- Email: [email protected]
Counterimmunoelectrophoresis in Hemophilus influenzae Type b epiglottitis and pericarditis
Volume 86 Number 4
B r i e f clinical a n d laboratory observations
brother nor his other siblings had serum antibodies to the dove.
REFERENCES 1. Reed CS: Hypersensitivity pneumonitis, Postgrad Med 51:120, 1972. 2. Ouchterlony O, and Nilsson CA: Immunodiffusion and immunoelectrophoresis, in Weir DM, editor: Handbook of experimental immunology, 2, Oxford, 1973, Blackwell Scientific Publication, pp 1-39. 3. Deodhar SD: Cellular immunology, Chicago, 1974, American Society of Clinical Pathologists. 4. Reiss JS, Weiss NS, Payette KM, and Starcimas J: Childhood pigeon breeders' disease, Ann Allerg 32:208, 1974.
Counterimmunoelectrophoresis in Hemophilus influenzae Type b epiglottitis and pericarditis Edward W. P. Smith, M.D.,* New Haven, Conn., and David L. Ingrain, M.D., Boston, Mass.
COUNTERIMMUNOELECTROPHORESIS is a rapid, sensitive, and specific method which can detect the presence of certain bacterial polysaccharide antigens. C o u n t e r i m m u n o e l e c t r o p h o r e s i s has b e e n used to detect polyribophosphate, the principal capsular polysaccharide antigen of Hemophilus influenzae type b, in patients with meningitis and arthritis caused by this organism.l, 2 We undertook these studies to determine whether detection of PRP could be used as a diagnostic technique in epiglottitis and pericarditis due to H. inf l u e n z a e type b. From the Department of Pediatrics, Yale University School of Medicine, Yale-New Haven Hospital, and the Department o f Pediatrics, Harvard Medical School, Children's Hospital Medical Center. Presented in part at the American Pediatric Society and the Society for Pediatric Research Annual Meeting in Washington, D. C., May, 1974. Supported in part by National Institutes o f Health Grant No. HD-O0177-0 7 and National Institutes o f Health, National Institute of A llergy and Infectious Disease Grant No. 1-RO1-AH2239-01. *Repr~ntaddress:Duke UniversityMedical Center, Department of Pediatrics,Divisionof Infectious Diseases, Durham, N. C. 27710.
571
5. Barboriak J J, Sosman AJ, and Reed CE: Serological studies in pigeon breeders' disease, J Lab Clin Med 65:600, 1965. 6. rink JN, Barboriak JJ, Sosman AJ, Bukosky RJ, and Arkins JA: Antibodies against pigeon serum proteins in pigeon breeders, J Lab Clin Med 71:20, 1968. 7. Moore VL, rink JN, Barboriak JJ, RuffLL, and Schlueter DP: Immunologic events in pigeon breeders' disease, J Allergy Clin Immunol 53:319, 1974. 8. Pepys J: Hypersensitivity diseases of the lungs due to fungi and organic dusts; Basel, 1969, S. Karger, p 102. 9. Pepys J: Immunologic approaches in pulmonary disease caused by inhaled materials, Ann NY Acad Sci 221:27, 1974.
MATERIALS
AND METHODS
Hyland Laboratories (Cosa Mesa, Calif.)Austigen II agar plates, electrophoresis power supply, and buffer (pH 8.2) were used. The well nearest the cathode was filled with s e r u m or pericardial fluid and the well nearest the anode was filled with (NH4) 2 SO4precipitated rabbit antisera to H. influenzae type b. The antisera did not form precipitin bands with overnight broth cultures of pneumococcal Serotypes 6, 15, 29, or 35 but did with E s c h e r i c h i a coli Easter. We did not absorb the cross-reacting antibody because this strain does not cause epiglottitis or purulent pericarditis. Counterimmunoelectrophoresis was performed for 30 minutes at 30 milliamps. After cooling at 8 ~ C for 30 additional minutes, the plates were examined for precipitin lines in a darkened room with the aid of an indirect light source.
Abbreviations used CIE: counterimmunoelectroptaoresis PRP: polyribop hosp hate
Serial dilutions of a known concentration of PRP in glycine buffered saline (0.05M, pH 8.6) were run to d e t e r m i n e the sensitivity of the system. T h e s e also served as positive controls. A portion of an agar plate with purified PRP in concentrations from 1,200 ng/ml to 2.5 ng/ml in the wells on the right and //. influenzae type b antisera in the wells opposite is presented in Fig. 1. After cooling for 30 minutes the last line is visible at 5 ng PRP/ml. After cooling for 10 hours the sensitivity of the system is greater t h a n 2.5 n g / m l b u t less t h a n 5 n g / m l ( n o t shown).
572
Brief clinical and laboratory observations
The Journal of Pediatrics April197 5
Table I. Duration of fever in patients with epiglottitis and quantitation of antigenemia in the five positive patients Patient C.M. R.L. F.D. B.S. M.R. D.B. J.P.
D.M.
Duration .fever (hr)
Serum PRP (ng/ml)
3 4 5 4 8 20 24 36
None detected None detected None detected ( 5 ("trace") 5 10 80 160
Table II. Quantitation of PRP in sera and pericardial fluids of patients with H. influenzae type b pericarditis PRP Patient
Pericardialfluid (tzg/ml)
Serum (izglml)
T.L.
585
1.28 (Day 1) 0.39 (Day 3) Positive 1.28
W.M. S.C. Fig. 1. Precipitin lines after CIE using known concentrations of PRP and H. in[luenzae type b antiserum in an agar plate. The last visible band is at 5 ng of PRP/ml. To quantitate the PRP in clinical samples, CIE was performed on serial dilutions of each specimen. The last dilution which gave a visible band was multiplied by 5 ng/ml to calculate the quantity of PRP in that specimen. PATIENT
SELECTION
The sera of eight patient s, 3 to 8 years of age, with/-/. influenzae type b epiglottitis were collected at the time of hospital admission prior to antibiotic therapy and tested immediately or after storage at - 7 0 ~ C. The diagnosis of epiglottitis was confirmed by the presence of a swollen, red epiglottis and by positive cultures for 11. influenzae type b from blood and epiglottis in all eight patients. Six p a t i e n t s u n d e r w e n t t r a c h e o t o m y a n d two r e q u i r e d orotracheal intubation. All received intravenous ampicillin at 200-400 mg/kg/day. There were no deaths. There were two groups of negative controls. Group 1 consisted of sera from ten normal children. Group 2 c o n s i s t e d of a c u t e sera f r o m ten c h i l d r e n , aged 7 months to 7 years of age, with proved viral croup.
19,000 Positive
Three patients with H. influenzae type b pericarditis ranging in age from 2 to 4 years were also studied. The diagnosis was made by clinical, roentgenographic, and electrOcardiographic findings consistent with pericarditis. Cultures of either blood or pericardial fluid were positive for H. influenzae type b in all patients. All were treated with intravenous ampicillin. Anterior pericardiectomy was performed in two patients and the third patient underwent pericardotomy. Five samples of pericardial fluid obtained at postmortem examination from children who died of causes other than pericarditis served as controls. RESULTS I m m u n o p r e c i p i t i n b a n d s b e c a m e positive in sera from four of eight patients with epiglottitis after a brief period of cooling. A fifth specimen became positive after cooling for 10 hours and will be referred to as "trace." All sera from each control group were negative. Additional laboratory and clinical observations were made to determine whether there existed any relationship to the presence or absence of PRP in these sera. There was no relation between the presence of PRP and
Volume 86 Number 4
age of the patient, degree of leukocytosis, height of fever, or the severity or length of respiratory distress. However, it appeared that CIE-positive patients had a longer d u r a t i o n of f e v e r than C I E - n e g a t i v e p a t i e n t s (Table I). The duration of fever in the three CIE-negative patients ranged from 3 to 5 hours prior to presentation in the emergency room when serum samples were obtained. In the four patients whose sera were positive, the duration of fever was 8 to 36 hours. The patient who was trace positive had a fever of 4 hours duration. The last column in Table I represents the quantitation of PRP in the positive sera. As the duration of fever increased from four to 36 hours, the serum concentration of PRP increased from less than 5 ng/ml to 160 rig/ ml. Specimens of per!cardial fluid from the three patients with pericarditis obtained by needle aspiration and simultaneous samples of serum were all markedly positive by CIE. These specimens were collected after 4 days of intravenous cephalothin therapy in Patient T. L., 2 days of intravenous ampicillin and oxacillin in Patient W. M., and 1 day of intravenous ampicillin and oxacillin in Patient S. C. In two patients (T. L. and W. M.) both the Gram stain and culture of the pericardial fluid were positive, while in the third patient (S.C.) the G r a m stain and culture were both negative. Blood cultures, obtained prior to initiation of antibiotic therapy, were positive in all three patients. No blood cultures were d r a w n at the time o f p e r i c a r d i o c e n t e s i s . A l l c o n t r o l specimens of pericardial fluid were CIE negative. Two samples of pericardial fluid and three of the sera were available for quantitation of PRP. The results are summarized in Table II. The concentrations of PRP in Table II are expressed as micrograms per milliliter, thus representing a 1,000-fold increase over the amounts in Fig. 1 and Table I. The specimens of pericardial fluid contained 585 ~g PRP/ml and 19,000 /xg/ml. Patient T.L.'s s e r u m on a d m i s s i o n c o n t a i n e d 1.28 /xg/ml, decreasing to 0.39/zg/ml on the third hospital day. Patient S. C.'s serum on admission also contained 1.28/xg/ ml. The remaining two specimens (W. M.'s serum and S. C.'s pericardial fluid) were markedly positive ()0.67 /~g/ml.) but sufficient amounts were unavailable for further quantitation. DISCUSSION Over three decades ago, Alexander and associates 3 felt that demonstration of the type-specific carbohydrate of H. influenzae type b in the serum of patients with epiglottitis was an immediate index of severity as
Brief clinical and laboratory observations
573
well as proof of etiology. Using CIE, sera from five of eight of our patients with epiglottitis contained small, b u t detectable, amounts of PRP and thus this method also permits an early etiologic diagnosis using type-specific antibody. Although the amount of PRP in the sera of our patients did not correspond to the severity of the clinical course, there appeared to be a direct correlation with the duration of fever. Using broth cultures, Anderson 4 has shown that the concentration of PRP depends on the n u m b e r of organisms present as well as their g r o w t h p h a s e a n d t h e metabolic c o n d i t i o n s of the medium. A similar situation may exist in epiglottitis. It is likely that our CIE-negative patients, all with a febrile course 5 hours or less, were so briefly septicemic as to p r e c l u d e p r o d u c t i o n o f d e t e c t a b l e a m o u n t s of PRP. Counterimmunoelectrophoresis was a useful adjunct in the etiologic diagnosis of epig!ottitis, but quantitation of PRP did not provide a reliable index of the severity of this infection. Purulent pericarditis due to 11. influenzae type b is an u n c o m m o n infection, although its incidence may be on the increase. 5 Rapid etiologic diagnosis is essential, but is often hindered by previous antibiotic therapy. Using CIE all samples of pericardial fluid and sera of our three patients contained large quantities of PRP in spite of varying intervals of antecedent antimicrobial treatment, and thus was a valuable diagnostic technique. In the patient with simultaneously obtained pericardiat fluid and serum (T. L.), the amount of PRP in the pericardial fluid was 500 times greater than that of the serum, suggesting that the pericardial sac may function as a depot for large amounts of antigen. The authors wish to thank Dr. Howard A. Pearson" and Dr. David H. Smith for their advice and support, Dr. Ralph E. Haynes for providing control sera, and Gretchen Umbach for secretarial assistance. REFERENCES
1. Ingram DL, Anderson P, and Smith DH: Countercurrent immunoelectrophoresis in the diagnosis of systemic diseases caused by Hemophilus influenzae type b, J PEDIATR 81:1156, 1972. 2. Coonrod JD, and Rytel MW: Determination of etiology of bacterial meningitis by counter immunoelectrophoresis, Lancet 1:1154, 1972. 3. Alexander HE, Ellis C, and Leidy G: Treatment of typespecific Hemophilus influenzae infections in infancy and childhood, J PEDIATR20:673, 1942. 4. Anderson P: Personal communication, 1974. 5. Echeverria P, Smith EWP, Ingram DL, Sade RM, and Gardner P: Hemophilus influenzae b pericarditis in children, Pediatrics (in press).
B r i e f clinical a n d laboratory observations
brother nor his other siblings had serum antibodies to the dove.
REFERENCES 1. Reed CS: Hypersensitivity pneumonitis, Postgrad Med 51:120, 1972. 2. Ouchterlony O, and Nilsson CA: Immunodiffusion and immunoelectrophoresis, in Weir DM, editor: Handbook of experimental immunology, 2, Oxford, 1973, Blackwell Scientific Publication, pp 1-39. 3. Deodhar SD: Cellular immunology, Chicago, 1974, American Society of Clinical Pathologists. 4. Reiss JS, Weiss NS, Payette KM, and Starcimas J: Childhood pigeon breeders' disease, Ann Allerg 32:208, 1974.
Counterimmunoelectrophoresis in Hemophilus influenzae Type b epiglottitis and pericarditis Edward W. P. Smith, M.D.,* New Haven, Conn., and David L. Ingrain, M.D., Boston, Mass.
COUNTERIMMUNOELECTROPHORESIS is a rapid, sensitive, and specific method which can detect the presence of certain bacterial polysaccharide antigens. C o u n t e r i m m u n o e l e c t r o p h o r e s i s has b e e n used to detect polyribophosphate, the principal capsular polysaccharide antigen of Hemophilus influenzae type b, in patients with meningitis and arthritis caused by this organism.l, 2 We undertook these studies to determine whether detection of PRP could be used as a diagnostic technique in epiglottitis and pericarditis due to H. inf l u e n z a e type b. From the Department of Pediatrics, Yale University School of Medicine, Yale-New Haven Hospital, and the Department o f Pediatrics, Harvard Medical School, Children's Hospital Medical Center. Presented in part at the American Pediatric Society and the Society for Pediatric Research Annual Meeting in Washington, D. C., May, 1974. Supported in part by National Institutes o f Health Grant No. HD-O0177-0 7 and National Institutes o f Health, National Institute of A llergy and Infectious Disease Grant No. 1-RO1-AH2239-01. *Repr~ntaddress:Duke UniversityMedical Center, Department of Pediatrics,Divisionof Infectious Diseases, Durham, N. C. 27710.
571
5. Barboriak J J, Sosman AJ, and Reed CE: Serological studies in pigeon breeders' disease, J Lab Clin Med 65:600, 1965. 6. rink JN, Barboriak JJ, Sosman AJ, Bukosky RJ, and Arkins JA: Antibodies against pigeon serum proteins in pigeon breeders, J Lab Clin Med 71:20, 1968. 7. Moore VL, rink JN, Barboriak JJ, RuffLL, and Schlueter DP: Immunologic events in pigeon breeders' disease, J Allergy Clin Immunol 53:319, 1974. 8. Pepys J: Hypersensitivity diseases of the lungs due to fungi and organic dusts; Basel, 1969, S. Karger, p 102. 9. Pepys J: Immunologic approaches in pulmonary disease caused by inhaled materials, Ann NY Acad Sci 221:27, 1974.
MATERIALS
AND METHODS
Hyland Laboratories (Cosa Mesa, Calif.)Austigen II agar plates, electrophoresis power supply, and buffer (pH 8.2) were used. The well nearest the cathode was filled with s e r u m or pericardial fluid and the well nearest the anode was filled with (NH4) 2 SO4precipitated rabbit antisera to H. influenzae type b. The antisera did not form precipitin bands with overnight broth cultures of pneumococcal Serotypes 6, 15, 29, or 35 but did with E s c h e r i c h i a coli Easter. We did not absorb the cross-reacting antibody because this strain does not cause epiglottitis or purulent pericarditis. Counterimmunoelectrophoresis was performed for 30 minutes at 30 milliamps. After cooling at 8 ~ C for 30 additional minutes, the plates were examined for precipitin lines in a darkened room with the aid of an indirect light source.
Abbreviations used CIE: counterimmunoelectroptaoresis PRP: polyribop hosp hate
Serial dilutions of a known concentration of PRP in glycine buffered saline (0.05M, pH 8.6) were run to d e t e r m i n e the sensitivity of the system. T h e s e also served as positive controls. A portion of an agar plate with purified PRP in concentrations from 1,200 ng/ml to 2.5 ng/ml in the wells on the right and //. influenzae type b antisera in the wells opposite is presented in Fig. 1. After cooling for 30 minutes the last line is visible at 5 ng PRP/ml. After cooling for 10 hours the sensitivity of the system is greater t h a n 2.5 n g / m l b u t less t h a n 5 n g / m l ( n o t shown).
572
Brief clinical and laboratory observations
The Journal of Pediatrics April197 5
Table I. Duration of fever in patients with epiglottitis and quantitation of antigenemia in the five positive patients Patient C.M. R.L. F.D. B.S. M.R. D.B. J.P.
D.M.
Duration .fever (hr)
Serum PRP (ng/ml)
3 4 5 4 8 20 24 36
None detected None detected None detected ( 5 ("trace") 5 10 80 160
Table II. Quantitation of PRP in sera and pericardial fluids of patients with H. influenzae type b pericarditis PRP Patient
Pericardialfluid (tzg/ml)
Serum (izglml)
T.L.
585
1.28 (Day 1) 0.39 (Day 3) Positive 1.28
W.M. S.C. Fig. 1. Precipitin lines after CIE using known concentrations of PRP and H. in[luenzae type b antiserum in an agar plate. The last visible band is at 5 ng of PRP/ml. To quantitate the PRP in clinical samples, CIE was performed on serial dilutions of each specimen. The last dilution which gave a visible band was multiplied by 5 ng/ml to calculate the quantity of PRP in that specimen. PATIENT
SELECTION
The sera of eight patient s, 3 to 8 years of age, with/-/. influenzae type b epiglottitis were collected at the time of hospital admission prior to antibiotic therapy and tested immediately or after storage at - 7 0 ~ C. The diagnosis of epiglottitis was confirmed by the presence of a swollen, red epiglottis and by positive cultures for 11. influenzae type b from blood and epiglottis in all eight patients. Six p a t i e n t s u n d e r w e n t t r a c h e o t o m y a n d two r e q u i r e d orotracheal intubation. All received intravenous ampicillin at 200-400 mg/kg/day. There were no deaths. There were two groups of negative controls. Group 1 consisted of sera from ten normal children. Group 2 c o n s i s t e d of a c u t e sera f r o m ten c h i l d r e n , aged 7 months to 7 years of age, with proved viral croup.
19,000 Positive
Three patients with H. influenzae type b pericarditis ranging in age from 2 to 4 years were also studied. The diagnosis was made by clinical, roentgenographic, and electrOcardiographic findings consistent with pericarditis. Cultures of either blood or pericardial fluid were positive for H. influenzae type b in all patients. All were treated with intravenous ampicillin. Anterior pericardiectomy was performed in two patients and the third patient underwent pericardotomy. Five samples of pericardial fluid obtained at postmortem examination from children who died of causes other than pericarditis served as controls. RESULTS I m m u n o p r e c i p i t i n b a n d s b e c a m e positive in sera from four of eight patients with epiglottitis after a brief period of cooling. A fifth specimen became positive after cooling for 10 hours and will be referred to as "trace." All sera from each control group were negative. Additional laboratory and clinical observations were made to determine whether there existed any relationship to the presence or absence of PRP in these sera. There was no relation between the presence of PRP and
Volume 86 Number 4
age of the patient, degree of leukocytosis, height of fever, or the severity or length of respiratory distress. However, it appeared that CIE-positive patients had a longer d u r a t i o n of f e v e r than C I E - n e g a t i v e p a t i e n t s (Table I). The duration of fever in the three CIE-negative patients ranged from 3 to 5 hours prior to presentation in the emergency room when serum samples were obtained. In the four patients whose sera were positive, the duration of fever was 8 to 36 hours. The patient who was trace positive had a fever of 4 hours duration. The last column in Table I represents the quantitation of PRP in the positive sera. As the duration of fever increased from four to 36 hours, the serum concentration of PRP increased from less than 5 ng/ml to 160 rig/ ml. Specimens of per!cardial fluid from the three patients with pericarditis obtained by needle aspiration and simultaneous samples of serum were all markedly positive by CIE. These specimens were collected after 4 days of intravenous cephalothin therapy in Patient T. L., 2 days of intravenous ampicillin and oxacillin in Patient W. M., and 1 day of intravenous ampicillin and oxacillin in Patient S. C. In two patients (T. L. and W. M.) both the Gram stain and culture of the pericardial fluid were positive, while in the third patient (S.C.) the G r a m stain and culture were both negative. Blood cultures, obtained prior to initiation of antibiotic therapy, were positive in all three patients. No blood cultures were d r a w n at the time o f p e r i c a r d i o c e n t e s i s . A l l c o n t r o l specimens of pericardial fluid were CIE negative. Two samples of pericardial fluid and three of the sera were available for quantitation of PRP. The results are summarized in Table II. The concentrations of PRP in Table II are expressed as micrograms per milliliter, thus representing a 1,000-fold increase over the amounts in Fig. 1 and Table I. The specimens of pericardial fluid contained 585 ~g PRP/ml and 19,000 /xg/ml. Patient T.L.'s s e r u m on a d m i s s i o n c o n t a i n e d 1.28 /xg/ml, decreasing to 0.39/zg/ml on the third hospital day. Patient S. C.'s serum on admission also contained 1.28/xg/ ml. The remaining two specimens (W. M.'s serum and S. C.'s pericardial fluid) were markedly positive ()0.67 /~g/ml.) but sufficient amounts were unavailable for further quantitation. DISCUSSION Over three decades ago, Alexander and associates 3 felt that demonstration of the type-specific carbohydrate of H. influenzae type b in the serum of patients with epiglottitis was an immediate index of severity as
Brief clinical and laboratory observations
573
well as proof of etiology. Using CIE, sera from five of eight of our patients with epiglottitis contained small, b u t detectable, amounts of PRP and thus this method also permits an early etiologic diagnosis using type-specific antibody. Although the amount of PRP in the sera of our patients did not correspond to the severity of the clinical course, there appeared to be a direct correlation with the duration of fever. Using broth cultures, Anderson 4 has shown that the concentration of PRP depends on the n u m b e r of organisms present as well as their g r o w t h p h a s e a n d t h e metabolic c o n d i t i o n s of the medium. A similar situation may exist in epiglottitis. It is likely that our CIE-negative patients, all with a febrile course 5 hours or less, were so briefly septicemic as to p r e c l u d e p r o d u c t i o n o f d e t e c t a b l e a m o u n t s of PRP. Counterimmunoelectrophoresis was a useful adjunct in the etiologic diagnosis of epig!ottitis, but quantitation of PRP did not provide a reliable index of the severity of this infection. Purulent pericarditis due to 11. influenzae type b is an u n c o m m o n infection, although its incidence may be on the increase. 5 Rapid etiologic diagnosis is essential, but is often hindered by previous antibiotic therapy. Using CIE all samples of pericardial fluid and sera of our three patients contained large quantities of PRP in spite of varying intervals of antecedent antimicrobial treatment, and thus was a valuable diagnostic technique. In the patient with simultaneously obtained pericardiat fluid and serum (T. L.), the amount of PRP in the pericardial fluid was 500 times greater than that of the serum, suggesting that the pericardial sac may function as a depot for large amounts of antigen. The authors wish to thank Dr. Howard A. Pearson" and Dr. David H. Smith for their advice and support, Dr. Ralph E. Haynes for providing control sera, and Gretchen Umbach for secretarial assistance. REFERENCES
1. Ingram DL, Anderson P, and Smith DH: Countercurrent immunoelectrophoresis in the diagnosis of systemic diseases caused by Hemophilus influenzae type b, J PEDIATR 81:1156, 1972. 2. Coonrod JD, and Rytel MW: Determination of etiology of bacterial meningitis by counter immunoelectrophoresis, Lancet 1:1154, 1972. 3. Alexander HE, Ellis C, and Leidy G: Treatment of typespecific Hemophilus influenzae infections in infancy and childhood, J PEDIATR20:673, 1942. 4. Anderson P: Personal communication, 1974. 5. Echeverria P, Smith EWP, Ingram DL, Sade RM, and Gardner P: Hemophilus influenzae b pericarditis in children, Pediatrics (in press).