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Rapid detection of neonatal group B streptococcal infections by latex agglutination
10 4
Brief clinical and laboratom' observations
The Journal of Pediatrics January 1980
Brief clinical and laboratory observations Rapid detection of neonatal group B streptococcal infections by latex agglutination Patricia I. Bromberger, M.D.,* Barry Chandler, M.D., Horace Gezon, M.D., and James E. Haddow, M.D., Scarborough, Maine
ACCURATE AND RAPID METHODS for identifying group B streptococcal infection in newborn infants are in great demand because early antibiotic treatment may improve morbidity and mortality. We report preliminary results using a latex agglutination test to diagnose GBS infections and compare them with the standard counterimmunoelectroph0resis technique. METHODS
The subjects were infants admitted to the Maine Medical Center and Central Maine Medical Center nurseries with suspected infection. Urine, blood, and cerebrospinal fluid samples were collected simultaneously with cultures, after informed consent had been obtained. Body fluid samples were refrigerated or frozen until tested. Urine was centrifuged to remove particulate matter and concentrated 25 times using a Minicon B-15 concentrator (Amicon, Lexington, Mass.). CSF was centrifuged only when red cells were present. Group B and type-specific rabbit antisera were supplied by Dr. Hazel Wilkinsen, Center for Disease Control, Atlanta, Ga. Subsequently, we prepared group and typespecific antisera in rabbits using the methods of Lancefield.' Group B streptococci isolated from patients were grouped and typed according to the Lancefield capillary precipitin technique? The whole rabbit antiserum was serially fractionated with 18% and 16% sodium sulfate, reconstituted in an From the Department of Pediatrics, Maine Medical Center, Department of Pediatrics, Central Maine Medical Center, the Foundation for Blood Research, and Atlantic Antibodies, Inc. Supported in part by a grant from the Department of Pediatrics, Maine Medical Center. *Reprint address: Foundationfor Blood Research, P.O. Box 426, Searborough, ME 04074.
equivalent volume of 0.9% saline, and dialyzed against saline overnight at 4~ Serum electrophoresis was performed to check for recovery of globulins. The optimally reactive dilution for globulin was 1:40. One part globulin diluted in glycine-buffered saline, pH 8.2, was mixed with one part latex particles, 0.8 lu (Difco, Detroit, Mich.) and incubated at 37~ for two hours. Two parts 0.1% bovine serum albumin in glycine-buffered saline with 0.15% sodium azide were added. The latex slide agglutination test was performed as described by NewmanY; a reaction 2 + or greater was considered positive. All test reactions were compared with positive and negative controls. A control latex suspension made from normal rabbit globulin was used to detect false positive reactions. Abbreviations used GBS: group B streptococcal LA: latex agglutination CIE: counterimmunoelectrophoresis CSF: cerebrospinal fluid The CIE procedure used 1% agarose in sodium barbital buffer, pH 8.6. Electrophoresis was carried out at 30 mA for 30 minutes. After incubation for 30 minutes at 4~ plates were read without magnification. To increase sensitivity, plates were soaked overnight in 0.45% saline, dehydrated, and stained with Buffalo Black. RESULTS A total of 125 patients was studied, 104 of whom were found to have negative blood, urine, and CSF bacterial cultures. Group B streptococcus was cultured from the blood of 11 infants (8 type III, 1 type Ia, 1 type Ib, 1 type Ic); three o f these infants had meningitis. The remaining ten infants were infected with a variety of organisms including Escherichia coli, group D streptococcus, alpha-
0022-3476/80/010104+03500.30/0 9 1980 The C. V. Mosby Co.
Volume 96 Number 1
hemolytic streptococcus, Haemophilus influenzae, meningococcus, and pneumococcus. As shown in Table I, latex agglutination identified group B antigen in the urine of all 11 patients infected with GBS, and seven had antigen detectable in unconcentrated urine. In these same samples, CIE detected group B antigen in eight of the 11, but only after urine had been concentrated in seven of the eight. Both CIE and latex agglutination identified group B antigen in the CSF in the three cases of meningitis. Only two of eight serum samples contained measurable amounts of group B antigen by LA; CIE was negative in all eight. The urine, CSF, and serum specimens in the 104 uninfected infants and in the ten infants with other bacterial infections were also tested for group B antigen. No false positive reactions were found using either technique, although occasional 1 + nonspecific agglutination reactions occurred when testing urine in the latex system. Crystals and mucus in the urine caused 3 to 4 + reactions, emphasizing the need for centrifugation prior to testing. Table II contains the results of type-specific antigen analysis carried out in the body fluid specimens from infants with GBS infections. LA identified type-specific antigen in the CSF of two infants with meningitis; the other CSF, blood, and urine specimens were negative for type-specific antigen. CIE detected type specific antigen in the CSF from all cases of meningitis, in one of eight serum specimens, and in five of 11 urine samples. All urine samples with measurable type-specific antigen also had group B antigen via CIE analysis. In the five patients with type-specific antigen in the urine, the type always correlated with that of the infecting organism. Latex agglutination appeared to be more sensitive than CIE in identifying group B antigen. We compared the sensitivities of the two methods by serially diluting body fluid specimens, broth cultures, and crude antigen extract (Table III, Column A). LA always detected group B antigen at higher dilutions than did CIE, confirming impressions gained from testing patient ~amples. Similarly, the sensitivities of the two methods for detecting type-specific antigen were compared (Table llI, Column B). CIE was always more sensitive than LA in detecting the presence of the type-specific antigen. DISCUSSION Latex agglutination and CIE have most often been used to detect the polysaccharide antigens produced by Haemophilus influenzae, pneumococcus, and meningococcus. With rare exception, ~ latex agglutination appears to be the more sensitive of the two methods. ~ ~-'; Until now, most studies involving group B streptococcal antigen detection have been carried out with CIE. In the present
Brief clinical and laboratory observations
10 5
Table I. Group B antigen detection comparison of LA and CIE in 11 newborn infants with GBS infections
~ody fluid
No. specimens
LA +
CIE +
It 11 7 8
7 11 3* 2
l 8 3* 0
Urine Unconcentrated Concentrated CSF Serum *Patients with meningitis.
Table IL Type-specific antigen in l l patients with GBS disease: comparison of LA and CIE
Body
No.
fluid
specimens
LA +
CIE +
Concentrated urine CSF
ll
0
7
2* (Ib,III) 0
5 (ia,ltl) 3* (Ib,III) l (Ill)
Serum
8
*Patients with meningitis.
Table II1. Comparison of sensitivity of LA and CIE* Column A group B antigen
Specimen 50X concentrated urine No. I~50X concentrated urine No. 2? CSF No. 17 CSF No. 2~ Whole cell culture Antigen extract
LA
I CIE
Column B type-specific antigen LA
I CIE
128
8
512
64
-
6d
64 512 512 8,000
4 64 "4 128
2 2 8
2 8 4 128
-
1
*All numbers in table = reciprocal of highest dilution at which test positive. tPatients with GBS disease.
study, the latex agglutination technique has been compared with CIE for detection of GBS antigen not only because it may be more sensitive, but also because it requires no specialized equipment or personnel, has reagents which remain stable for many months, and produces test results within minutes. In the present study, group B antigen, the polysaccharide common to all group B organisms, could be detected more sensitively by latex agglutination than by CIE. All infants infected with GBS were identified by the latex agglutination test performed on urine specimens. Other
10 6
Brief clinical and laboratory observations
studies using CIE for detecting group B antigen have identified between 50 to 100% of infected infants. 7-1~Our own CIE success rate of 70% falls within the range o f those published results. F o r infants with GBS sepsis, urine is the body fluid which most frequently contains detectable group B antigen, and CSF can be added to that list when meningitis is also present. In the present study, CIE has proven more sensitive than LA in measuring type-specific antigen. T h i s is in contrast to a report of 12 infants with type I l l GBS meningitis, in w h o m latex agglutination was more sensitive than CIE in detecting type lII antigen in CSF. 11 This difference may be due to different antibody strengths or to a difference in the latex particle sensitization method. The identifiable presence of type-specific antigen in body fluids did not increase the n u m b e r of infants with GBS disease identified by immunologic means in our study. In spite of the fact that we did not encounter false positive latex agglutination reactions, there is greater potential for false positive reactions with LA than with CIE. In serum, nonspecilic agglutination reactions occur when IgM antibodies are present, and these can be counteracted by adding dithiothreitol) Nonspecific agglutination reactions occur occasionally in CSF and may be avoided by heating the sample. 2 False positive reactions may occur both in latex and CIE systems; a cross-reaction has been reported in CSF from a patient with meningitis due to S. pneumoniae when tested with latex particles sensitized with antiserum to type III GBS, 11 and certain strains of group G streptococcus are known to cross-react with group B. 1-~ The latex agglutination test appears to be more sensitive than CIE for the detection of group B antigen in the urine of infants with GBS infections and has identified all infants with proven infection in this series.
The Journal of Pediatrics January 1980
Our thanks to Drs. George Halle tt and John Serrage, Department of Pediatrics, Maine Medical Center, and to the nurses and house staff of the Neonatal Intensive Care Unit. REFERENCES 1. Lancefield RC, McCarty M, and Everly WN: Multiple mouse protective antibodies directed against group B streptococci, J Exp Med 142:165, 1975. 2. Newman RB, Stevens RX, and Gaafar ttA: Latex agglutination test for the diagnosis of Haemophilus influenzae meningitis, J Lab Clin Med 76:107, 1970. 3. Coonrad JD, and Rylko-Bauer: Latex agglutination in the diagnosis of pneumococcal infection, J Clin Microbiol 4:168, 1976. 4. Whittle HC, Egler LJ, Tugwell P, and Greenwood BM: Rapid bacteriologic diagnosis of pyogenic meningitis by latex agglutination, Lancet 2:619, 1974. 5. Ward JI, Siber GR, Scheifele DW, and Smith DH: Rapid diagnosis of Hemophilus influenzae type b infections by latex particle agglutination and counterrimmunoelectrophoresis, J PEDIAXR93:37, 1978. 6. Severin WPJ: Latex agglutination in the diagnosis of meningococcal meningitis, J Clin Pathol 25:1079, I972. 7. Rhodes PG, and Hall RT: Countercurrent immunoelectrophoresis (CIE) of urine: A rapid diagnostic tool for group B streptococcal sepsis in the neonate, J PZD~ATR 91:833, 1977. 8. Siegel JD, and McCracken GH: Detection of group B streptococcal antigens in body fluids of neonates, J PEDIATR 93:491, 1978. 9. Shakelford PG, and Stechenberg BW: Countercurrent immunoelectrophoresis (CIE) in group B streptococcal disease, Pediatr Res 11:505, 1977. 10. Edwards MS, and Baker CJ: Prospective diagnosis of early onset group B streptococcal infection by countercurrent immunoelectrophoresis, J PEDIATR94:286, 1979. 11. Edwards MS, and Baker CJ: Comparison of antigen detection by latex particle agglutination and counterimmunoelectrophoresis in Type III, group B streptococcal meningitis, 18th lnterscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, Georgia October 1-4, 1978. 12. Curtis SN and Krause RM: Antigenic relationships between groups B and G streptococci, J Exp Med 120:629, 1964.
Defective primary dentition in survivors of neonatal mechanical ventilation Fergus M.B. Moylan, M.D.,* Edward B. Seldin, D.M.D., M.D., Daniel C. Shannon, M.D., and I. David Todres, M.D., Boston, Mass.
THERE HAVE BEEN numerous reports on the incidence of dental caries in young children, their association with *Reprint address: New England Medical Center Hospital, 171 Harrison Ave., Boston, MA 02111.
ingestion of sugar, and the characteristic distribution of teeth affected. 1' '-' Other studies have shown an increased incidence of defective dentition in subjects with serious neurologic deficits? ~ We have seen a large n u m b e r of young children with abnormal dentition, the etiology of
0022-3476/80/010106+03500.30/0 9 1980 The C. V. Mosby Co.
Brief clinical and laboratom' observations
The Journal of Pediatrics January 1980
Brief clinical and laboratory observations Rapid detection of neonatal group B streptococcal infections by latex agglutination Patricia I. Bromberger, M.D.,* Barry Chandler, M.D., Horace Gezon, M.D., and James E. Haddow, M.D., Scarborough, Maine
ACCURATE AND RAPID METHODS for identifying group B streptococcal infection in newborn infants are in great demand because early antibiotic treatment may improve morbidity and mortality. We report preliminary results using a latex agglutination test to diagnose GBS infections and compare them with the standard counterimmunoelectroph0resis technique. METHODS
The subjects were infants admitted to the Maine Medical Center and Central Maine Medical Center nurseries with suspected infection. Urine, blood, and cerebrospinal fluid samples were collected simultaneously with cultures, after informed consent had been obtained. Body fluid samples were refrigerated or frozen until tested. Urine was centrifuged to remove particulate matter and concentrated 25 times using a Minicon B-15 concentrator (Amicon, Lexington, Mass.). CSF was centrifuged only when red cells were present. Group B and type-specific rabbit antisera were supplied by Dr. Hazel Wilkinsen, Center for Disease Control, Atlanta, Ga. Subsequently, we prepared group and typespecific antisera in rabbits using the methods of Lancefield.' Group B streptococci isolated from patients were grouped and typed according to the Lancefield capillary precipitin technique? The whole rabbit antiserum was serially fractionated with 18% and 16% sodium sulfate, reconstituted in an From the Department of Pediatrics, Maine Medical Center, Department of Pediatrics, Central Maine Medical Center, the Foundation for Blood Research, and Atlantic Antibodies, Inc. Supported in part by a grant from the Department of Pediatrics, Maine Medical Center. *Reprint address: Foundationfor Blood Research, P.O. Box 426, Searborough, ME 04074.
equivalent volume of 0.9% saline, and dialyzed against saline overnight at 4~ Serum electrophoresis was performed to check for recovery of globulins. The optimally reactive dilution for globulin was 1:40. One part globulin diluted in glycine-buffered saline, pH 8.2, was mixed with one part latex particles, 0.8 lu (Difco, Detroit, Mich.) and incubated at 37~ for two hours. Two parts 0.1% bovine serum albumin in glycine-buffered saline with 0.15% sodium azide were added. The latex slide agglutination test was performed as described by NewmanY; a reaction 2 + or greater was considered positive. All test reactions were compared with positive and negative controls. A control latex suspension made from normal rabbit globulin was used to detect false positive reactions. Abbreviations used GBS: group B streptococcal LA: latex agglutination CIE: counterimmunoelectrophoresis CSF: cerebrospinal fluid The CIE procedure used 1% agarose in sodium barbital buffer, pH 8.6. Electrophoresis was carried out at 30 mA for 30 minutes. After incubation for 30 minutes at 4~ plates were read without magnification. To increase sensitivity, plates were soaked overnight in 0.45% saline, dehydrated, and stained with Buffalo Black. RESULTS A total of 125 patients was studied, 104 of whom were found to have negative blood, urine, and CSF bacterial cultures. Group B streptococcus was cultured from the blood of 11 infants (8 type III, 1 type Ia, 1 type Ib, 1 type Ic); three o f these infants had meningitis. The remaining ten infants were infected with a variety of organisms including Escherichia coli, group D streptococcus, alpha-
0022-3476/80/010104+03500.30/0 9 1980 The C. V. Mosby Co.
Volume 96 Number 1
hemolytic streptococcus, Haemophilus influenzae, meningococcus, and pneumococcus. As shown in Table I, latex agglutination identified group B antigen in the urine of all 11 patients infected with GBS, and seven had antigen detectable in unconcentrated urine. In these same samples, CIE detected group B antigen in eight of the 11, but only after urine had been concentrated in seven of the eight. Both CIE and latex agglutination identified group B antigen in the CSF in the three cases of meningitis. Only two of eight serum samples contained measurable amounts of group B antigen by LA; CIE was negative in all eight. The urine, CSF, and serum specimens in the 104 uninfected infants and in the ten infants with other bacterial infections were also tested for group B antigen. No false positive reactions were found using either technique, although occasional 1 + nonspecific agglutination reactions occurred when testing urine in the latex system. Crystals and mucus in the urine caused 3 to 4 + reactions, emphasizing the need for centrifugation prior to testing. Table II contains the results of type-specific antigen analysis carried out in the body fluid specimens from infants with GBS infections. LA identified type-specific antigen in the CSF of two infants with meningitis; the other CSF, blood, and urine specimens were negative for type-specific antigen. CIE detected type specific antigen in the CSF from all cases of meningitis, in one of eight serum specimens, and in five of 11 urine samples. All urine samples with measurable type-specific antigen also had group B antigen via CIE analysis. In the five patients with type-specific antigen in the urine, the type always correlated with that of the infecting organism. Latex agglutination appeared to be more sensitive than CIE in identifying group B antigen. We compared the sensitivities of the two methods by serially diluting body fluid specimens, broth cultures, and crude antigen extract (Table III, Column A). LA always detected group B antigen at higher dilutions than did CIE, confirming impressions gained from testing patient ~amples. Similarly, the sensitivities of the two methods for detecting type-specific antigen were compared (Table llI, Column B). CIE was always more sensitive than LA in detecting the presence of the type-specific antigen. DISCUSSION Latex agglutination and CIE have most often been used to detect the polysaccharide antigens produced by Haemophilus influenzae, pneumococcus, and meningococcus. With rare exception, ~ latex agglutination appears to be the more sensitive of the two methods. ~ ~-'; Until now, most studies involving group B streptococcal antigen detection have been carried out with CIE. In the present
Brief clinical and laboratory observations
10 5
Table I. Group B antigen detection comparison of LA and CIE in 11 newborn infants with GBS infections
~ody fluid
No. specimens
LA +
CIE +
It 11 7 8
7 11 3* 2
l 8 3* 0
Urine Unconcentrated Concentrated CSF Serum *Patients with meningitis.
Table IL Type-specific antigen in l l patients with GBS disease: comparison of LA and CIE
Body
No.
fluid
specimens
LA +
CIE +
Concentrated urine CSF
ll
0
7
2* (Ib,III) 0
5 (ia,ltl) 3* (Ib,III) l (Ill)
Serum
8
*Patients with meningitis.
Table II1. Comparison of sensitivity of LA and CIE* Column A group B antigen
Specimen 50X concentrated urine No. I~50X concentrated urine No. 2? CSF No. 17 CSF No. 2~ Whole cell culture Antigen extract
LA
I CIE
Column B type-specific antigen LA
I CIE
128
8
512
64
-
6d
64 512 512 8,000
4 64 "4 128
2 2 8
2 8 4 128
-
1
*All numbers in table = reciprocal of highest dilution at which test positive. tPatients with GBS disease.
study, the latex agglutination technique has been compared with CIE for detection of GBS antigen not only because it may be more sensitive, but also because it requires no specialized equipment or personnel, has reagents which remain stable for many months, and produces test results within minutes. In the present study, group B antigen, the polysaccharide common to all group B organisms, could be detected more sensitively by latex agglutination than by CIE. All infants infected with GBS were identified by the latex agglutination test performed on urine specimens. Other
10 6
Brief clinical and laboratory observations
studies using CIE for detecting group B antigen have identified between 50 to 100% of infected infants. 7-1~Our own CIE success rate of 70% falls within the range o f those published results. F o r infants with GBS sepsis, urine is the body fluid which most frequently contains detectable group B antigen, and CSF can be added to that list when meningitis is also present. In the present study, CIE has proven more sensitive than LA in measuring type-specific antigen. T h i s is in contrast to a report of 12 infants with type I l l GBS meningitis, in w h o m latex agglutination was more sensitive than CIE in detecting type lII antigen in CSF. 11 This difference may be due to different antibody strengths or to a difference in the latex particle sensitization method. The identifiable presence of type-specific antigen in body fluids did not increase the n u m b e r of infants with GBS disease identified by immunologic means in our study. In spite of the fact that we did not encounter false positive latex agglutination reactions, there is greater potential for false positive reactions with LA than with CIE. In serum, nonspecilic agglutination reactions occur when IgM antibodies are present, and these can be counteracted by adding dithiothreitol) Nonspecific agglutination reactions occur occasionally in CSF and may be avoided by heating the sample. 2 False positive reactions may occur both in latex and CIE systems; a cross-reaction has been reported in CSF from a patient with meningitis due to S. pneumoniae when tested with latex particles sensitized with antiserum to type III GBS, 11 and certain strains of group G streptococcus are known to cross-react with group B. 1-~ The latex agglutination test appears to be more sensitive than CIE for the detection of group B antigen in the urine of infants with GBS infections and has identified all infants with proven infection in this series.
The Journal of Pediatrics January 1980
Our thanks to Drs. George Halle tt and John Serrage, Department of Pediatrics, Maine Medical Center, and to the nurses and house staff of the Neonatal Intensive Care Unit. REFERENCES 1. Lancefield RC, McCarty M, and Everly WN: Multiple mouse protective antibodies directed against group B streptococci, J Exp Med 142:165, 1975. 2. Newman RB, Stevens RX, and Gaafar ttA: Latex agglutination test for the diagnosis of Haemophilus influenzae meningitis, J Lab Clin Med 76:107, 1970. 3. Coonrad JD, and Rylko-Bauer: Latex agglutination in the diagnosis of pneumococcal infection, J Clin Microbiol 4:168, 1976. 4. Whittle HC, Egler LJ, Tugwell P, and Greenwood BM: Rapid bacteriologic diagnosis of pyogenic meningitis by latex agglutination, Lancet 2:619, 1974. 5. Ward JI, Siber GR, Scheifele DW, and Smith DH: Rapid diagnosis of Hemophilus influenzae type b infections by latex particle agglutination and counterrimmunoelectrophoresis, J PEDIAXR93:37, 1978. 6. Severin WPJ: Latex agglutination in the diagnosis of meningococcal meningitis, J Clin Pathol 25:1079, I972. 7. Rhodes PG, and Hall RT: Countercurrent immunoelectrophoresis (CIE) of urine: A rapid diagnostic tool for group B streptococcal sepsis in the neonate, J PZD~ATR 91:833, 1977. 8. Siegel JD, and McCracken GH: Detection of group B streptococcal antigens in body fluids of neonates, J PEDIATR 93:491, 1978. 9. Shakelford PG, and Stechenberg BW: Countercurrent immunoelectrophoresis (CIE) in group B streptococcal disease, Pediatr Res 11:505, 1977. 10. Edwards MS, and Baker CJ: Prospective diagnosis of early onset group B streptococcal infection by countercurrent immunoelectrophoresis, J PEDIATR94:286, 1979. 11. Edwards MS, and Baker CJ: Comparison of antigen detection by latex particle agglutination and counterimmunoelectrophoresis in Type III, group B streptococcal meningitis, 18th lnterscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, Georgia October 1-4, 1978. 12. Curtis SN and Krause RM: Antigenic relationships between groups B and G streptococci, J Exp Med 120:629, 1964.
Defective primary dentition in survivors of neonatal mechanical ventilation Fergus M.B. Moylan, M.D.,* Edward B. Seldin, D.M.D., M.D., Daniel C. Shannon, M.D., and I. David Todres, M.D., Boston, Mass.
THERE HAVE BEEN numerous reports on the incidence of dental caries in young children, their association with *Reprint address: New England Medical Center Hospital, 171 Harrison Ave., Boston, MA 02111.
ingestion of sugar, and the characteristic distribution of teeth affected. 1' '-' Other studies have shown an increased incidence of defective dentition in subjects with serious neurologic deficits? ~ We have seen a large n u m b e r of young children with abnormal dentition, the etiology of
0022-3476/80/010106+03500.30/0 9 1980 The C. V. Mosby Co.