Sullivan, Nicolaides & Partners, Taringa,3 Queensland, Australia. Received 22 October 1996/Returned for modification 9 December 1996/Accepted 24 January ...
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1997, p. 384–386 1071-412X/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 4, No. 3
Combined Determination of Coxiella burnetii-Specific Immunoglobulin M (IgM) and IgA Improves Specificity in the Diagnosis of Acute Q Fever PETER DEVINE,1* CATHERINE DOYLE,2
AND
GEOFF LAMBKIN3
1
Clinical Sciences, University of Queensland, Herston, PanBio Pty Ltd., Windsor,2 and Sullivan, Nicolaides & Partners, Taringa,3 Queensland, Australia Received 22 October 1996/Returned for modification 9 December 1996/Accepted 24 January 1997
Immunoglobulin M (IgM) and IgA responses in patients with acute Q fever were compared by enzyme-linked immunosorbent assay. An increase in both IgM and IgA was observed in paired sera from all 19 patients with acute Q fever, and both IgM and IgA levels showed good correlation with complement fixation test titers. Paired sera from 23 patients with infections other than Q fever were also tested. IgM levels were elevated in three of these patients, while IgA levels were elevated in three different patients (87% specificity for either IgM or IgA). As no patients in the disease control group showed elevated levels of both IgM and IgA, definition of a positive result as elevated levels of both IgM and IgA improved specificity to 100% without a decrease in sensitivity. This study indicates that detection of specific IgA is a useful adjunct to that of IgM in the diagnosis of acute Q fever. peroxidase or anti-human IgA–peroxidase (100 ml/well) and, after another wash, a 10-min incubation with tetramethylbenzidine substrate (100 ml/well). The reaction was stopped by the addition of 100 ml of 1 M phosphoric acid per well, and the strips were read at 450 nm with a microtiter plate reader. Positivity was determined by comparison to a reference serum (cutoff calibrator). A positive sample was defined as having a sample absorbance/calibrator absorbance ratio (ELISA ratio) of $1.0, and a negative sample was one with a ratio of ,1.0. CFTs were performed in microtiter plates by standard procedures (1). A commercially available phase-II C. burnetii (Nine Mile strain) CFT antigen (CSL, Melbourne, Australia)
Coxiella burnetii is the causative agent of Q fever, a worldwide zoonosis. Acute infection in humans presents as a febrile, self-limiting disease marked by symptoms including fever, headache, myalgia, muscle cramps, hepatitis, and respiratory complications. As isolation of the organism is hazardous, the most common method of diagnosis is through the detection of specific antibody in serum, with enzyme-linked immunosorbent assay (ELISA) considered the most sensitive method (5). Elevation of C. burnetii-specific immunoglobulin M (IgM) levels is commonly used to diagnose acute disease, with antibody appearing shortly after the onset of symptoms and being detected for up to 3 months (3). However, some patients with diseases other than Q fever may show elevation in assays for C. burnetii-specific IgM. The majority of patients with acute Q fever have also been reported to show an IgA response to phase II antigen (9), though IgA has not been used as a marker for routine diagnosis of disease. The present study was designed to investigate the use of IgA as an adjunct to IgM in the diagnosis of Q fever. Paired serum samples (n 5 84) collected 6 to 35 days apart from 19 patients with acute C. burnetii (Q fever) infection and 23 patients with other infections (6 patients infected with Mycoplasma pneumoniae, 6 with Epstein-Barr virus [EBV], 6 with cytomegalovirus [CMV], and 5 with Bordetella pertussis) were tested by a C. burnetii-specific complement fixation test (CFT) and commercial ELISAs for IgM and IgA (PanBio Pty Ltd., Brisbane, Australia). In addition, 40 sera with CFT titers of antibody to C. burnetii ranging from 8 to 256, and 149 sera from asymptomatic blood donors, were also tested by ELISA. Q fever IgM and IgA ELISAs were performed according to the manufacturer’s instructions. Briefly, serum was diluted 1/100 in the diluent provided (containing goat anti-human IgG) and incubated for 15 min at room temperature to remove any competing IgG and rheumatoid factor before transfer to phase II C. burnetii antigen-coated microwells for 20 min at room temperature (100 ml/well). After a wash with phosphatebuffered saline containing 0.05% Tween 20, bound IgM or IgA was detected via a 20-min incubation with anti-human IgM–
FIG. 1. Comparison of Q fever ELISA results for IgA and IgM with results of the Q fever CFT. Individual assay values are represented by circles (IgA) or squares (IgM). The mean ELISA ratios (sample absorbance/cutoff calibrator absorbance) are represented by horizontal bars. There was a significant correlation between ELISA ratio and CFT titer (regression analysis) for both IgA (r2 5 0.3341, P , 0.0001) and IgM (r2 5 0.2937, P , 0.0001).
* Corresponding author. Mailing address: University of Queensland, B Floor, Clinical Sciences Building, Royal Brisbane Hospital, Herston 4029, Queensland, Australia. Phone: 61-7-33655202. Fax: 61-7-33655203. 384
VOL. 4, 1997
NOTES
TABLE 1. Comparison of IgA and IgM responses in paired sera from patients with acute Q fever Serum sample (days)a
CFT titer b
385
TABLE 2. IgA and IgM responses in paired sera from patients with infections other than Q fever
ELISA ratio IgA
IgM
1A 1B (13)
ND $256
0.4 6.9
0.4 7.4
2A 2B (8)
ND 16
0.4 7.2
0.2 6.7
3A 3C (7)
ND ND
0.6 1.3
0.3 3.3
Serum sample
ELISA ratio IgA
IgM
CMV 1A 1B
0.3 0.8
0.2 0.9
2A 2B
0.1 0.1
0.4 0.8
3A 3B
0.2 0.3
0.2 0.9
4A 4B
0.4 0.9
0.2 0.4
5A 5B
0.2 0.4
0.2 0.5
6A 6B
0.3 0.3
0.3 0.5
EBV 7A 7B
0.3 0.3
0.36 0.35
8A 8B
0.6 1.1
0.10 0.39
9A 9B
0.5 0.9
0.20 0.46
10A 10B
0.1 0.1
0.10 0.47
11A 11B
1.5 2.0
0.27 0.59
12A 12B
0.1 0.1
0.18 0.22
0.1 0.2
0.3 0.2
4A 4B (6)
2 128
0.4 2.6
0.3 6.4
5A 5B (13)
2 32
0.7 3.2
0.1 6.7
6A 6B (22)
2 $128
0.2 4.7
0.7 7.6
7A 7B (20)
2 64
0.4 1.2
0.9 6.2
8A 8B (20)
2 128
0.5 5.0
0.1 6.5
9A 9B (22)
2 32
0.8 1.7
0.2 5.0
10A 10B (10)
2 128
0.3 2.0
0.2 8.0
11A 11B (17)
ND 64
0.3 1.9
0.2 7.4
12A 12B (7)
2 128
0.3 1.2
0.3 8.0
13A 13B (10)
2 .128
0.2 2.6
0.3 6.8
14A 14B (29)
2 128
0.2 4.7
0.1 6.7
15A 15B (11)
ND 64
0.9 3.1
0.2 7.2
14A 14B
0.2 0.2
0.1 0.1
16A 16B (12)
ND 2
0.2 1.5
0.2 3.2
15A 15B
0.5 0.5
0.3 0.2
17A 17B (20)
ND 16
0.4 1.1
0.4 8.0
16A 16B
0.4 0.5
0.1 1.7
18A 18B (9)
ND .128
0.2 4.5
0.1 6.6
17A 17B
0.4 0.2
0.1 0.2
2 64
1.4 3.6
5.4 6.8
18A 18B
0.2 1.0
0.2 0.6
0.1 0.1
0.3 0.2
20A 20B
0.1 0.1
0.1 1.2
21A 21B
0.1 0.0
0.1 2.9
22A 22B
0.0 0.1
0.1 0.1
23A 23B
0.0 0.0
0.1 0.1
19A 19B (17) a b
Days between collections of paired sera. ND, not done.
was used. Serum samples were started at a dilution of 1:8 and titrated twofold, with a control serum also tested at this dilution to ensure that there was no anticomplementary activity. The endpoint was defined as the highest titer with $75% fixation. A fourfold-or-higher increase between two serum samples was considered indicative of acute Q fever (3, 8). For a single serum sample, a titer of $16 was considered positive. A significant increase in both IgA and IgM ELISA ratios, from ,1.0 in the first sample to $1.0 in the second sample, was
M. pneumoniae 13A 13B
B. pertussis 19A 19B
386
NOTES
demonstrated in paired sera from all 19 patients with acute Q fever (Table 1). The IgA and IgM ratios were also well correlated with results of the CFT for Q fever (Fig. 1). Of sera with CFT titers of 8.0 (n 5 10), one showed elevation in the IgA ELISA and five showed elevation in the IgM ELISA, while significantly more patients with CFT titers of $16 showed elevated IgA and IgM levels (Fisher’s exact test, P , 0.0001 for IgA and P 5 0.0004 for IgM). All but 1 of 30 patients with CFT titers of $16 showed elevated IgA levels (sensitivity 5 97%), and all patients with CFT titers of $16 showed elevated IgM levels in the ELISA (sensitivity 5 100%). Subsequently, 29 of 30 patients with CFT titers of $16 showed elevated levels of both IgA and IgM (sensitivity 5 97%). Paired sera from patients with CMV (n 5 6), EBV (n 5 6), M. pneumoniae (n 5 6), and B. pertussis (n 5 5) infections were also tested in the Q fever IgA and IgM ELISAs (Table 2). The Q fever IgA ELISA showed elevated levels in two patients with EBV infection and one patient with M. pneumoniae infection, while the Q fever IgM ELISA showed elevated levels in one patient with M. pneumoniae infection and two patients with B. pertussis infection (specificity of either ELISA 5 87%). It is of particular interest that elevation of IgA and IgM levels did not occur in the same patients, indicating that the combined use of IgA and IgM ELISAs (a positive result taken as elevated levels of both IgA and IgM) gave no false-positive results (specificity 5 100%). Elevation of IgA levels was also observed in 5 of 149 (3.4%) asymptomatic blood donors, while 4 (2.7%) of these patients showed elevated levels in the IgM ELISA (data not shown). As was also seen in the disease control group, no patients in the asymptomatic control group showed elevated levels of both IgA and IgM. Consequently, the combined testing of IgM and IgA levels in the diagnosis of acute Q fever does not lead to a significant decrease in sensitivity but significantly improves the specificity of diagnosis. Elevated levels of IgA specific for C. burnetii phase-I antigen have been well accepted as an indicator of chronic Q fever (4, 6), but there have been contradictory reports regarding IgA responses to phase II antigen during acute infection. An IgA response to phase-II antigen could not be demonstrated in acute-phase serum from Japanese children with Q fever pneu-
CLIN. DIAGN. LAB. IMMUNOL.
monia (7), while another report indicated that only 12.5% of patients showed IgA responses (2). However, in another study an IgA response to phase II antigen was demonstrated in 90% of patients with acute Q fever (9), in close agreement with the results of this study. These differences may have been due to the strains of C. burnetii used or to the assay systems used. This study has demonstrated that the combined use of C. burnetiispecific IgM and IgA ELISAs improves the specificity of diagnosis of acute Q fever without compromising sensitivity. Combined use of these markers should lead to improved accuracy in the diagnosis of this disease. REFERENCES 1. Bradstreet, C. M. P., and C. E. D. Taylor. 1962. Technique of complement fixation test applicable to the diagnosis of virus diseases. Mon. Bull. Minist. Health Public Health Lab. Serv. 21:96–104. 2. Embil, J., J. C. Williams, and T. J. Marrie. 1990. The immune response in a cat-related outbreak of Q fever as measured by the indirect immunofluorescence test and the enzyme-linked immunosorbent assay. Can. J. Microbiol. 36:292–296. 3. Field, P. R., J. G. Hunt, and A. M. Murphy. 1983. Detection and persistence of specific IgM antibody to Coxiella burnetii by enzyme-linked immunosorbent assay: a comparison with immunofluorescence and complement fixation tests. J. Infect. Dis. 148:477–487. 4. Peacock, M. G., R. N. Philip, J. C. Williams, and R. S. Faulkner. 1983. Serological evaluation of Q fever in humans: enhanced phase I titers of immunoglobulins G and A are diagnostic for Q fever endocarditis. Infect. Immun. 41:1089–1098. 5. Pe´ter, O., G. Dupuis, M. G. Peacock, and W. Burgdorfer. 1987. Comparison of enzyme-linked immunosorbent assay and complement fixation and indirect fluorescent-antibody tests for detection of Coxiella burnetii antibody. J. Clin. Microbiol. 25:1063–1067. 6. Raoult, D., J. Urvolgyi, J. Etienne, M. Roturier, J. Puel, and H. Chaudet. 1988. Diagnosis of endocarditis in acute Q fever by immunofluorescence serology. Acta Virol. 32:70–74. 7. To, H., N. Kako, G. Q. Zhang, H. Otsuka, M. Ogawa, O. Ochiai, S. V. Nguyen, T. Yamaguchi, H. Fukushi, N. Nagaoka, M. Akiyama, K. Amano, and K. Hirai. 1996. Q fever pneumonia in children in Japan. J. Clin. Microbiol. 34:647–651. 8. Williams, J. C., M. R. Johnston, M. G. Peacock, L. A. Thomas, S. Stewart, and J. L. Portis. 1984. Monoclonal antibodies distinguish phase variants of Coxiella burnetii. Infect. Immun. 43:421–428. 9. Worswick, D., and B. P. Marmion. 1985. Antibody responses in acute and chronic Q fever and in subjects vaccinated against Q fever. J. Med. Microbiol. 19:281–296.
