University Community Engagement Conference

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University Community Engagement Conference 23-26
November,2009, Bayview Hotel, Penang, Malaysia

• .

Sustainable diagnostics for the third world
countries
Asma Ismail PhD Deputy Vice-Chancellor, Research
and Innovation and Professor, Institute for
Research in Molecular Medicine (INFORMM),
Universiti Sains Malaysia, 11800 Penang,Malaysia
A child dies every 15 seconds from a water-borne
disease
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Introduction to Diagnostics

• When we are sick, diagnostics have to be
  performed so that we know what is the cause of
  the problem in order to provide effective
  management to patients.
• Despite the availability of diagnostics in the
  market, we still do not have adequate number of
  diagnostic tests for the underdeveloped countries
  because
• Diagnostics that have been developed do not
  address the disease needs of developing and/or
  underdeveloped countries.
• Even if the diagnostics are available, they
  cannot be used since they need trained personnel
  to perform the test
• Available but cannot be used due to the lack of
  proper infrastructure/facilities
• Available but they are too expensive.

Developing Malaysian Indigenous Technology
platforms
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Introduction

• Herein lies the real challenge of RD to provide
  solutions to the neglected people especially in
  the area of health.
• There is a need to make sure that the diagnostics
  offered have sustainable qualities.

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RD approaches to diagnostics

• In the area of diagnostics, research need to be
  performed with the client in mind so that the
  product developed is relevant
• Create diagnostics that would be in demand at
  least for 10 years
• Technology chosen should be innovative and if
  possible should use indigenous technology
  platforms.
• Developed products should be used to enhance
  K-economy of the country and as well as enhance
  the quality of life of the people.

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What kind of diagnostics?
Advise from a Nobel Laureate
If we work on research topics that the West is
not interested in, we will always be 20 years
ahead. If we work on topics that the West is
interested in, we will always be 20 years
behind. ...... Ahmad Zewail
BLUE OCEAN STRATEGY Kim,W.C. and Mauborgne,R.
(2005).
1999 Nobel Prize in Chemistry
Linus Pauling Professor of Chemical Physicsand
Professor of Physics at Cal Tech
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Working from Z to A Market foresight
requirements for rapid diagnostics
Criteria for design and development

• Rapid
• Specific
• Sensitive
• Easy to perform
• Built-in controls
• Cost effective
• Transported without cold chain

Patent indigenous technology platforms
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Application of Blue Ocean Strategy to detection
of typhoid carriers

• Typhoid is a disease mainly among the
  underdeveloped countries and remains a public
  health problem with 21 million typhoid cases and
  200 000 deaths annually mainly among children1.
• Typhoid is caused by the bacteria Salmonella
  enterica serovar Typhi which is transmitted via
  food handlers who are carriers.
• WHO defines a chronic carrier as one who
  continues to excrete S. Typhi in stools 1 year
  after the onset of acute typhoid fever.
• Excretion within less than a year is considered
  as a transient carrier.

• Crump, J.A., S.P. Luby and E.D. Mintz. 2004. The
  global burden of typhoid fever. Bulletin of the
  WHO. 82(5) 346-353.
• WHO (2003) Backgroud document The diagnosis,
  treatment and prevention of typhoid fever.
  Communicable Disease Surveillance and Response
  Vaccines and Biologicals WHO/VB/03.07.

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Carrier diagnosis

• Available methods to diagnose for typhoid
  carriers
• Antibody detection test by means of Vi antigen
  (not available commercially)
• Culture method
• Stools
• Rectal swabs
• Bile
• PCR assay is not yet available the market.
• Carrier detection by means of stool or rectal
  swab culture is only 1-5 due to the intermittent
  release of the organism and low culture isolation
  rate from stool 2. For acute typhoid, the
  sensitivity is higher for stool culture at 25.
• Bile culture for carrier status can deliver
  sensitivity of gt90 but is traumatic to patients.
• Hence asymptomatic carriers continue to
  perpetuate the disease.
• If typhoid carriers can be detected and treated,
  we would be able to effectively control the
  spread of typhoid

WHO (2003) Backgroud document The diagnosis,
treatment and prevention of typhoid fever.
Communicable Disease Surveillance and Response
Vaccines and Biologicals WHO/VB/03.07.
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Focus and benefit of research

• Detection of typhoid carriers is not easy due to
  the lack of effective lab tests for carriers.
• If we can design diagnostic tests to detect for
  carriers
• We can provide treatment to the carriers
• We create a carrier registry and study the
  carriers himself/herself (Fundamental and
  clinical studies)
• We can isolate S.Typhi from the carriers,
  sequence its DNA and compare that to those
  isolated from acute cases (Molecular studies)
• We can monitor and help reduce transmission of
  the disease effectively in the community and
• Reduces health cost to the government
• Many studies (goldmine for research) can be done
  if we can detect for carriers

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Study on Carriers of typhoid in Kelantan
USM Health campus

• In comparison to our neighbouring countries,
  Typhoid is not a big problem in Malaysia
  (incidence rate 3/100,000 population or 300 to
  400 cases/year). We do not have multi-resistant
  strains and diagnosis of the disease can be done
  via culture and serological method.
• Typhoid however is a problem Kelantan since it is
  endemic here.
• Despite outbreaks of typhoid in the state, no
  carriers have EVER been confirmed since they
  could not culture S.typhi from the stools
  obtained from suspected carriers.

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Incidence rate of typhoid fever per 100,000
population in Malaysia (2000-2005) (Jabatan
Kesihatan Kelantan, 2005)
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Discovery of 50KDa protein
Commercialization Moving to the global market

• INFORMM and the School of Medical Sciences at USM
  Health campus have created breakthroughs in
  typhoid research
• Makes sense for us to take up the challenge of
  developing diagnostics for carriers by
  collaborating with the Kelantan State Health, MOH
  to see if together, we can help to control the
  spread of typhoid in Kelantan state.

• Outputs
• 33 Publications
• 7 Patents 34 pending
• Created spin-off biotech company
• Creation of 500 jobs
• Supported local industries
• Generated income to country,university,
• Inventors
• Won gt57 awards
• RM14 million grants obtained

Pakistan India Philippines South
Africa Guam Papua New Guinea Thailand
Vietnam Egypt Turkey United Arab
Republic Indonesia Bangladesh China Sudan Cameroon
Nigeria USA
SOLD to 18 countries
Global Distribution of the Kits
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Strategies for development of new methods using
the 50KDa protein and the gene encoding for the
protein to detect carriers

• Antibody detection (screening test)
• Detecting for the presence of IgA and IgG among
  suspected typhoid carriers by means of a
  serological, dot EIA test, TYPHIDOT C.
• Antigen detection (confirmatory test)
• Culture using fixed weight volume of stools
  isolated from among food handlers and suspected
  carriers
• PCR test to detect S. Typhi from stools of
  suspected carriers using EZ TYPHI PCR
• Samples
• Stools and serum are collected from people in
  the community who had previously had typhoid more
  that 1 year ago, or among foodhandlers during a
  typhoid outbreak

Culture method
EZ TYPHI PCR
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Typhidot C Results IgA and IgG
Result is POSITIVE when the dot is equal or
darker than the positive control
IgA
IgG
IgA pos control
IgG pos control


USD 2/test Results produced in 3 hours



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Create new technology platforms for molecular
diagnostics
EZ TYPHI PCR using 50kDa DNA sequence
USD 1
USM invention M. Ravichandran et al
Boil sample to obtain DNA
3 min
40 min
80 min
100bp DNA ladder marker
Sample DNA water (2 steps)
PCR product 415bp
Add 2 ul lysate 18 ul water
Amplication of target genes
Gel electrophoresis analysis

• No cold storage
• Only two pipetting steps
• Does not need PCR skilled personnels
• Cheap (USD 10 to now USD 1)

• Highly sensitive
• and specific (100)
• Cost effective
• USD 1

Duration Approx 2.5 to 3 hours
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TYPHIDOT C results

Test is considered highly probable a carrier if
patient is IgG pos IgA pos (17.17) Test is
considered highly probable a carrier if patient
is IgA pos only (5.56) Test is suggestive of
carrier/convalescence if IgG pos only
(30.98) Test is suggestive of not a carrier
if IgG neg, IgA neg (46.29) Last
update d 1st May 2009
Possible screening tool
Possible carriers 53.71
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Correlation of 100 between Typhidot, Culture
PCR



TYPHIDOT 100 backed culture and PCR
Typhidot-M negative, not an acute case IgG
inactivator used removed IgG and RFM
Key / Not applicable
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Summary of culture and PCR results
First typhoid carrier registry for Kelantan
Total number of S. Typhi and S. Paratyphi
isolated from stools of suspected carriers and
food handlers, and water samples
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Conclusion

• The improved stool sampling method (fixed weight
  sampling method) was successful in detecting
  typhoid carriers in Kelantan.
• The EZ Typhi PCR test could match the culture
  results suggesting our PCR test could be an
  alternative method. Culture still needs to be
  done to detect for antimicrobial sensitivity.
• Developed the first carrier registry for Kelantan
• The TYPHIDOT C was shown to be a potential
  screening tool to detect for possible carriers.
  Those positive can be further analysed for
  culture and PCR. This would be a more
  cost-effective approach rather than culturing
  stool samples and performing PCR for all contacts
  and suspects.
• The success of detecting typhoid carriers will
  create an impact to public health. The TYPHIDOT C
  assay can be used to screen for typhoid carriers
  among food handlers and immigrants and further
  confirmed by culture and PCR.
• We will continue to screen stools and sera till
  end of research duration to create a
  comprehensive carrier registry.
• Screening among food handlers will continue to be
  done to curb typhoid outbreak in Kelantan.

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Success Story impact to the Kelantan community

• Based on investigative study results of
  suspected typhoid carriers via Typhidot C ,
  improved culture method and EZ Typhi PCR,
  Kelantan State Health Department took several
  actions as follows
• Provided treatment to those individuals who have
  shown stool culture and PCR positives
• Provided treatment to those individuals whom
  their sera have shown IgA and IgG positives only
  IgA positives and only IgG positives.
• As a result of the actions taken by the Kelantan
  State Health Department led by Dr. Lila P. Mohd
  Meeran and Dr. Hani Mat Hussin, the number of
  typhoid cases are decreasing tremendously from
  2006- July 2009.

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IMPACT

• This is an example of a collaborative effort
  between the University and MOH that
• had a direct benefit to the Kelantan community
• By lowering the number of typhoid cases in
  Kelantan we had inadvertently reduced the overall
  number of typhoid cases in Malaysia.

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Thank you

• asma_at_usm.my
• www.usm.my/ri

Prof Prabha- INFORMM AP Phua Kia Kien -INFORMM Dr
Lila P. Mohamed Meeran -MOH Dr Hani Mat Hussain
-MOH Dr Sharina Dir -MOH Dr. Aziah Ismail -
INFORMM Dr Kirnpal Kaur- School of Medicine Siti
Norazura Mohamad- INFORMM Amy Amilda Anthony-
INFORMM Elis- (NFORMM) Prof M. Ravichandran
(AIMST) Prof John Wain- Health Protection Agency,
UK Dr Sattheesh, Health Protection Agency,
UK Prof Ataharul, Univ of Dhaka
Children from the BRAC village