Rapid Diagnosis of Group B Streptococcal Infection Utilizing a Commercially Available Latex Agglutination Assay

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Rapid

Diagnosis

of Group

B Streptococcal

Infection

Utilizing

a Commercially

Available

Latex

Agglutination

Assay

Charles A. Friedman, MD, David F. Wender, MD, and

John E. Rawson, MD

From the Hinds General Hospital, Mississippi Baptist Medical Center, and Department of Pediatrics, University of Mississippi Medical School, Jackson

ABSTRACT. The Weilcogen Strep B latex assay rapidly identifies all cases of culture-positive sepsis and menin-gitis and may be more sensitive than standard culture techniques for identifying group B Streptococcus disease and assessing the degree of severity. The quantitation of antigen concentration combined with the peripheral

WBC count proves helpful in predicting poor outcome. Pediatrics 1984;73:27-30; septicemia, group B Streptococ-cus.

Group B Streptococcus (GBS) infection in new-horns can be rapidly progressive and is often lethal.’ Accurate diagnosis and early therapy is essential. Antihiotic therapy is often begun empirically on the basis of clinical suspicion of infection before the

results of laboratory cultures are known. Tradition-ally, significant neonatal infection by GBS or other bacterial pathogens has been implied by the early detection of nonspecific acute-phase reactants2” or

an abnormal peripheral WBC count.4 Although

these findings are useful in identifying patients at

high risk for infection, they do not offer diagnostic

specificity.

A latex particle agglutination assay

(Wellcogen Strep B, Burroughs Wellcome) to de-tect the GBS polysaccharide antigen is now corn-merically available. We evaluated the diagnostic accuracy of this assay in a series of infants with suspected bacterial sepsis. To assess further the clinical usefulness of the assay, the relationships between antigen concentration, neutrophil counts, and survival were studied.

Received for publication March 10, 1983; accepted June 10, 1983.

Reprint requests to (C.A.F.) Hinds General Hospital, 1850 Chad-wick Dr, Jackson, MS 39204.

American Academy of Pediatrics.

MATERIALS AND METHODS

Subjects

The study group consisted of 98 newborns Un-dergoing evaluation for neonatal sepsis at Hinds General Hospital and Mississippi Baptist Medical Center between January 1981 and September 1982. All patients had a blood culture and a complete blood count (CBC) with differential as a part of the initial sepsis evaluation, and most patients had ear, cord, or gastric aspirate cultures. Lumbar punctures were performed on 15 ofthe 98 newborns, and fluids were analyzed for Gram stain, culture, glucose, pro-tein, cell count, and differential. Serum and urine from all patients and CSF from the 15 infants who had a lumbar puncture were saved for GBS antigen

assay. Serial CBCs were obtained in all infants and repeat GBS antigen determinations were performed when initial antigen determinations or blood cul-tures were positive for GBS. Antigen determina-tions were not routinely repeated if initially nega-tive and if initial blood cultures remained sterile. All infants were treated with ampicillin and gen-tamicin, or penicillin and gentamicin intravenously according to standard therapy.5 Antibiotics were continued for at least ten days in infants with blood culture-proven bacterial sepsis or meningitis. When bacterial sepsis was strongly suspected on clinical and laboratory grounds despite negative blood cul-tures, antibiotics were continued for seven to ten days. All infants who were GBS antigen positive with or without an accompanying blood culture

positive for GBS were changed to penicillin only and treated for 14 days.

Latex Assay

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Bur-28

GROUP

B STREPTOCOCCAL

INFECTION

roughs Wellcome (Dr Max Moody, Research

Tn-angle Park, NC). Polystyrene latex particles coated

with antibody to the group-specific carbohydrate antigen of GBS were mixed in a standard phos-phate-buffered solution with a suspension of strep-tococcal extract antigen as a positive control.

Se-rum, urine, and CSF were tested immediately after

collection (in most cases) or were frozen at -20#{176}C until assayed for the presence of the GBS antigen.

Antibody-free latex particles served as a negative

control to rule out nonspecific agglutinations. Urine samples were concentrated 25-fold using a Minicon B-iS concentrator (Amicon Corp, Lexington, MA). Samples demonstrating clumping agglutinations

within three minutes were considered positive for the GBS antigen. No agglutination or fine

granu-lanity was judged negative. Samples with equivocal agglutinations were heated to 100#{176}Cfor five mm-utes and retested.

Serial tenfold dilutions of 27 sera positive for GBS antigen were assayed to find the highest titer

giving

latex agglutination.

Data and Statistical Analysis

Neutropenia was defined as less than 2,500 gran-ulocytes pen microliter. The ratio of immature to

total neutrophils was calculated and a ratio of less than 0.14 was considered abnormal.6 The

x2

test

for proportional differences was used for statistical

comparisons.

RESULTS

Correlation of Latex Assay with Blood

Culture-Proven GBS Disease

Twenty-one newborns had blood cultures

posi-tive for GBS, ten newborns had blood cultures

positive for another organism, and 67 newborns had negative blood cultures (Table 1). Seven of the bacteremic patients had foci of infection identified

on admission: five patients with pneumonia, one

patient with peniorbital cellulitis, and 1 patient with

septic arthritis.

All 21 infants with GBS-positive blood cultures had GBS antigen detected in the serum. Urine

specimens were assayed for GBS in 19 ofthe infants

with GBS-positive blood cultures and were positive in all 19 patients. Four of the 21 patients with

positive blood culture and positive GBS antigen

had late-onset disease (onset after 2 weeks of age).

In two of these patients, the urine and serum were

initially negative at the time of presentation, but both had positive GBS antigen when retested at six and 12 hours, respectively. The other two infants

with late-onset disease and all the infants with early-onset disease had serum and urine positive

TABLE 1. Group B Streptococcus

Positivity in Serum and Blood Cult Newborns with Suspected Sepsis*

Antigen ure (BC)

(GBS Ag) Results in

BC BC Positive BC Totals Positive for Orga- Negative

for GBS nism Other for GBS than GBS

GBSAg 21 2t 11 34

positive

GBSAg 0 8 56 64

negative

Totals 21 10 67 98

* GBS antigen positivity (group B streptococcal antigen

positive) as measured by Wellocogen Strep B latex agglu-tination assay (Burroughs Wellcome). Sensitivity for blood culture positivity on presentation was 19/21, or 90%, and 21/21, or 100% when retested.

t One blood culture positive for group G Streptococcus, one blood culture positive for Staphylococcus aureus but patient’s rectal culture was positive for GBS. (Specificity

for GBS among all 31 positive blood cultures is 29/31, or

94%).

:1:Includes one patient with serology positive for syphilis.

for GBS antigen at the time of presentation. Ten of the 21 patients with positive blood culture and antigen had serum and urine retested several times during the 2-week antibiotic treatment. Three of these ten patients converted to GBS antigen

nega-tive during the 2-week treatment period. In the

other seven patients serum and urine remained

positive at the end of the 2 weeks of antibiotic

treatment. Five of these seven patients had antigen titers equal to or greater than 1:10 in the initial serum specimen. One of these seven patients had urine tested for GBS antigen 5 months after

treat-ment, and urine was GBS antigen positive at that time.

Thirteen patients with GBS-negative blood

cul-tures had GBS antigen present in urine; 11 of the 13 patients also had GBS antigen present in serum (Table 1). In one of the 13 infants, a group G

Streptococcus that cross-reacted serologically with GBS7 was isolated from blood and CSF, both of

which gave positive GBS antigen reactions. In an-other infant, a blood culture grew Staphylococcus aureus, but GBS was recovered from a rectal

cul-ture. Four of the 13 patients had additional cultures (ear, cord, rectal, or gastric aspirate) positive for

GBS. In two of these four patients, the mother had

been treated prior to delivery with antibiotics. The

clinical features of the 1 1 patients with serum pos-itive GBS antigen but with negative blood cultures did not differ from clinical features of the 21 pa-tients with positive GBS antigen and positive blood cultures.

GBS antigen was absent in eight infants whose

blood cultures were positive for other organisms:

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Escherichia coli (two patients), Listeria monocyto-genes (two patients), Salmonelki enteritidis (one patient), Streptococcus viridans (one patient), and

S pneumoniae (one patient). An eighth infant with congenital syphilis was also found to be GBS anti-gen negative (Table 1). In a ninth infant who had necrotizing entercolitis, peritoneal fluid and stools were positive for Clostridium perfringens, but blood, CSF, and urine cultures were negative along with the GBS antigen test.

Among 56 patients with negative GBS antigen and blood culture, 20 patients exhibited equivocal agglutination reactions which became negative on heating. All GBS antigen-positive sera and urine tested remained positive when heated to 100#{176}Cfor five minutes.

Correlation of Latex Assay with CSF

Culture-Proven GBS

Of the 15 infants who had lumbar punctures,

seven had CSF specimens that were GBS antigen positive (Table 2). Of these seven specimens, four were culture positive for GBS, a fifth was culture positive for a cross-reacting group G Streptococcus, and two were culture negative. CSF findings were suggestive of meningitis in these two infants with negative cultures. In one infant, Gram positive cocci

were seen on CSF Gram stain, and the CSF

con-tamed 46 WBCs per microliter (100% polymorpho-nuclear cells). In the other infant, the CSF con-tamed 2,200 WBCs (59% polymorphonuclear cells). In the latter case, the mother had been treated with ampicillin prior to delivery. Eight other CSF sam-ples were culture negative and GBS antigen nega-tive and had negative CSF Gram stains and normal CSF chemistries and cell counts.

TABLE 2. Group B Streptococcus Positivity in CSF and CSF Culture R

with Suspected Meningitis*

Antigen esults in

(GBS Ag) Newborns

CSF CSF Culture Culture Positive for Positive Organism for GBS Other Than

GBS

CSF Culture Negative

Totals

GBSAg 4 it

positive

GBSAg 0 0

negative

2t

8

7

8

Totals 4 1 iO 15

* GBS Ag positivity (group B streptococcal antigen pos-itive) as measured by Wellcogen Strep B latex aggluti-nation assay (Burroughs Welicome).

t CSF culture positive for a cross-reacting group G St rep-tococcus.

t CSF chemistries and/or Gram stain suggestive of

men-ingitis in both patients.

TABLE 3. Combination of Both Neutropenia and High-Titer Group B Streptococcus (GBS) Antigen Con-centration*

Present Absent Total

Death 6 0 6

Survivors 5 16 21

Totals ii 16 27

* Neutropenia is defined by criteria of Manroe et al.8

GBS antigen concentration was measured by serial dilu-tions of patients’ sera to determine the highest dilution

giving positive reaction by Wellcogen Strep B latex

ag-glutination assay (Burrough Welicome). High titer GBS antigen concentration = serum dilution 1:10, giving a positive antigen reaction by Strep B latex assay.

Correlation with Neutrophil Counts and Outcome

The relationship between GBS antigen concen-tration (measured by serial serum dilutions),

neu-trophil counts and survival was assessed in 27 pa-tients with positive GBS antigen. When initially

evaluated, the six infants who would not survive had both neutropenia and a serum sample positive for GBS antigen in 1:10 dilution. Three patients who died at 5,

7,

and 12 hours of age were positive

at 1:50, 1:1,000, and 1:10,000 dilutions, respectively. Only five of the 21 survivors in this group had both neutropenia and serum positive at 1:10 dilution

(x2

< .01) (Table 3). The combination of neutro-penia and serum GBS antigen positivity at equal to or greater than 1:10 dilution was more predictive of

poor outcome than neutropenia alone or GBS

an-tigen positivity alone. Elevation of the ratio of

immature-to-total neutrophils,8 even when

com-bined with high-titer antigen concentration, was not predictive of poor outcome.

One-volume exchange transfusions were per-formed on four patients with high serum antigen concentrations (1:10,000, 1:100, and two patients with 1:10) and neutropenia. Exchange transfusion had no effect on the antigen concentration titer in two patients who subsequently died; in the other

two patients, the antigen could not be detected 30

minutes postexchange but reappeared by six hours. These two infants survived.

DISCUSSION

The commerically available Wellcogen Strep B

antigen assay enabled correct diagnosis of all 21 culture-positive patients with GBS disease. In 19 of these 21 patients, the latex assay was positive on initial testing (initial sensitivity of 90%), with the remaining two patients becoming positive at six and 12 hours after initial presentation. The test also enabled determination of the presence of the

GBS antigen in 11 infants with negative cultures. We treated these 11 infants with antibiotics

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30 GROUP

B

STREPTOCOCCAL INFECTION

whether these infants were truly infected or simply exposed to antigen through colonization with GBS.

In 31 patients with positive blood cultures (GBS

and other organisms), the latex test was

appropri-ately positive or negative in 29 patients (specificity

of 94%). Group G Streptococcus, a known cross-reacting organism,7 was interpreted as GBS in se-rum, urine, and CSF of one patient. In another patient whose serum was positive for GBS antigen, the blood culture grew Staphylococcus aureus, al-though GBS was cultured from the rectum.

Previous studies have shown that latex particle

agglutination is generally more sensitive than

coun-terimmune electrophoresis assay for the detection

of both GBS9”#{176} and Haemophilus influenzae.’1”2

Latex particle agglutination assay may be able to

detect as little as 60 to 70 ng/mL of GBS antigen, compared with at least 1 tg of antigen per milliliter

of concentration detected by counterimmune elec-trophoresis (M. Moody, personal communication, 1982).

Our study points out that GBS antigen positivity

in fluids of patients with suspected infection allows

a specific diagnosis to be made before the results of

blood and CSF cultures are known. The specificity

of the latex agglutination assay for GBS antigen

contrasts with the nonspecific identification of in-fants at high-risk for GBS disease reported by Manroe et al.6’8 Although early diagnosis and

spe-cific therapy were instituted on the basis of GBS

antigen positivity in 34 patients, six patients died

(17.6%): four patients died of GBS pneumonia and

septicemia, one of intraventricular hemorrhage, and one of pulmonary hemorrhage. Standard blood cultures obtained after institution of antibiotics were sterile, regardless of ultimate outcome.

Because the findings of neutropenia and high antigen concentrations (measured by serial serum dilution) are more indicative of poor outcome than either finding alone, early identification of these

high-risk factors may allow early intervention with

specific antibiotic or other therapy such as WBC

transfusions,’3 or anti-GBS-specific antibody.’4

ACKNOWLEDGMENTS

We thank Dr Max Moody of Burroughs Wellcome for

his continuing help in this project. We acknowledge and

appreciate reviews of this manuscript by Dr Arnold

Smith, Infectious Disease Division, Children’s

Or-thopedic Hospital, Seattle, and Dr Robert Daum, Infec-tious Disease Division, Department of Pediatrics, Tulane

University, New Orleans.

REFERENCES

1. Siegel JD, McCracken GH: Sepsis neonatorum. N Engl J Med 1981;304:642

2. Sabel K-G, Hanson LA: The clinical usefulness of C-reactive protein (CRP) determinations in bacterial meningitis and septicemia in infancy. Acta Paediatr Scand 1974;63:381 3. Adler SM, Denton RL: The erythrocyte sedimentation rate

in the newborn period. J Pediatr 1975;86:942

4. Boyle RI, Chandler BD, Stonestreet BS, et al: Early iden-tification of sepsis of infants with respiratory distress. Pe-diatrics 1978;62:744

5. McCracken GH, Nelson JD, in Antimicrobial Therapy for Newborns. New York, Grune & Stratton, 1977

6. Manroe BL, Weinberg AG, Rosenfeld CR, et al: The neo-natal blood count in health and disease. J Pediatr 1979;95:89 7. Curtis SN, Krause RM: Antigenic relationships between

groups B and G streptococci. J Exp Med 1964;120:629 8. Manroe BL, Rosenfeld CR, Weinberg AG, et al: The

differ-ential leukocyte count in the assessment and outcome of early-onset neonatal group B streptococcal disease. J Pe-diatr 1977;91:632

9. Webb BJ, Baker CJ: Commerical latex agglutination test for rapid diagnosis of group B streptococcal infection in infants. J Clin Microbiol 1980;12:442

10. Bromberger P1, Chandler B, Gezon H, et al: Rapid detection of neonatal group B streptococcal infections by latex agglu-tination. J Pediatr 1980;96:104

11. Daum RS, Siber GR, Kaon JS, et al: Evaluation of a com-merical latex particle agglutination test for rapid diagnosis of Haemophilus influenzae type B infection. Pediatrics

1982;69:466

12. Ward JI, Siber GR, Scheifele DW, et al: Rapid diagnosis of Hemophilu.s influenzae type B infections by latex aggluti-nation and counterimmune electrophoresis. J Pediatr

1978;95:37

13. Christensen RD, Rothstein G, Anstall HB, et al: Granulo-cyte transfusions in neonates with bacterial infection, neu-tropenia, and depletion of mature marrow neutrophils. Pe-diatrics 1982;70:1

14. Santos JI, Shigoeka AO, Rote NS, et al: Protective efficacy of a modified immune serum globulin in experimental group B streptococcal infection. J Pediatr 1981;99:873-879

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1984;73;27

Pediatrics

Charles A. Friedman, David F. Wender and John E. Rawson

Available Latex Agglutination Assay