Journal of Infection (2014) xx, 1e4
www.elsevierhealth.com/journals/jinf
Immunogenicity of the Q fever skin test Teske Schoffelen a,*, Tineke Herremans b, Tom Sprong c,d, Marrigje Nabuurs-Franssen c, Jos W.M. van der Meer a, Leo A.B. Joosten a, Mihai G. Netea a, Henk A. Bijlmer b, Marcel van Deuren a a
Department of Internal Medicine, Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands b Centre for Infectious Disease Control, National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands c Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, P.O. Box 9015, 6500 GS Nijmegen, The Netherlands d Department of Internal Medicine, Canisius Wilhelmina Hospital, P.O. Box 9015, 6500 GS Nijmegen, The Netherlands Accepted 8 March 2014 Available online - - -
KEYWORDS Q fever; Coxiella burnetii; Skin test; Immunogenicity; Booster effect; Interferon-gamma; Serology; Diagnostic test
Summary Objectives: The Q fever skin test is used to measure cell-mediated immunity to Coxiella burnetii in pre-vaccination screening to exclude individuals with pre-existing immunity. We investigated whether this in-vivo test influences subsequent measurements of immune response. Methods: We assessed the humoral and cellular immune responses before, and 6 and 12 months after skin testing in 63 individuals who were not vaccinated because of either a positive skin test or positive serology in screening. IgG anti-C. burnetii antibodies were measured using immune-fluorescence assay (IFA). The cellular immune response was assessed by measuring invitro C. burnetii-specific interferon (IFN)-g production in blood. Results: Of the 35 subjects with a positive skin test and negative serology, 15/35 (43%) showed seroconversion at 6 months, and 7/32 (22%) seropositivity at 12 months. The mean SE specific IFN-g production in this group increased from 185 88 pg/mL (at baseline) to 422 141 pg/mL at 6 months (P Z 0.009) and 223 91 pg/mL at 12 months (P Z 0.17). Of the 28 subjects with positive serology (and unknown skin test results), 21/28 (75%) showed an increase in IgG anti-phase I titres at 6 months, and 11/25 (44%) at 12 months. The mean SE specific IFN-g production was significantly increased at 6 months, but not at 12 months.
* Corresponding author. Radboud University Medical Center, Department of Internal Medicine, Internal Postal Code 463, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: þ31 24 3618819; fax: þ31 24 3541734. E-mail address: [email protected] (T. Schoffelen). 0163-4453/$36 ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinf.2014.03.008 Please cite this article in press as: Schoffelen T, et al., Immunogenicity of the Q fever skin test, J Infect (2014), http://dx.doi.org/ 10.1016/j.jinf.2014.03.008
2
T. Schoffelen et al. Conclusions: Q fever skin testing causes higher antibody titres and higher in-vitro IFN-g to C. burnetii, and therefore affects subsequent Q fever diagnostics. ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Introduction Q fever is caused by Coxiella burnetii, an intracellular Gram-negative bacterium. Initial infection often goes unnoticed, but in a minority of cases chronic infection develops and can present as endocarditis or vascular wall infection, which leads to high morbidity and mortality.1 Infection is normally diagnosed by serology, by measuring IgM and IgG antibodies against the two different antigenic phases of C. burnetii. Q fever skin testing is used to measure in vivo cellmediated immunity to C. burnetii. Its use is limited to pre-vaccination screening, to exclude individuals with pre-existing immunity who would be at increased risk of hypersensitivity reactions to the vaccine.2 Major drawbacks of the skin test are that it requires specialized personnel, needs a second visit to read, and can be troublesome for the tested subjects. Even more important, from an immunological point of view and with consequences for (epidemiological) follow up, is that in-vivo skin testing, by injection of C. burnetii antigens, may induce a cellular and humoral immune response and thus possibly modifies subsequent immunologic measurements. Between 2007 and 2010, the Netherlands faced the largest Q fever outbreak ever reported, with more than 4000 notified human cases of acute Q fever.3 In June 2010, the Dutch health authorities decided to offer vaccination to all individuals at risk for chronic Q fever living in the area of the epidemic. The resulting screening and vaccination took place between January 2011 and July 2011.4 In the current study, we evaluated the impact of skin testing on the measurement of both humoral and cellular C. burnetii specific immune responses in a group of 63 individuals who were screened in a Q fever vaccination campaign and were not subsequently vaccinated.
Subjects and methods Setting The study was performed during a Q fever vaccination campaign in the Netherlands (JanuaryeJuly 2011) and the year thereafter. Before Q fever vaccination, all 1786 eligible individuals were screened by skin testing and Q fever serology: 210 were found to be skin test positive (but seronegative) and 181 were seropositive (with unknown skin test results, because they were not invited for reading). In accordance with the recommendations, both groups were excluded from vaccination because of the increased risk of side effects.2 Individuals from these excluded groups were asked to participate in the postvaccination study. The study was approved by the Central Committee on Research on Human Subjects (CCMO). After informed consent, blood was collected just before, and 6 and 12 months after skin testing to measure the
humoral and cellular specific immune responses to C. burnetii.
Q fever skin test The commercially available Q-vax skin test (CSL, Australia) was used.5 It contains diluted formaldehydeinactivated C. burnetii Henzerling strain phase I-antigens (10 times lower concentration than in Q-vax vaccine). One dose of Q-vax skin test (lot# 0996-06501) was diluted 30 times and 0.1 mL was administered intracutaneously in the forearm according to the product information.6 The local reaction to the skin test was read after seven days, but only if serology was negative. Any localized induration was considered to be positive and led to exclusion from vaccination.
Q fever serology The humoral immune response was assessed by a commercially available immunofluorescence assay (IFA; Focus Diagnostics, Cypress, USA). A positive serology was defined as anti-phase I or phase II C. burnetii IgM/IgG titre 1:32. An increase of titres was defined by two-fold increase or more.
C. burnetii specific IFN-g production Cellular immune response was assessed by measuring invitro C. burnetii specific IFN-g production in whole blood as previously described.7 Net IFN-g production was expressed as the concentration of IFN-g in the C. burnetii stimulated sample minus that in the negative control sample. The assay was considered positive when net IFN-g production was 32 pg/mL, based on previous findings.7
Statistics Mean standard error (SE) IFN-g production was calculated and compared using a paired samples t-test. Median serological titres were calculated for groups and the Wilcoxon matched-pairs signed rank test was used to compare median serological titres at different time points. P < 0.05 was considered significant.
Results Sixty-three individuals (mean age [SD] of 66 [11] years, 76% male) were asked to participate in the study: 35/63 with a positive skin test and 28/63 with positive serology and unknown skin test results, at screening. These were selected from the larger group of eligible individuals, because they lived close to the health care centre where blood was collected. At twelve months, 32/35 and 25/28 of these individuals were available for repeated blood tests.
Please cite this article in press as: Schoffelen T, et al., Immunogenicity of the Q fever skin test, J Infect (2014), http://dx.doi.org/ 10.1016/j.jinf.2014.03.008
Immunogenicity of the Q fever skin test
3
Figure 1 Individual immune responses, both serological and interferon-gamma (IFN-g), of all study subjects before (t Z 0) and at 6 and 12 months after Q fever skin test. The subjects with negative serology but positive skin test (n Z 35) are depicted separately from those with positive serology and unknown skin test (n Z 28) in pre-vaccination screening. Serological response is shown as the anti-C. burnetii phase I and II IgG titre, as measured with immunofluorescence assay. C.b.-induced IFN-g is the net in-vitro IFN-g production in pg/mL after stimulation of blood with C. burnetii Nine Mile minus IFN-g in the unstimulated sample.
Individual immune responses at baseline, 6 and 12 months are shown in Fig. 1. Of the 35 individuals with a positive skin test and negative serology, 15/35 (43%) showed seroconversion after 6 months, and 7/32 (22%) were seropositive after 12 months. The proportions with positive results in the IFN-g assay were 19/35 (54%) at baseline, 26/35 (74%) after 6 months and 22/ 32 (69%) after 12 months. The mean SE specific IFN-g production in this group increased from 185 88 pg/mL (at baseline) to 422 141 pg/mL at 6 months (P Z 0.009) and 223 91 pg/mL at 12 months (P Z 0.17) after skin testing. In the 28 individuals with positive serology in prevaccination screening, an increase of IgG anti-phase I and II antibody titre was observed after the skin test: at 6 months, 21/28 (75%) had an increase in phase I IgG titre, 14/28 (50%) in phase II IgG titre; at 12 months, 11/25 (44%) in phase I IgG and 4/25 (16%) phase II IgG titre. The increase of median titre was 2-fold after 6 months (median phase I IgG from 1:64 to 1:128, P Z 0.0001; phase II IgG from 1:128 to 1:256, P Z 0.001) and this significant increase largely disappeared after 12 months (median phase I IgG 1:64 P Z 0.04; phase II IgG 1:64 P Z 0.20). The proportions with positive results in the IFN-g assay were 22/28 (79%) at baseline, 23/28 (82%) after 6 months and 22/25 (88%) after 12 months. The IFN-g production in this group increased from 301 100 pg/mL (at baseline) to 918 296 pg/mL at 6 months (P Z 0.039) and 500 271 pg/mL 12 months (P Z 0.598) after skin testing.
Discussion Our results showed that the intracutaneous Q fever test, containing a low dose of Q-vax antigen, induces clear C. burnetii-specific immune responses in about half of the
tested individuals with a positive Q fever skin test. In addition, a median 2-fold increase in antibody titre was found in patients with positive serology in pre-vaccination screening. These effects were measurable after 6 months, but seemed to wane at 12 months. Since all individuals in this study had evidence of pre-existing immunity (either a positive skin test or positive serology), this immune response should be considered as a boosting effect. Strikingly, the proportion of individuals who seroconverted at 6 months after a positive skin test (43%), was almost as high as what was previously reported at 6 months after Q fever vaccination with a 300 times higher dose of individuals with a negative skin test (46%).8 In addition, the previously reported mean SE IFN-g production in vaccinated individuals was 22 4 pg/mL (at baseline), 141 21 pg/mL at six months and 94 15 pg/mL at 12 months after Q fever vaccination. Thus, while the IFN-g production was already higher in the skin-test positive subjects than in the vaccinated individuals, vaccination reached similar levels as in the skin-test positive subject at baseline, while the latter reached a mean IFN-g production of more than twice as high. In individuals who were seropositive in pre-vaccination screening, skin testing also led to a boosting effect, increasing the anti-C. burnetii IgG titres as measured after 6, but not after 12 months. A similar boosting effect is described for the tuberculin skin test (TST) on subsequent interferon-gamma release assay (IGRA) responses, often used in combination to diagnose latent tuberculosis.9 This phenomenon makes it difficult for clinicians to discriminate between boosting and conversion of any increase in the IGRA response after TST. The immunogenetic effect of the Q fever skin test has been described before, but has never been quantified. Intracutaneous administration of undiluted Q fever wholecell vaccine has been tried in imprisoned volunteers in
Please cite this article in press as: Schoffelen T, et al., Immunogenicity of the Q fever skin test, J Infect (2014), http://dx.doi.org/ 10.1016/j.jinf.2014.03.008
4 the early 1960s.10 The antibody response appeared to be related to the number of doses of vaccine given, but was more delayed and reached lower titres than after subcutaneous administration. Moreover, five out of six skin test positive individuals, who subsequently were not vaccinated, seroconverted in the follow-up. However, the problem of repeated injection of whole-cell vaccines, is the increased risk for developing adverse reactions. To overcome this problem, Waag et al. used a chemical extraction procedure to develop a vaccine with good immunogenicity and reduced adverse effects upon serial use. They showed in non-immune volunteers that a second subcutaneous vaccination with a chloroform-methanol residue phase I vaccine, 3e6 months after a priming subcutaneous dose, can mount serological and cellular response, whereas one dose did not.11 In this respect, the intracutaneous Q fever test can be seen as a kind of vaccination. In rabies prophylaxis, intradermal administration of cell culture vaccines is an accepted alternative to the standard intramuscular route.12 It only takes one-tenth of the dose that is used intramuscularly to obtain an adequate humoral response.13 The effective skin-associated immune response is most likely due to the extensive local network of dendritic cells and the skin-draining lymph nodes to generate effector T and B cells, leading to long-term protective immunity. We believe that clinicians, immunologists and epidemiologists should be aware of the phenomenon that individuals who have been skin tested, as is the current practice in Q fever pre-vaccination screening, may develop higher anti-phase I C. burnetii IgG antibody titres and a more robust in-vitro IFN-g release. This may lead to false diagnosis of re-infection or even possible chronic Q fever. We also suggest that an in-vitro test, like the C. burnetii specific IFN-g production assay, should be further investigated as an alternative for the Q fever skin test for assessing preexisting immunity.
Acknowledgements This work was supported by Netherlands Organisation for Health Research and Development [grant numbers 205530001, 205520002 to T.Sc. and a Vici grant to M.G.N.].
T. Schoffelen et al.
References 1. Parker NR, Barralet JH, Bell AM. Q fever. Lancet 2006; 367(9511):679e88. 2. Marmion B. Q fever: the long journey to control by vaccination. Med J Aust 2007;186(4):164e6. 3. van der Hoek W, Schneeberger PM, Oomen T, WegdamBlans MC, Dijkstra F, Notermans DW, et al. Shifting priorities in the aftermath of a Q fever epidemic in 2007 to 2009 in The Netherlands: from acute to chronic infection. Euro Surveill 2012;17(3):20059. €mke HC, 4. Isken LD, Kraaij-Dirkzwager M, Bondt PE, Ru Wijkmans C, Opstelten W, et al. Implementation of a Q fever vaccination program for high-risk patients in the Netherlands. Vaccine 2013;31(23):2617e22. 5. Ackland JR, Worswick DA, Marmion BP. Vaccine prophylaxis of Q fever. A follow-up study of the efficacy of Q-Vax (CSL) 1985e1990. Med J Aust 1994;160(11):704e8. 6. Q-VAX Q fever vaccine and Q-VAX skin test. Product information e TGA approved. Australia: CSL Biotherapies; 2008. 7. Schoffelen T, Joosten LA, Herremans T, de Haan AF, € mke HC, et al. Specific interferon g detecAmmerdorffer A, Ru tion for the diagnosis of previous Q fever. Clin Infect Dis 2013; 56(12):1742e51. 8. Schoffelen T, Herremans T, Sprong T, Nabuurs-Franssen M, Wever PC, Joosten LA, et al. Limited humoral and cellular responses to Q fever vaccination in older adults with risk factors for chronic Q fever. J Infect 2013;67(6):565e73. 9. van Zyl-Smit RN, Pai M, Peprah K, Meldau R, Kieck J, Juritz J, et al. Within-subject variability and boosting of T-cell interferon-gamma responses after tuberculin skin testing. Am J Respir Crit Care Med 2009;180(1):49e58. 10. Bell JF, Luoto L, Casey M, Lackman DB. Serologic and skin-test response after Q fever vaccination by the intracutaneous route. J Immunol 1964;93:403e8. 11. Waag DM, England MJ, Bolt CR, Williams JC. Low-dose priming before vaccination with the phase I chloroform-methanol residue vaccine against Q fever enhances humoral and cellular immune responses to Coxiella burnetii. Clin Vaccine Immunol 2008;15(10):1505e12. 12. WHO. Rabies vaccine: WHO position paper. Wkly Epidemiol Rec 2010;85(32):309e20. 13. Briggs DJ, Banzhoff A, Nicolay U, Sirikwin S, Dumavibhat B, Tongswas S, et al. Antibody response of patients after postexposure rabies vaccination with small intradermal doses of purified chick embryo cell vaccine or purified Vero cell rabies vaccine. Bull World Health Organ 2000;78(5):693e8.
Please cite this article in press as: Schoffelen T, et al., Immunogenicity of the Q fever skin test, J Infect (2014), http://dx.doi.org/ 10.1016/j.jinf.2014.03.008
www.elsevierhealth.com/journals/jinf
Immunogenicity of the Q fever skin test Teske Schoffelen a,*, Tineke Herremans b, Tom Sprong c,d, Marrigje Nabuurs-Franssen c, Jos W.M. van der Meer a, Leo A.B. Joosten a, Mihai G. Netea a, Henk A. Bijlmer b, Marcel van Deuren a a
Department of Internal Medicine, Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands b Centre for Infectious Disease Control, National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands c Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, P.O. Box 9015, 6500 GS Nijmegen, The Netherlands d Department of Internal Medicine, Canisius Wilhelmina Hospital, P.O. Box 9015, 6500 GS Nijmegen, The Netherlands Accepted 8 March 2014 Available online - - -
KEYWORDS Q fever; Coxiella burnetii; Skin test; Immunogenicity; Booster effect; Interferon-gamma; Serology; Diagnostic test
Summary Objectives: The Q fever skin test is used to measure cell-mediated immunity to Coxiella burnetii in pre-vaccination screening to exclude individuals with pre-existing immunity. We investigated whether this in-vivo test influences subsequent measurements of immune response. Methods: We assessed the humoral and cellular immune responses before, and 6 and 12 months after skin testing in 63 individuals who were not vaccinated because of either a positive skin test or positive serology in screening. IgG anti-C. burnetii antibodies were measured using immune-fluorescence assay (IFA). The cellular immune response was assessed by measuring invitro C. burnetii-specific interferon (IFN)-g production in blood. Results: Of the 35 subjects with a positive skin test and negative serology, 15/35 (43%) showed seroconversion at 6 months, and 7/32 (22%) seropositivity at 12 months. The mean SE specific IFN-g production in this group increased from 185 88 pg/mL (at baseline) to 422 141 pg/mL at 6 months (P Z 0.009) and 223 91 pg/mL at 12 months (P Z 0.17). Of the 28 subjects with positive serology (and unknown skin test results), 21/28 (75%) showed an increase in IgG anti-phase I titres at 6 months, and 11/25 (44%) at 12 months. The mean SE specific IFN-g production was significantly increased at 6 months, but not at 12 months.
* Corresponding author. Radboud University Medical Center, Department of Internal Medicine, Internal Postal Code 463, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: þ31 24 3618819; fax: þ31 24 3541734. E-mail address: [email protected] (T. Schoffelen). 0163-4453/$36 ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinf.2014.03.008 Please cite this article in press as: Schoffelen T, et al., Immunogenicity of the Q fever skin test, J Infect (2014), http://dx.doi.org/ 10.1016/j.jinf.2014.03.008
2
T. Schoffelen et al. Conclusions: Q fever skin testing causes higher antibody titres and higher in-vitro IFN-g to C. burnetii, and therefore affects subsequent Q fever diagnostics. ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Introduction Q fever is caused by Coxiella burnetii, an intracellular Gram-negative bacterium. Initial infection often goes unnoticed, but in a minority of cases chronic infection develops and can present as endocarditis or vascular wall infection, which leads to high morbidity and mortality.1 Infection is normally diagnosed by serology, by measuring IgM and IgG antibodies against the two different antigenic phases of C. burnetii. Q fever skin testing is used to measure in vivo cellmediated immunity to C. burnetii. Its use is limited to pre-vaccination screening, to exclude individuals with pre-existing immunity who would be at increased risk of hypersensitivity reactions to the vaccine.2 Major drawbacks of the skin test are that it requires specialized personnel, needs a second visit to read, and can be troublesome for the tested subjects. Even more important, from an immunological point of view and with consequences for (epidemiological) follow up, is that in-vivo skin testing, by injection of C. burnetii antigens, may induce a cellular and humoral immune response and thus possibly modifies subsequent immunologic measurements. Between 2007 and 2010, the Netherlands faced the largest Q fever outbreak ever reported, with more than 4000 notified human cases of acute Q fever.3 In June 2010, the Dutch health authorities decided to offer vaccination to all individuals at risk for chronic Q fever living in the area of the epidemic. The resulting screening and vaccination took place between January 2011 and July 2011.4 In the current study, we evaluated the impact of skin testing on the measurement of both humoral and cellular C. burnetii specific immune responses in a group of 63 individuals who were screened in a Q fever vaccination campaign and were not subsequently vaccinated.
Subjects and methods Setting The study was performed during a Q fever vaccination campaign in the Netherlands (JanuaryeJuly 2011) and the year thereafter. Before Q fever vaccination, all 1786 eligible individuals were screened by skin testing and Q fever serology: 210 were found to be skin test positive (but seronegative) and 181 were seropositive (with unknown skin test results, because they were not invited for reading). In accordance with the recommendations, both groups were excluded from vaccination because of the increased risk of side effects.2 Individuals from these excluded groups were asked to participate in the postvaccination study. The study was approved by the Central Committee on Research on Human Subjects (CCMO). After informed consent, blood was collected just before, and 6 and 12 months after skin testing to measure the
humoral and cellular specific immune responses to C. burnetii.
Q fever skin test The commercially available Q-vax skin test (CSL, Australia) was used.5 It contains diluted formaldehydeinactivated C. burnetii Henzerling strain phase I-antigens (10 times lower concentration than in Q-vax vaccine). One dose of Q-vax skin test (lot# 0996-06501) was diluted 30 times and 0.1 mL was administered intracutaneously in the forearm according to the product information.6 The local reaction to the skin test was read after seven days, but only if serology was negative. Any localized induration was considered to be positive and led to exclusion from vaccination.
Q fever serology The humoral immune response was assessed by a commercially available immunofluorescence assay (IFA; Focus Diagnostics, Cypress, USA). A positive serology was defined as anti-phase I or phase II C. burnetii IgM/IgG titre 1:32. An increase of titres was defined by two-fold increase or more.
C. burnetii specific IFN-g production Cellular immune response was assessed by measuring invitro C. burnetii specific IFN-g production in whole blood as previously described.7 Net IFN-g production was expressed as the concentration of IFN-g in the C. burnetii stimulated sample minus that in the negative control sample. The assay was considered positive when net IFN-g production was 32 pg/mL, based on previous findings.7
Statistics Mean standard error (SE) IFN-g production was calculated and compared using a paired samples t-test. Median serological titres were calculated for groups and the Wilcoxon matched-pairs signed rank test was used to compare median serological titres at different time points. P < 0.05 was considered significant.
Results Sixty-three individuals (mean age [SD] of 66 [11] years, 76% male) were asked to participate in the study: 35/63 with a positive skin test and 28/63 with positive serology and unknown skin test results, at screening. These were selected from the larger group of eligible individuals, because they lived close to the health care centre where blood was collected. At twelve months, 32/35 and 25/28 of these individuals were available for repeated blood tests.
Please cite this article in press as: Schoffelen T, et al., Immunogenicity of the Q fever skin test, J Infect (2014), http://dx.doi.org/ 10.1016/j.jinf.2014.03.008
Immunogenicity of the Q fever skin test
3
Figure 1 Individual immune responses, both serological and interferon-gamma (IFN-g), of all study subjects before (t Z 0) and at 6 and 12 months after Q fever skin test. The subjects with negative serology but positive skin test (n Z 35) are depicted separately from those with positive serology and unknown skin test (n Z 28) in pre-vaccination screening. Serological response is shown as the anti-C. burnetii phase I and II IgG titre, as measured with immunofluorescence assay. C.b.-induced IFN-g is the net in-vitro IFN-g production in pg/mL after stimulation of blood with C. burnetii Nine Mile minus IFN-g in the unstimulated sample.
Individual immune responses at baseline, 6 and 12 months are shown in Fig. 1. Of the 35 individuals with a positive skin test and negative serology, 15/35 (43%) showed seroconversion after 6 months, and 7/32 (22%) were seropositive after 12 months. The proportions with positive results in the IFN-g assay were 19/35 (54%) at baseline, 26/35 (74%) after 6 months and 22/ 32 (69%) after 12 months. The mean SE specific IFN-g production in this group increased from 185 88 pg/mL (at baseline) to 422 141 pg/mL at 6 months (P Z 0.009) and 223 91 pg/mL at 12 months (P Z 0.17) after skin testing. In the 28 individuals with positive serology in prevaccination screening, an increase of IgG anti-phase I and II antibody titre was observed after the skin test: at 6 months, 21/28 (75%) had an increase in phase I IgG titre, 14/28 (50%) in phase II IgG titre; at 12 months, 11/25 (44%) in phase I IgG and 4/25 (16%) phase II IgG titre. The increase of median titre was 2-fold after 6 months (median phase I IgG from 1:64 to 1:128, P Z 0.0001; phase II IgG from 1:128 to 1:256, P Z 0.001) and this significant increase largely disappeared after 12 months (median phase I IgG 1:64 P Z 0.04; phase II IgG 1:64 P Z 0.20). The proportions with positive results in the IFN-g assay were 22/28 (79%) at baseline, 23/28 (82%) after 6 months and 22/25 (88%) after 12 months. The IFN-g production in this group increased from 301 100 pg/mL (at baseline) to 918 296 pg/mL at 6 months (P Z 0.039) and 500 271 pg/mL 12 months (P Z 0.598) after skin testing.
Discussion Our results showed that the intracutaneous Q fever test, containing a low dose of Q-vax antigen, induces clear C. burnetii-specific immune responses in about half of the
tested individuals with a positive Q fever skin test. In addition, a median 2-fold increase in antibody titre was found in patients with positive serology in pre-vaccination screening. These effects were measurable after 6 months, but seemed to wane at 12 months. Since all individuals in this study had evidence of pre-existing immunity (either a positive skin test or positive serology), this immune response should be considered as a boosting effect. Strikingly, the proportion of individuals who seroconverted at 6 months after a positive skin test (43%), was almost as high as what was previously reported at 6 months after Q fever vaccination with a 300 times higher dose of individuals with a negative skin test (46%).8 In addition, the previously reported mean SE IFN-g production in vaccinated individuals was 22 4 pg/mL (at baseline), 141 21 pg/mL at six months and 94 15 pg/mL at 12 months after Q fever vaccination. Thus, while the IFN-g production was already higher in the skin-test positive subjects than in the vaccinated individuals, vaccination reached similar levels as in the skin-test positive subject at baseline, while the latter reached a mean IFN-g production of more than twice as high. In individuals who were seropositive in pre-vaccination screening, skin testing also led to a boosting effect, increasing the anti-C. burnetii IgG titres as measured after 6, but not after 12 months. A similar boosting effect is described for the tuberculin skin test (TST) on subsequent interferon-gamma release assay (IGRA) responses, often used in combination to diagnose latent tuberculosis.9 This phenomenon makes it difficult for clinicians to discriminate between boosting and conversion of any increase in the IGRA response after TST. The immunogenetic effect of the Q fever skin test has been described before, but has never been quantified. Intracutaneous administration of undiluted Q fever wholecell vaccine has been tried in imprisoned volunteers in
Please cite this article in press as: Schoffelen T, et al., Immunogenicity of the Q fever skin test, J Infect (2014), http://dx.doi.org/ 10.1016/j.jinf.2014.03.008
4 the early 1960s.10 The antibody response appeared to be related to the number of doses of vaccine given, but was more delayed and reached lower titres than after subcutaneous administration. Moreover, five out of six skin test positive individuals, who subsequently were not vaccinated, seroconverted in the follow-up. However, the problem of repeated injection of whole-cell vaccines, is the increased risk for developing adverse reactions. To overcome this problem, Waag et al. used a chemical extraction procedure to develop a vaccine with good immunogenicity and reduced adverse effects upon serial use. They showed in non-immune volunteers that a second subcutaneous vaccination with a chloroform-methanol residue phase I vaccine, 3e6 months after a priming subcutaneous dose, can mount serological and cellular response, whereas one dose did not.11 In this respect, the intracutaneous Q fever test can be seen as a kind of vaccination. In rabies prophylaxis, intradermal administration of cell culture vaccines is an accepted alternative to the standard intramuscular route.12 It only takes one-tenth of the dose that is used intramuscularly to obtain an adequate humoral response.13 The effective skin-associated immune response is most likely due to the extensive local network of dendritic cells and the skin-draining lymph nodes to generate effector T and B cells, leading to long-term protective immunity. We believe that clinicians, immunologists and epidemiologists should be aware of the phenomenon that individuals who have been skin tested, as is the current practice in Q fever pre-vaccination screening, may develop higher anti-phase I C. burnetii IgG antibody titres and a more robust in-vitro IFN-g release. This may lead to false diagnosis of re-infection or even possible chronic Q fever. We also suggest that an in-vitro test, like the C. burnetii specific IFN-g production assay, should be further investigated as an alternative for the Q fever skin test for assessing preexisting immunity.
Acknowledgements This work was supported by Netherlands Organisation for Health Research and Development [grant numbers 205530001, 205520002 to T.Sc. and a Vici grant to M.G.N.].
T. Schoffelen et al.
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Please cite this article in press as: Schoffelen T, et al., Immunogenicity of the Q fever skin test, J Infect (2014), http://dx.doi.org/ 10.1016/j.jinf.2014.03.008
