A 60-year-old caucasian woman was admitted to the hospital complaining of pleuritic chest pain, chills and fever accompanied by fatigue and malaise. Two weeks prior to admission, the patient suffered from tonsillitis and was treated with feneticillin prescribed by her general practitioner. Examination of her chest revealed decreased breath sounds and crackles in the left posterior lung base, and a chest radiograph showed a consolidation of the left lung base. Laboratory studies revealed a WBC count of 8200/µL (54% neutrophils, 36% lymphocytes, 4% monocytes, and 2% eosinophils), hematocrit of 35%, an erythrocyte sedimentation rate of 51 mm/h and a C-reactive protein value (CRP) of 144 mg/l. Results of liver function tests were within the normal range. Small spectrum antibiotic therapy with intravenous amoxicillin (1000 mg per 6 hours) was started. The patient seemed to respond after initiating antibiotic therapy; her condition became better and CRP-values dropped to 75 mg/l on day 3 after hospital admission. She was discharged home four days after admission. Three sets of blood cultures were taken at admission but no organisms were cultured. Bacterial cultures for bacteria and fungi of sputum remained negative. Sputum culture on Legionella-specific media was not performed. An immunochro- matographic membrane test (Binax NOW; Binax, USA) to detect L. pneumophila se- rogroup 1 soluble antigens in urine had been performed, but the result was negative. Four days after hopital admission, results of ELISA for L. pneumophila seroroup 1-7 (Virion/Serion, Würzburg, Germany) demonstrated negative levels of IgM (74 U/ ml) and IgG (< 10 U/ml) antibodies (reference ranges IgM/IgG: negative < 120/50, equivocal 120-140/50-70, positive > 140/70). Two weeks later, tests revealed marked levels of complement fixating (CF; Virion/Serion, Würzburg, Germany) antibodies to
C. burnetii (titer of < 1 : 10 in the acute phase serum, and ≥ 1 : 160 in the convalescent serum sample). CF tibodies to influenza A and B viruses, adenovirus, coronavirus, Mycoplasma pneumoniae, respiratory syncytial virus and Chlamydophila psittaci were negative. Because the patient had always lived in an urban setting, without con- tact with animals except for a domestic dog, the diagnosis Q fever was thought to be very unlikely. The clinical microbiologist noticed the increased IgM level of 74 U/ml and suspected a serological cross-reaction between L. pneumophila and C. burnetii. Serology for L. pneumophila was repeated. Three weeks after hospital admission, results of ELISA for L. pneumophila demonstrated increased levels of IgM (93 U/ ml) and after four weeks, levels of IgM increased further above the cut-off value (204 U/ml). IgG levels remained negative (<10 U/ml) at both three and four weeks. In addition, samples were tested for L. pneumophila serogroup 1 (VIRCELL, S.L., Santa Fe, Granada, Spain [4]) and C. burnetii IgM and IgG antibodies by an indirect immunofluorescent assay (in-house assay). Using acute and convalescent serum, IgG titers were 1:16 and 1:64 respectively, and IgM titers 1:128 and 1:256 respectively for L. pneumophila. IgG antibodies against C. burnetii were absent in the acute serum and 1:64 in the convalescent serum, IgM titers were 1:64 in both the acute and convalescent serum sample.
DISCUSSION
The background frequency of elevated anti-L. pneumophila titers in the normal pop- ulation in The Netherlands has been found to be very low. In a study using 480 blood samples from a serum bank established in a nation-wide population based survey, the prevalance of measured IgM levels between 50 and 94 U/ml was only 2.7% [5]. Seropositivity for IgG is much more common, with 5.4% seropositivity (>70 U/ml) and another 4.4% with borderline increased IgG titer (50-70 U/ml). Cross-reactive antibodies with L. pneumophila are occasionally found in patients with respiratory tract infections (Bordetella pertussis, Haemophilus influenzae, Chlamydophila pneu- moniae) and infections other than respiratory tract, including Pseudomonas spp., Bacteroides spp., Campylobacter spp., Salmonella typhi and Neisseria meningitidis [6,7]. Serological cross-reactions between Legionella spp. and Coxiella spp. have been reported previously [6,8,9]. One report described a patient with Q fever pneu- monia and cross-reacting antibodies to L. pneumophila sg 4, demonstrated by a cross absorption study [8]. Musso et al. found that 34.5% of Q fever patients had a significant titer of antibodies against L. micdadei at levels significantly higher than those found in the healthy population, using microimmunofluorescence assay [6]. Cross-reactions involved IgG antibodies and were demonstrated by a cross-adsorption study and protein immunoblotting. Western blot analysis performed indicated that cross-reactions were probably due to both protein and lipopolysaccharide antigens. It should be remembered, however, that these cross-reactions are the exceptions and that the specificity of most commercial used ELISAs is high [4,5].
Serological Testing for Legionnaires’ Disease and Cross-reactions with Coxiella burnetii 41
Serological cross-reactions between L. pneumophila and C. burnetii are in fact not surprising given the phylogenetic similarity between the two species. Both these bacteria are in the gamma subdivision of the proteobacteria, distantly placed from Rickettsiae which belong in the alpha subdivision. Sequencing of the C. burnetii 16S rRNA and genome has identified substantial homology with L. pneumophila [10,11,12]. These organisms have similar intracellular lifestyles and may utilize com- mon genes to infect their host. In addition, the C. burnetii antigen has also been reported to express on its surface epitopes of the so-called common bacterial antigen and eukaryotic chaperoning protein [13,14], which has extensive homology with proteins from other prokaryotes, such as L. pneumophila [15].
The presence of cross-reacting antibodies in patients with CAP emphasizes the need for cautious interpretation of antibody titers to members of the family Coxiella and Legionella. L. pneumophila and C. burnetii are both gram-negative bacteria po- tentially responsible for similar clinical syndromes. Although the clinical condition of the patient described was benign and probably self-limiting, both Q fever and LD are able to cause severe illness with high mortality. A misdiagnosis of either condition may lead to incorrect and ineffective treatment. Tetracycline is the standard treat- ment for Q fever and is less effective for LD [16]; erythromycin and quinolones are standard treatment for LD and is probably ineffective for Q fever [17]. In addition, failure to recognize cases of LD will hamper the detection of potential clusters or outbreaks of LD.
ACKNOWLEDgEMENTS:
We thank Ria Woudenberg (Laboratory for Medical Microbiology, Rijnmond Zuid, Rotterdam, The Netherlands) for performing the C. burnetii indirect immunofluo- rescence assay.
REFERENCES
1. van der Eerden, M.M., F. Vlaspolder, C.S. de graaff, T. groot, W. Bronsveld, H.M. Jansen, and W.g. Boersma. 2005. Value of intensive diagnostic microbiological investigation in low-
and high-risk patients with community-acquired pneumonia. Eur. J. Clin. Microbiol. Infect. Dis. 24:241-249.
2. Fields, B. S., R. F. Benson, and R. E. Besser. 2002. Legionella and Legionnaires’ disease: 25
years of investigation. Clin. Microbiol. Rev. 15:506-526.
3. Parker, N.R., J.H. Barralet, and A.M. Bell. 2002. Q fever. Lancet. 367:679-688.
4. Diederen, B.M., J.A. Kluytmans, M.F. Peeters. 2006. Evaluation of Vircell enzyme-linked im-
munosorbent assay and indirect immunofluorescence assay for detection of antibodies against Legionella pneumophila. Clin. Vaccine. Immunol. 13:361-364.
5. Boshuizen, H.C., J.W. Den Boer, H. de Melker, J.F. Schellekens, M.F. Peeters, J.A. van Vliet, and M.A. Conyn-van Spaendonck. 2003. Reference values for the SERION classic ELISA for
detecting Legionella pneumophila antibodies. Eur. J. Clin. Microbiol. Infect. Dis. 22:706- 708.
6. Musso, D., and D. Raoult. 1997. Serological cross-reactions between Coxiella burnetii and Legionella micdadei. Clin. Diagn. Lab. Immunol. 4:208-212.
7. Edelstein, P.H., R.M. McKinney, R.D. Meyer, M.A. Edelstein, C.J. Krause, and S.M. Fine- gold. 1999. Immunologic diagnosis of Legionnaires’ disease: cross-reactions with anaerobic
and microaerophilic organisms and infections caused by them. J. Infect. Dis. 141:652-655.
8. Dwyer, D.E., V.L. gibbons, L.M. Brady, and A.L. Cunningham. 1999. Serological reaction to Legionella pneumophila group 4 in a patient with Q fever. J. Infect. Dis. 158:499-500. 9. Finidori, J.P., D. Raoult, N. Bornstein, and J. Fleurette. 1992. Study of cross-reaction between
Coxiella burnetii and Legionella pneumophila using indirect immunofluorescence assay and immunoblotting. Acta Virol. 36:459-465.
10. Maurin, M., and D. Raoult. 1999. Q fever. Clin. Microbiol. Rev. 12:518-553.
11. Seshadri, R, I.T. Paulsen, J.A. Eisen, et al. 2003. Complete genome sequence of the Q-fever
pathogen Coxiella burnetii. Proc. Natl. Acad. Sci. USA. 100:5455-5460.
12. Vogel, J.P. 2004. Turning a tiger into a house cat: using Legionella pneumophila to study Coxiella burnetii. Trends Microbiol. 12:103-105.
13. Vodkin, M.H., and J.C. Williams. 1988. A heat shock operon in Coxiella burnetii produces a
major antigen homologous to a protein in both mycobacteria and Escherichia coli. J. Bacte- riol. 170:1227-1234.
14. Williams, J.C., T.H. Hoove, D.M. Waag, N. Bhatnagar, C.R. Bolt, and g.H. Scott. 1999.
Antigenic structure of Coxiella burnetii: a comparison of lipopolysaccharide and protein antigens as vaccines against Q fever. Ann N Y Acad Sci. 590:370-380.
15. Hoffman, P.S., L. Houston, and C.A. Butler. 1990. Legionella pneumophila htpAB heat shock
operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila- infected HeLa cells. Infect. Immun. 58:3380-3387.
16. yeaman, M.R., L.A. Mitscher, and O.g. Baca. 1987. In vitro susceptibility of Coxiella bur- netii to antibiotics, including several quinolones. Antimicrob. Agents. Chemother. 31:1079- 1084.