Download Project description BIO3 "Application of DNA chip technology for the

Project description BIO3
"Application of DNA chip technology for the development of diagnostic kits for rapid
detection of drug resistant tuberculosis in Southeast Asia"
The emergence of multi-drug resistant (MDR) tuberculosis (TB) is one of the main
challenges for TB control worldwide. Vietnam is among the 22 countries with highest TB
burden in the world. Results from the recent National TB Drug Resistance survey
showed that MDR-TB accounted for 3% among new cases and 19% in retreated cases.
Even though drug susceptibility testing (DST) is essential for proper treatment, in most
high-burden setting including Vietnam, capacity for diagnosis of drug resistant TB is
limited. The method used for DST is conventional culture, which takes many weeks to
get results and only limited at national referent hospitals due to its high cost, complexity
and labour intensiveness.
In addition to the above problem, the pandemic of HIV infection has brought
considerable change to the epidemiology of mycobacterial infections over the world
because of the association with increased infections due to non-tuberculous
Mycobacteria (NTM). In Vietnam, about 4% of the total annual 180,000 new tuberculosis
(TB) cases are HIV seropositive patients. The diagnosis of TB patients is mostly based on
clinical symptoms and acid fast bacillus (AFB) smear microscopy, a test that cannot
distinguish NTM infection from M. tuberculosis infection. This problem can lead to
diagnosis misclassification, especially for patients with pulmonary TB-like symptoms
caused by NTM. Patients can be, thus, miss-diagnosed as having pulmonary TB. Since
pulmonary manifestations account for 94% of cases of NTM diseases, a large proportion
of HIV patients with NTM infection may get treatments with anti-tuberculosis regimens.
Such treatments expose the patients to improper regimens because the susceptibility of
NTM to anti-TB drugs is different to M. tuberculosis. Even though the most important
method for the identification of NTM isolates is culture, the lack of resources and
expertise in health care system creates barrier to culture diagnosis. Diagnosis of NTM
diseases is therefore often neglected in Vietnam.
The research team has obtained grant from AIRD for carrying out research on molecular
epidemiology of TB in Southeast Asia (Vietnam, Laos and Cambodia), in which we are
investigating the evolution of drug resistant M. tuberculosis in the region and we will
determine the prevalence of genetic mutations involved in resistance to the first line
anti- tuberculosis drugs. We will also determine the most commonly found
opportunistic NTM species that cause disease manifestation in HIV seropositive
patients. The outcomes of the project will provide essential epidemiological and genetic
information for the development of diagnosis tests dedicated to the detection of drug
resistant TB and to the characterization of NTM infections. From the data achieved from
our project, we will design genetic markers based on drug resistance mutation patterns
obtained for each single first line drug and for the different drugs in combination. We
will also design genetic markers specific for the different NTM species. Since, it is largely
known that drug resistance and population genetic composition are dependent to the
ecosystems in which they evolve (drug pressure, health care systems, populations, etc...)
and that the genotypes of the circulating strains vary greatly from population to
population, the tests developed based on specific information of local populations will
be more efficient than the ones that developed elsewhere.
As a complement to this already existing project, we propose the present project with
the aim to apply DNA chip technology for developing diagnostic kits, first for the rapid
detection of drug resistant tuberculosis, and second for the identification of the most
commonly found NTM species responsible for disease manifestations in immunocompromised patients. Since a microarray can contain tens of thousands of probes, an
array experiment can accomplish many genetic tests in parallel. Therefore it is possible
to develop a single chip for the identification of M. tuberculosis and NTM species and for
the determination of drug resistant profile of the clinical MTB isolates. With DNA chip
technology the whole procedure would take only few hours to get the results and with a
significant decrease of the global cost.
The principle for the DNA chip production will include several major steps. First, we will
design the oligonucleotide probes based on the genetic markers that we identify.
Second, silicon chips will be coated with the designed oligonucleotides. Third, we will
develop procedure for the detection of the targeted sequences. The detection will
include the amplification of the targeted sequence, the labelling of the amplified
products and the hybridization of labelled amplified products with the probes on the
chips. The hybridization will be detected and quantified by visualization, measurement
and quantification of the labels. Finally, we will carry out research to determine the
sensitivity and specificity of the produced kits compared to the results obtained with the
gold standard solid culture methods.
Because the designed genetic markers will be tailored to the genetic patterns of the
regional MTB and NTM populations, the detection kits should have high sensitivity and
specificity. In addition, if the kits can be produced locally, the price will be cheaper
compared to the imported ones and affordable to Vietnamese patients as well as
patients in other developing countries such as Laos and Cambodia. The project is of
highly scientific and medical relevance and practical interest and will contribute to the
health care and to the development of technology in Vietnam and in the Region.
Moreover, these developments, in the framework of USTH grant, will build up the role of
USTH in the field of biomedicine in Vietnam and in the Region.ms to build a research
laboratory viable in the long-term for them.