From Bench to Bed Side: Routine to researchMelioidosis research experience

Melioidosis Research Center, KKU, E-mail: melioid@kku.ac.th 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