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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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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
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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.
38
CONTACT
SPRINT-TB MANAGER
Dr. Kristina Rutkute
+65 6601 5371
SPRINT-TB DIRECTOR
Prof. Nicholas Paton
+65 6772 6988
SYMPOSIUM ORGANISERS
SPRINT-TB
National University of Singapore
Center for Translational Medicine
MD6, Level 15
14 Medical Drive
Singapore 117599
www.sprinttb.org
SYMPOSIUM WEBSITE
www.tuberculosis.sg