Rose Bengal and counterimmunoelectrophoresis, two tests that detect antibodies against different structural antigens, when carried out on spinal fluid permitted rapid diagnosis of human Brucella meningitis. The Rose Bengal test was positive in five out of five patients studied, and counterimmunoelectrophoresis was positive in all but one. The Brucella meningitis was characterized by an increase of immunoglobulin G in the cerebrospinal fluid

Delgado, G

Díaz, R

Maraví-Poma, E

Rivero, A

PubMed

Diaz, R. (Ramón)

Maravi-Poma, E. (Enrique)

Delgado, G. (Gabriel)

Rivero, A. (Antonio)

Dadun, University of Navarra

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1978, p. 236-2370095-1 137/78/0007-0236$02.00/0Copyright © 1978 American Society for MicrobiologyVol. 7, No. 2Printed in U.S.A.Rose Bengal Plate Agglutination andCounterimmunoelectrophoresis Tests on Spinal Fluid in theDiagnosis of Brucella MeningitisRAMON DIAZ,'* ENRIQUE MARAVI-POMA,2 GABRIEL DELGADO,2 AND ANTONIO RIVERO2Departments ofMicrobiology and Neurology, Clinica Universitaria, University of Navarra, Pamplona,Spain,' and Department ofInternal Medicine, Residencia Sanitaria "Virgen del Camino,"Pamplona, Spain2Received for publication 27 June 1977Rose Bengal and counterimmunoelectrophoresis, two tests that detect antibod-ies against different structural antigens, when carried out on spinal fluid permittedrapid diagnosis of human Brucella meningitis. The Rose Bengal test was positivein five out of five patients studied, and counterimmunoelectrophoresis was posi-tive in all but one. The Brucella meningitis was characterized by an increase ofimmunoglobulin G in the cerebrospinal fluid.Since the diagnostic utility of the Rose Ben-gal (RB) and counterimmunoelectrophoresis(CIEP) tests in human brucellosis (5) is now wellestablished, we were interested in whether thesetests could be used to detect Brucella antibodiesin cerebrospinal fluid (CSF) of patients withBrucella meningitis, to establish a rapid diag-nosis of this complication. Several authors (2,8-10) have reported that there is an increase ofimmunoglobulin G (IgG) in CSF of patients withpurulent and tuberculous meningitis. To ourknowledge no report has been made on thebehavior of IgG in CSF of patients with Brucellameningitis. In this study, we have also investi-gated the concentration of IgG in CSF from suchpatients.The CSF studied was obtained from five pa-tients with brucellosis, confirmed by seroagglu-tination, Coombs, complement fixation (CF-B),RB, and CIEP tests. All these patients had, inaddition, clinical evidence of meningitis. ControlCSF was selected from seven patients with bru-cellosis, confirmed by blood cultures and byserology but not showing any symptoms of men-ingitis, and was used both in the serological testand for the determination of the concentrationof IgG.Brucella abortus 99 antigen was prepared andemployed in seroagglutination Coombs, and CF-B tests according to Alton et al. (1). The RB testwas carried out as described by Morgan et al.(7). The antigen for this test was obtained fromthe Central Veterinary Laboratory, Weybridge,England. The soluble antigens employed in im-munodiffusion and CIEP were lipopolysaccha-ride (LPS) extracted from B. melitensis 16Mand protein antigen (P) extracted from B. meli-tensis 115; methods of extraction, gel diffusion,and CIEP have been described previously (5).Complement fixation test with P antigen (CF-P)was also carried out, using 5 jig of P antigen ineach antigen-serum mixture.IgG concentration was determined by the ra-dial immunodiffusion method of Mancini (6),using plates of ultra-low concentration obtainedfrom ICL Scientific, Foundation Valley, Calif.The range of IgG concentration that could bedetected varied between 3 and 15 mg/100 ml.The CSF samples of patients with symptoms ofmeningitis were diluted to produce results withinthe range of the reference serum, and the resultswere multiplied by the dilution factor. For anti-Brucella antibody adsorption, heat-killed cellsuspensions of B. melitensis 16M and B. oviswere centrifuged, and packed volumes of 0.1 mlwere mixed with 0.5 ml of CSF and placed at37°C for 2 h. The mixture was centrifuged, andthe supernatant CSF was removed and tested.In the same way, the CSF was adsorbed bymixing 0.5 ml with 2.5 mg of lyophilized P anti-gen.Table 1 summarizes the results obtained bythe serological methods and the concentrationof IgG in CSF. The five samples from patientswith meningitis symptoms were positive in theRB test, and all but one developed precipitationlines in less than 1 h when examined by CIEPusing P antigen. The CSF that was positive inCIEP was also positive in the CF-P test. Noneof the control CSF samples from patients withbrucellosis without meningitis symptoms werepositive in any of the serological tests employed.The results of adsorption show that B. meli-tensis 16M cells, but not P antigen or the rough236TABLE 1. Results of serological and bacteriological tests and concentration ofIgG in CSF from patientswith clinical symptoms of Brucella meningitisReciprocal of titers CIEP' IgG concen-Patient no. (no. of Culture trationRB SATh Coombs CF-B' CF-P' lines) (mg/100B~~~~~~~~~~~~~~~~~~~~~~~ml)1 2 20 1.280 128 16 2 B. melitensis 27.2biotype 22 1 10 320 32 16 2 NDe 23.23 4 40 160 32 <2 0 Negative' 13.24 4 40 320 ND" ND" 2 NDe ND"5 8 80 2.560 256 128 5 Negativef 50a CIEP with P antigen.bSAT, Seroagglutination test.CF test with whole cell smooth B. abortus 99.d CF test with P antigen.eND, Not done.f Patients were under antibiotic treatment when the sample for the culture was obtained." Not done because the sample was lost.culture of B. ovis, were capable of removingantibodies active in the RB, seroagglutination,Coombs, and CF-B tests. On the other hand,antibodies active in CIEP and CF-P were onlyremoved by P antigen. Our previous work (3, 4)demonstrated that B. ovis does not remove an-tibodies to LPS specific for the smooth phase ofthe genus Brucella and also that LPS is theantigenic component that plays the most impor-tant role in the RB, seroagglutination, andCoombs tests. Those results and our presentfindings show that in the CSF of patients withBrucella meningitis there are antibodies againsttwo different structural antigenic components,which are detectable by RB and CIEP. On theother hand, the CF test can be used to detectantibodies against LPS and P antigens.To confirm the existence of antibodies againstthe LPS antigen in CSF, the CSF of patients no.1, 2, and 5 was concentrated five times by lyoph-ilization. Double immunodiffusion using theLPS antigen against the concentrated CSF gaverise to the characteristic precipitin line of theLPS antigen (4).The results of the test to determine the IgGconcentrations in the CSF showed that in pa-tients with brucellosis, but without symptoms ofmeningitis, the range of IgG values was less orequal to 3 mg/100 ml. However, in patients withBrucella meningitis the levels of IgG were sig-nificantly higher, these values varying between13.2 and 50 mg/100 ml. These results indicatedthat the determination of IgG concentration inCSF could be of value in the diagnosis of Bru-cella meningitis in patients suspected of havingbrucellosis.LITERATURE CITED1. Alton, G. G., L. M. Jones, and D. E. Pietz. 1975.Laboratory techniques in brucellosis. World Health Or-ganization, Geneva.2. Buchanan, N., and G. Macnab. 1972. Cerebrospinalfluid complement and immunoglobulins in meningitisand encephalitis. S. Afr. Med. J. 46:1376-1382.3. Diaz, R., L. M. Jones, D. Leong, and J. B. Wilson.1968. Surface antigens of smooth brucellae. J. Bacteriol.96:893-901.4. Diaz, R., and D. Levieux. 1972. R6le respectif en serol-ogie de la brucellosis bovine des antigenes et des im-munoglobulines G, et G2 dans les tests d'agglutination,de Coombs et au Rose Bengale ainsi que dans le phen-omene de zone. C. R. Acad. Sci. 274:1593-1596.5. Diaz, R., E. Maravi-Poma, and A. Rivero. 1976. Com-parison of counterimmunoelectrophoresis with otherserological tests in the diagnosis of human brucellosis.Bull. W.H.O. 53:417-424.6. Mancini, G., 0. Carbonara, and J. F. Heremans. 1965.Immunochemical quantitation of antigens by simpleradial diffusion. Immunochemistry 2:235-254.7. Morgan, W. J. B., D. J. MacKinnon, J. R. Lawson,and G. A. Cullen. 1969. The Rose Bengal plate agglu-tination test in the diagnosis of brucellosis. Vet. Rec.85:636-641.8. Noya, M., J. Castillo, A. Alvarez-Prechous, and M.Lema. 1976. Modificacion de los niveles de IgG delliquido cefalorraquideo durante el curso de las menin-gitis. Rev. Clin. Esp. 140:217-221.9. O'Toole, R. D., M. D. Seattle, G. F. Thornton, M. K.Mukherjee, and K. N. Neogy. 1971. Cerebrospinalfluid immunoglobulins in bacterial meningitis. A possi-ble role for antibody in pneumococcal meningitis. Arch.Neurol. 25:218-224.10. Smith, H., B. Bannister, and M. J. O'Shea. 1973.Cerebrospinal-fluid immunoglobulins in meningitis.Lancet ii:591-593.NOTES 23 7Voi,. 7,

Rose Bengal plate agglutination and counterimmunoelectrophoresis tests on spinal fluid in the diagnosis of Brucella meningitis

Universidad de Navarra

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Rose Bengal plate agglutination and counterimmunoelectrophoresis tests on spinal fluid in the diagnosis of Brucella meningitis.

Rose Bengal plate agglutination and counterimmunoelectrophoresis tests on spinal fluid in the diagnosis of Brucella meningitis.

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