From Bench to Bed Side: Routine to research Melioidosis research experience Melioidosis Research...
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From Bench to Bed Side: Routine to researchMelioidosis research experience
Melioidosis Research Center, KKU, E-mail: [email protected] http://web.kku.ac.th/~melioid
Research questions
We need the rapid diagnosis?, Why The organism has been known as a grate
immitator 80-90% of septicemic cases die within 1-2
weeks Conventional bacterial culture need at least
72 hrs Over usage of antibiotic cost 300 millions
Baht/year
Burkholderia pseudomallei
Isolation & Identification Culture Hemo 3-7 days Ashdown selective medium (Ashdown 1979,
Wuthiekanun et al., 1990) API 20 NE commercial kits (Dance et al., 1989) Minitek disc (B. pseudomallei and B. cepacia)
(Ashdown, 1992) Blood culture techniques (Wuthiekanun et al., 1990) Monoclonal antibodies (Rugdech et al., 1995)
Gram’s stain B.pseudomallei
Burkholderia pseudomallei
โคโลนี� B. pseudomallei แสดง hemolysis บนี blood agar
Burkholderia pseudomallei
โคโลนี�เหี่��ยวย�นีของ
B. pseudomallei
Laboratory Diagnosis
Laboratory diagnosis : Conventional methods :
Bacterial cultivation required 72 h. Serological methods :
Antibody detection.Antigen detection.
Molecular Biology methods:Specific DNA probe.PCR.
Detection of specific antibodies and/or antigens to B. pseudomallei in serum or clinical specimens in suspected patients.
Antibody and Antigen Detection (TRF Project)
Sirisinha Sirisinha et alet al., 2000.., 2000.
Antibody Detection (TRF Project)
Sirisinha Sirisinha et alet al., 2000.., 2000.
Antigen Detection (TRF Project)
Sirisinha Sirisinha et alet al., 2000., 2000..
Combined Ab and Ag Detection
Sirisinha Sirisinha et alet al., 2000., 2000..
What we need more?
Rapid test 1 day identification Rapid test 10 min (bed side)
Latex agglutination
Using monoclonal antibodies Specific t0 200 Kda exopolysaccharide Specific to B. pseudomallei not B.
thailandensis and others
Anuntagool, et al, 2000
Latex Agglutination
Test of latex
1369 culture-positive specimens, 204 specimens were culture-positive for
B. pseudomallei. Of those, 194 (95%) were positive by
MAb-LA and the type of blood culture system did not affect positivity rates.
95.1% sensitivity, 99.7% specificity and 98.8% and 99.2% for positive and negative predictive values, respectively.
Anuntagool, et al, 2000
Further test
The method was evaluated in a clinical situation on 396 hemocultures positive for bacterial growth
of which 75 cultures were positive for B. pseudomallei by conventional biochemical tests.
The sensitivity and specificity of the MAb-LA test were 95% and 100%, respectively.
The positive and negative predictive values were 100% and 99%.
Samosornsuk et al, 1999
Development
Latex agglutination Test from culture Test directly from blood
blood hemoculture Turbid or growth
Latex agglutination 2 min
Test available
Distribute to oversea (Hong Kong) Distribute throughout Thailand Plan to train to all public health staff in all
area of Thailand
Development to new Technology
Phage display techniques
Patent registration
Specific antibodies to Detect antigen Detect antibodies
Undergoing project