JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2895-2896 0095-1137/91/122895-02$02.00/0 Copyright C 1991, American Society for Microbiology

Vol. 29, No. 12

Comparison of Commercially Available Group B Streptococcal Latex Agglutination Assays DAVID P. ASCHER,* SAM WILSON, AND GERALD W. FISCHER Department of Pediatrics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road,

Bethesda, Maryland 20814-4799 Received 28 May 1991/Accepted 28 August 1991

Detection of group B streptococcus (GBS) antigen in urine by latex particle agglutination (LPA) may facilitate the rapid diagnosis of GBS sepsis. We sought to compare three commercial LPA assays with specimens that were spiked with type-specific antigen, group-specific antigen, or type III organisms. There were sensitivity differences between the assays, but the Bactigen assay performed best, detecting as little as 1 ng of GBS group-specific antigen per ml in urine and as few as 105 CFU of GBS type III organisms per ml in urine, serum, and cerebrospinal fluid.

Streptococcus agalactiae, the group B streptococcus (GBS), is the major cause of neonatal sepsis and meningitis in the United States (1). Detection of GBS antigen in urine or other body fluids by latex particle agglutination (LPA) may facilitate the rapid diagnosis of GBS sepsis. GBS LPA assays have been reported to be 88 to 100% sensitive and 81 to 100% specific (2, 4, 6, 8, 10, 12, 13) and are commercially available in three assays: Wellcogen (Wellcome Diagnostics, Research Triangle Park, N.C.), Directigen (Becton-Dickinson Microbiology Systems, Cockeysville, Md.), and Bactigen (Wampole Laboratories, Cranbury, N.J.). The present study was designed to compare the sensitivities of these commercially available assays by using known amounts of GBS group-specific antigen, GBS type-specific antigen, and two type III organisms. Streptococcal antigens. One milligram of either the GBS group-specific antigen or GBS antigen specific for type Ia, lb, II, or III was added to 1 ml of sterile normal saline. GBS group-specific and GBS type-specific antigen were prepared from supernatant fluids of GBS grown in 70-liter fermentors and were considered partially purified. The culture media and antigen preparations were performed by the methods of Carey et al. (5) and Gotschlich et al. (7). Tube dilutions (1:10) were serially made to a concentration of 10-11 g. Streptococcal strains. GBS strains SS878 and Norris (virulent, type III clinical isolates) were prepared as previously described (9). The bacterial concentration was determined spectrophotometrically and then adjusted to a final concentration of 108 bacteria per ml in 0.9% saline. One milliliter of bacterial suspension was added to 4 ml of either serum, urine, or cerebrospinal fluid. The actual number of CFU used was subsequently confirmed by serial dilution and quantitative plating. Commercial assays. Reagents were warmed to room temperature, and specimens were prepared according to the procedures recommended by the manufacturer. Assays were performed according to the directions for each product. For each LPA assay, the limit of sensitivity was determined by spiking (i) sterile normal saline with GBS groupspecific or GBS type-specific antigen, (ii) serum and urine with GBS group-specific antigen, and (iii) serum, urine, and cerebrospinal fluid with type III Norris and type III SS878 *

Corresponding author.

GBS in mid-log-phase growth. Fresh sterile male adult sera, cerebrospinal fluid, and urine (subjects were not receiving antibiotics) were obtained and spiked with 1 mg of GBS group-specific antigen. Serial 1:10 dilutions were made in the respective body fluids to determine endpoints. Each assay was run in duplicate with different lots, without substantially different results. Comparisons of limits of sensitivity for each assay are summarized in Tables 1 and 2. The limit of sensitivity for Bactigen and Directigen assays was 1 ng of GBS groupspecific antigen per ml in normal saline and between 105 and 107 CFU of type III organisms per ml of body fluids. The Bactigen assay was generally 100 times more sensitive than the Wellcogen LPA assay in all specimen fluids. A difference between assays was considered substantial if the difference was by a factor of 100 or greater. GBS LPA is a method of diagnosis based on the immunologic detection of the group-specific carbohydrate antigen in body fluids. Its advantages over culture are that it is rapid and may be positive after antibiotic therapy has begun. The three commercial GBS LPA assays (Wellcogen, Directigen, and Bactigen) all consist of rabbit anti-GBS polysaccharide polyclonal antibodies on latex beads, though their exact formulations are proprietary. The three assays demonstrated different sensitivities, depending on which body fluid was tested. All three assays detected antigen in the nanogram range, but the equivalent number of organisms detected was in the range of 105 to 107 bacteria per ml. In a previous rhesus monkey study with GBS type III strains SS878 (9), the Wellcogen LPA assay was positive in 12% of 17 amniotic fluids with a mean colony count of 7.6 x 106 GBS CFU/ml and in 94% of 17 samples with a mean level of GBS of 2.1 x 108 CFU/ml. The serum LPA was positive in only 17% of 18 rhesus monkey infants, of which 15 were bacteremic. In 1983, Hemming et al. (9) also demonstrated that the limit of sensitivity for the Wellcogen LPA assay was 80 ,ug of GBS antigen per ml, 0.125 ,ug of purified group B carbohydrate per ml, and 2.4 x 106 CFU of GBS strain SS878 per ml. Wasilauskas and Hampton (14) previously reported the following limits of sensitivity for purified GBS antigen: Wellcogen, 1.6 ng/ml; Directigen, 50 nglml; and Bactigen, 1.6 ng/ml. When the assay results were compared against results for five GBS-culture-positive cerebrospinal fluid 2895

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TABLE 1. Summary of latex assay comparison Spiked fluid and antigen or body fluid

Limit of sensitivity (ng/ml) of:

Wellcogen

Bactigen

Directigen

1

Group

10,000 10,000 10,000 1,000 100

Serum Urine

10 100

Type Type Type Type

la lb Il III

100 1,000 10 10 1

100 1,000 100 100 1

2 0.1 100

0.01 1

a Fluid 1, normal saline with group- and type-specific antigen; fluid 2, body fluids with group-specific antigen.

specimens by serial twofold dilutions, the Wellcogen assay was approximately two to four times more sensitive than the Bactigen assay, which was approximately 10 times more sensitive than the Bactigen assay, which was approximately 10 times more sensitive than the Directigen assay. Furthermore, McGowan and James (11) compared these three assays against twelve GBS-culture-positive cerebrospinal fluid specimens: Bactigen detected 11 of 12, Directigen detected 5 of 12, and Wellcogen detected 8 of 12. However, the latter two studies used a Directigen assay that consisted of a mouse monoclonal antibody that had known sensitivity problems (4). The Directigen assay was changed in 1988 to a rabbit polyclonal antibody assay that apparently has improved sensitivity (4). Antigen preparations of known concentrations (108 bacteria per ml) of seven GBS (five serotypes) resulted in a reactive Directigen (4) test. Although similar studies have been published or have appeared as abstracts, the present study utilizes the current modified Directigen reagent and challenges all three products with identical test solutions. Our results comparing the three assays with group-specific antigen in urine and serum are consistent with findings of Hemming et al. (9) but different from the findings of Wasilauskas and Hampton (14). The method of preparation of the antigen solutions was not noted in the other investigations comparing the three GBS LPA assays, and thus the limits of sensitivities for the TABLE 2. Summary of latex assay comparison

Type III GBS spike and body fluid Norris Serum Urine CSF

SS878 Serum Urine CSF

Limit of sensitivity (CFU/ml) of:

Wellcogen

Bactigen

Directigen

107

î05 î05 105

105

î07

107

107 107 107

105 105 105

a Both strains were in log phase. CSF, cerebrospinal fluid.

105

105

106 106 107

antigen preparations may not be comparable. The clinical implications for the differences in sensitivities among these three assays are not clear, and these assays have yet to be studied with large numbers of clinical isolates representing different serotypes and strains of GBS. Nevertheless, the insensitivity of the LPA assays in detecting whole organisms may lead to false-negative results for patients with invasive GBS disease, as noted by other investigators (3, 9, 10, 12). We generally found that the Bactigen assay was more sensitive than the Wellcogen or Directigen assays when urine, sera, and cerebrospinal fluid spiked with GBS groupspecific antigen or type III GBS whole organisms were compared. Substantial sensitivity differences may exist among the three commercially available GBS LPA assays, as well as a surprising insensitivity in detecting actual live organisms in cerebrospinal fluid, urine, and serum. REFERENCES 1. Anthony, B. F., and D. M. Okada. 1977. The emergence of group B streptococci in infections of the newborn infant. Annu. Rev. Med. 28:355-369. 2. Baker, C. J., and M. A. Rench. 1983. Commercial latex agglutination for detection of group B streptococcal antigen in body fluids. J. Pediatr. 102:393-395. 3. Becker, J. A., D. P. Ascher, J. Mendiola, and G. W. Fischer. 1991. The poor sensitivity of urine latex agglutination testing in neonates with group B streptococcal bacteremia. Pediatr. Res. 29:280A. 4. Bleeker, D. L., M. J. Zimbro, M. B. Erbe, and J. E. Mortensen. 1989. Polyclonal anti-group B Streptococcus latex antigen detection test. Pediatr. Infect. Dis. J. 8:251-252. 5. Carey, R. B., T. K. Eisenstein, G. D. Shockman, T. F. Greber, and R. M. Swenson. 1980. Soluble group- and type-specific antigens from type III group B Streptococcus. Infect. Immun. 28:195-203. 6. Friedman, C. A., D. F. Wender, and J. E. Rawson. 1984. Rapid diagnosis of group B streptococcal infection utilizing a commercially available latex agglutination assay. Pediatrics 73:27-30. 7. Gotschlich, E. C., T. Y. Liu, and M. Artenstein. 1969. Human immunity to the meningococcus. J. Exp. Med. 129:1349-1365. 8. Hamoudi, A. C., M. J. Marcon, H. J. Cannon, and R. E. McClead. 1983. Comparison of three major antigen detection methods for the diagnosis of group B streptococcal sepsis in neonates. Pediatr. Infect. Dis. J. 2:432-435. 9. Hemming, V. G., W. T. London, L. P. Smith, B. L. Curfman, G. W. Fischer, and J. L. Sever. 1983. Detection of group B streptococcal antigens in amniotic fluid of rhesus monkeys. J. Clin. Microbiol. 17:1127-1131. 10. Ingram, D. L., D. M. Suggs, and A. W. Pearson. 1982. Detection of group B streptococcal antigen in early-onset and late-onset group B streptococcal disease with the Wellcogen Strep B latex agglutination test. J. Clin. Microbiol. 16:656-658. 11. McGowan, K. L., and L. D. James. 1989. Comparative evaluation of Directigen, Bactigen, and Wellcogen reagents for detection of bacterial antigens in CSF from children, abstr. C-19, p. 396. Abstr. 89th Annu. Meet. Am. Soc. Microbiol. 1989. 12. Rabalais, G. P., D. R. Bronfin, and R. S. Daum. 1987. Evaluation of a commercially available latex agglutination test for rapid diagnosis of group B streptococcal infection. Pediatr. Infect. Dis. J. 6:177-181. 13. Sanchez, P. J., J. D. Siegel, N. B. Cushion, and N. Threlkeld. 1990. Significance of a positive urine group B streptococcal latex agglutination test in neonates. J. Pediatr. 116:601-606. 14. Wasilauskas, B. L., and K. D. Hampton. 1986. Bacterial antigen detection: the sensitivity of rapid diagnostic tests, abstr. C-161, p. 354. Abstr. 86th Annu. Meet. Am. Soc. Microbiol. 1986.

Comparison of commercially available group B streptococcal latex agglutination assays.

Detection of group B streptococcus (GBS) antigen in urine by latex particle agglutination (LPA) may facilitate the rapid diagnosis of GBS sepsis. We s...
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