University –Community Engagement Conference
Bayview Hotel, Penang, Malaysia
diagnostics for the
third world countries
Asma Ismail PhD
Research and Innovation and
Professor, Institute for Research in
Molecular Medicine (INFORMM),
Universiti Sains Malaysia, 11800
A child dies every 15 seconds from a water-borne
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
– 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
– Available but they are too expensive.
• Herein lies the real challenge of R&D
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
R&D approaches to diagnostics
• In the area of diagnostics, research need to be performed
with the client in mind so that the product developed is
• Create diagnostics that would be in demand at least for 10
• 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.
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 1999 Nobel Prize
BLUE OCEAN STRATEGY
Linus Pauling Professor of Chemical Physics
and Professor of Physics at Cal Tech Kim,W.C. and Mauborgne,R. (2005).
Working from Z to A: Market foresight
requirements for rapid diagnostics
Criteria for design and development
•Easy to perform
•Transported without cold chain
Patent indigenous technology platforms
Application of Blue Ocean
Strategy to detection of typhoid
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
1. Crump, J.A., S.P. Luby and E.D. Mintz. 2004. The global burden of typhoid fever. Bulletin of the WHO.
2. WHO (2003) Backgroud document: The diagnosis, treatment and prevention of typhoid fever.
Communicable Disease Surveillance and Response Vaccines and Biologicals WHO/V&B/03.07.
Available methods to diagnose for typhoid carriers:
Antibody detection test by means of Vi antigen (not available commercially)
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 . For acute typhoid, the sensitivity is higher for stool culture
Bile culture for carrier status can deliver sensitivity of >90% 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/V&B/03.07.
Focus and benefit of
• 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
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
• 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
Incidence rate of typhoid fever per 100,000 population in
Malaysia (2000-2005) (Jabatan Kesihatan Kelantan, 2005)
Commercialization: 50KDa protein
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 Outputs
for carriers by collaborating with the Kelantan State Health, MOH to
see if together, we can help to control the spread of typhoid in •33 Publications
•7 Patents + 34
Kelantan state. pending
Pakistan biotech company
•Creation oflocal jobs
South Africa industries
•Generated income to
Papua New Guinea country,university,
Egypt •Won >57 awards
Turkey •RM14 million grants
United Arab Republic obtained
Nigeria SOLD to 18
USA Global Distribution of the Kits countries
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 Culture method
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
PCR test to detect S. Typhi from stools of
suspected carriers using EZ TYPHI PCR
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
EZ TYPHI PCR
Typhidot C Results – IgA and IgG
Result is POSITIVE when the dot
is equal or darker than the IgG
IgA pos control
IgG pos control
Results produced in 3 hours
Create new technology
platforms for molecular EZ TYPHI PCR USD 1
using 50kDa DNA
Boil sample USM invention
3 min M. Ravichandran et al 40 min
to obtain DNA
Sample Add 2 ul lysate
+ 18 ul water
of target Gel electrophoresis
Only two pipetting steps
Does not need PCR skilled
Cheap (USD 10 to now USD 1) •Highly sensitive
and specific (100%)
Duration: Approx 2.5 to 3 hours •Cost effective
TYPHIDOT C results
Samples Size No. of positive No. of negative
results with results with
(N = 594) Typhidot® IgG Typhidot® IgG
No. of positive 102 33
Typhidot® IgA (17.17%) (5.56%)
No. of negative 184 275
Typhidot® IgA (30.98%) * (46.29%)
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 Possible (46.29%)
Possible carriers = 53.71%
Last update d: 1st May 2009
Correlation of 100% between Typhidot, Culture & PCR
Samples Lab No. IgM Grade IgG Grade IgA Grade
CR0044 Neg / Pos 4+ Pos 4+ *
CR0063 Neg / Neg / Pos 2+ *
CR0388 Neg / Pos 2+ Neg / *
CR0419 Neg / Pos 2+ Neg /
FH0006/08 Neg / Pos 4+ Pos 4+
100% backed culture
and PCR FH0939/08 Neg / Pos 4+ Pos 2+
FH1843/08 Neg / Neg / Pos 1+
FH2125/08 Neg / Pos 4+ Pos 4+
FH2164/08 Neg / Pos 4+ Pos 1+
not an acute case FH2172/08 Neg / Neg / Pos 1+
IgG inactivator used FH3197/08 Neg / Pos 4+ Pos 4+
removed IgG and RFM
FH0033/09 Neg / Pos 4+ Pos 2+
FH0069/09 Pos 2+ (?) Pos 4+ Pos 4+
FH0075/09 Neg / Pos 2+ Neg /
Key : / = Not applicable
Summary of culture and PCR results
No of samples S. Typhi S. Paratyphi B
Stool samples of
suspected carriers 0
Stool samples of
7 + 3 = 10
food handlers 0
Water samples 1
Total isolates 15 1
Total number of S. Typhi and S. Paratyphi isolated from stools of
suspected carriers and food handlers, and water samples
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.
Success Story – impact to the
• 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
– 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.
• This is an example of a collaborative effort
between the University and MOH that
had a direct benefit to the Kelantan
• By lowering the number of typhoid cases
in Kelantan we had inadvertently reduced
the overall number of typhoid cases in
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
Prof M. Ravichandran (AIMST)
Prof John Wain- Health Protection
Agency, UK Children from the BRAC village
Dr Sattheesh, Health Protection Agency,
Prof Ataharul, Univ of Dhaka