Counterimmunoelectrophoresis in the Diagnosis of

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1977, p Copyright C 1977 Americn Society for Microbiology Vol. 5, No. 4 Printed in U.S.A. Counterimmunoelectrophoresis in the Dignosis of Bcteril Meningitis HANNE COLDING* AND INGA LIND Neisseri Deprtment nd Deprtment ofdignostic Bcteriology, Sttens Seruminstitut, nd Deprtment of Clinicl Bcteriology, Institute of Medicl Microbiology, University of Copenhgen, 00 Copenhgen, Denmrk* Received for publiction 12 November 1976 The im of the present study ws to investigte whether counterimmunoelectrophoresis (CIE) would fcilitte the rpid, etiologicl dignosis of bcteril meningitis when used in prllel with other routine methods in medicl bcteriologicl lbortory. Of 3,674 consecutive specimens of cerebrospinl fluid (CSF) received t the Deprtment of Dignostic Bcteriology, Sttens Seruminstitut, 283 specimens (ech representing one ptient) were selected for exmintion by CIE on the bsis of the following criteri: bcteri or pleocytosis or both by microscopy or positive culture or both. CLE ws performed with ntiser to Neisseri meningitidis (groups A, B, nd C), Streptococcus pneumonie (omniserum nd pools A to I), nd Hemophilus influenze type b. Antigen ws detected in 57% (72/126) of specimens in which cultures reveled these three kinds of microorgnisms in CSF nd in 12% (17/139) of the culture-negtive specimens. CSF specimens from ptients with bcteril meningitis cused by other species were ll negtive in COE, except four, three of which contined Escherichi coli ntigen recting with ntiserum to N. meningitidis group B nd one E. coli ntigen recting with ntiserum to H. influenze type b. Specific dignosis ws chieved in 60% (170/283) of the specimens studied nd could be estblished within 1 h in 85% (145/170) b,y the combined results of microscopy nd COE. Ten specimens, nine of which showed rection with ntiserum to N. meningitidis group A, were positive by COE only. Counterimmunoelectrophoresis (CIE) ws developed for use in forensic medicine s rpid method requiring only smll mounts of rectnts (6). The technique ws further elborted for the detection of Au ntigen (9, 15) nd hs lso proved useful in the detection of bcteril ntigens in cerebrospinl fluid (CSF) or serum (3, 5, 7, 8, 11). The im of the present study ws to investigte whether CEE would mke ny contribution to the rpid, etiologicl dignosis of bcteril meningitis when used in prllel with routine methods in medicl bcteriologicl lbortory. MATERIALS AND METHODS Specimens. During the period 1 September 1973 to 31 December 1974, totl of 3,674 specimens of CSF were received for bcteriologicl exmintion t the Deprtment of Dignostic Bcteriology, Sttens Seruminstitut. All specimens in which n etiologicl dignosis hd been estblished by routine culturl procedures or which hd shown bcteri or pleocytosis or both by microscopy were stored t -20 C for future exmintion by CGE. For specimens contining Streptococcus pneumonie or Hemophilus influenze type b, the specificity of microscopy findings ws regulrly confirmed by the performnce of cpsulr rections on the CSF specimens. Ech of the 283 CSF specimens studied represented one ptient. Storge for up to 2 yers did not seem to cuse ny deteriortion of the ntigens involved, s judged by repeted exmintion of selected positive specimens. CIE. The equipment employed for immunoelectrophoresis ws from Dnsk Lbortorieudstyr A/S, Copenhgen. In principle, the method used ws tht described by Coonrod nd Rytel (4). Glss slides (10 by 10 cm) were coted with 15 ml of 1% grose (Litex, btch number AGS, 105 AX) dissolved in Veronl buffer (ph 8.2, ionic strength 0.05) contining M ethylenediminetetrcetic cid. The sme buffer ws used in the buffer reservoir of the electrophoresis pprtus. Prllel rows of wells, 4 mm in dimeter, were punched out on the slides t distnce of 3 mm from ech other. Ech well ws filled with 10,ul of the rectnt. The wells contining the ntiser were on the nodic side of the electrophoresis chmber. The slides were ttched to the buffer reservoir by pper wicks (Whtmn no. 17) nd subjected to constnt voltge of 2.5 V/cm for 1 h t room temperture. The slides were exmined for precipittion bnds ginst drk bckground in oblique trnsillumintion nd then stined for protein with Coomssie brillint blue 405

2 406 COLDING AND LIND (20). (Ech experiment included known culture-positive CSF specimens in which the relevnt ntigen hs been demonstrted previously by CIE [positive controls].) Antiser. Rbbit ntiser ginst Neisseri meningitidis groups A nd C were produced s described previously for ntiser to N. gonorrhoee (13). Horse ntiserum ginst N. meningitidis group B ws produced by John B. Robbins, Food nd Drug Administrtion, Bethesd, Md., nd received vi Frits Orskov, Sttens Seruminstitut. Rbbit ntiser ginst H. influenze type b nd S. pneumonie were provided by Ern Lund, Sttens Seruminstitut. The set of pneumococcl ntiser comprised omniserum, which is pool of ll 83 types, pools A to I inclusive, nd relevnt type/group ntiser. The following commercilly vilble ntiser were used in preliminry experiments: rbbit ntiser to N. meningitidis group A (Difco, btch no ), N. meningitidis group B (btch no ), N. meningitidis group C (btch no ), nd H. influenze type b (btch no ). Serologicl grouping of isolted strins of N. meningitidis nd S. pneumonie. Strins ofn. meningitidis were exmined by mens of cogglutintion method (12) dpted for serologicl grouping of meningococci. So fr, this test hs only been estblished for groups A nd C. All other strins were designted nongroupble (NG). Strins of S. pneumonie were exmined by Ern Lund by the Neufeld cpsulr rection method. RESULTS CIE technique. The setup used for exmintion of the totl mteril ws chosen fter series of preliminry experiments in which ll specimens previously found positive by culture were investigted by vrious modifictions of CIE. Ech experiment consisted of four slides, the wells of which were filled ccording to the sme digrm. Electrophoresis ws run for 1 h (two slides) nd 2 h (two slides). One set of slides ws exmined immeditely fter COE nd then stined. The second set of slides ws stored for h t 100C, reexmined, nd then stined. As compred with the results obtined fter 1 h, no further specimens from ptients with N. meningitidis or H. influenze type b meningitis becme positive fter electrophoresis for 2 h nd/or storge of the slides for h. For ptients with S. pneumonie meningitis, the number of CSF specimens in which ntigen could be detected by CIE fter 1 h, with or without subsequent storge of the slides, is shown in Tble 1. With ll the ntiser used, prticulrly the omniserum, the number of positive findings ws higher fter storge for h. The number of specimens tht becme positive incresed further when pools A to I were used in ddition. By extending the electrophoresis by 2 h, two of these specimens lso becme J. CLIN. MICROBIOL. positive with omniserum. For prcticl resons, ll specimens were exmined primrily with omniserum nd electrophoresis for 1 h. Subsequently, ll CIE-positive specimens nd ll specimens from which S. pneumonie hd been isolted by culture were exmined by mens of pools A to I nd relevnt group/type ntiser. The sensitivity of CIE ws slightly enhnced by stining for protein (Tble 2). All precipittes seen before stining were recognized fter stining. Antiser. Antiser ginst N. meningitidis Groups A nd C gve distinct precipittion lines with homologous group-specific polyscchride ntigens (received vi Alice Reyn s gift from Emil Gotschlich, The Rockefeller University, New York). They showed no cross-rections with the other group-specific ntigens, nd in exmintion of culture-positive specimens, they gve single distinct precipittion lines only with those from which strin of the homologous group ws isolted. Of ll ntiser ginst N. meningitidis group B tested, only tht provided by J. B. Robbins gve distinct precipittion line with group B TABLE 1. Antigen detection in specimens of CSF from ptients with S. pneumonie meningitis No. positive with ntiserum ginst S. pneumonie' CIE performed Totl no. Omnise- Pools Group/ for (h) rum type S U S U S U Results of CIE performed for 1 h compred with those obtined by CIE for 1 h nd subsequent storge of the slides for h t 10 C. b S, Stined; U, unstined. TABLE 2. Demonstrtion of bcteril ntigens in 283 specimens of CSF by culture, microscopy, nd CIE No. of specimens positive No. of by Culture result speci- CIE mens mie ex- Micros- Stined Unstined Culture positive N. meningitidis S. pneumonie H. influenze E. coli Other bcteri Culture negtive

3 VOL. CVE 5, 1977 IN DIAGNOSIS OF BACTERIAL MENINGIS 407 polyscchride. This ntiserum precipitted group-specific polyscchrides B nd C nd gve blurred unspecific precipittes in gret number of specimens. Furthermore, three out of five specimens from ptients with E. coli meningitis gve strong, distinct precipittion lines. Therefore, the interprettion of positive result obtined by mens of ntiserum to N. meningitidis group B ws bsed on the microscopy reding vilble t the sme time. With the routine setup, the pneumococcl ntiser gve precipittes only with specimens from ptients with S. pneumonie meningitis. Antiserum to H. influenze type b gve single distinct precipitte, with the mjority of CSF specimens contining the homologous ntigen. One out of five specimens from ptients with E. coli meningitis gve distinct precipitte with this ntiserum. Blurred unspecific precipittes occurred occsionlly fter storge of the slides for h. The commercil ntiser tested gve either fewer positive results thn those found by mens of the ntiser described bove or none t ll. Therefore, these ntiser were not used in this study. Comprison of the results obtined by culture, microscopy, nd CIE. Used s the only method, culture (Tble 2) reveled the pthogen in the highest proportion of cses of meningitis in which the dignosis could be estblished, viz., 85% (144/170). Fifty-seven percent (72/126) of the culture-positive cses of meningitis due to N. meningitidis, S. pneumonie, nd H. influenze type b contined demonstrble group- or type-specific ntigens. The frequency of CIE-positive results within these three groups differed significntly: N. meningitidis, 35/64; S. pneumonie, 14/32; H. influenze type b, 23/30 (X2 = 7.17, F = 2, P < 0.05). Microscopy offered dignosis in more cses (104/126) thn did CIE (72/126). Put together, these two methods yielded correct dignosis within 1 h in 85% (145/170) of the cses or 51% (145/283) of the specimens exmined. The results obtined for the culture-positive CSF specimens re shown in detil in Tble 3. Four specimens negtive by microscopy were positive by CIE. For ll CIE-negtive specimens tht hd shown lrge numbers of bcteri by microscopy, CIE ws repeted with twofold dilutions of CSF. The negtive results were confirmed, nd thus they could not be due to excess of ntigen. Four out of five specimens from which E. coli ws isolted showed positive CIE rections. Three gve strong, distinct precipittes with ntiserum to N. meningitidis group B, nd one gve precipitte with ntiserum to H. influenze type b. These results re referred to in Discussion. Specimens from 13 ptients with infections due to other bcteril species were negtive by CIE (Streptococcus spp. [81, Stphylococcus ureus [2], Listeri monocytogenes [1], Klebsiell oxytoc, [1], nd Acinetobcter clcoceticus [1]). The results obtined for specimens positive by CIE nd/or microscopy nd negtive by culture re shown in Tble 4. Fourteen of seventeen CIE-positive specimens in this group gve positive rection with ntiserum to N. meningitidis. Another two specimens gve precipittes with both pneumococcl omniserum, pool A, nd ntiserum ginst group. One specimen gve positive rection with ntiserum ginst H. influenze type b. A totl of 10 specimens negtive by both microscopy nd culture were positive by CIE. Detection of group/type-specific ntigens by CIE. For culture-positive specimens, complete greement ws found between the serologicl grouping of the isolted strins nd the CIE grouping. The mjority of cses of meningococcl meningitis were cused by N. meningitidis group A. Five of nineteen specimens from which NG strins were isolted were TABLE 3. Comprison of the results obtined by culture, microscopy, nd CIE in the dignosis of bcteril meningitis: culture-positive CSF specimens No. culture + Etiology Totl Microscopy + Microscopy - no. CIE + CIE - CIE + CIE - N. meningi tidis S. pneumo nie H. influ enze E. coli Other bcte ri +, Positive; -, negtive. TABLE 4. Comprison of the results obtined by culture, microscopy, nd CIE in the dignosis of bcteril meningitis: Culture-negtive CSF specimens No. culture - Etiology Totl no. Microscopy + Microscopy - CIE+ CIE - CIE + N. meningitidis S. pneumonie H. influenze Other bcteri , Negtive; +, positive.

4 408 COLDING AND LIND found to contin group B ntigen (Tble 5). The correltion between the CIE dt for CSF specimens nd the results of serotyping of isolted strins of S. pneumonie is shown in Tble 6. Strins of type 7F were isolted from five, nd type 14 strin ws isolted from one, of the CIEnegtive specimens. Since it hs been reported tht 7F nd 14 re positively chrged when the ph vries between 5 nd 8 (11), CIE ws repeted with CSF pplied to the nodic well. The results remined negtive. DISCUSSION The mjority of investigtors compring different methods for the detection of the specific pthogen in bcteril meningitis hve found bcteriologicl culture procedures to be the most sensitive (3, 7, 19, ). CEE is rpid method nd is lso sensitive method, in tht it is possible to detect the presence of 0.02 to 0.5 gg of cpsulr polyscchride ntigens per ml (4, 7, 8). Another dvntge is tht results re independent of the presence of living microorgnisms, which mens tht CIE cn be used for specimens contining formlin or obtined TABLE 5. Detection ofgroup-specific N. meningitidis ntigen in CSF by CIE No. of No. CIE positive with ntiserum ginst N. men- Culture result speci- ingitidis mens group group group A B C Culture-positive group A NG C Culture negtive TABLE 6. Correltion between CIE dt obtined fter 1 h nd serotypes of S. pneumonie strins isolted by culture from CSF of 32 ptients Antise- No. of Serotypes of S. pneumonie specimens CIE +b CIE -_ A 6 4, F, C(3) 4 B 11 6A(2), 6B, 8(3), 19A, 6B, 19A 19F(2) C 6 7F(5), 31 D 0 E 2 1OA(2) F 1 41A G 1 29G H 4 23F(3) 14 I 1 38 rum pool One strin of ech type ws found unless indicted by figure in prentheses. b +, Positive; -, negtive. J. CLIN. MICROBIOL. from ptients lredy under tretment with ntibiotics, nd for specimens tht hve tken long time to rrive by mil. In this study, the results obtined by CGE for selected specimens of CSF were compred with those obtined by direct microscopy nd culture. The pthogen ws detected in 85% of confirmed cses of bcteril meningitis by mens of bcteriologicl culture procedures, in 77% by direct microscopy, nd in 55% by CGE. By using the combintion microscopy nd CGE, etiologicl dignosis ws chieved within 1 h in the sme number of cses s ws obtined by culture lone (85%). CIE ws positive in 57% (72/ 126) of the culture-positive cses. CGE offered dignosis in 12% (17/139) of cses with negtive culture. Nine out of ten specimens lso found negtive by direct microscopy showed rection with ntiserum to N. meningitidis group A (Tble 4). This mens tht, used s the only method, CGE gve the specific dignosis in the sme number of cses of group A meningococcl meningitis s culture nd tht n increse of bout 30% in the number of confirmed cses could be obtined by using both methods. Antiserum to N. meningitidis group B gve strong precipittion rections with three out of five specimens from ptients with E. coli meningitis. The group B ntigen is known to be identicl with E. coli cpsulr polyscchride Kl (10), which frequently occurs in strins cusing neontl meningitis (16). In ll three cses, direct microscopy reveled grm-negtive rods, so the mistke of inititing tretment pproprite for meningococcl meningitis could be voided. Serologicl cross-rectivity between H. influenze nd E. coli s shown with one specimen hs been demonstrted previously by Ouchterlony nlysis of bcteril extrcts (). Serologicl cross-rectivity between enteric bcteri nd N. meningitidis groups A nd C nd S. pneumonie types I nd III hs lso been described (17), but cross-rective ntigens hve not been detected in ny of the CSF specimens studied. There ws complete greement in this study between serologicl grouping of the isolted strins of N. meningitidis nd S. pneumonie nd tht obtined by CIE performed on the corresponding CSF specimens. In cses of meningococcl meningitis, this informtion is obtined t n erlier stge thn usul, nd group B ntigen ws detected in 5 of 19 specimens from which NG strins hd been isolted. CSF specimens from ptients with infections due to S. pneumonie types 7F nd 14, which occur quite frequently in Denmrk (14), were ll negtive by CGE. Since the present investigtion ws completed, buffer which permits

5 VOL. CV1E 5, 1977 IN DIAGNOSIS OF BACTERIAL MENINGmS 409 the detection of type 7F ntigen by mens of CIE hs been described (1). In conclusion, CEE yielded n increse of 6% (10/170) in the number of confirmed cses of bcteril meningitis when used in prllel with routine methods in well-estblished bcteriologicl lbortory. The verge trnsport time for the specimens exmined ws less thn 24 h. Under conditions less optiml for the culturl procedures, CIE cn be n even more useful supplement, especilly with respect to the dignosis of group A meningococcl meningitis. Furthermore, the dignostic vlue of CEE cn be extended by exmintion of both CSF nd serum for the presence of bcteril ntigens (2). However, the vlue of CIE depends entirely on the qulity ofthe ntiser employed nd, due to serologicl cross-rectivity between species cusing meningitis, e.g., between N. meningitidis group B ntigen nd E. coli cpsulr polyscchride Kl, the interprettion of some positive findings requires tht the result of direct microsocopy be vilble t the sme time. LITERATURE CITED 1. Anhlt, J. P., nd P. K. W. Yu Counterimmunoelectrophoresis of pneumococcl ntigens: improved sensitivity for the detection of types VII nd XIV. J. Clin. Microbiol. 2: Anonymous Dignosis nd prognosis in pyogenic meningitis. Lncet 1: Coonrod, J. D., nd M. W. Rytel Determintion of etiology of bcteril meningitis by counterimmunoelectrophoresis. Lncet 1: Coonrod, J. D., nd M. W. Rytel Detection of type-specific pneumococcl nxtigens by counterimmunoelectrophoresis. I. Methodology nd immunologic properties of pneumococcl ntigens. J. Lb. Clin. Med. 81: Coonrod, J. D., nd M. W. Rytel Detection of type-specific pneumococcl ntigens by counterimmunoelectrophoresis. II. Aetiologic dignosis ofpneumococcl pneumonie. J. Lb. Clin. Med. 81: Culliford, B. J Precipitin rections in forensic problems. Nture (London) 201: Edwrds, E. A Immunologic investigtions of meningococcl disese. I. Group-specific Neisseri meningitidis ntigens present in the serum of ptients with fulminnt meningococcemi. J. Immunol. 106: Edwrds, E. A., P. M. Muehl, nd R. 0. Peckinpugh Dignosis of bcteril meningitis by counterimmunoelectrophoresis. J. Lb. Clin. Med. 80: Gocke, D. J., nd C. Howe Rpid detection of ustrli ntigen by counterimmunoelectrophoresis. J. Immunol. 104: Ksper, D. L., J. L. Winkelhke, W. D. Zollinger, B. L. Brndt, nd M. S. Artenstein Immunochemicl similrity between polyscchride ntigens ofescherichi coli 07:K1(L):NM nd group B Neisseri meningitidis. J. Immunol. 110: Kenny, G. E., B. B. Wentworth, R. P. Besley, nd H. M. Foy Correltion of circulting cpsulr polyscchride with bcteremi in pneumococcl pneumoni. Infect. Immun. 6: Kronvll, G A rpid slide-gglutintion method for typing pneumococci by mens of specific ntibody dsorbed to protein A-contining stphylococci. J. Med. Microbiol. 6: Lind, I Identifiction ofneisseri gonorrhoee by mens of fluorescent ntibody technique. Act Pthol. Microbiol. Scnd. 70: Lund, E Types of pneumococci found in blood, spinl fluid nd pleurl exudte during period of 15 yers ( ). Act Pthol. Microbiol. Scnd. 78: Prince, A. M., nd K. Burke Serum heptitis ntigen (SH): rpid detection by high voltge immunoelectroosmophoresis. Science 169: Robbins, J. B., G. H. McCrcken, E. C. Gotschlich, F. Orskov, I. 0rskov, nd L. A. Hnson Escherichi coli Kl cpsulr polyscchride ssocited with neontl meningitis. N. Engl. J. Med. 290: Robbins, J. B., R. L. Myerowitz, J. K. Whisnnt, M. Argmn, R. Schneerson, Z. T. Hndzel, nd E. C. Gotschlich Enteric bcteri cross-rective with Neisseri meningitidis groups A nd C nd Diplococcus pneumonie types I nd mi. Infect. Immun. 6: Schneerson, R., M. Brdshw, J. K. Whisnnt, R. L. Myerowitz, J. C. Prke, Jr., nd J. B. Robbins An Escherichi coli ntigen cross-rective with the cpsulr polyscchride of Hemophilus influenze type b: occurrence mong known serotypes, nd immunochemicl nd biologic properties ofe. coli ntiser towrd H. influenze type b. J. Immunol. 1 '8: Tobin, B. M., nd D. M. Jones Immunoelectroosmophoresis in the dignosis of meningococcl infections. J. Clin. Pthol. 25: Weeke, B Generl remrks on principles, equipment nd procedure, p In A mnul of quntittive imnmunoelectrophoresis. Universitetsforlget, Oslo.. Whittle, H. D., B. M. Greenwood, N. McD. Dvidson, A. Tomkins, P. Tugwell, D. A. Wrrell, A. Zlin, A. D. M. Bryceson, E. H. 0. Prry, M. Brueton, M. Duggn, J. M. V. Oomen, nd A. D. Rjkovic Meningococcl ntigen in dignosis nd tretment of group A meningococcl infections. Am. J. Med. 58: