Identification of specific antigens of Pseudomonas pseudomallei and evaluation of their efficacies for diagnosis of melioidosis

Abstract

Current methods for the diagnosis of melioidosis are based on bacteriological culture. A number of serological tests currently available lack specificity and sensitivity. This is largely due to the use of crude antigens which results in a significant cross-reactivity with sera from individuals infected with other bacteria. In this study five different antigens were prepared and evaluated for their potential usefulness in diagnosis of melioidosis. These included a 19.5-kDa antigen which was previously shown to be specific by Western blotting (immunoblotting), a crude cell extract, a veronal extract, a 39.0-kDa antigen, and an immunoaffinity-purified antigen. All antigens were used for detecting antibody in sera from patients with septicemic melioidosis by indirect enzyme-linked immunosorbent assay. The results were compared with those obtained with sera from patients with other bacterial infections and normal sera from areas where the infection is and is not endemic.

Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

“…That is, probably, the reason why sensitivity of the IHA test in the septicaemic group of our patients was low (70%). The detection of IgM antibody was not tested by ELISA in this study; however, with a partially purified antigen, Anuntagool et al [3] found that the detection of IgG antibody by ELISA was more reliable than that of IgM antibody in acutephase sera. This conclusion was contrary to that of Ashdown [I 51.…”

“…A glycolipid spot can also be obtained on thinlayer chromatograms of the extractable lipids from B. cepacia and B. mallei (data not shown). The problem was the same: as that encountered with the 19.5kDa antigen [3], h.owever, the amount of glycolipid from B. mallei was too small to make its purification possible. The existence of GL in these two bacteria is not unexpected because B. pseudomallei and B. cepacia belonged to the same RNA homology group [ 131, DNA-DNA homology value between B. pseudomallei and B. mallei was more than 90% and the sequences of 1174 bases of 16s rRNA of the two species matched completely [ 141.…”

“…cannot be based solely on clinical symptoms but requires bacteriological examinations including culture confirmation. Unfortunately, the latter is usually time-consuming (minimum 2 days) [2] and the result is often too late to be useful for appropriate early treatment [3]. Moreover, the prognosis of acute septicaemic melioidosis depends on early diagnosis and appropriate treatment [I].…”

“…One reason for this phenomena was the use of impure antigens. A partially purified protein antigen (19.5 kDa) was reported to be highly sensitive and specific for the diagnosis of melioidosis [3], however, due to its protein nature, the stability has not been guaranteed and satisfactory techniques for the preparation of this antigen make it difficult for use in most endemic areas. In comparison with IHA, the ELISA generally gives higher sensitivity and specificity, however, in some melioidosis patients, ELISA shows negative results in IHA-positive sera [2].…”

Rapid antigen detection assay for identification of Burkholderia (Pseudomonas) pseudomallei infection

“…The more widely used serological methods for antibody detection are difficult to interpret, particularly in areas where the infection is endemic, where the background antibody levels may be elevated. This drawback still persists even when more-purified antigens are used for detection (2,5). The quantitation of specific IgM antibody, as carried out in several laboratories (6), is not completely satisfactory because false positives still occur.…”