European Heart Journal (1986) 7, 1062-1066

Serological diagnosis of Q fever endocarditis

G. DUPUIS*, O. PETER*, R. LiJTHYf, J. NICOLETJ, M. PEACOCK§ AND W. BURGDORFER§

* Division of Clinical Microbiology and Infectious Diseases, Valais Central Institute, Sion, ̂ Division ofInfectious Diseases, University Hospital, Zurich, \Institute of Veterinary Bacteriology, University of Bern,Bern, Switzerland and ̂ Laboratory of Pathobiology, Rocky Mountain Laboratories, National Institute of

Allergy and Infectious Diseases, Hamilton, Montana, U.S.A.

KEY WORDS: Q fever, Coxiella burnetii, rickettsial endocarditis.

The diagnosis of Q fever endocarditis cannot be made by bacterial cultures and necessitates serologicalidentification of specific antibodies to Coxiella burnetii which stimulates mainly the production of anti-phaseII antibodies during the acute disease, but primarily anti-phase I antibodies in endocarditis. Indirect micro-immunofluorescence allows rapid detection of specific IgA, IgG andlgM. The results of serological analyses of191 acute cases of Q fever were compared with those of 8 cases o/Coxiella burnetii endocarditis. All sera wereevaluated by complement fixation and microimmunqfluorescence tests. The highest titre differences betweenprimary Q fever and Q fever endocarditis were observed with anti-phase I IgA and IgG antibodies measured bymicroimmunofluorescence followed by anti-phase I antibodies measured by complement fixation tests. Anti-phase I IgG andlgM titres were consistently higher than anti-phase II titres in endocarditis. The reverse is truein acute Q fever. In addition, anti-phase I IgA appeared to be diagnostic for Coxiella burnetii endocarditis.Accordingly we recommend the testing of these specific IgA, IgG, andlgM by microimmunofluorescence incases of culture-negative endocarditis. These tests could also prove useful for following the development ofCoxiella burnetii endocarditis in patients under treatment.

Introduction

Primary or acute Q fever is an influenza-likeinfection, frequently accompanied by pneumonia.It normally follows a benign course, althoughprogression to a chronic stage, Coxiella burnetiiendocarditis, may occur and presents a dangerouscomplication'11. Although only about 200 cases havebeen reported so far, Q fever endocarditis appearsto occur more frequently than assumed. Most caseshave been discovered in the United Kingdom andAustralia'2"4'.

The frequency of endocarditis after primary Qfever is estimated to be in the order of 1 to 2%, but sofar no work has been done to confirm this. Thelatency period between the acute or primary phase,which has often gone undiagnosed, and endocarditisis still unknown, with estimates ranging from 2 to 20years. Although more often diagnosed in middle-aged men, Q fever endocarditis can also affectwomen and children'5!. Clinically, it presents itself

Submitted for publication on the 6 May 1986.

Address for correspondence: G. Dupuis, Valais Central Institute,CH-1951 Sion 3/Switzerland.

as a culture-negative endocarditis, with patientssuffering from fever, tachycardia, heart murmur,hepatomegaly and splenomegaly. Often there is ahistory of pre-existing mitral or aortic valvularlesion'67'. Nonspecific laboratory examinationsoften reveal a leucocytosis with anaemia andthrombopenia, hypergammaglobulinaemia andpathologic liver tests'8!, with a mortality rate of upto 50%, even with medical and surgical treatment, itis imperative to diagnose these cases at the earliestpossible time to shorten delay of effective antibiotictherapy. Isolation of C. burnetii from routine bloodcultures is not possible, and only serology allows aclinical diagnosis. Inoculation of guinea pigs withvalvular tissues taken during surgery frequentlyallows a definite confirmation of the diagnosis.

Unique to C. burnetii is its antigenic phase vari-ation. The virulent phase I is isolated from natural orlaboratory infections of animals and man, whereasthe avirulent phase II occurs during serial passageof C. burnetii in immunologically incompetenthosts, such as embryonated hen eggs or cell tissueculture systems. Phase I to II variation seems tocorrelate with smooth and rough lipopolysaccharidechanges'9). Specific high levels of anti-phase I anti-bodies are normally found in chronic Q fever

0195-668X, 86,121062+05 S02.00,0 i 1986 The European Society of Cardiology

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Q fever endocarditis 1063

(endocarditis), whereas specific anti-phase IIantibodies predominate during primary Q fever.

The serological test most commonly used up tothe present time has been the complement fixation(CF) test, with anti-phase I antibody titres greaterthan 1:200 being considered diagnostic of C.burnetii endocarditis. However, cases have beendiscovered with titres below this level''!. Among themore sensitive serological tests developed during thelast few years, is the microimmunofluorescence test(IFA), which allows the rapid and easy detection ofspecific IgA, IgG and IgM antibodies to phase I andphase II C. burnetiim.

This paper deals with a comparative serologicalanalysis of the CF and IFA tests in 8 cases of Q feverendocarditis and in 191 cases of acute Q fever!"'.

Materials and methods

The sera of the 8 patients suffering from culture-negative endocarditis were obtained from theDepartments of Internal Medicine of the UniversityHospitals at Zurich and Berne and from thePediatric Service of the University Hospital atGeneva. The diagnosis of Q fever endocarditis foreach of these cases was established between 1980 and1984 according to the classically known criterionof an anti-phase I antibody titre greater than 1:200in CF. The IFA analyses (IgA, IgG, IgM, to C.burnetii phases I and II) were performed fromfrozen serum specimens in late 1984. Only results ofthe first sample from each patient, i.e. before anyantibiotic therapy, are reported here.

A short description of the patients condition atthe time of the diagnosis is presented. Patient 1 wasadmitted for surgical correction of a ventricularseptal defect. Follow-up clinical investigationrevealed an endocarditis with fever, hepatospleno-megaly, and negative blood cultures. A liver biopsyshowed a granulomatous hepatitis. Guinea pigsinoculated with the patient's blood developed anti-bodies to C. burnetii. The patient's serum showedhigh CF anti-phase I antibody titre. Patients 2 and 3were admitted for a fever of unknown origin, andwith aortic regurgitation. Investigations revealedan endocarditis, negative blood cultures and a highCF anti-phase I antibody titre. Patient 4 was admit-ted for surgical correction of a tetralogy of Fallot.Clinical investigation revealed an endocarditis withfever, hepatosplenomegaly, negative blood culturesand a high CF anti-phase I antibody titre. Patient5 underwent an aortic valve replacement for stenosisin November 1979. In June 1982, he was admitted

for fever of unknown origin. The follow-up clinicalinvestigations revealed endocarditis, negative bloodcultures and a high CF anti-phase I antibody titre.He was treated with cotrimoxazole and rifampicin.In January 1983, he underwent a second aorticvalve replacement for regurgitation. C. burnetiiwas isolated from heart valve tissue. Patient 6 wasadmitted for a fever of unknown origin. Follow-upclinical investigations revealed an endocarditis,negative blood cultures and a high CF anti-phase Iantibody titre. Patient 7 presented a serologicallyconfirmed acute Q fever in 1973. He underwent afirst aortic valve replacement for regurgitation in1977 and a second in April 1981. He was admitted inNovember 1981 for a fever of unknown origin withheart failure. Follow-up clinical investigationsrevealed an endocarditis, negative blood culturesand a high CF anti-phase I antibody titre. Patient 8underwent in January 1985 an aortic, mitral andtricuspid valve replacement for rheumatic heartdisease. Clinical investigations revealed an endo-carditis with fever, negative blood cultures and ahigh CF anti-phase I antibody titre.

The 191 sera from patients with acute Q feverwere sent to us by physicians in Le Chable during anepidemic in the Val de Bagnes (Valais) in theautumn of 1983. All 191 sera were examined by CFand IFA (IgA, IgG, IgM) tests for anti-phase I andII antibodies to C. burnetipw. Sera of 12 patientssuffering from streptococcal endocarditis were alsoexamined for these antibodies.

Complement fixation test (CF)

The micro-method was performed as describedby the Centers for Disease Control (CDC, Atlanta,GA, U.S.A)i'4i except that the starting serumdilution was 1:10 and the sensitized erythrocyteconcentration was 2-5%. The antigen C. burnetiiphase I was of strain Herzberg (Veterinaria AG,Zurich, Switzerland) and the antigen C. burnetiiphase II was of strain Nine Mile (Virion AG,Ruschlikon, Switzerland). Serum, antigen, comple-ment (Virion AG) and haemoly tic system (Boehring,Marburg, Germany), were prepared in veronalbuffer (Oxoid BR 16). The end point was defined asthe highest dilution with ^ 75% fixation. Resultsare expressed as reciprocal titres (for example 200for the 1:200 dilution).

Indirect microimmunofluorescence test (IFA)

The IFA tests were performed according to the

1064 G. Dupuis et al.

Table 1 Reciprocal C. bumetii titres in patients with Q-fever endocarditis obtained with the complement fixation test and themicroimmunofluorescence test using antigens of phase I and II

Patient

1234567g

Average

Complement fixation

I

640320

1280640640640640640

679

II

16012801280640

201280640640

742

IgG

I

4096020480409602048020480409602048020480

28 160

Microimmunofluorescence

II

6402048020480

25602560

2048051205120

9680

IgM

I

320640640

80640640160160

410

II

206403208040

3208020

188

IgA

I

16012802560

160160320

2560320

940

II

2040

32020

128020

1601280

390

Rheumatoidfactor

+++++++++

method described by Philip et al.liS]. The C. bumetiiphase I antigen (Nine Mile strain) had been passed307 times in guinea pigs, cloned from primarychicken embryo cell culture plaques and passedtwice in chicken embryos. The C. bumetii phase IIantigen (strain Nine Mile) had been passed 90 timesin chicken embryos, cloned from primary chickenembryo cell culture plaques and passed three timesin chicken embryos. Antigens of C. bumetii phase Iand II were applied side by side to microscope slideswith a pen over a premarked 18-dot template. Weused FITC (fluorescein isothiocyanate) goat anti-human IgM and IgG conjugates (Biomerieux,France), and rabbit anti-human IgA conjugate(Dakopatts, Denmark). IgM reactive sera weretested for rheumatoid factor with the rheumatoidfactor tube test 'Roche' (Roche Diagnostica, Basel,Switzerland).

Results

Table I shows individual and average titres forsera taken at the moment of diagnosis from the 8cases of C. bumetii endocarditis. Phase I CF titreswere consistently above 200, with an average titre of679. Anti-phase I IgG titres by IFA were consist-ently higher with an average titre of 28 160.

Figure 1 illustrates the quantitative relationshipbetween the average titres for the 191 cases of acute Qfever and for the 8 cases of C. bumetii endocarditis.The highest titre differences between primary Qfever and Q fever endocarditis were observed withanti-phase I IgA and IgG antibodies measured by

IFA, followed by anti-phase I antibodies measuredbyCF.

Table 2 compares the range of titres obtainedwith the two tests for the 8 cases of endocarditis andthe 191 cases of acute Q fever. Antibodies to phase Iin CF as well as anti-phase I IgA and IgG in IFAtests were never present at significant levels inprimary Q fever but were very high in endocarditis.An overlap of elevated titres was regularly observedin phase II titres. Phase I IFA titres were alwaysequal or superior to phase II titres except for IgA incases 5 and 8. Rheumatoid factor was positive in all8 cases.

In the 12 sera from patients suffering from strep-tococcal endocarditis, no anti-phase I or II IgA orIgM against C. bumetii were detected. In the serafrom 3 patients only residual anti-phase I and IIIgG were found. No rheumatoid factor wasdetected.

Discussion

Antibody titres to C. bumetii phase I, measuredby CF, were always greater than 200 in all eightcases of endocarditis. In contrast, none of the 191cases of primary Q fever presented a titre greaterthan 20. This confirms the validity of the diagnosticcriterion of phase I antibody titres in the diagnosisof Q fever endocarditis. However, titres of less than200, do not exclude this diagnosis!'). Antibody titresto C. bumetii phase II by CF cannot distinguishendocarditis from primary Q fever because they areoften at the same level.

Q fever endocarditis 1065

25600 -

12800 -

6460

3200

1600

800

400

200

100

28160

17

9680

1384

Primary 0 fever

0 fever endocardi t is

9 4 0

410

7 0 5

n208

1188

6 7 9 74 2

3 9 0

172

Phosel PhaseH Phase I Phase Hi Phase I Phase Dl Phase 1 PhaseD IIgG IgM IgA CF

Figure 1 Average reciprocal C. burnetii titres in patients with primary Q fever (191 cases)and Q fever endocarditis (8 cases) obtained with the complement fixation test (CF) andmicroimmunofiuorescence test (IgG, IgM, IgA) using antigens of phase I and II.

Table 2 Range of reciprocal C. burnetii titres in patientswith Q fever endocarditis and primary Q-fever obtained withthe complement fixation test (CF) and the microimmuno-fiuorescence test (IFA) using antigens of phase I and II

Serologic test Endocarditis(N=8)

Primary Q fever(#=191)

•• CFICFIIIFA IgG I

• IgG II

IFA IgM I• IgM II

IFA IgA IIgA IIRheumatoid factor

320-128020-1280

20480-40960640-20480

80-640<20-640

160-2560<20-1280

40-320

<10-2040-1280

<2<M0320-10 240

<20-64080-10 240

<20<20-320

<20

With IFA, anti-phase I IgG antibodies can bedetected at very high dilutions in endocarditis only;the average titre for the 8 cases was approximately28 000 whereas it was 17 in the 191 cases of acute Qfever. Anti-phase I IgG appears to be the most

sensitive discriminator, since none of the 8 cases ofendocarditis had a level below 20 480, whereas noneof the 191 cases of primary Q fever had a level above40. Because anti-phase II IgG are raised in both theacute and the chronic disease state, they are of nodifferential diagnostic value. Moreover, in eachindividual case, anti-phase I IgG titre was alwaysequal or higher than anti-phase II IgG titre in endo-carditis, and greatly lower in primary Q fever. Ourresults correlate well with those reported by Peacocket a/."0' for their 5 cases of Q fever endocarditis.

With IFA, anti-phase I IgM are usually present atslightly higher levels than those of anti-phase II IgMin cases of endocarditis; the reverse is true in acutecases. However, due to the presence of rheumatoidfactors in cases of C. burnetii endocarditis, theclinical significance of these IgM titres is uncertain.On the other hand, the rheumatoid factor beingabsent in acute Q fever, IgM is diagnostic in thateasel'2!.

Elevated anti-phase I IgA titres were found inall 8 patients and appear to be diagnostic for C.burnetii endocarditis. In contrast, such titres were

7066 G. Dupuis et al.

undetectable in the 191 cases of acute Q fever. Onthe other hand, anti-phase II IgA antibodies invariable titres were found in both clinical conditionsas reported by others'16'. Consequently, only anti-phase I IgA is a useful indicator for suspected casesof C. burnetii endocarditis.

No significant levels of IgA, IgG and IgM anti-Cburnetii phase I or II were found in the sera of the 12persons with streptococcal endocarditis.

In addition to the CF test, the clinician can nowuse the IFA test, a sensitive, rapid and simplemethod, to establish the diagnosis of C. burnetiiendocarditis. In accordance with Peacock et a/.'10'we found that the most sensitive parameters for thediagnosis of C. burrtetii endocarditis are anti-phaseI IgA and IgG. Another possible advantage of theseindicators identified by IFA would be their earlierappearance in sera from patients developing a C.burnetii endocarditis. Regular checks of all the casesof acute Q fever we diagnosed in 1983 will possiblyconfirm this. The level of these indicators duringcontrol checks of cases of C. burnetii endocarditisunder treatment could also prove useful.

We would like to thank the cooperation of M. P. Glauser(CHUV, Lausanne), D. Laufer (Hopital Cantonal, Geneve),F. Burkhardt (Universitat Bern, Bern), M. F. Paccaud(Institut d'Hygiene, Geneve), L. Matter (Mikrobiologie,St-Gall) and M. C. Mottiez (ICHV, Sion). This work wassupported by a grant from the Swiss Foundation forCardiology.

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