Comparison of a new, rapid enzyme-linked immunosorbent assay with latex particle agglutination for the detection of Haemophilus influenzae type b infections

Abstract

A new, rapid enzyme-linked immunosorbent assay (ELISA) for the detection of polyribosylribitol phosphate of Haemophilus influenzae type b was compared with a commercially available latex particle agglutination (LPA) system (Bactigen; Wampole Laboratories, Cranbury, N.J.). By adding specimens and the anti-poly-ribosyIribitol phosphate immunoglobulin-enzyme conjugate to the solid phase in a single step, it was possible to complete the ELISA procedure in 30 min. The ELISA was capable of detecting 0.3 ng of polyribosylribitol phosphate per ml in cerebrospinal fluid, 0.6 ng/ml in urine, and 1.2 ng/ml in serum; the in vitro sensitivity of LPA in these body fluids was 0.6, 0.3, and 0.3 ng/ml, respectively. Both procedures detected polyribosylribitol phosphate in specimens from 25 patients with bacteriologically confirmed H. influenzae type b infections. The specificity of ELISA appeared to be superior to that of LPA. ELISA was positive in only one of seven patients who had a positive LPA test and a clinical illness that was not compatible with haemophilus infection. Moreover, five patients with bacteriologically confirmed infections due to other pathogens (Streptococcus pneumoniae type 14 [two patients], Neisseria meningitidis group C, Escherichia coli K100, and Staphylococcus aureus) had false-positive LPA tests; only two (E. coli and S. aureus) were positive by ELISA. A total of 108 samples from 61 patients who had no evidence of haemophilus infections were negative by both procedures. The ELISA is a rapid, sensitive, and specific alterdiative to LPA for the detection of haemophilus polyribosylribitol phosphate.

Laboratory diagnosis of bacterial meningitis.

“…Unfortunately, meningococcal antigen is sensitive to heat and, therefore, might not be detectable by LA after specimens have been heated to 100°C (160). Cross-reactions between a commercial H. influenzae LA reagent and S. pneumoniae, N. meningitidis group C, S. aureus, and Escherichia coli have been reported (84). In addition, urine can contain urethral flora that can cause false-positive results due to antigenic cross-reactions (44).…”

Rapid bacterial antigen detection is not clinically useful

“…Several factors, however, limit the clinical utility of bacterial antigen tests and make their routine use controversial. These factors include a decline in the incidence of Hib invasive disease following the advent of specific vaccination (35), imperfect antigen specificity (4,6,8,11,13,21,23,25,28,30,32), misleading positive results due to detection in urine or CSF of circulating Hib antigen after vaccination (7,15,36,43,44), contamination of urine with skin flora (3,40), and, importantly, the common failure of physicians to respond to rapidly generated diagnostic test results (26). In this review of over 5,000 LA tests, we found no evidence of clinical utility to support their current common use and instead found a high incidence of confounding results.…”

Rapid diagnosis of severe Haemophilus influenzae serotype b infections by monoclonal antibody enzyme immunoassay for outer membrane proteins

“…In fact, no positive reactions were noted in CSF, serum, or urine specimens collected from children infected with bacteria other than H. influenzae type b or from children without evidence of bacterial infection, whereas on three occasions a false-positive reaction was the result of the LA test in three cases of infection with S. pneumoniae. Recently, Macone et al (19) reported that five patients infected with a pathogen other than H. influenzae serotype b had a positive Bactigen-LA test. These five cases were represented by S. pneumoniae type 14 (two cases), N. meningitidis group C (one case), E. coli K-100 (one case), and Staphylococcus aureus (one case).…”

“…With the commercial kit recommendations, one can find that heating the specimen at 80°C for 5 min is required to eliminate these inconclusive reactions. It was also reported that the use of polyclonal antibodies in the EIA system for the detection of H. influenzae type b antigens could yield a false-positive result (19,25). Such nonspecific reactions and methods to minimize them have been reviewed by Yolken (31).…”

“…These include counterimmunoelectrophoresis (CIE) (7,13,14,18,20), latex agglutination (LA) (10,21,24,28,30), coagglutination (COA) (10,26,27,29,30), and enzyme-linked immunosorbent assay (8,11,15,19,25). The immunodiagnostic reagents used for the detection of Haemophilus influenzae serotype b antigen (polyribose phosphate [PRP]) show false-positive results with Escherichia coli, Streptococcus pneumoniae, and other bacteria (3,9,12,13,19,23,25). Most of these cross-reactions are due to the nonspecificity of the polyclonal antisera used.…”