From Bench to Bed Side: Routine to research Melioidosis research experience Melioidosis Research Center, KKU,

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 บน blood agar Burkholderia pseudomallei โคโลนีเหี่ยว ย่นของ B. pseudomal lei

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 et al., Sirisinha et al., 2000.

Antibody Detection (TRF Project) Sirisinha et al., Sirisinha et al., 2000.

Antigen Detection (TRF Project) Sirisinha et al., 2000.

Combined Ab and Ag Detection Sirisinha et al., 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 hemocultureTurbid 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

Molecular Biology Methods  PCR.  PCR (Rattanathongkom et al., 1996) Sensitivity of 0.5 fg of genomic DNA or 1 bacterial cell/ml.