ABSTRACT
Burkholderia pseudomallei is the causative agent of melioidosis. In northeast Thailand, this gram-negative bacterium is a major cause of mortality from septicemia. The definitive diagnosis of this disease is made by bacterial culture. In this study, we produced a monoclonal antibody (MAb) specific to the 30-kDa protein of B. pseudomallei by in vivo and in vitro immunization of BALB/c mice with a crude culture filtrate antigen. The MAb could directly agglutinate with all 243 clinical isolates of B. pseudomallei but not with other gram-negative bacteria, except for one strain of Burkholderia mallei. However, the MAb cross-reacted with the gram-positive Bacillus sp. and Streptococcus pyogenes. B. pseudomallei in brain heart infusion broth (BHIB) subcultured from a BacT/Alert automated blood culture system could be identified by simple agglutination with this MAb assay. The sensitivity and specificity of direct agglutination compared to the "gold standard," the culture method, were 94.12 and 98.25%, respectively. However, the MAb adsorbed to polystyrene beads or latex particles directly identified the bacterium in blood culture specimens and in BHIB subcultured from a BacT/Alert automated blood culture system. The sensitivity of the latex agglutination test was 100% for both blood culture and BHIB specimens. The specificity was 85.96 and 96.49% for the blood culture and BHIB specimens, respectively. The specificity could be increased if the nonspecific materials in the blood culture broths were eradicated by centrifugation at low speeds. Thus, a combination of blood culture and the agglutination method could be used for the rapid diagnosis of melioidosis in the routine bacteriological laboratory. This method could speed up detection of the bacterium in blood culture by at least 2 days, compared to the conventional bacterial culture method. In addition, the MAb is stable at room temperature for 2 weeks and at 4, -20, and -70 degrees C for at least 1 year. The latex reagent was stable for at least 6 months at 4 degrees C.
Subject(s)
Antibodies, Monoclonal , Bacteriological Techniques , Burkholderia pseudomallei/immunology , Burkholderia pseudomallei/isolation & purification , Melioidosis/diagnosis , Agglutination Tests/statistics & numerical data , Animals , Antibodies, Bacterial , Antigens, Bacterial/blood , Bacteriological Techniques/statistics & numerical data , Blood/microbiology , Cross Reactions , Drug Stability , Evaluation Studies as Topic , Humans , Indicators and Reagents , Latex Fixation Tests/statistics & numerical data , Melioidosis/immunology , Mice , Sensitivity and SpecificityABSTRACT
Throat swab cultures and indirect hemagglutination assay (IHA) for Pseudomonas pseudomallei were done in 1000 randomly selected children at a large hospital in northeast Thailand. During 18 months, 17 children with melioidosis were admitted (0.46% of pediatric admissions excluding neonates born in the hospital). Throat swab was positive for P. pseudomallei in 8 of these but in none of 1000 control children. IHA seroprevalence rose at a conversion rate of 24% per year, from 12% in those 1-6 months old to a plateau at approximately 80% after age 4 years. No control child < 4 had an IHA titer > 1:160. The median titer in children with melioidosis was 1:80 (range, negative [in3]-1:5120). Specificity of IHA declined with age, but high titers (> or = 1:640) remained diagnostically useful. Thus, throat carriage of P. pseudomallei indicates active melioidosis. There is no evidence for an asymptomatic carrier state in children. Environmental exposure to P. pseudomallei in endemic areas begins when the child becomes mobile.
Subject(s)
Burkholderia pseudomallei/isolation & purification , Carrier State/diagnosis , Melioidosis/diagnosis , Pharynx/microbiology , Adolescent , Age Factors , Antibodies, Bacterial/blood , Burkholderia pseudomallei/immunology , Child , Child, Preschool , Hemagglutination Tests , Humans , Infant , Melioidosis/microbiology , Prospective Studies , Random AllocationABSTRACT
Eighty-one fresh isolates of Pseudomonas pseudomallei from melioidosis patients were subjected to the analysis for the fatty acid composition by gas-liquid chromatography (GLC) and pH-dependent pattern of nonspecific phosphatase activity. All the test strains were identical in the GLC profile showing the three peaks of characteristic hydroxy acids (3-OH 14:0, 2-OH 16:0, 3-OH 16:0) and the two prominent peaks of cyclopropane acids (17:0 delta, 19:0 delta). They had also basically the same pH-dependent curves of the enzymatic activity with paranitrophenyl phosphate as substrate, showing two to three peaks or shoulders only in the acidic side of the curve. These two biochemical characteristics could differentiate P. pseudomallei distinctly from P. aeruginosa, but not from P. cepacia.
