Serological Diagnosis of Human Melioidosis with Indirect Hemagglutination and Complement Fixation Tests

Abstract

An indirect hemagglutination (IHA) test and a complement fixation (CF) test were evaluated from test results on sera from 212 human melioidosis patients of which 119 were culturally proved cases. Significant antibody titers (IHA titers of 1:40 or greater and CF titers of 1:4 or greater) were demonstrated with either test in all except five patients. IHA and CF titers ranged as high as 1:20,480 and 1:1,024, respectively. Antibodies were usually demonstrated by both tests 1 week after onset of disease. Transient seronegative reactions during the course of disease were seen in sera of approximately 19% of the patients with either IHA and CF but rarely with both tests. High titers in either test were obtained by the third week of disease and reached maximum levels in 4 to 5 months. Titers usually were detectable for 9 or more months. Antibodies were detected by IHA and CF tests in 80 to 100% of the sera obtained at various time intervals from 9 months to 2 or more years after disease onset. Antibody persistence occurred in patients who had a short disease course, as well as in patients with prolonged, complicated infections. The IHA test had excellent specificity when evaluated with normal human sera and diverse antimicrobial sera from hyperimmunized rabbits and human patients. The CF antigen appeared to contain common antigens with some but not all types of Pseudomonas aeruginosa . The specificity of the CF antigen could be enhanced without appreciable effect on its sensitivity by use of a titer of 1:8 in lieu of 1:4 as a criterion for a significant reaction. Either test could be used advantageously for the laboratory diagnosis of melioidosis.

Comparison of Melioidosis Indirect Hemagglutination Assay between Three Testing Laboratories in Australia

“…Detailed descriptions of this test are available within performing laboratory standard operating procedures (internal documents). These methods are based on work published previously ( 7 , 18 – 21 ).…”

Laboratory diagnosis of melioidosis: Past, present and future

“…There are many antibody detection formats currently in use. [89][90][91] These tests are generally unstandardized ''in house'' assays. The performance characteristics of these serological tests are ambiguous as most studies involve small groups of patients and investigators compare their assays against an imperfect gold standard (bacterial culture).…”

“…Sheep erythrocytes are sensitized with crude antigen derived from local clinical B. pseudomallei strains. 89,90 The sensitized erythrocytes are then added to serial dilutions of heat-inactivated patient sera. The IHA titer is the highest dilution of patient serum that causes distinct agglutination of erythrocytes.…”

“…93 False positive IHA titers may occur in patients with systemic P. aeruginosa infections. 90 Acutely bacteremic patients appear to be more likely to have negative IHA, limiting the usefulness of this assay for diagnosis of severe melioidosis. 93,94 This paradoxical seronegativity may reflect defective humoral immune responses in these patients although negative titers may simply reflect the early presentation.…”

Human immune responses to Burkholderia pseudomallei characterized by protein microarray analysis (53.3)

“…IHA titers of all samples were determined using a passive hemagglutination assay kit (Center for Immunodiagnostic Production). Briefly, 2-fold serial dilutions of plasma samples (1:10 to 1:1280) were incubated with heat-killed B. pseudomalleisensitized sheep erythrocytes for 2 h at 25 o C, and the IHA titer was read in comparison with unsensitized sheep red blood cells [19]. Five recombinant B. pseudomallei proteins previously shown to be immunoreactive in melioidosis patients were generated using the RTS-100 system.…”