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C-reactive protein and Limulus amebocyte lysate assay in diagnosis of bacterial meningitis
Volume 108 Number 3
7. Slifkin M, Gil GM. Evaluation of the Culturette Brand Ten-Minute Group A Strep ID technique. J Clin MicrobioI 1984;20:12. 8. Chang M J, Mohla C. Ten-minute detection of group A streptococci in pediatric throat swabs. J Clin Microbiol 1985;21:258. 9. Miceika BG, Vitous AS, Thompson KD. Detection of group A streptococcal antigen directly from throat swa'bs with a ten-minute latex agglutination test. J Clin Microbiol 1985;21:467. 10. Berkowitz CD, Anthony BF, Kaplan EL, et al. A cooperative study of latex agglutination to identify group A streptococcal antigen in throat swabs of patients with acute pharyngitis. J PEDIATR 1985;107:89. 11. Godsey J, Schulman R, Eriquez L. The hydrolysis of
Clinical and laboratory observations
423
L-pyrrolidonyl-/%naphthylamide as an aid in the rapid identification of Streptococcus pyogenes, S. avium, and group D enterococci. Program and Abstracts of the 81st Annual Meeting of the American Society of Microbiology, Dallas, March 1 to 6, 1981, p 276. 12. Facklam RR, Thacker LG, Fox B, et al. Presumptive identification of streptocci with as new test system. J Clin Microbiol 1982;15:987. 13. Wasilauskas BL, Hampton KD. Evaluation of the StreptA-Fluor identification method for group A streptococci. J Clin Microbiol 1984;20:1205. 14. Szewczuk A, Mulczyk M. Pyrrolidonyl peptidase in bacteria: the enzyme from Bacillus subtilis. Eur J Biochem 1969; 8:63.
C-reactive protein and Limulus amebocyte lysate assay in diagnosis of bacterial meningitis Joseph Eiden, M.D., and Robert H. Yolken, M.D. From the Division of Infectious Diseases, D e p a r t m e n t of Pediatrics, The Johns Hopkins M e d i c a l School, Baltimore
On initial presentation, nonbacterial meningitis is frequently difficult to distinguish clinically from bacterial meningitis, and commonly used laboratory tests such as C S F glucose and protein determinations and total leukocyte count with differential count can yield ambiguous results. ~ Corrall et a12 found that C-reactive protein was elevated in the C S F of all patients with bacterial meningitis and was useful in distinguishing bacterial from nonbacterial meningitis. In the same study, the L i m u l u s amebocyte lysate assay of bacterial endotoxin in the C S F also predicted infection with gram-negative bacteria. However, other investigators have found varying degrees of sensitivity of C R P for the diagnosis of bacterial meningitis. 3-6 W e thus undertook a prospective study to evaluate the predictive value of C R P and L A L in conjunction with standard C S F laboratory determination for distinguishing bacterial from aseptic meningitis. Supported by Contract 1 A122680 from the National Institutes of Health and by the Eudowood Division of Pediatric Infectious Disease. Presented in part at the annual meeting of the Society for Pediatric Research, Washington, D.C., May 9, 1985. Submitted for publication July I, 1985; accepted Sept. 10, 1985. Reprint requests: Robert H. Yolken, M.D., Chief, Division of Infectious Diseases, Department of Pediatrics, The Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205.
METHODS C S F specimens were obtained between November 1982 and September 1984 from 250 infants and children younger than 18 years of age. All patients were undergoing initial evaluation for suspected meningitis at the Children's Medical and Surgical Center of The Johns Hopkins Medical Institutions. At the time of diagnostic lumbar puncture, informed consent was requested for obtaining additional C S F for study. Patients with ventricular shunts and those for whom informed consent could not be obtained were excluded from study. CRP EU LAL PMN
C-reactive protein Endotoxin units Limutus amebocyte Iysate [assay] Polymorphonuclear lymphocyte
Laboratory tests of CSF. C S F from all patients was Gram stained and cultured for bacteria according to routine procedures of clinical bacteriology laboratories. Specimens were also evaluated for total leukocyte count with differential count, and glucose and protein levels, in the clinical pathology laboratories. In some instances, quantity of C S F was insufficient to allow performance of all tests. The normal range of protein concentration in C S F of neonates and infants was obtained from the report of Sharff et al. 7 C S F glucose <40 m g / # L was considered abnormal and indicative of bacterial infection.
424
Clinical and laboratory observations
The Journal of Pediatrics March 1986
T a b l e . Sensitivity, specificity, and predictive value of laboratory tests of CSF in patients with CSF pleocytosis
Gram stain
Glucose* (mg/dl)
Sensitivity~ n 11/16 9/14 % 69 64 SpecificityII n 28/28 26/28 % I00 93 Predictive value of positive test n 11/11 9/11 % 100 82 Predictive value of negative test n 28/33 26/31 % 85 84
Glucose or protein or Gram stain or CRP
Proteini(mg/dl)
CRP
LAL
>20%
>60%
Glucose or protein or Gram stain
10/14 71
14/17 82
6/6 ~ 100
13/14 93
10/14 71
13/14 93
14/14 100
25/28 89
27/28 96
29/29 100
10/28 36
21/28 75
23/28 82
22/28 79
10/13 77
14/15 93
6/6 100
13/31 42
10/17 59
13/18 72
14/20 70
25/29 86
27/30 90
29/29 100
10/11 90
21/31 84
23/24 96
22/22 100
PMNs
*CSF glucose<40 mg/dl consideredpositiveresult. tProtein abovenormalrange for age consideredpositiveresult. w for patientswith meningitiscaused by gram-negativeorganisms. :~Positivetests/tests performed. HNegativetests/tests performed.
Specimens for CRP and LAL assay. Specimens were placed in separate sterile pyrogen-free tubes and immediately refrigerated at 4 ~ C. Specimens were tested within 12 hours or frozen at - 7 0 ~ C for later testing. C-reactive protein. A qualitative latex agglutination assay for CRP (CR-test, Hyland Laboratories, Costa Mesa, Calif.) was used to assay 50 #L aliquots of CSF in individual wells on a glass slide, as described by Corrall et al. z A CSF specimen was considered positive if specific agglutination was noted. Detection of endotoxin for gram-negative bacteria. Bacterial endotoxin was detected with LAL test kits (Pyrogent, Mallincrodt, Inc., St. Louis). An individual test consisted of a tube containing dried LAL capable of detecting 0.03 E U / m l endotoxin. A test for endotoxin was considered positive if a clot formed in the tube after incubation at 37 ~ C for 1 hour after the addition of 250 #L CSF. RESULTS
Patients with bacterial meningitis. Bacterial pathogens were isolated from CSF by standard microbiologic methods in 17 patients. Infecting organisms included Haemophilis influenzae type b (nine patients), Neisseria meningitidis group B (three), group B streptococcus (two), Streptococcus pneumoniae (two), and Listeria monocytogenes (one). CRP was positive in 14 (82%) of 17 patients with bacterial meningitis. Negative results were observed with CSF from a 1-week-old neonate with group B streptococcus and from an ll-month-old infant with H.
influenzae b meningitis. In an 18-month-old child with H. influenzae b meningitis, the initial CRP determination was negative, but the CRP was positive at 1:2, 1:4, and 1:8 dilutions of CSF, indicating that this specimen was negative because of a prozone phenomenon. The other two false-negative specimens, however, were negative on testing at similar dilutions. Sufficient CSF was available to permit LAL assay in 10 cases of bacterial meningitis. LAL was positive in all six in which meningitis was caused by gram-negative bacteria. Four specimens from patients with gram-positive bacteria were negative for endotoxin by LAL assay. Patients with CSF pleocytosis and negative bacterial culture. Forty-six patients had >_ 10 W B C / m m 3 CSF and negative bacterial cultures. Of these 46, four had CSF specimens contaminated by blood as noted by visual inspection. Fourteen additional patients received antibiotics prior to lumbar puncture. CRP and LAL assays were performed on the remaining 28 specimens. No endotoxin was detected by LAL in any specimen (0 of 25) in which there was sufficient CSF for assay. The latex agglutination test for CRP gave negative results in 27 of 28 instances. The false-positive CRP assay occurred in a three-year old boy with meningitis, fever, and maculopapular rash. The patient's blood and, CSF cultures remained negative, and he was discharged from the hospital after 3 days with a diagnosis of aseptic meningitis. Patients without pleocytosis. None of the 204 patients with <10 WBC/mm 3 were found to have bacterial menin-
Volume 108 Number 3
gitis. The CRP assay was negative in 185 of 187 such cases in which an adequate specimen was available for testing. One of the two false-positive CRP assays occurred in an 18-month-old boy with chicken pox and febrile seizures. CSF glucose and protein and leukocyte count were all within normal limits, and the Gram stain was negative. The other false-positive CRP was obtained from a 6year-old boy with pneumonia. Lumbar puncture was performed because of complaint of neck stiffness. Blood and CSF cultures were negative and CSF laboratory values were within normal limits. The LAL assay was negative in 175 of 176 cases in which an adequate sample was available for assay. Sensitivity, specificity, and predictive value of laboratory assays of CSF in evaluation of bacterial and nonbacteriai meningitis (Table). The utility of the CSF leukocyte count in the differentiation of bacterial and nonbacterial meningitis was evaluated in 224 patients. Excluded were those patients with visible blood contamination of CSF (four patients), insufficient CSF for WBC count (eight), or negative cultures but prior antibiotic therapy (14). All patients in whom a pathogen was cultured from CSF also had an elevated CSF WBC count. The sensitivity of CSF pleocytosis in detecting bacterial meningitis was thus 100% in this study. However, 28 patients had elevated CSF WBC count (>_ 10 WBC/mm 3) with negative culture. The predictive value of CSF pleocytosis for bacterial meningitis was thus only 38%. In terms of the utility of the determination of the percentage of polymorphonuclar leukocytes, no value could be found that resulted in good levels of both sensitivity and specificity. For example, although 13 of 14 patients with bacterial meningitis had >20% PMN in CSF specimens (93% sensitivity), 10 of 28 patients with pleocytosis and without bacterial meningitis also had >20% PMNs (36% specificity). On the other hand, if a level of >60% PMNs was used the sensitivity and specificity were 71% and 75%, respectively. For the remainder of the CSF laboratory measurements, sensitivity, specificity, and predictive values were calculated for those patients initially found to have elevated CSF WBC count (Table). None of the routine tests (glucose and protein concentration, Gram stain) had a sensitivity >71%. When the three routine assays were considered as a group, the sensitivity was 93% in 14 patients. The CRP test was more sensitive than either Gram stain, glucose, or protein, but the CRP test also did not achieve 100% sensitivity in detecting bacterial meningitis in patients with CSF pleocytosis. However, when the results of the CRP determinations were combined with those of the three routine assays, all patients with bacterial meningitis were detected by at least one of the assays. In
Clinical and laboratory observations
425
one instance, CRP was positive but Gram stain was negative and glucose and protein were normal in CSF from a 10-month-old girl whose blood and CSF cultures grew H. influenzae b. That patient was initially thought to have an upper respiratory tract infection, but she returned to the clinic with persistent fever, and a lumbar puncture was performed. The patient was subsequently admitted and given treatment after a lumbar puncture revealed a CSF total leukocyte count of 658 W B C / m m 3 and a differential count of 36% PMNs. When CSF Gram stain, glucose, protein, and CRP were considered as a test battery in patients with CSF pleocytosis, the predictive value of negative results was 100%. In addition, the predictive value of positive results of this test battery was 70%, as compared to 38% if CSF pleocytosis alone was used to detect bacterial meningitis. The LAL assay proved to be 100% sensitive (six of six) in predicting the presence of gram-negative bacteria in CSF. Twenty-five patients with CSF pleocytosis and negative culture were also negative for endotoxin by LAL test. Four other patients with positive CSF cultures for gram-positive organisms did not have detectable levels of endotoxin in the CSF. DISCUSSION The LAL assay for gram-negative endotoxin has not proved useful in predicting the presence of gram-negative sepsis 8'9 but has been reported as helpful in detecting meningitis caused by gram-negative bacteria? ~ In our study the utility of the LAL assay was assessed in conjunction with other laboratory tests of CSF. Difficulty was encountered in obtaining enough additional CSF (250 tzL) from neonates and infants to allow performance of the LAL assay. The test for endotoxin was sensitive in those instances in which it was used, but the CRP was also positive in the same cases. The LAL assay, then, did not appear to increase significantly the detection of bacterial meningitis in this study, but it was useful in distinguishing gram-positive from gram-negative meningitis. Using CSF pleocytosis as the sole criterion for beginning antibiotic therapy in the patients in this study, more patients with aseptic meningitis (28) than with bacterial meningitis (17) would be hospitalized and receive treatment. If the physician had decided to treat only when an abnormality was detected by laboratory evaluation of glucose and protein or results of Gram stain, initiation of antibiotic therapy would have been delayed in one of the patients with bacterial meningitis. However, an abnormality in either CSF glucose, protein, Gram stain, or CRP correctly predicted bacterial meningitis in all instances. The specificity of this battery of four tests was 79%, and the predictive value of a positive assay 70% in patients with
4 26
Clinical and laboratory observations
CSF pleocytosis. With use of this battery we might expect to avoid antibiotic therapy in more than half of patients with CSF pleocytosis while maintaining a high degree of sensitivity for the detection of bacterial meningitis. The analysis of CSF CRP by latex agglutiation is rapid and may be easily performed in even the smallest laboratories. However, published reports have disagreed about the sensitivity of CSF CRP in predicting bacterial meningitis. Corrall et al. 2 found that CRP was elevated in the CSF of 24 of 24 patients with culture-proved bacterial meningitis but iia only two of 32 patients with nonbacterial meningitis. WalterspieP later noted that CSF specimens from four of 10 children with H. influenzae meningitis were negative by the latex agglutination test for CRP. The CSF from neonates with bacterial meningitis was tested for CRP by Philip and Baker 4 using a sensitive laser nephelometry technique. Those investigators found elevated CRP levels in only two of 11 patients with cultureproved bacterial meningitis. Benjamin et al? also reported a low sensitivity (66%) when laser nephelometry was used to detect CRP in the CSF of patients with bacterial meningitis. However, Abramson et al.6 found that in 72 of 74 patients with bacterial meningitis CRP was detectable by the latex agglutination assay. In our study, 14 (82%) of 17 patients with bacterial meningitis had detectable CSF CRP. Moreover, in one of these patients results were false-negative because of the prozone phenomenon, which was detected by further dilution of the specimen. Although a number of factors (e.g., specimen handling, age of the patient, underlying disease) might explain the divergent results reported for the sensitivity of the CRP assay, our study and two others z,6 reported the highest sensitivities with use of the same latex agglutination assay (CR-test).
The Journal of Pediatrics March 1986
Our data indicate that the CRP assay can increase the sensitivity of detection of bacterial meningitis without significant loss of specificity. The assay is easily performed, and with appropriate dilutions to avoid the prozone phenomenon it should be seriously considered as an addition to the assays routinely used in the evaluation of suspected bacterial meningitis. REFERENCES 1. ModlinJF. Coxsackie and ecbovirus. In: Mandel GL, Douglas RG Jr, Bennett JE, eds. Principles and practices of infectious diseases, 2nd ed. New York: John Wiley, 1985. 2. Corrall JC, Pepple JM, Moxon ER, Hughes WT. C-reactive protein in spinal fluid of children with meningitis. J PEDIATR 1981;99:365-369. 3. Walterspiel JN. C-reactive protein in cerebrospinal fluid. Pediatr Infect Dis 1983;2:194. 4. Phillip AGS, Baker CJ. Cerebrospinal fluid C-reactive protein in neonatal meningitis. J PEDIATR1983;102:715~717. 5. Benjamin DR, Olpheim KE, Brewer L. ls C-reactive protein useful in the management of children with suspected bacterial meningitis? Am J Clin Pathol 1984;81:779-792. 6. Abramson JS, Hampton KD, Baba S, Wasilauskas BL, Maron MJ. The use of C-reative protein from cerebrospinal fluid for differentiating meningitis from other central nervous system diseases. J Infect Dis 1985;151;854-858. 7. Sharff LD, Platt LH, McCracker GH Jr. Cerebrospinal fluid examination in neonates:comparison of high risk=infantswith and without meningitis. J PEDIATR1976;88:473-477. 8. Stumacher RJ, Kovnat MJ, McCabe WR. Limitations of the usefulness of the Limulus assay for endotoxim N Engl J Med 1973;288:1261-1264. 9. Elin RJ, Robinson RA, Levine AS, Wolff SM. Lack of clinical usefulness of the Limulus test in the diagnosis of endotoxemia. N Engl J Med 1975;293:521-524. 10. Ross S, Rodriguez W, Controni G, Korengold G, Watson S, Khan W. Limulus lysate test for gram-negative bacterial meningitis. JAMA 1975;233:1366-1369.
Childhood acquired immune deficiency syndrome manifesting as acrodermatitis enteropathica Timothy K. Tong, M.D., Lori R. A n d r e w , M.D., A b e y Albert, M.D., a n d John J. Mickell, M.D. From the Department of Pediatrics, Division of Pediatric Critical Care, Children's Medical Center, Medical College of Virginia, Richmond
The diagnosis of acquired immune deficiency syndrome is more difficult in the pediatric population because of the Submitted for publication July 8, 1985; accepted Sept. 17, 1985. Reprint requests: John J. Mickell, M.D., MCV Station Box 530, Richmond, VA 23298.
number of immunologic disorders seen in this age group. Another disease that may resemble childhood AIDS in clinical, biochemical, and immunologic findings is acrodermatitis enteropathica. The symptoms include eczematous lesions, especially in the perioral and perineal areas, alopecia, conjunctivitis, chronic diarrhea, stomatitis, and intercurrent bacterial and candidal infections. The cause of
7. Slifkin M, Gil GM. Evaluation of the Culturette Brand Ten-Minute Group A Strep ID technique. J Clin MicrobioI 1984;20:12. 8. Chang M J, Mohla C. Ten-minute detection of group A streptococci in pediatric throat swabs. J Clin Microbiol 1985;21:258. 9. Miceika BG, Vitous AS, Thompson KD. Detection of group A streptococcal antigen directly from throat swa'bs with a ten-minute latex agglutination test. J Clin Microbiol 1985;21:467. 10. Berkowitz CD, Anthony BF, Kaplan EL, et al. A cooperative study of latex agglutination to identify group A streptococcal antigen in throat swabs of patients with acute pharyngitis. J PEDIATR 1985;107:89. 11. Godsey J, Schulman R, Eriquez L. The hydrolysis of
Clinical and laboratory observations
423
L-pyrrolidonyl-/%naphthylamide as an aid in the rapid identification of Streptococcus pyogenes, S. avium, and group D enterococci. Program and Abstracts of the 81st Annual Meeting of the American Society of Microbiology, Dallas, March 1 to 6, 1981, p 276. 12. Facklam RR, Thacker LG, Fox B, et al. Presumptive identification of streptocci with as new test system. J Clin Microbiol 1982;15:987. 13. Wasilauskas BL, Hampton KD. Evaluation of the StreptA-Fluor identification method for group A streptococci. J Clin Microbiol 1984;20:1205. 14. Szewczuk A, Mulczyk M. Pyrrolidonyl peptidase in bacteria: the enzyme from Bacillus subtilis. Eur J Biochem 1969; 8:63.
C-reactive protein and Limulus amebocyte lysate assay in diagnosis of bacterial meningitis Joseph Eiden, M.D., and Robert H. Yolken, M.D. From the Division of Infectious Diseases, D e p a r t m e n t of Pediatrics, The Johns Hopkins M e d i c a l School, Baltimore
On initial presentation, nonbacterial meningitis is frequently difficult to distinguish clinically from bacterial meningitis, and commonly used laboratory tests such as C S F glucose and protein determinations and total leukocyte count with differential count can yield ambiguous results. ~ Corrall et a12 found that C-reactive protein was elevated in the C S F of all patients with bacterial meningitis and was useful in distinguishing bacterial from nonbacterial meningitis. In the same study, the L i m u l u s amebocyte lysate assay of bacterial endotoxin in the C S F also predicted infection with gram-negative bacteria. However, other investigators have found varying degrees of sensitivity of C R P for the diagnosis of bacterial meningitis. 3-6 W e thus undertook a prospective study to evaluate the predictive value of C R P and L A L in conjunction with standard C S F laboratory determination for distinguishing bacterial from aseptic meningitis. Supported by Contract 1 A122680 from the National Institutes of Health and by the Eudowood Division of Pediatric Infectious Disease. Presented in part at the annual meeting of the Society for Pediatric Research, Washington, D.C., May 9, 1985. Submitted for publication July I, 1985; accepted Sept. 10, 1985. Reprint requests: Robert H. Yolken, M.D., Chief, Division of Infectious Diseases, Department of Pediatrics, The Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205.
METHODS C S F specimens were obtained between November 1982 and September 1984 from 250 infants and children younger than 18 years of age. All patients were undergoing initial evaluation for suspected meningitis at the Children's Medical and Surgical Center of The Johns Hopkins Medical Institutions. At the time of diagnostic lumbar puncture, informed consent was requested for obtaining additional C S F for study. Patients with ventricular shunts and those for whom informed consent could not be obtained were excluded from study. CRP EU LAL PMN
C-reactive protein Endotoxin units Limutus amebocyte Iysate [assay] Polymorphonuclear lymphocyte
Laboratory tests of CSF. C S F from all patients was Gram stained and cultured for bacteria according to routine procedures of clinical bacteriology laboratories. Specimens were also evaluated for total leukocyte count with differential count, and glucose and protein levels, in the clinical pathology laboratories. In some instances, quantity of C S F was insufficient to allow performance of all tests. The normal range of protein concentration in C S F of neonates and infants was obtained from the report of Sharff et al. 7 C S F glucose <40 m g / # L was considered abnormal and indicative of bacterial infection.
424
Clinical and laboratory observations
The Journal of Pediatrics March 1986
T a b l e . Sensitivity, specificity, and predictive value of laboratory tests of CSF in patients with CSF pleocytosis
Gram stain
Glucose* (mg/dl)
Sensitivity~ n 11/16 9/14 % 69 64 SpecificityII n 28/28 26/28 % I00 93 Predictive value of positive test n 11/11 9/11 % 100 82 Predictive value of negative test n 28/33 26/31 % 85 84
Glucose or protein or Gram stain or CRP
Proteini(mg/dl)
CRP
LAL
>20%
>60%
Glucose or protein or Gram stain
10/14 71
14/17 82
6/6 ~ 100
13/14 93
10/14 71
13/14 93
14/14 100
25/28 89
27/28 96
29/29 100
10/28 36
21/28 75
23/28 82
22/28 79
10/13 77
14/15 93
6/6 100
13/31 42
10/17 59
13/18 72
14/20 70
25/29 86
27/30 90
29/29 100
10/11 90
21/31 84
23/24 96
22/22 100
PMNs
*CSF glucose<40 mg/dl consideredpositiveresult. tProtein abovenormalrange for age consideredpositiveresult. w for patientswith meningitiscaused by gram-negativeorganisms. :~Positivetests/tests performed. HNegativetests/tests performed.
Specimens for CRP and LAL assay. Specimens were placed in separate sterile pyrogen-free tubes and immediately refrigerated at 4 ~ C. Specimens were tested within 12 hours or frozen at - 7 0 ~ C for later testing. C-reactive protein. A qualitative latex agglutination assay for CRP (CR-test, Hyland Laboratories, Costa Mesa, Calif.) was used to assay 50 #L aliquots of CSF in individual wells on a glass slide, as described by Corrall et al. z A CSF specimen was considered positive if specific agglutination was noted. Detection of endotoxin for gram-negative bacteria. Bacterial endotoxin was detected with LAL test kits (Pyrogent, Mallincrodt, Inc., St. Louis). An individual test consisted of a tube containing dried LAL capable of detecting 0.03 E U / m l endotoxin. A test for endotoxin was considered positive if a clot formed in the tube after incubation at 37 ~ C for 1 hour after the addition of 250 #L CSF. RESULTS
Patients with bacterial meningitis. Bacterial pathogens were isolated from CSF by standard microbiologic methods in 17 patients. Infecting organisms included Haemophilis influenzae type b (nine patients), Neisseria meningitidis group B (three), group B streptococcus (two), Streptococcus pneumoniae (two), and Listeria monocytogenes (one). CRP was positive in 14 (82%) of 17 patients with bacterial meningitis. Negative results were observed with CSF from a 1-week-old neonate with group B streptococcus and from an ll-month-old infant with H.
influenzae b meningitis. In an 18-month-old child with H. influenzae b meningitis, the initial CRP determination was negative, but the CRP was positive at 1:2, 1:4, and 1:8 dilutions of CSF, indicating that this specimen was negative because of a prozone phenomenon. The other two false-negative specimens, however, were negative on testing at similar dilutions. Sufficient CSF was available to permit LAL assay in 10 cases of bacterial meningitis. LAL was positive in all six in which meningitis was caused by gram-negative bacteria. Four specimens from patients with gram-positive bacteria were negative for endotoxin by LAL assay. Patients with CSF pleocytosis and negative bacterial culture. Forty-six patients had >_ 10 W B C / m m 3 CSF and negative bacterial cultures. Of these 46, four had CSF specimens contaminated by blood as noted by visual inspection. Fourteen additional patients received antibiotics prior to lumbar puncture. CRP and LAL assays were performed on the remaining 28 specimens. No endotoxin was detected by LAL in any specimen (0 of 25) in which there was sufficient CSF for assay. The latex agglutination test for CRP gave negative results in 27 of 28 instances. The false-positive CRP assay occurred in a three-year old boy with meningitis, fever, and maculopapular rash. The patient's blood and, CSF cultures remained negative, and he was discharged from the hospital after 3 days with a diagnosis of aseptic meningitis. Patients without pleocytosis. None of the 204 patients with <10 WBC/mm 3 were found to have bacterial menin-
Volume 108 Number 3
gitis. The CRP assay was negative in 185 of 187 such cases in which an adequate specimen was available for testing. One of the two false-positive CRP assays occurred in an 18-month-old boy with chicken pox and febrile seizures. CSF glucose and protein and leukocyte count were all within normal limits, and the Gram stain was negative. The other false-positive CRP was obtained from a 6year-old boy with pneumonia. Lumbar puncture was performed because of complaint of neck stiffness. Blood and CSF cultures were negative and CSF laboratory values were within normal limits. The LAL assay was negative in 175 of 176 cases in which an adequate sample was available for assay. Sensitivity, specificity, and predictive value of laboratory assays of CSF in evaluation of bacterial and nonbacteriai meningitis (Table). The utility of the CSF leukocyte count in the differentiation of bacterial and nonbacterial meningitis was evaluated in 224 patients. Excluded were those patients with visible blood contamination of CSF (four patients), insufficient CSF for WBC count (eight), or negative cultures but prior antibiotic therapy (14). All patients in whom a pathogen was cultured from CSF also had an elevated CSF WBC count. The sensitivity of CSF pleocytosis in detecting bacterial meningitis was thus 100% in this study. However, 28 patients had elevated CSF WBC count (>_ 10 WBC/mm 3) with negative culture. The predictive value of CSF pleocytosis for bacterial meningitis was thus only 38%. In terms of the utility of the determination of the percentage of polymorphonuclar leukocytes, no value could be found that resulted in good levels of both sensitivity and specificity. For example, although 13 of 14 patients with bacterial meningitis had >20% PMN in CSF specimens (93% sensitivity), 10 of 28 patients with pleocytosis and without bacterial meningitis also had >20% PMNs (36% specificity). On the other hand, if a level of >60% PMNs was used the sensitivity and specificity were 71% and 75%, respectively. For the remainder of the CSF laboratory measurements, sensitivity, specificity, and predictive values were calculated for those patients initially found to have elevated CSF WBC count (Table). None of the routine tests (glucose and protein concentration, Gram stain) had a sensitivity >71%. When the three routine assays were considered as a group, the sensitivity was 93% in 14 patients. The CRP test was more sensitive than either Gram stain, glucose, or protein, but the CRP test also did not achieve 100% sensitivity in detecting bacterial meningitis in patients with CSF pleocytosis. However, when the results of the CRP determinations were combined with those of the three routine assays, all patients with bacterial meningitis were detected by at least one of the assays. In
Clinical and laboratory observations
425
one instance, CRP was positive but Gram stain was negative and glucose and protein were normal in CSF from a 10-month-old girl whose blood and CSF cultures grew H. influenzae b. That patient was initially thought to have an upper respiratory tract infection, but she returned to the clinic with persistent fever, and a lumbar puncture was performed. The patient was subsequently admitted and given treatment after a lumbar puncture revealed a CSF total leukocyte count of 658 W B C / m m 3 and a differential count of 36% PMNs. When CSF Gram stain, glucose, protein, and CRP were considered as a test battery in patients with CSF pleocytosis, the predictive value of negative results was 100%. In addition, the predictive value of positive results of this test battery was 70%, as compared to 38% if CSF pleocytosis alone was used to detect bacterial meningitis. The LAL assay proved to be 100% sensitive (six of six) in predicting the presence of gram-negative bacteria in CSF. Twenty-five patients with CSF pleocytosis and negative culture were also negative for endotoxin by LAL test. Four other patients with positive CSF cultures for gram-positive organisms did not have detectable levels of endotoxin in the CSF. DISCUSSION The LAL assay for gram-negative endotoxin has not proved useful in predicting the presence of gram-negative sepsis 8'9 but has been reported as helpful in detecting meningitis caused by gram-negative bacteria? ~ In our study the utility of the LAL assay was assessed in conjunction with other laboratory tests of CSF. Difficulty was encountered in obtaining enough additional CSF (250 tzL) from neonates and infants to allow performance of the LAL assay. The test for endotoxin was sensitive in those instances in which it was used, but the CRP was also positive in the same cases. The LAL assay, then, did not appear to increase significantly the detection of bacterial meningitis in this study, but it was useful in distinguishing gram-positive from gram-negative meningitis. Using CSF pleocytosis as the sole criterion for beginning antibiotic therapy in the patients in this study, more patients with aseptic meningitis (28) than with bacterial meningitis (17) would be hospitalized and receive treatment. If the physician had decided to treat only when an abnormality was detected by laboratory evaluation of glucose and protein or results of Gram stain, initiation of antibiotic therapy would have been delayed in one of the patients with bacterial meningitis. However, an abnormality in either CSF glucose, protein, Gram stain, or CRP correctly predicted bacterial meningitis in all instances. The specificity of this battery of four tests was 79%, and the predictive value of a positive assay 70% in patients with
4 26
Clinical and laboratory observations
CSF pleocytosis. With use of this battery we might expect to avoid antibiotic therapy in more than half of patients with CSF pleocytosis while maintaining a high degree of sensitivity for the detection of bacterial meningitis. The analysis of CSF CRP by latex agglutiation is rapid and may be easily performed in even the smallest laboratories. However, published reports have disagreed about the sensitivity of CSF CRP in predicting bacterial meningitis. Corrall et al. 2 found that CRP was elevated in the CSF of 24 of 24 patients with culture-proved bacterial meningitis but iia only two of 32 patients with nonbacterial meningitis. WalterspieP later noted that CSF specimens from four of 10 children with H. influenzae meningitis were negative by the latex agglutination test for CRP. The CSF from neonates with bacterial meningitis was tested for CRP by Philip and Baker 4 using a sensitive laser nephelometry technique. Those investigators found elevated CRP levels in only two of 11 patients with cultureproved bacterial meningitis. Benjamin et al? also reported a low sensitivity (66%) when laser nephelometry was used to detect CRP in the CSF of patients with bacterial meningitis. However, Abramson et al.6 found that in 72 of 74 patients with bacterial meningitis CRP was detectable by the latex agglutination assay. In our study, 14 (82%) of 17 patients with bacterial meningitis had detectable CSF CRP. Moreover, in one of these patients results were false-negative because of the prozone phenomenon, which was detected by further dilution of the specimen. Although a number of factors (e.g., specimen handling, age of the patient, underlying disease) might explain the divergent results reported for the sensitivity of the CRP assay, our study and two others z,6 reported the highest sensitivities with use of the same latex agglutination assay (CR-test).
The Journal of Pediatrics March 1986
Our data indicate that the CRP assay can increase the sensitivity of detection of bacterial meningitis without significant loss of specificity. The assay is easily performed, and with appropriate dilutions to avoid the prozone phenomenon it should be seriously considered as an addition to the assays routinely used in the evaluation of suspected bacterial meningitis. REFERENCES 1. ModlinJF. Coxsackie and ecbovirus. In: Mandel GL, Douglas RG Jr, Bennett JE, eds. Principles and practices of infectious diseases, 2nd ed. New York: John Wiley, 1985. 2. Corrall JC, Pepple JM, Moxon ER, Hughes WT. C-reactive protein in spinal fluid of children with meningitis. J PEDIATR 1981;99:365-369. 3. Walterspiel JN. C-reactive protein in cerebrospinal fluid. Pediatr Infect Dis 1983;2:194. 4. Phillip AGS, Baker CJ. Cerebrospinal fluid C-reactive protein in neonatal meningitis. J PEDIATR1983;102:715~717. 5. Benjamin DR, Olpheim KE, Brewer L. ls C-reactive protein useful in the management of children with suspected bacterial meningitis? Am J Clin Pathol 1984;81:779-792. 6. Abramson JS, Hampton KD, Baba S, Wasilauskas BL, Maron MJ. The use of C-reative protein from cerebrospinal fluid for differentiating meningitis from other central nervous system diseases. J Infect Dis 1985;151;854-858. 7. Sharff LD, Platt LH, McCracker GH Jr. Cerebrospinal fluid examination in neonates:comparison of high risk=infantswith and without meningitis. J PEDIATR1976;88:473-477. 8. Stumacher RJ, Kovnat MJ, McCabe WR. Limitations of the usefulness of the Limulus assay for endotoxim N Engl J Med 1973;288:1261-1264. 9. Elin RJ, Robinson RA, Levine AS, Wolff SM. Lack of clinical usefulness of the Limulus test in the diagnosis of endotoxemia. N Engl J Med 1975;293:521-524. 10. Ross S, Rodriguez W, Controni G, Korengold G, Watson S, Khan W. Limulus lysate test for gram-negative bacterial meningitis. JAMA 1975;233:1366-1369.
Childhood acquired immune deficiency syndrome manifesting as acrodermatitis enteropathica Timothy K. Tong, M.D., Lori R. A n d r e w , M.D., A b e y Albert, M.D., a n d John J. Mickell, M.D. From the Department of Pediatrics, Division of Pediatric Critical Care, Children's Medical Center, Medical College of Virginia, Richmond
The diagnosis of acquired immune deficiency syndrome is more difficult in the pediatric population because of the Submitted for publication July 8, 1985; accepted Sept. 17, 1985. Reprint requests: John J. Mickell, M.D., MCV Station Box 530, Richmond, VA 23298.
number of immunologic disorders seen in this age group. Another disease that may resemble childhood AIDS in clinical, biochemical, and immunologic findings is acrodermatitis enteropathica. The symptoms include eczematous lesions, especially in the perioral and perineal areas, alopecia, conjunctivitis, chronic diarrhea, stomatitis, and intercurrent bacterial and candidal infections. The cause of