asisstant Professor Department of Chemistry

National University of Singapore

Singapore

Shu Sin received his PhD from Harvard University, and

continued his postdoctoral training at Harvard Medical

School. In 2011 he joined NUS and an Assistant Professor

in Chemistry. Assistant Professor Chng is an expert in outer membrane biogenesis in

bacteria. His interests in mycobacteria primarily lie in the understanding of mycolic acid

transport and assembly.

Flipping Threhalose Monomycolate Across the Inner Membrane in

Mycobacteria

The mycobacterial outer membrane (OM) is characterized by the presence of mycolic

acids, which are C60-C90 long, branched chain fatty acids, either existing in the forms of

trehalose mono- and diesters, or covalently attached to peptidoglycan via arabinogalactan

polysaccharides. Being the major component in the OM, mycolic acids make this bilayer

extremely hydrophobic and render the membrane impervious to many antibiotics. Mycolic

acids are synthesized in the cell as trehalose monomycolates (TMMs), and have to be

translocated across the inner membrane (IM) and the aqueous periplasm before being

functionalized onto the cell wall. While the biosynthetic pathway of TMM and the final steps

of assembly at the OM have been well characterized, how TMM is transported across the

IM and periplasm are still not clear. Recently, an essential IM protein MmpL3 has been

implicated in TMM transport across the cell envelope and is believed to be inhibited by

multiple pharmacophores. Here, we present direct biochemical evidence for the function of

MmpL3 in flipping TMM across the IM. Furthermore, we demonstrate that a couple of

potential MmpL3 inhibitors tested may directly target the TMM flippase. Our work provides

fundamental insights into mycolic acid transport and validates MmpL3 as a viable target for

the development of new antibiotics against mycobacterial infections.

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Associate Professor Department of Pharmacy National University of Singapore

Singapore

Mei Lin obtained her PhD in antimalarial drug discovery

from NUS. Dr Go is a member of the NUS Drug

Development Unit and Medicinal Chemistry Program.

Mei Lin’s research interests are in the area of designing and synthesizing novel

compounds with the aim of optimizing biological activity, physicochemical profiles and

understanding their modes of action by structure-activity relationship studies and

biochemical/ pharmacological approaches.

Membrane Targeting Antimycobacterials – Really?

The concept of damaging the membrane as a therapeutic strategy has received limited

attention, largely due to concerns that agents that disrupt bacterial membranes would lack

selectivity and perturb mammalian plasma membranes as well. Yet, the availability of potent

and selective membrane targeting agents would be a boon to the treatment of TB as it

would offer an effective means by which persistent dormant mycobacteria could be

eradicated and treatment times shortened. I will discuss the antimycobacterial activities of

indoles bearing a cationic amphiphilic motif and a positively charged substituted

aminomethyl arm. A detailed SAR revealed that potent and selective killing of mycobacteria

resided in several 4-fluoro or 6-methoxy 1-octyl indoles in which the basic nitrogen is

embedded in a non-aromatic spiro ring system. These compounds induces changes in the

thermotropic profile of phospholipid vesicles suggestive of localization within the

hydrophobic core of the bilayer. Bacteriological mechanism of action investigations

revealed cell membrane permeabilization and depolarization in M. bovis BCG. These

changes preceded cell death indicating that the loss in membrane integrity was not an

epiphenomenon. Several potent analogs showed monophasic kill kinetics, demonstrated a

low spontaneous resistance mutation frequency and upregulated the cell envelope stress-

inducible promoter piniBAC indicating that envelope-related targets may also be involved

in antimycobacterial activity.

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New ClpP1P2 Inhibitors with Reduced Activity Against the Human

Proteasome: the Next Generation of TB Drugs?

This talk will discuss modifications of mycobacterial ClpP1P2 inhibitors to reduce

the activity against the human proteasome.

Brian W Dymock is an Associate Professor at

NUS and Deputy Director of the NUS Drug

Development Unit (DDU). Brian’s PhD in

organic synthesis was at Southampton and Glasgow Universities, UK. Brian

worked as a medicinal chemist in Roche UK (antivirals) and Vernalis (anticancer

HSP90 inhibitor NVP-AUY922 now in Phase 2). Brian then moved to Evotec UK,

followed by S*BIO Pte Ltd in Singapore as Head of Chemistry in 2006. S*BIO

discovered and developed 5 compounds into the clinic for oncology and

autoimmune diseases, all of which were outlicensed. In December 2011 Brian

joined NUS in the Pharmacy Department. His research focuses on the discovery

and development of biologically active small molecules primarily for cancer. He

has a special interest in designed multiple ligands and fragment screening and is

in the process of establishing a fragment screening platform in NUS. The NUS

DDU was set up in 2011 to add value to life sciences research in NUS through

development of drug discovery projects, testing of novel compounds in internal

assays and advising on commercial aspects.

Associate Professor Department of Pharmacy

National University of Singapore

Singapore

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Researcher Department of Microbiology &

Immunology

National University of Singapore

Singapore

Pooja is a Research Assistant in A/Prof. Thomas

Dick’s lab at NUS where she first started off as

Research Staff during the initial phase of setting

up the Antibacterial drug discovery laboratory and subsequently continued to

pursue her PhD. Her research interests include identifying drug modes of action

and bacterial cell death mechanisms. Her PhD work focused on uncovering the

mechanisms of action of key sterilizing TB drug, pyrazinamide.

Pyrazinamide: Old Drug, New Mechanisms of Resistance

Pyrazinamide (PZA) is a critical component of first and second line treatment of

tuberculosis (TB), yet its mechanism of action largely remains an enigma. We

carried out a genetic screen to isolate M. bovis BCG mutants resistant to

pyrazinoic acid (POA), the bioactive derivative of PZA, followed by whole genome

sequencing of 26 POA resistant strains. We found resistance conferring mutations

in two pathways: missense mutations in aspartate decarboxylase panD, involved in

synthesis of the essential acyl carrier coenzyme A (CoA), and frameshift mutations

in the vitro non-essential polyketide synthase genes mas and ppsA-E, involved in

the synthesis of the virulence factor phthiocerol dimycocerosate (PDIM).

Emergence of resistance through the loss of a virulence factor in vitro may explain

the lack of clear molecular patterns in PZA resistant clinical isolates, other than

mutations in the prodrug converting enzyme. The apparent interference of POA

with virulence pathways may contribute to the drug’s excellent in vivo efficacy

compared to its modest in vitro potency.

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Prof. Brian Angus is the Director of the Oxford Centre for Clinical Tropical

Medicine. He joined the Nuffield Department of Clinical Medicine in 1993 and

originally worked in Thailand and Ghana studying pharmacokinetics in severe

malaria and melioidosis. His research focus is now on clinical trials in influenza,

HIV, Chronic Fatigue Syndrome, C. difficile-Associated Diarrhoea and typhoid.

He is Clinical Tutor in Medicine and Associate Professor and Reader in Infectious

Diseases at the University of Oxford. He holds an honorary Senior Clinical

Scientist tltle at the MRC CTU.

Professor University of Oxford

Oxford, UK

Human Challenge Studies in Enteric Fever

We have established a human challenge study with volunteers given S. typhi or

paratyphi in order to investigate pathophysiology and vaccine efficacy.

KEYNOTE SPEAKER

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Dr. Fukushima has been a TB pulmonologist for

more than 30 years and has served as the Chief

Consultant to the Nagasaki TB Control Program

since 2000. He is has been the Director of the Respiratory Division since 2005 and Deputy

Director of the Japanese Red Cross Nagasaki Genbaku Isahaya Hospital since 2010. Dr.

Fukushima’s TB diagnostics research includes registration trials using QuantiFERON-Plus, the

latest generation interferon gamma release assay, as well as comparing diagnostic aids in

active and latent TB. Dr. Fukushima is a council member of Japan Society for Tuberculosis

and Japan Respiratory Society. He has belonged to Japan’s TB Expert group (led by JATA-

RIT) since 2000 and is also a Clinical Professor in the School of Medicine of Nagasaki

University.

Clinical Comparison Study of QFT and T-SPOT in the Detection of LTBI and

Active Pulmonary TB

Interferon-γ release assays (IGRAs) have revolutionized the diagnosis of tuberculosis (TB)

infecton by using blood samples and minimizing influence from nontuberculous

mycobacteria and BCG vaccination. Two IGRAs are currently available; QuantiFERON®-TB

Gold (QFT) 1) and T-SPOT®.TB (T-SPOT) 2). However, independent studies on clinical

performance of QFT compared to the different methodologies of T-SPOT for the detection

of latent TB infection (LTBI), early active TB, and advanced TB are lacking. The objective of

this study is to compare the sensitivity of QFT and T-SPOT using different methodologies

through a head-to-head comparison study using evidence of early disease by CT scanning

or direct detection of Mycobacterium TB by culture or PCR.

Professor Nagasaki Genbaku Isahaya Hospital

Nagasaki, Japan

KEYNOTE SPEAKER

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Dr Catherine Ong is Associate Consultant in Infectious

Diseases of National University Hospital and Visiting

Consultant in the Singapore Tuberculosis Control Unit.

She was awarded the competitive NRF-MOH

Healthcare Research Scholarship to pursue TB host

immunopathology research with Professor Jon

Friedland at Imperial College London. Catherine was awarded the Presidential Award prize

at the American Society for Leukocyte Biology, the Keystone Symposia Global Health

Travel Award by Bill and Melinda Gates Foundation, the International Investigator Award by

the Infectious Diseases Society of America, and the Exxon-Mobil NUS Research Fellowship.

In 2015, she was awarded the National Medical Research Council Transition Award to

further her career as a clinician-scientist as a Principal Investigator examining host factors

causing pathology in TB affecting the central nervous system. Her areas of interest in TB

include host-pathogen interactions, biomarker discovery and host-directed therapies.

Assistant Professor Department of Medicine

National University of Singapore

Singapore

Targeting Tuberculosis and Tissue Destruction

Pulmonary cavitation, the hallmark of established disease, is characterized by high bacillary

burden. Cavitation is associated with delayed sputum culture conversion, emergence of

drug resistance, and transmission of infection. The host immunological reaction to M.

tuberculosis drives the development of pulmonary cavities. TB is characterized by a matrix

degrading phenotype in which the activity of proteases are unopposed by their specific

tissue inhibitors. The role of the host immune system such as neutrophils in TB will be

explored as there are evidence that immune cells play both a protective role as well as in

host tissue destruction. Consequently, immunomodulatory therapies in preclinical and

clinical trials may be useful adjuncts in treating TB. Strategies targeting the neutrophil and

proteases have the potential to improve cure rates, reduce transmission and decrease

morbidity and mortality.

15

Dr. Molton received his medical training at Fremantle

Hospital in Western Australia and proceeded to

advanced specialist training in Infectious Diseases at the

Alfred Hospital in Melbourne and National University

Hospital in Singapore (NUH). He is in charge of clinical

matters within the NUH Division of Infectious Diseases in his role as Clinical Director, and

oversees research within the Division of Advanced Internal Medicine as Research Director.

During this time he has pursued his interest in tropical and travel medicine, attaining a

Certificate in Travel Health and spending 3 months in Peru studying for the Gorgas

Diploma in Tropical Medicine and Hygiene. He has performed volunteer clinics in

Cambodia with NUS Medical School, and was part of a medical volunteer team that

travelled to the Philippines with the Singapore Red Cross following Typhoon Haiyan. Dr.

Molton’s research interests include TB imaging and enhancing treatment adherence.

TB Treatment in the Age of Smartphones

Suboptimal medication adherence for infectious diseases such as tuberculosis (TB) results

in poor clinical outcomes and ongoing infectivity. The current system of Directly Observed

Therapy (DOT) for TB has a number of limitations. We developed an integrated

smartphone-based DOT system to provide regular medication reminders and facilitate

video recording of pill ingestion at predetermined timings each day, for upload and later

review by a healthcare worker. We evaluated the system in a prospective study of

adherence to a dietary supplement. by the smartphone system and by pill count. We

have demonstrated the feasibility, acceptability and accuracy of a smartphone-based

adherence support and monitoring system. The system has the potential to supplement

and support the provision of DOTS for TB and also to improve adherence in other

conditions.

Consultant Infectious Diseases Division

National University Hospital

Singapore

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Research Fellow Department of Medicine

National University of Singapore

Singapore

Developing Animal Model Relapse Platforms for Testing Sterilizing Activity

of Drug Combinations for Tuberculosis

Success in the field of Tuberculosis has been largely slowed down due to the dearth of

animal models that mimic the human-like pathology in order to evaluate novel drugs/

regimens for the treatment of this deadly disease. To develop a better approach to TB

animal models that may help prioritize regimens for use in clinical trials, we aim to test a

number of mouse TB relapse models and further adjust and refine them to produce

relapse rates equivalent to those seen in TB clinical trials. The approach shall also

incorporate PET-based imaging of mice so that they can be followed for relapse over time,

again resembling the approach of clinical trials more closely than the usual method of

sacrificing animals randomly to check for TB relapse. Currently, we are working with

C3HeB/FeJ mouse model of Tuberculosis, which develops human-like pathology after TB

infection. We are working to tweak this model to overcome its current limitations by

evaluating different infection routes before we can use it in the relapse model.

Rupangi is a research fellow at the Department of

Medicine, National university of Singapore. Her

research interests are focused on host-pathogen

interactions in the context of tuberculosis and drug

discovery against Mycobacterium tuberculosis. She is currently working to develop a better

approach to TB animal models to help prioritize regimens for use in clinical trials. Besides,

she is working on an ex vivo model - the whole blood bactericidal activity (WBA), for

measuring the combined effects of administered drugs, host factors and strain factors on

mycobacterial sterilization.

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NOTES

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Mu-Lu Wu1, Martin Gengenbacher1, Chuu Ling Chan1, Jade Chiu Shan Chung3,

Swaine Chen3,4, Hans-Joachim Mollenkopf5, Stefan H.E. Kaufmann2, Thomas

Dick1

1Department of Microbiology and Immunology, Yong Loo Lin School of

Medicine, National University of Singapore, Singapore; 2Department of Immunology, Max

Planck Institute for Infection Biology, Berlin, Germany; 3Genome Institute of Singapore,

Singapore; 4Department of Medicine, Yong Loo Lin School of Medicine, National

University of Singapore, Singapore; 5Core Facility Microarray/Genomics, Max Planck

Institute for Infection Biology, Berlin, Germany

POSTERS

Extremely Drug Resistant Small-Cell Survival Morphotype in Mycobacteria

Mycobacteria, generally believed to be non-sporulating, are well known to survive shock

starvation in saline for extended periods of time in a non-replicating state without any

apparent morphological changes. Here we uncover that mycobacteria can undergo cellular

differentiation by exposing Mycobacterium smegmatis to mild starvation conditions. Traces

of various carbon sources in saline triggered the development of a novel small resting cell

(SMRC) morphotype. Development of SMRCs could also be observed for other

mycobacteria, suggesting evolutionary conservation of this differentiation pathway.

Fluorescence microscopic analyses showed that development of SMRCs progresses via

septated multi-nucleoided cell intermediates, which divide to generate mono-nucleoided

SMRCs. Intriguingly, saline shock-starved large resting cells (LARCs), which did not show

cell size or surface changes when observed by scanning electron microscopy, remodeled

their internal structure to septated multi-nucleoided cells, similar to the intermediates seen

during SMRC development. Comparative transcriptome analyses of SMRC and LARC

development showed large consistency with our biological observations. These results

suggest that mycobacteria harbor a starvation-induced differentiation program that differs

from the canonical cell division cycle, in which at first septated multi-nucleoided cells are

generated. Under zero-nutrient conditions bacteria terminate development at this stage as

LARCs. In the presence of traces of a carbon source, these multi-nucleoided cells continue

differentiation into mono-nucleoided SMRCs. Both SMRCs and LARCs exhibited extreme

antibiotic tolerance. SMRCs showed increased long-term starvation survival, which was

associated with the presence of lipid inclusion bodies. Interestingly, the stringent response

regulator relA appeared to play the role of cellular differentiation other than a starvation

survival factor in this process.

1

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Pyrazinamide Resistance is Caused by Two Distinct Mechanisms:

Prevention of Coenzyme A Depletion and Loss of PDIM Synthesis

Pyrazinamide (PZA) is a critical component of first and second line treatment of

tuberculosis (TB), yet its mechanism of action largely remains an enigma. We carried out a

genetic screen to isolate Mycobacterium bovis BCG mutants resistant to pyrazinoic acid

(POA), the bioactive derivative of PZA, followed by whole genome sequencing of 26 POA

resistant strains. Rather than finding mutations in fatty acid synthase FAS I and ribosomal

protein S1 / RpsA, we found resistance conferring mutations in two pathways: missense

mutations in aspartate decarboxylase panD, involved in synthesis of the essential acyl

carrier coenzyme A (CoA), and frameshift mutations in the vitro non-essential polyketide

synthase genes mas and ppsA-E, involved in the synthesis of the virulence factor

phthiocerol dimycocerosate (PDIM). The POA-resistant strains fell into different

phenotypic classes based on their resistance levels to POA, which corresponded with their

respective genotypic classes. Probing for cross resistance to two structural analogs of

POA, nicotinic acid and benzoic acid, showed that the analogs share the PDIM but not the

CoA related mechanism of action with POA. Sequencing ten POA resistant

Mycobacterium tuberculosis H37Rv isolates confirmed the presence of at least two distinct

mechanisms of resistance to the drug. Emergence of resistance through the loss of a

virulence factor in vitro may explain the lack of clear molecular patterns in PZA resistant

clinical isolates, other than mutations in the prodrug converting enzyme. The apparent

interference of POA with virulence pathways may contribute to the drug’s excellent in vivo

efficacy compared to its modest in vitro potency.

2 Pooja Gopal, Michelle Yee, Jickky Sarathy, Jian Liang Low, Martin Gengenbacher,

Thomas Dick

Department of Microbiology and Immunology, Yong Loo Lin School of

Medicine, National University of Singapore, Singapore

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Phenotypic and Genotypic Analyses of Pyrazinamide Resistance in

Mycobacterium tuberculosis Clinical Isolates

Pyrazinamide (PZA) was discovered in the 1950s and has since been playing an essential

role as a key treatment-shortening drug in tuberculosis (TB) chemotherapy. Yet, the

mechanism of action/resistance of PZA (a pro-drug) and its active form, pyrazinoic acid

(POA) remains poorly understood. This translates into challenges in accurately diagnosing

PZA resistance in the clinical setting. Phenotypic methods, the gold standard in diagnosing

TB drug resistance, are limited for PZA because of the low potency of the drug in vitro and

the uncertain predictive value of the current growth assays. Molecular methods (detecting

mutations in pncA gene as PncA is involved in conversion of PZA into POA) lack sensitivity

as some clinical isolates are shown to be PZA resistant with wildtype pncA, suggesting

alternative mechanism(s) of PZA/POA resistance. In order to address these shortcomings

in current diagnostic methods, we study the correlation between various growth / growth

inhibition assays and genetic polymorphisms of PZA resistant and susceptible

Mycobacterium tuberculosis clinical isolates. The various growth / growth inhibition assays

include classical broth dilution method vs. BACTEC MGIT 960. The broth dilution assays

include media with different pH. Furthermore, MICs are determined for PZA and POA. First

results of our ongoing analyses will be discussed.

3 Michelle MK Yee1,2, Pooja Gopal1, Rick TH Ong2, Yik-Ying Teo2, Martin

Gengenbacher1, Thomas Dick1

1Department of Microbiology and Immunology, Yong Loo Lin School of

Medicine, National University of Singapore, Singapore; 2Saw Swee Hock

School of Public Health, National University of Singapore; Singapore

21

4 Devika Mukherjee1, Hanxun Zou2, Shouping Liu2,3, Roger Beuerman2,3,4, Thomas

Dick1

1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine,

National University of Singapore, Singapore; 2Singapore Eye Research Institute, Singapore; 3SRP Neuroscience and Behavioural Disorders, Duke-NUS Graduate Medical School,

Singapore; 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National

University of Singapore, Singapore

Membrane Targeting Drugs Against Mycobacteria- a Road Less Travelled

Aims: It is well known that the major issues with TB treatment are resistance and

persistence. Drugs with novel mechanisms of action are needed to help address these

issues. We test the hypothesis that targeting the cytoplasmic membrane may be an

effective way to kill persister mycobacteria as well as delay the emergence of resistance.

Methods: In vitro activity of AM-0016, a novel semi-synthetic xanthone-based

antibacterial, was assessed against growing and persister mycobacteria. Resistance

mutation frequencies were also determined. Membrane potential was assessed

biochemically and electron microscopic analyses were carried out.

Results: AM-0016 rapidly sterilized growing tubercle bacillus cultures and displayed strong

bactericidal activity against hypoxic and INH induced persister bacteria. Spontaneous

resistance mutation frequency was lower than 10-8

. Exposure to AM-0016 resulted in rapid

collapse of the membrane potential. Imaging revealed deformation of the cell envelope.

Conclusions: Targeting the cytoplasmic membrane may be an attractive approach to

eliminate persister mycobacteria and slow down the emergence of genetic drug

resistance.

22

Fragment-Based Mycobacterium tuberculosis Whole Cell Screen Delivers

Hits for Drug Discovery

Mycobacterium tuberculosis is a leading health threat killing 1.5 million people annually

and chemotherapy against tuberculosis (TB) gets increasingly challenging due to the

spread of drug resistance. Therefore, new drugs with a novel mechanism of action are

urgently needed. Current high throughput screening approaches are facing high attrition

rates of compounds often associated with toxicity and unfavorable pharmacokinetic profile.

Here, we introduce a new screening approach using a library of 1000 chemically diversified

fragment compounds, which generally possess desirable physiochemical and

pharmacokinetics properties. At a concentration of 1 mM, the growth inhibition of M.

tuberculosis H37Rv measured by turbidity delivered 84 (8.4%) primary hits at 70% cut off.

Of the hits, 33 compounds showed at least 50% growth inhibition at 500µM in dose

response assays, which is comparable with the MIC50 of pyrazinamide, a fragment first line

anti-TB drug. Fifteen compounds out of the 33 hits were showing activity against the

clinically relevant non-tuberculosis mycobacteria species, Mycobacterium avium and

Mycobacterium abscessus, exhibiting at least 50% growth inhibition at 500µM. Further

profiling of the hits for membrane toxicity (hemolysis assay) and cytotoxicity (HepG2, THP-

1) identified 12 compounds with < 50% cytotoxicity at 500µM concentration. Our findings

indicate that fragment libraries are valuable resources for Mtb drug discovery delivering

low molecular weight hits with attractive physiochemical properties.

Dereje Abate Negatu1,, Joe Jiajun Liu2, Courtney C. Aldrich3, Martin

Gengenbacher2, Thomas Dick1, 3

1 Department of Microbiology and Immunology, Yong Loo Lin School of

Medicine, National University of Singapore, Singapore; 2 BSL-3 Core Facility, Yong Loo Lin

School of Medicine, National University of Singapore, Singapore; 3 Department of

Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minnesota, USA

5

23

Caseinolytic Protease Gene Regulator (ClgR) is a Substrate for the

Mycobacterial Clp Protease

Mycobacterial caseinolytic protease ClpP1P2 is a degradative protease thatrecently gained

interest as a genetically and pharmacologically validated drugtarget for tuberculosis. The

first whole cell active ClpP1P2 inhibitor, bortezomib,is currently undergoing lead

optimization to introduce selectivity for the bacterial target. Our knowledge about

ClpP1P2 regulation and substrate specificity that isrecognized in these to-be-degraded

are limited. Here, employing reporter strainsof Mycobacterium bovis BCG, we show that

inhibition of ClpP1P2 by bortezomibresults in increased transcription of the clpP1, clgR

and acr2 genes. Via redfluorescent protein (RFP) fusions, we showed that ClgR, the

transcriptional activatorfor clpP1P2 and acr2, is a substrate of ClpP1P2 and that the C-

terminalexposure of APVVSLAVA is required for degradation. Accumulation of

ClgRappears to be toxic to bacilli, suggesting post-translational regulation of ClgRvia

ClpP1P2 protease is essential for cellular integrity of mycobacterium and amolecular

mechanism how inhibition of ClpP1P2 protease activity by bortezomibexerts whole cell

antimicrobial activity.

6 Yoshiyuki Yamada1, Thomas Dick2

1Department of Medicine, Yong Loo Lin School of Medicine, National

University of Singapore, Singapore; 2Department of Microbiology and

Immunology, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore

24

Combining the Shotgun and Sniper Approach to Target the Mycobacterial

Cell Wall

Discovering new drugs for tuberculosis (TB) is like playing darts with spaghetti: promising

lead molecules that are potent enzyme inhibitors but cannot penetrate the mycobacterial

cell. Our goal is to identify attractive targets with their associated whole-cell active lead

compounds for development of new clinical candidates. To achieve this, we have employed

a pathway-based whole-cell screen approach, which allows screening against a defined

pathway inside the bacterial cell using the double-membrane barrier as a filter. Of particular

interest is the cell wall biosynthesis pathway because it is well validated as a drug target.

Our study design employs a two-tier screening strategy for hit identification. First, a high-

throughput screen of over 70,000 chemically diverse scaffolds for growth inhibitors of

Mycobacterium bovis BCG (M. bovis BCG), which led to the selection of 18 compounds.

Second, a pathway-based screen of the primary hits in a M. bovis BCG reporter strain

carrying a cell wall stress inducible promoter, iniBAC fused to the mCherry reporter gene.

Differential expression of the reporter gene allowed us to identify a new class of a cell wall

targeting compound, E11. The compound is non-cytotoxic and has a minimum inhibitory

concentration (MIC50) of about 10µM against M. bovis BCG and M. tuberculosis. Currently,

we are carrying out efficacy studies against persisters and target deconvolution by chemical

genetics (resistance generation coupled with whole genome sequencing).

7 Annanya Shetty1, Umayal Lakshmanan2, Choong Ling2, Anders Poulsen2, Jeffrey

Hill2, Yoshiyuki Yamada1, Thomas Dick1

1Department of Microbiology and Immunology, Yong Loo Lin School of

Medicine, National University of Singapore, Singapore; 2Experimental and Therapeutics

Centre A*STAR, Singapore

25

Wilfried Moreira, Dinah binte Aziz, Thomas Dick

Department of Microbiology & Immunology, Yong Loo Lin School of Medicine,

National University of Singapore, Singapore

Boromycin Kills Mycobacterial Persisters without Detectable Resistance

Boromycin is a boron-containing polyether macrolide antibiotic isolated from

Streptomyces antibioticus. It was shown to be active against Gram positive bacteria and to

act as an ionophore for potassium ions. The antibiotic is ineffective against Gram negative

bacteria where the outer membrane appears to block access of the molecule to the

cytoplasmic membrane. Here we asked whether Boromycin is active against

Mycobacterium tuberculosis which, similar to Gram negative bacteria, possesses an outer

membrane. The results show that Boromycin is a potent inhibitor of mycobacterial growth

(MIC50 = 80 nM) with strong bactericidal activity against growing and non-growing drug

tolerant persister bacilli. Exposure to Boromycin resulted in a rapid loss of membrane

potential, reduction of the intracellular ATP level and leakage of cytoplasmic protein.

Consistent with Boromycin acting as a potassium ionophore, addition of KCl to the

medium blocked its antimycobacterial activity. In contrast to the potent antimycobacterial

activities of the polyether macrolide, its cytotoxicity and haemolytic activity were low (CC50

= 30 uM, HC50 = 40 uM) with a selectivity index of more than 300. Spontaneous resistant

mutants could not be isolated suggesting a mutation frequency of less than 10-9 / CFU.

Taken together, the results suggests that targeting the mycobacterial membrane / ion

gradient may be an attractive chemotherapeutic intervention level to kill otherwise drug

tolerant persister bacilli, and to slow down the development of genetic antibiotic

resistance.

8

26

Adam Hotra1,2,3, Manuel Suter4, Goran Biuković4, Priya Ragunathan1, Subhashri

Kundu4, Thomas Dick4 and Gerhard Grüber1

1School of Biological Sciences, Nanyang Technological University, Singapore; 2School of Physical and Mathematical Sciences, Nanyang Technological

University, Singapore; 3Nanyang Institute of Technology in Health and Medicine,

Interdisciplinary Graduate School, Nanyang Technological University, Singapore; 4Department of Microbiology and Immunology, Yong Loo Lin School of Medicine,

National University of Singapore, Singapore

Deleting a Unique Polypeptide γ-Loop of Mycobacterial F-ATP Synthase

Affects its ATP Catalysis and H+-Pumping

The discovery of the drug bedaquiline, which specifically targets mycobacterial F-ATP

synthase, proved that the enzyme is essential for both aerobically growing and dormant

mycobacteria (1,2). In mycobacteria the enzyme catalyses ATP synthesis by the catalytic

domain (α3/β3 subunits) at the expense of the torque energy that is generated by the

rotating transmembrane H+-pump (a/c9 subunits) and the connecting central stalk γ/ε

subunits. The reversible reaction, a weak or so called “latent” ATP hydrolysis was also

observed in some of the fast growing mycobacteria (3,4). Previously, we described the

solution structure of mycobacterial subunit γ (γ1-204) and its unique polypeptide loop γ166-

179, the latter was assigned by a sequence analysis in the close proximity of rotating c-ring

(5). Here we determined whether the loop γ166-179 affects ATP catalysis and subsequent H+-

pumping (6). We performed functional studies of F-ATP synthase containing γ (wild type) or

γ without the loop (Δγ166-179), using inverted membrane vesicles (IMV) of strain M.

smegmatis mc2 155 and a set of different F-ATP synthase inhibitors. The overall reduction

of ATP hydrolysis in the presence of different inhibitors, confirmed that the wild type (WT) F

-ATP synthase has a low ATP hydrolytic activity. An increase of the ATP hydrolytic activity by

34% and the reduction of the ATP synthesis activity by 54% were observed in IMV of the

mutant lacking the loop, as compared with WT F-ATP synthase. So far, ATP hydrolysis

driven H+-pumping was not observed in mycobacteria (4). However, deletion of the γ-loop

resulted in coupling of ATP hydrolysis and H+-pumping (6). In summary, these results

suggest that the γ-loop plays a distinctive role in regulating and coupling of ATP synthesis/

hydrolysis with H+-pumping.

9

27

Bedaquiline Targets the ε Subunit of Mycobacterial F-ATP Synthase

The tuberculosis drug Bedaquiline inhibits mycobacterial F-ATP synthase by binding to its

c subunit. Using purified ε subunit of the synthase and spectroscopy we previously

demonstrated that the drug interacts with this protein near its unique tryptophan residue.

Here we show that replacement of ε’s tryptophan with alanine resulted in Bedaquiline

hyper susceptibility of the bacteria. Overexpression of wild type ε subunit caused

resistance. These results suggest that the drug targets the ε subunit.

Subhashri Kundu1, Goran Biuković1, Gerhard Grüber2, Thomas Dick1

1Department of Microbiology and Immunology, Yong Loo Lin School of

Medicine, National University of Singapore, Singapore; 2School of

Biological Sciences, Nanyang Technological University, Singapore 10

28

Comparison of Imaging Modalities in Tuberculosis

Aims: PET scanning offers the chance to assess the metabolic activity of lesions, and may

reflect more closely the biological efficacy of drugs. PET-MRI eliminates the radiation dose

associated with CT leaving just that of the PET scan resulting in 75% reduction in total

radiation dose. The aim of this study was to compare various PET, MRI and CT options for

imaging TB with a view to assessing their potential for clinical or research applications in

quantifying the burden of TB.

Methods: Patients with confirmed pulmonary TB were recruited from 2 hospitals in

Singapore. Clinical assessment was performed. Sputum was sent for for acid fast smear,

culture and GeneXpert. A standard posterior-anterior chest X-ray (CXR) was performed.

PET-MRI was performed using a Siemens Biograph mMR PET-MR scanner. In select cases

PET-CT was performed using a Siemens Biograph mCT PET-CT scanner.

Results: We recruited 20 patients: mean age was 56 years; 75% were male. 40% reported

cough, 50% weight loss, 15% fever and 20% night sweats. The median time between

commencing TB treatment and the study visit was 13.5 days. 45% had bilateral disease on

CXR, 30% had cavitation, 15% had pleural effusion (all unilateral), 15% had

lymphadenopathy and 35% had nodules. Mean lung involvement was 17.8%.

Ten patients had combined PET/MR and PET/CT. A total of 108 PTB lesions were detected

on PET/MR and 112 on PET/CT. The four lesions that were not observed on PET/MR all

had low mean SUVs (≤ 1.3).

Conclusions: PET MRI appears to offer a number of advantages over CXR in detecting TB

and monitoring response to treatment for little extra radiation exposure. We saw evidence

of more extensive disease on PET-MRI than CXR in most patients. PET/MR is qualitatively

comparable to PET/CT, with all but 4 lesions seen on PET/CT also being visualised on PET/

MR.

James S. Molton1, 2, Benjamin A. Thomas3, Pang Yan1, Padmasayee

Papineni2, John J. Totman3, Tow Keang Lim1, 2 , Roland Jureen4, Nicholas

I. Paton1, 2

1University Medicine Cluster, National University Health System, Singapore; 2Department of

Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 3A*STAR-NUS Clinical Imaging Research Centre, Singapore; 4Department of Laboratory

Medicine, Microbiology Unit, National University Hospital, Singapore

11

29

James S. Molton1, 2, Benjamin A. Thomas3, Pang Yan1, , Claire Naftalin2,

John J. Totman3, Tow Keang Lim1, 2 , Bin Eng Cynthia Chee4, Yee Tang

Sonny Wang4, Nicholas I. Paton1, 2

1University Medicine Cluster, National University Health System, Singapore; 2Department of

Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 3A*STAR-NUS Clinical Imaging Research Centre, Singapore; 4Tuberculosis Control Unit,

Tan Tock Seng Hospital, Singapore

Sub-Clinical Tuberculosis Detected by PET-MRI in Household Contacts of

Individuals with Smear Positive Pulmonary Tuberculosis

Introduction: Traditionally there are considered to be several possible outcomes following

exposure to TB: no infection, latent infection and active clinical disease. We sought to

determine whether there is evidence of subclinical disease activity in heavily exposed

healthy TB contacts using PET/MRI.

Methods: Contacts living with an individual with smear-positive TB for ≥1 month underwent

clinical assessment, IGRA, chest X-ray (CXR) and PET/MRI scan. PET/MRI was performed

using a Siemens Biograph mMR PET/MR scanner one hour after injection with ~150 MBq

18F-FDG. Images were systematically reviewed. Standardized uptake value (SUV) analysis

was performed for each abnormal lesion.

Results: 30 household contacts (40% IGRA positive) of 20 index patients were enrolled. CXR

was abnormal (minor upper lobe scarring) in one contact. PET/MRI scan was abnormal in

23%, predominantly FDG uptake in hilar lymph nodes or lung apices (SUVmax 1•6 to 3•9).

Abnormal MRI findings were seen in 17% (enlarged lymph nodes, minimal pleural effusions

and focal parenchymal signal abnormalities).

Discussion: We found evidence of subclinical TB amongst heavily-exposed, asymptomatic

household contacts. These may represent a sub-group at higher risk of later reactivation.

PET-based imaging may provide important insights into the natural history of sub-clinical

TB that may not be available from structural imaging methods.

12

30

Francesca Leek1, Claire Naftalin

2, Benjamin Thomas

1, Pang Yan

2,

John Totman1, David Townsend

1, Nicholas Paton

2 13

1A*STAR-NUS Clinical Imaging Research Centre, Singapore; 2

Department of

Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

68Ga-DOTANOC PET/MR for Imaging Pulmonary Lesions in Tuberculosis

Introduction: PET/MR imaging allows assessment of the metabolic activity of lesions (PET) as

well as structural anatomy of pulmonary pathology (MRI). The standard radiotracer for PET

is 18F-fluorodeoxyglucose (FDG), which labels metabolically active cells; due to the non-

specific uptake of FDG there is a need to identify alternative PET tracers to localise and

characterise pulmonary tuberculosis (TB) lesions.

68Ga-DOTA-I-NaI3-octreotide (DOTANOC) is a somatostatin analogue PET ligand which

binds with high affinity to somatostatin receptor-subtype 2, the most prevalent receptor

subset in granulomatous tissue such as TB. Although TB lesions have been identified using

somatostatin analogue tracers using SPECT, no study has previously utilised PET.

This study assesses the ability of 68Ga-DOTATNOC PET/MR to detect pulmonary lesions in

subjects with active TB; the uptake will be compared to that of FDG PET/MR.

Methods: For this pilot study, five patients with microbiologically confirmed pulmonary TB

are to be recruited within 28 days of starting TB therapy. A 68Ga-DOTANOC dynamic PET/

MR scan will be performed followed by an FDG dynamic PET/MR scan within 48 hours.

The two PET ligands are to be compared both qualitatively and quantitatively to determine

the suitability of 68Ga-DOTANOC for detecting active TB lesions.

Results: Preliminary qualitative analysis of the four subjects scanned to date suggests a

more focal uptake of 68Ga-DOTANOC in large regions of active lung when compared with

a more homogeneous FDG uptake. 68Ga-DOTANOC identified additional lesions which

had no FDG uptake resulting in the percentage of active lung being greater in the 68Ga-

DOTANOC scans.

Conclusion: This exploratory study confirms that 68Ga-DOTANOC PET detects pulmonary

TB lesions and may be of use in the localisation and characterisation of lesions. The efficacy

of 68Ga-DOTANOC in identifying subclinical disease is to be investigated in a larger cohort

of high risk contacts of patients with TB.

31

Meera Gurumurthy1, Rupangi Verma1, Claire M. Naftalin1, Kim Hor

Hee1, Qingshu Lu2, Kin Hup Tan1, Simi Issac3, Wenwei Lin1, Angelia

Tan4, Kok-Yong Senga1, Lawrence Soon-U Lee1, Nicholas I. Paton1

1Department of Medicine, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore; 2Singapore Clinical Research Institute, Singapore; 3BSL-3 Core

Facility, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 4Investigational Medicine Unit, National University Health System, Singapore

14

Activity of Faropenem with and without Rifampicin against Mycobacterium

tuberculosis: Evaluation in a Whole Blood Bactericidal Activity Trial

Background : Faropenem has in vitro activity against Mycobacterium tuberculosis (Mtb) and

shows synergy with rifampicin. We tested this in a whole-blood bactericidal activity (WBA)

trial.

Methods: We randomised healthy volunteers to receive a single oral dose of faropenem

(600mg; with amoxicillin/clavulanic acid 500mg/125mg; FAR-AMC n=8); rifampicin (10mg/

kg; RIF n=14); or the combination (RIF-FAR-AMC n=14). Blood was drawn at intervals to 8

hours post-dose. Drug levels were measured using liquid chromatography-tandem mass

spectrometry. WBA was measured by inoculating blood samples with Mtb and estimating

the change in bacterial colony forming units (CFU) after 72 hours.

Results: There was no activity in the FAR-AMC group (cumulative WBA 0.06ΔlogCFU;

p=0.99 vs zero change). There was a trend favouring the RIF-FAR-AMC group at 8 hours

(cumulative WBA -0.57±0.09 and -0.60±0.10ΔlogCFU in RIF and RIF-FAR-AMC respectively;

p=0.113) that was significant in the first hour post-dose (p=0.023). Faropenem Cmax and

AUC were 5.4mg/L and 16.2h*mg/L respectively, and MIC against Mtb H37Rv was 5-10

mg/L.

Conclusions: Faropenem is not active when used alone, possibly due to inadequate plasma

levels relative to MIC. However, there was evidence of modest synergy with rifampicin that

may merit further testing in clinical trials.

Trial registration: ClinicalTrials.gov (NCT02393586)

32

Claire M. Naftalin1, Rupangi Verma1, Meera Gurumurthy1, Qingshu Lu2,

Benjamin Yeo Chaik Meng1, Kin Hup Tan1, Wenwei Lin1, Buduo Yu3,

Veronique Dartois4, Nicholas I. Paton1

1Department of Medicine, Yong Loo Lin School of Medicine, National

University of Singapore, Singapore; 2Singapore Clinical Research Institute, Singapore; 3nvestigational Medicine Unit, National University Hospital, Singapore; 4Public Health

Research Institute Centre, New Jersey Medical School, Rutgers, USA

15 Evaluating Systemic Pyrazinoic Acid Boosted with Allopurinol against

Mycobacterium tuberculosis: Application of a Whole Blood Bactericidal

Activity Model

Background: Co-administering pyrazinamide (PZA) with allopurinol increases systemic

levels of the active metabolite, pyrazinoic acid (POA), by inhibiting the enzyme xanthine

oxidase. We performed a Whole-blood Bactericidal Activity (WBA) trial to determine

whether increasing host-derived POA levels enhances anti-mycobacterial activity.

Methods: 12 healthy volunteers were randomized to take either PZA 10mg/kg or PZA

25mg/kg. At the 1st study visit, participants took a single dose of PZA. At the 2nd visit (7

days later), PZA plus allopurinol 100mg was given. Blood was drawn at intervals to 48 hours

post PZA dose. Drug levels were measured using liquid chromatography-tandem mass

spectrometry. WBA was measured by inoculating blood samples with Mtb and estimating

the change in bacterial colony forming units (CFU) after 72 hours.

Results: Co-administration of PZA with allopurinol produced a large increase in the POA

AUC(0-48) (62.9h*µg/mL versus 93.4 h*µg/mL for PZA alone versus PZA+allopurinol

respectively; P < 0.001) and in POA Cmax (2.8 µg/mL versus 4.0 µg/mL P<0.001). The

mean cumulative WBA at 8 hours post-dose was 0.05±0.06ΔlogCFU with 10mg/kg PZA

alone and -0.01±0.06ΔlogCFU with 25mg/kg PZA alone. There was no effect of allopurinol

co-administration on cumulative WBA for PZA doses combined (0.02±0.07ΔlogCFU for

PZA alone versus 0.00 ±0.07ΔlogCFU for PZA plus allopurinol; difference -0.01, 95% (CI -

0.06 to 0.03) ΔlogCFU; P = 0.49). WBA and POA concentrations showed a significant

relationship (P <0.0001) , higher drug levels corresponding to greater bactericidal activity.

Conclusions: Our study demonstrated little bactericidal activity of PZA in the WBA.. Co-

administration of allopurinol with PZA significantly boosted plasma levels of POA without

corresponding increase in WBA. This may be due to the conditions of the WBA assay not

being conducive to demonstrating anti-mycobacterial activity of PZA, or the possibility of

the host-generated POA being less effective than POA generated inside bacteria.

33

1 National University Singapore, Singapore; 2 Medical Research Council

Clinical Trials Unit at UCL, United Kingdom; 3 Singapore Clinical Research Institute,

Singapore; 4 Duke-NUS, Singapore

TRUNCATE-TB: Design and Implementation of a Strategic Clinical Trial in

Asia

Two-thirds of the global TB burden is estimated to be in Asia. TRUNCATE-TB (two-month

regimens using novel combinations to augment treatment effectiveness for drug-sensitive

tuberculosis (DS-TB)) is a randomised, open-label, multi-arm, multi-stage (MAMS), parallel

group strategy trial. The trial will be implemented at clinical sites in Philippines, Thailand,

Indonesia and Singapore, with the goal of building clinical trial capacity and collaborations

regionally.

The primary aim of the trial is to determine whether a strategy of treating pulmonary DS-TB

for 2 months with one of a number of novel combination regimens and re-treating relapse

with a 6 month course of standard treatment will be non-inferior to the WHO-standard 6

month treatment/8-month re-treatment approach in terms of TB sputum culture status at 2

years after randomisation. Secondary aims are to determine whether there are advantages

of the TRUNCATE-management strategy from the patient perspective (e.g. quality of life)

and programme perspective (e.g. adherence, drug resistance).

Adult patients will be randomised to receive 6 months’ standard treatment as a control, or

to one of a number of boosted 2-month regimens chosen for their enhanced sterilising

activity (combinations of standard drugs with new or repurposed drugs: high-dose

rifampicin, linezolid, clofazimine, bedaquiline, rifapentine, or levofloxacin).

16 Padmasayee Papineni1, Pang Yan1Patrick Phillips2, Qingshu Lu3, Yin Bun

Cheung4, Andrew Nunn2, Nicholas Paton1

34

17 Huipeng Neo1,2,4, 1Shanshan Ji, Federico Torta1, Anne Bendt1, Cynthia

Chee5, Yee Tang Wang5, Pavanish Kumar3, Amit Singhal3, Gennaro De

Libero3, Markus R Wenk1

1Singapore Lipidomics Incubator (SLING), National University of Singapore, Singapore;

2Department of Biological Sciences, Faculty of Science, Singapore; 3Singapore Immunology

Network (SigN), A*STAR, Singapore; 4Agilent Technologies Singapore Pte Ltd, Singapore; 5Tuberculosis Unit, Tan Tock Seng Hospital, Singapore

Lipid Variations in Human Plasma of Tuberculosis Patients

Eradication of tuberculosis (TB) remains challenging with lengthy treatment durations and

the existence of drug resistant Mycobacterium tuberculosis (Mtb). As human pathogens

continue to infect and acquire resistance to current anti-microbial drugs, recent focus has

turned to potential host-directed therapeutics in search for novel treatment strategies.

Pathogens such as Mtb make use of host lipids as building blocks and influence the host

cell physiology to enable their survival and replication. Hence, detection of changes in the

host lipidome during TB progression may enable novel and precise diagnostic tools as well

as potentially providing insights into host-pathogen interactions. To test this hypothesis, we

aimed to identify changes in the human plasma lipidome of active TB patients in

comparison with the lipidome of healthy controls and latent individuals. Analyses were

performed by liquid chromatography mass spectrometry using targeted analysis for the

major classes of lipids. The lipid classes that showed the most significant differences (p <

0.05) were molecular species of ceramides, sphingomyelins, plasmalogen and ether

phosphatidylethanolamines. These lipids were significantly lower in the plasma of active TB

patients. Validation of these potential disease biomarkers in longitudinal studies with

human plasma samples from active TB patients who were on established TB drug treatment

over a period of six months was carried out and will be discussed.

35

Obtaining Quality RNA from Intracellular Mycobacterium tuberculosis

from Clinical Samples for Gene Expression Profiling

Transcriptomics approaches have shown promise as potential biomarkers for TB. The

Whole Blood Bactericidal Assay (WBA) is an ex vivo PK/PD model that measures the

bactericidal activity of study drug (s) in the context of the host immune system and

bacterial strain virulence.1 The bacterial transcriptional signatures from clinical samples

(whole blood cultures and sputum) would be useful tools to evaluate the efficacy of

potential TB drug candidates(s). One of the key challenges is to obtain sufficient starting

material for gene expression profiling, given the variable and low bacterial load at time of

sample collection and after drug treatment. Moreover, given the nature of mixed host-

pathogen clinical samples, host RNA would make up majority of the extracted RNA

population. Our overall aim of this project is to develop a RNA extraction method that

would allow us to obtain quality Mycobacterium tuberculosis RNA from clinical samples for

gene expression profiling. This work describes the resolution of 2 challenges:

Mycobacterial cell lysis and bacterial RNA enrichment and stabilization. We were able to

detect as low as 5x10^2 CFU of Mycobacterium bovis bacillus Calmette-Guerin (BCG) in

whole blood cultures by quantitative PCR. By associating the CT values with the

corresponding CFUs, we established a standard curve. The sequencing of these samples is

underway to evaluate for quality, depth and coverage.

Philip Kam Weng Kwan1,, Wenwei Lin1,, Paola Florez de Sessions2,

Ahmad Naim Nazri Mohamed2, Nicholas Paton,1, Martin Lloyd Hibberd2,3

1Department of Medicine, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore; 2 Genome Institute of Singapore, Agency for Science, Technology

and Research, Singapore; 3Department of Pathogen Molecular Biology, London School of

Hygiene & Tropical Medicine, United Kingdom

18

36

James S. Molton1, 2, Yan Pang1, Zhuochun Wang3, Boqin Qiu3, Pei Wu3,

Afifah Rahman Shepherd1, Wei Tsang Ooi3, Nicholas I. Paton1, 2

1University Medicine Cluster, National University Health System,

Singapore; 2Department of Medicine, Yong Loo Lin School of Medicine, National

University of Singapore, Singapore; 3School of Computing, National University of

Singapore, Singapore

19 Development and Evaluation of a Smart-Phone-Based System for

Implementing Directly Observed Therapy

Objectives: Suboptimal medication adherence for infectious diseases such as tuberculosis

(TB) results in poor clinical outcomes and ongoing infectivity. The current system of Directly

Observed Therapy (DOT) for TB has a number of limitations. We aimed to develop and

evaluate a smartphone-based system to facilitate remotely observed therapy via

transmission of videos rather than in-person observation.

Design: We developed an integrated smartphone and web-based system to provide

regular medication reminders and facilitate video recording of pill ingestion at

predetermined timings each day, for upload and later review by a healthcare worker. We

evaluated the system in a single arm, prospective study of adherence to a dietary

supplement. Healthy volunteers were recruited through an online portal. Entry criteria

included age ≥21 and owning an iOS or Android based device. Participants took a dietary

supplement pill once, twice or three-times a day, for 2 months. We instructed them to

video each pill taking episode using the system.

Outcome: Adherence as measured by the smartphone system and by pill count.

Results: 42 eligible participants were recruited (median age 24; 86% students). Videos were

classified as received - confirmed pill intake (3475, 82.7% of the 4200 videos expected),

received - uncertain pill intake (16, <1%), received - fake pill intake (31, <1%), not received

– technical issues (223, 5.3%) or not received – assumed non-adherence (455, 10.8%).

Overall median estimated participant adherence by MIST was 90.0%, similar to that

obtained by pill count (93.8%). There was a good relationship between participant

adherence as measured by MIST and by pill count (Spearmans rs 0.66, p<0.001).

Conclusions: We have demonstrated the feasibility, acceptability and accuracy of a

smartphone-based adherence support and monitoring system. The system has the

potential to supplement and support the provision of DOTS for TB and also to improve

adherence in other conditions.

37

NOTES

38

CONTACT

SPRINT-TB MANAGER

Dr. Kristina Rutkute

kristina_rutkute@nuhs.edu.sg

+65 6601 5371

SPRINT-TB DIRECTOR

Prof. Nicholas Paton

nick_paton@nuhs.edu.sg

+65 6772 6988

SYMPOSIUM ORGANISERS

SPRINT-TB

National University of Singapore

Center for Translational Medicine

MD6, Level 15

14 Medical Drive

Singapore 117599

sprint-tb@nus.edu.sg

www.sprinttb.org

SYMPOSIUM WEBSITE

www.tuberculosis.sg

39

40

Copyright © 2016 SPRINT-TB. All rights reserved.

PLATINUM SPONSOR:

EVENT SUPPORTED BY:

SPRINT-TB-16-017