Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia.

A monoclonal antibody-based latex agglutination (MAb-LA) test was employed for the rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. These patients were admitted to 12 hospitals in the northeastern part of Thailand which is a region known to be endemic for melioidosis. Blood samples were collected and immediately added to the blood culture bottles which were incubated in either automated (five hospitals) or manual (seven hospitals) culture systems. Of a total of 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. The performance of the MAb-LA test on these specimens was highly satisfactory compared with culture detection and confirmation by biochemical test, with 95.1% sensitivity, 99.7% specificity and 98.8% and 99.2% for positive and negative predictive values, respectively. The method described is highly reproducible, simple to perform even by inexperienced laboratory personnel and does not require expensive or elaborate equipment.

Shedding of lipopolysaccharide and 200-kDa surface antigen during the in vitro growth of virulent Ara- and avirulent Ara+ Burkholderia pseudomallei.

Non-virulent Ara+ B. pseudomallei environmental isolates differ from virulent Ara- clinical isolates by their ability to assimilate L-arabinose and the absence of a 200 kDa antigen on their surface. The latter, present only on the Ara- isolates from either clinical or environmental origin, was recently demonstrated by its immunoreactivity with monoclonal antibody (MAb) 5F8. We recently demonstrated that lipopolysaccharide (LPS) from both biotypes were indistinguishable from one another with regard to SDS-PAGE profiles and immunoreactivities with immune sera. In this study, the shedding of LPS and 200-kDa antigen into the culture medium during the in vitro growth of Ara- was compared with that of its Ara+ counterpart, using MAb-based sandwich ELISAs. The results showed that the LPS shedding profiles from the two biotypes were similar to one another. This was in contrast to the situation with the 5F8-reactive antigen. The culture fluid of all Ara- isolates and none of the Ara+ isolates were found to react strongly with the MAb 5F8 during the early log phase of growth. However, during the late stationary phase, a trace amount of the 5F8-reactive material could also be detected in the culture fluid of the Ara+ isolates.

Antigenic heterogeneity of lipopolysaccharide among Burkholderia pseudomallei clinical isolates.

Burkholderia pseudomallei (BP) causes melioidosis, a potentially fatal human infection in the tropics. Clinical isolates from different geographical locations have similar morphological and biochemical characteristics. Although BP has been reported to possess 2 types of lipopolysaccharide (LPS) differing in the chemical structure of their O-polysaccharide (O-PS) component, earlier report demonstrated that the clinical strains exhibited identical LPS moieties. Recently, we reported antigenic similarity between the pathogenic (Ara-) and nonpathogenic (Ara+) biotypes. However, a few clinical isolates showed atypical SDS-PAGE profiles. In this study, LPS from 739 BP isolated from patients and animals in different geographical areas were extracted by proteinase K digestion method. Their SDS-PAGE profiles and their immunoreactivities with patients' sera and monoclonal antibody (MAb) to LPS were analyzed. The isolates showed 3 LPS patterns differing in the number and electrical mobility of bands in silver-stained gel. A majority of BP (711) isolates exhibited identical typical ladder pattern, 21 isolates showed atypical ladder pattern and 7 isolates did not exhibit ladder appearance. However, all LPS preparations exhibited similar endotoxic activity as determined by Limulus amebocyte lysate assay. On the other hand, there were no immunological cross reactivity between typical and atypical LPS, as judged from Western blot against homologous and heterologous sera from melioidosis patients from whom the typical and atypical LPS were isolated. Nevertheless, a Western blot profile of the typical LPS showed some variations when probed with MAb against BP LPS (9D5). Heat-killed bacteria from all LPS groups could similarly activate mouse macrophage cell line to produce nitric oxide (NO) and inducible NO synthase (iNOS).