Surface plasmon resonance immunosensor for rapid and specific diagnosis of melioidosis antibody.

Melioidosis, caused by Burkholderia pseudomallei, is a potentially fatal disease, which requires an accurate and rapid diagnosis. This paper reports on the highly sensitive and specific detection of melioidosis antibodies by surface plasmon resonance immunosensor. The sensing surface was immobilized with B. pseudomallei BipD protein via a 11-mercaptoundecanoic acid self-assembled monolayer. Under optimum conditions individual sera of melioidosis patients, non-melioidosis patients (negative) and blood donors (control) were analyzed at a dilution of 1:6,000 in 10 mM phosphate buffered saline pH 7.50. The cut-off value determined from the mean +/- 2SD of 20 control and 20 negative sera was 3.3 m degrees. At this cut-off both sensitivity and specificity were 100%. The system required only a short analysis (20 minutes) and regeneration time (12 minutes). In addition, one immobilization of the sensing surface could be reused more than 30 times. The advantages of the proposed method are savings in both time and cost of analysis, while at the same time providing excellent sensitivity and specificity.

Production and purification of Burkholderia pseudomallei BipD protein.

Type III secretion system (TTSS) clusters contain virulent genes of both animal and plant Gram-negative bacteria. The full length (933 bp) gene of bipD, a member of TTSS cluster of Burkholderia pseudomallei, was isolated by PCR amplification from B. pseudomallei DNA and cloned into pGEX 4T-1. GST-BipD fusion protein and BipD protein obtained by removal of GST using thrombin were employed to detect the presence of B. pseudomallei antibodies in sera obtained from melioidosis and non-melioidosis patients by immunoblotting. Sensitivity and specificity of BipD protein was 100% and 91.1%, respectively, whereas that of fusion protein was 77.8% and 90.0%, respectively.

Prospective study of the feasibility and effectiveness of a second-trimester quadruple test for Down syndrome in Thailand.

OBJECTIVE: To evaluate the feasibility and effectiveness of a quadruple test for Down syndrome in the second trimester of pregnancy in clinical settings in Thailand. METHODS: From October 2015 to September 2016, a prospective study was undertaken in 19 hospitals in Songkhla province, Thailand. Women with a singleton pregnancy of 14-18 weeks were enrolled and underwent the quadruple test. The risk cutoff value was set at 1:250. All women with a positive test (risk ≥1:250) were offered amniocentesis. Women were followed up until delivery. RESULTS: Among 2375 women, 206 (8.7%) had a positive quadruple test; 98 (47.6%) of these women voluntarily underwent amniocentesis. Overall, seven pregnancies were complicated with chromosomal abnormalities (2.9 cases in 1000), including four cases of Down syndrome (1.7 in 1000) and three of other abnormalities. The detection, false-positive, and accuracy rates of the quadruple test for Down syndrome were 75.0%, 8.6%, and 91.4%, respectively. CONCLUSION: The quadruple test was found to be a feasible and efficient method for screening for Down syndrome in the second trimester of pregnancy in a Thai clinical setting. The test should be performed for pregnant women before an invasive test for Down syndrome.

Prevalence of extended-spectrum beta-lactamases (ESBLs) produced in blood isolates of gram-negative bacteria in a teaching hospital in southern Thailand.

One hundred and eighty-three strains of gram-negative bacteria were isolated from 177 bacteremic patients during a 6-month period in 2004 at a teaching hospital in southern Thailand. Extended-spectrum beta-lactamases (ESBL) production was detected in K. pneumoniae, E. coli, and E. cloacae, at rates of 16/36 (44.4%), 3/59 (5.1%), and 2/13 (15.4%), respectively. All but one of the screened positive strains were also positive by both the combination disk method and the E-test. All of the K. pneumoniae strains that were resistant to ceftazidime by disk diffusion were demonstrated to be positive for ESBL production by both the combination disk and E-test methods. Most of the ESBL positive strains had a high MIC (more than 32 microg/ml) to ceftazidime. However, all the ESBL positive strains were sensitive to imipenem.

Searching for virulence Burkholderia pseudomallei genes by immunoscreening the lambda ZAPII expressed genomic library.

The lambdaZAP II expressed genomic library of B. pseudomallei was screened with pooled melioidosis serum preabsorbed with E. coli host cell. The positive clones were detected by using protein A-CDP-star chemiluminescence. All of 14 positive clones reacted with only the pooled absorbed melioidosis serum and not the pooled absorbed normal serum when tested with the plaque dot blot analysis. The expressed genes were detected by using a combination of immunoscreening, bioinformatics and molecular biology. At least six in vivo expressed genes were identified by this approach. Two were well known virulent genes, gmhA (a capsule biosynthetic gene) and bipD (type III secretion protein gene). Another two were genes coded for conserved hypothetical protein. The last two isolated genes were groEL (a chaperonine protein gene), and a gene encoding transmembrane protein.