Detection of Coxiella burnetii Antibody

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, June 1987, p /87/ $02.00/0 Copyright 1987, Americn Society for Microbiology Vol. 25, No. 6 Comprison of Enzyme-Linked Immunosorbent Assy nd Complement Fixtion nd Indirect Fluorescent-Antibody Tests for Detection of Coxiell burnetii Antibody O. PÉTER,»* G. DUPUIS,' M. G. PEACOCK,2 AND W. BURGDORFER2 Division of Clinicl Microbiology nd Infectious Diseses, Vlis Centrl Institute, CH-1951 Sion 3, Switzerlnd,' nd Lbortory of Pthobiology, Rocky Mountin Lbortories, Ntionl Institute of Allergy nd Infectious Diseses, Hmilton, Montn Received 8 December 1986/Accepted 4 Mrch 1987 An enzyme-linked immunosorbent ssy () ws developed to detect immunoglobulin G to Coxiell burnetii phse II. Serum smples from 213 ptients who hd hd Q fever 1 yer previously nd from 301 blood donors from six loclities in Switzerlnd were tested by nd by indirect fluorescent-ntibody (IFA) nd complement fixtion (CF) tests. The nd the IFA nd CF tests detected ntibody to C. burnetii in 202 (94.8%), 193 (96%), nd 166 (77.8%) of the 213 Q fever ptients, respectively. With the serum smples from blood donors, the yielded higher percentge of positive ser thn did the IFA nd CF tests. The high specificity of the three tests ws confirmed by nlyzing pired serum smples from 36 ptients suffering from cute pneumoni of virl or bcteril origin. In these cses, the serologicl results were negtive by the three tests, except for three Q-fever cses included s positive control. Q fever ws described for the first time in 1937 in Austrli (7). Since then, the worldwide distribution of the disese hs been recognized. Coxiell burnetii is n obligte intrcellulr rickettsil orgnism, which cuses Q fever in nimls nd humns. This bcterium ws nmed fter F. M. Burnet, who discovered the microorgnism in the spleens of experimentlly infected mice (7), nd fter H. R. Cox, who succeeded in culturing it in embryonted eggs (5). Humn infections result from contct with infected sheep, gots, or cttle or from infected plcents. Although infections in nimls re usully inpprent, prt from cusing more frequent bortions, the most common clinicl presenttion in humns is n influenzlike disese, often ccompnied by pneumoni (2). However, chronic disese, prticulrly endocrditis, my pper yers fter the primry episode (13, 22). The dignosis of both primry nd chronic Q fever by serology is desirble, since culturing the orgnism is hzrdous nd requires specilly equipped lbortories. Unique to C. burnetii is its ntigenic-phse vrition. The virulent phse I is isolted during nturl or lbortory infections of humns or nimls, wheres the virulent phse II develops during seril pssge in immunologiclly incompetent hosts, such s fertilized eggs or cell cultures. This phse trnsition seems to relte to some of the biologicl chrcteristics of the smooth-rough lipopolyscchride vrition (1). Serologiclly, nti-phse I ntibodies re present t high titers only during the chronic form of the illness, wheres nti-phse II ntibodies re lrgely predominnt in primry cute Q fever (18). In 1948 Gsell (12) nd in 1952 nd 1956 Wiesmnn et l. (23, 24) investigted few outbreks of Q fever in Switzerlnd. However, prt from those reports nd the one recently published by Bruppcher et l. (3), the incidence of Q fever in Switzerlnd ws not known. In the pst, the complement fixtion (CF) test ws used to detect ntibody to C. burnetii. Recently, we hve shown tht incidence of Q * Corresponding uthor fever my be higher thn estimted if the indirect fluorescent-ntibody (IFA) test is used rther thn the CF test (9). Since 1983, few ppers hve delt with immunoenzymtic tests for the dignosis of Q fever (8, 11, 21). In this pper, we describe the enzyme-linked immunosorbent ssy () nd compre it with the CF nd IFA tests to detect humn immunoglobulin G (IgG) to C. burnetii phse II. MATERIALS AND METHODS Serum smples. We received 301 blood smples collected in Switzerlnd by the Sion, Mrtigny, Genev, nd Bern blood trnsfusion centers of the Swiss Red Cross. As controls, 36 pired blood smples were collected in Sion from hospitlized ptients suffering from cute pneumoni of virl or bcteril origins (influenz virus, prinfluenz virus, picornvirus, respirtory syncytil virus, Epstein-Brr virus, Chlmydi psittci, nd Mycoplsm pneumonie); lso included were smples tken from three Q fever cses s positive control. Dignostic criteri for these infections were seroconversion or twofold or higher increse in the titer of CF or IFA tests, or both. Furthermore, we tested 213 blood smples collected in Switzerlnd from ptients who hd hd Q fever during lrge outbrek 1 yer previously (10). All these cses were dignosed by the CF test with the criteri described bove nd by the detection of specific IgM in the IFA test (19).. The smples were prepred in microtiter pltes. Optiml concentrtions of ntigen, serum, nd enzyme conjugte were determined by checkerbord titrtion. Purified C. burnetii phse Il (strin Nine Mile) (Commonwelth Serum Lbortories, Melbourne, Austrli) ws sonicted for 30 min. Ech well of flt-bottom polystyrene microtiter pltes ws coted with 100,ul of C. burnetii suspension diluted 1:200 in 1 M sodium crbonte buffer (ph 9.6). After incubtion for 3 h t 37 C, the pltes were kept t 4 C until needed. The pltes were wshed twice by running 400 ml of sline-tween (9% NCI, 05% Tween 20) in the wshing device (Virion Interntionl, Chm, Downloded from on December 15, 2018 by guest

2 1064 PÉTER ET AL. TABLE 1. CF, IFA, nd IgG to C. burnetii phse Il in blood donors No. of No. (%) of smples positive by: Loclity (cnton)' smples tested IFA CF Genev (GE)b (23.7) 9 (15.3) 8 (13.6) Romnshorn (TG) 30 9 (30) 5 (16.7) 2C ZollbrIck (BE) 30 5 (16.7) 4 (13.3) 4C Sembrncher (VS) (35.7) 14 (33.3) 12 (28.6) Leytron (VS) (32.9) 22 (31.4) 16 (22.2) Sion (VS)b (28.6) 14 (20) 11 (15.7) Totl 86 (28.6) 68 (22.6) 53 (17.6) GE, Genev; TG, Thurgu; b BE, Bern; VS, Vlis. Surrounding towns re included. C A totl of 30% of the ser were nticomplementry. Switzerlnd). The serum dilutions were prepred in phosphte-buffered sline-tween buffer (phosphte-buffered sline [ph 7.2], 01 M MgCl2, 1% Tween 20) (negtive control, 1:50 to 1:1,600; positive control, 1:100 to 1:12,800; test serum, 1:200), nd l ws dded to ech well. The serum smples to be screened were tested in duplicte, nd two wells were left free of serum, one serving s control for the conjugte nd the other serving s control for the substrte. The first incubtion ws done t 37 C for 1 h. The trys were wshed once with 400 ml of sline-tween nd 100,ul of lkline phosphtse-conjugted swine ntiserum to humn IgG, -y-chin specific (Orion, Finlnd), diluted 1:250 in phosphte-buffered sline-tween buffer ws then distributed to ll the wells except the substrte control. The plte ws incubted for 3 h t room temperture. After the plte ws wshed, 100,ul of substrte (p-nitrophenyl phosphte) diluted in diethnolmine buffer (6 M [ph 9.6]) ws dded to ll the wells, nd the plte ws kept in the drk t room temperture for 1 h. The rections were stopped by ddition of 25,ul of 3 N NOH per well, nd the opticl density (OD) ws red with the Virion reder; the OD of the substrte control well ws subtrcted. The fifth dilution (1:3,200) of the positive control ws clibrted by computer to n OD of 35 for ech plte. This vlue served s the cutoff. In this wy, we were ble to compre results mong pltes, even if the intensity of colored rections differed between runs. IFA nd CF tests. The IFA test ws performed by the technique described by Philip et l. (20) nd dpted to the C. burnetii ntigen (phse II, strin Nine Mile; Rocky Mountin Lbortories, Hmilton, Mont.) s described elsewhere (19). The serum smples were screened t dilution of 1:2 Positive serum smples were then diluted in twofold steps to 1:64 The serum smples were lso tested by the stndrd CF micrornethod (17) with n ntigen phse II, strin Nine Mile (Virion Interntionl), s described previously (19). Screening with this test ws done t 1:10 dilution. Positive serum smples were titrted twofold to 1:320 dilution. RESULTS Reproducibility of. The OD of the positive control verged 63 ± 07 (stndrd devition) t dilution of 1:20 In the sme wy, the OD of the negtive control verged t dilution of 1:20 Sensitivity. Tble 1 summrizes the percentges of positive serum smples from the 301 blood donors from different towns in Switzerlnd. They vry from 16.7 to 35.7%, from 13.3 to 33.3%, nd from 6.7 to 28.6% s detected by, IFA, nd CF, respectively. The reveled 86 positive smples of 301 tested (28.6%), wheres IFA detected 68 (22.6%) nd CF detected 53 (17.6%). The results of nd CF re not sttisticlly different (X2 = 3.2; 1 df) by the x2 method. It should be pointed out tht 10 nd 11 serum smples from Romnshorn nd Zollbruck, respectively, were nticomplementry in the CF test. The nd IFA (Fig. 1), s well s the nd CF (Fig. 2), were compred for 140 serum smples from Leytron nd Sion together (Tble 1). The reveled 43 positive smples, of which 9 were negtive by IFA (Fig. 1). Two smples found positive by IFA were negtive by. Of the sme 43 -positive smples, 19 were negtive by CF, nd 3 CF-positive smples were negtive by (Fig. 2). Of the 213 ptients hving hd Q fever 1 yer previously (Fig. 3 nd 4), 202 (94.8%) still hd ntibody to C. burnetii detectble by, 193 (96%) hd ntibody detectble by IFA, nd 166 (77.8%) hd ntibody detectble by CF. All the CF-positive serum smples were detected by, wheres three IFA-positive smples were negtive by. For this group of ptients, the x2 method shows tht the results of nd CF (X2 = 5.12; 1 df), but not those of nd IFA (x2 = 1.68; 1 df), re sttisticlly different. The results of IFA nd CF (X2 = 3.63; 1 df) were not significntly different. Of the 514 tested serum smples, 302 were found positive by t lest one test (Tble 2). Only one serum smple which ws positive by the IFA nd CF tests ws negtive by. In the sme wy, 13 smples tht were negtive by were positive by IFA or CF. Thus the detected 288 of totl of 302 positive serum smples (95.4%). For comprison, IFA nd CF reveled 260 (86.1%) nd 219 (72.5%) positive smples. The number positive by OD (405 mll) < 8 7, à I0 I20 4l >80 IFA FIG. 1. Comprison of OD nd IFA titers of ntibody to C. burnetii phse II in 140 serum smples from blood donors in Sion nd Leytron. Verticl nd horizontl brs indicte the threshold titer nd threshold OD, respectively. sil J. CLIN. MICROBIOL. Downloded from on December 15, 2018 by guest

3 VOL. 25, 1987 the three tests corresponded to 69.2% of ll the positive serum smples (209 of 302), nd 84.8% (256 of 302) of the smples positive by were confirmed by IFA or CF. Furthermore, there ws good correltion between incresing OD vlues nd incresing IFA titers. The correltion ws not s good between the OD vlues nd CF titers, bove ll for smples with high CF ntibody titers (Fig. 4). Specificity. The pired serum smples from the three ptients suffering from Q fever, which were included s control, showed seroconversion, the OD vlues vrying from 07 to 60 nd from 16 to 1.06 for the first two cses nd showing high increse for the third (41 to 92). On the other hnd, no significnt OD vritions were observed between the pired serum smples from ptients with cute pneumoni of other origin. However, the OD vlue of two pired serum smples vried from 33 to 38 (negtive to positive) nd the other vried from 36 to 28 (positive to negtive); however, ll four smples showed OD vlues close to the cutoff vlue of (stndrd devition). Similrly, we did not observe ny significnt vrition of the titers with the IFA nd CF tests, except for the three cute Q fever cses. DISCUSSION Our findings suggest tht the sensitivity of the is superior to those of the IFA nd CF tests. Indeed, the detected more positive serum smples from ptients who hd hd Q fever 1 yer previously (10) thn did the IFA or CF test. At 1 yer fter the cute episode, no ntibody ws detected by CF in 22.2% of the ptients, by IFA in 9.4% of the ptients, nd by in only 5.2% of the ptients. Sttisticl nlysis of the results by the x2 method showed tht the difference, between the nd the CF test only, OD (405 nm) 1.2. i 1. 1.c 9 7 fi 11 Il t >8O CF FIG. 2. Comprison of OD nd CF titers of ntibody to C. burnetii phse Il in 138 serum smples from blood donors in Sion nd Leytron. Verticl nd horizontl brs indicte the threshold titer nd threshold OD, respectively. FOR COXIELLA BURNETIl ANTIBODY 1065 OD (405 nid) 1.2. I s I * 1- f 1 * * :t s e. r.0 : Ir i L. ; >640 IFA FIG. 3. Comprison of OD nd IFA titers of ntibody to C. burnetii phse Il in 213 serum smples from ptients who hd hd Q fever 1 yer previously. Verticl nd horizontl brs indicte the threshold titer nd threshold OD, respectively. OD (405.nm) r'3. 1i8 qe1 Il L F S. i Se L 1r t t. 1 t : >80 CF TI TERS FIG. 4. Comprison of OD nd CF titers of ntibody to C. burnetii phse Il in 213 serum smples from ptients who hd hd Q fever 1 yer previously. Verticl nd horizontl brs indicte the threshold titer nd threshold OD, respectively. s 3* Downloded from on December 15, 2018 by guest

4 1066 PÉTER ET AL. J. CLIN. MICROBIOL. TABLE 2. Comprison of with IFA nd CF for detection of IgG to C. burnetii phse Il No. of positive smples by No. of positive smples by No. of No. of positive smples two tests one test No. of negtive smples tested by three testsspe -IFA -CF IFA-CF IFA CF p is significnt. Similr results were observed with blood donors, lthough the differences were not significnt. Such differences between nd the other tests were not surprising, becuse of the higher sensitivity of immunoenzymtic tests over CF or IFA for Q fever. This hs lso been reported by Kruss et l. (15) nd Roges nd Edlinger (21), nd it is known to occur with other rickettsie (4, 6, 14). In our study, the percentge of positive serum smples mong blood donors is not representtive of the Swiss popultion, since most of the donors cme from rurl loclities. Nevertheless, our results show tht cses of Q fever re more frequent thn hs been generlly ssumed. The vrious serologic tests used for Q fever (CF, IFA, microgglutintion, hemgglutintion, etc.) re highly specific, nd there re no known cross-rections between C. burnetii nd ny bcteril or virl microorgnisms (16). The limited study with our confirmed this specificity, since no significnt OD vritions were observed in serum smples from ptients suffering from pneumoni of other origin. The sme observtions were mde with IFA s the routine dignostic test during the pst 3 yers in our lbortory. At lest 50% of the positive serum smples from blood donors hd titers of 1:10 (threshold) by CF. An increse or decrese of titer of 1 dilution in this test could hlve or double the number of positive serum smples, depending on the sensitivity of the test. For IFA nd, on the other hnd, titers or OD vlues were spred over lrger scle nd only few vlues were close to the cutoff. Another problem, encountered with CF, is nticomplementry ctivity s recorded with serum smples from Romnshorn nd Zollbruck (Tble 1). Such rections seem to be relted to the nture s well s the storge conditions of the serum smples. Contminted smples used in the my give flse-positive rections, wheres they will not influence the IFA results. Although the IFA is simple to perform nd very economicl in the use of regents, reding is subjective nd is tiring when lrge number of ser re to be screened. Our results indicte tht for epidemiologicl survey, the is the test of choice. It is simple to perform nd is more sensitive thn the other tests used. The is lso useful for the dignosis of cute cses, s reported by Doller et l. (8) nd Field et l. (11). At present, we re evluting the for the dignosis of cute nd chronic Q fever by monitoring clss-specific IgG, IgM, nd IgA to phses I nd Il of C. burnetii. ACKNOWLEDGMENTS We thnk the physicins nd nurses of the Centre Médico-Socil in the Bgnes Vlley, s well s J. Amcker (Sion Hospitl), J. Petite (Mrtigny Hospitl), L. Perrin (Genev Cntonl Hospitl), nd P. Bchmnn (Schweizerische Rot Kreuz Bern) for their indispensble collbortion nd the technicins of the Deprtment of Serology, Institute Centrl des Hôpitux Vlisns, Sion, in prticulr M.-C. Mottiez nd M.-C. Pnntier, for their skillful technicl ssistnce. This work ws supported by grnt from the Fond Ntionl Suisse de l Recherche Scientifique (no ). LITERATURE CITED 1. Amno, K.-l., nd J. C. Willims Chemicl nd immunologicl chrcteriztion of lipopolyscchrides from phse I nd phse Il Coxiell burnetii. J. Bcteriol. 160: Bc, O. G., nd D. Pretsky Q fever nd Coxiell burnetii: model for host-prsite interctions. Microbiol. Rev. 47: Bruppcher, R., A. E. Metzler, J. Nicolet, H. V. Bertschinger, nd J. Gelzer Zur Q-Fieber-Prvlenz in der Schweiz. Eine seroepidemiologische Untersuchung durch den B-Dienst der Armee. Schweiz. Z. Milit. Med. 60: Clements, M. L., J. S. Dumler, P. Fiset, C. L. Wissemn, M. J. Snyder, nd M. M. Levine Serodignosis of Rocky Mountin spotted fever: comprison of IgM nd IgG enzymelinked immunosorbent ssys nd indirect fluorescent ntibody test. J. Infect. Dis. 148: Cox, H. R Cultivtion of rickettsie of the Rocky Mountin spotted fever, typhus nd Q fever groups in embryonic tissues of developing chicks. Science 94: Dsch, G. A., S. Hlle, nd A. Bourgeois Sensitive microplte enzyme-linked immunosorbent ssy for detection of ntibodies ginst the scrub typhus rickettsi (Rickettsi tsutsugmushi). J. Clin. Microbiol. 9: Derrick, E. H Q fever, new entity: clinicl fetures, dignosis, nd lbortory investigtion. Med. J. Aust. 2: Doôler, G., P. C. Doller, nd H.-J. Gerth Erly dignosis of Q fever: detection of immunoglobulin M by rdioimmunossy nd enzyme immunossy. Eur. J. Clin. Microbiol. 3: Dupuis, G., O. Péter, M.-C. Mottiez, nd M. Vouilloz Séro-prévlence de l fièvre Q humine en Suisse. Schweiz. Med. Wochenschr. 116: Dupuis, G., O. Péter, D. Pedroni, nd J. 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