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Editorial Commentary: A New Assay: Specific Interferon- Detection for the Diagnosis of Previous Q Fever

  • Australian Rickettsial Reference Laboratory, Australia
A New Assay: Specic Interferon-γDetection for the
Diagnosis of Previous Q Fever
Stephen R. Graves
Microbiology, Pathology NorthHunter, New South Wales Health Pathology and Australian Rickettsial Reference Laboratory, Geelong, Victoria, Australia
(See the Major Article by Schoffelen et al on pages 174251.)
Exposure to a microbe may be either
clinically apparent (ie, the patient is sick)
or not (ie, latent infection). In either
case, antibodies are usually synthesized
by the B-cell arm (humoral immunity) of
the patients adaptive immune response.
This is the marker of exposure detected
by most serological tests carried out in
diagnostic laboratories. However, the
other component of the adaptive immune
system, the T-cell (or cell-mediated) im-
mune response, is equally important to
the patients recovery and survival. How-
ever, this aspect of the immune response
is rarely used to diagnose infection as T
lymphocytes are involved, rather than
antibodies, and they are more difcult to
use in routine laboratory testing. Never-
theless, cell-mediated immunity (CMI) is
both sensitive and specic, both features
being the hallmarks of a good diagnostic
The article by Schoffelen et al [1]in
this issue of Clinical Infectious Diseases
uses the CMI arm of the patients adap-
tive immune response to diagnose Q
fever, or more correctly, to diagnose pre-
vious exposure to Coxiella burnetii, the
causative bacterium.
When T lymphocytes in the patient
posited in the skin, they are attracted to
the site and undergo antigen-stimulated
proliferation while in the process of at-
tackingthe invading microbe (antigen).
This produces an induration (lump) in the
skin. One of the key cytokines involved in
this process is interferon gamma (IFN-γ).
If the patients T lymphocytes are
exposed to antigen in vitro, IFN-γis pro-
duced and can be measured. This is the
basis of the assay developed by the
authors. It is a good assay (sensitive and
specic) but will probably never take
over from the current serological assays
for the simple reason that T lymphocytes
in blood are more difcult to work with
in the diagnostic laboratory than are anti-
bodies in serum. Nevertheless, it is an ad-
ditional diagnostic test and could be very
useful in certain patients with difcult-
to-interpret serology results.
It would be well worth developing as a
routine assay in diagnostic reference lab-
oratories in countries where Q fever is a
problemand this is probably everycoun-
try in the world, except New Zealand
(where no cases have ever been diag-
nosed). Countries that have periodic out-
breaks, such as the Netherlands and
Germany, may nd such an assay to
be a very valuable additional tool in
controlling Q fever. Countries with high
levels of background Q fever (but rarely
large outbreaks because of different local
animal husbandry practices), such as Aus-
tralia, may also nd it useful. Unfortunate-
ly, where large distances are involved in
transporting patient samples to the diag-
nostic laboratory, the assay may not be
useful, as the patients T lymphocytes (as
distinct from their antibodies) may not
survive the long trip to the laboratory. For
this new assay, it would be important to
know the maximum time available to get
blood from the patient to the laboratory.
In a related diagnostic test for tuberculo-
sis, it is 12 hours, greatly limiting the use-
fulness of the assay in Australia.
Interestingly, a similar assay was used
many years ago in Australia when the
human Q fever vaccine (QVAX) was being
developed and tested, to show that vacci-
nees developed a CMI response [26].
Schoffelen et als results show that skin
testing, serological testing, and their new
T-cell in vitro assay give essentially the
same diagnostic results, which is very
pleasing. One area of diagnostic difculty
in Q fever is the accurate diagnosis of the
postQ fever fatigue syndrome.There
appears to be no serological pattern that
correlates with it. It would be interesting
to know if there were an INF-γrelease
assay that could diagnose it. That would
be of great practical benet to the prac-
ticing medical microbiologist. Would
Australian doctors and laboratories use
Received 12 February 2013; accepted 19 February 2013;
electronically published 5 March 2013.
Correspondence: Stephen R. Graves, Microbiology, Patholo-
gy NorthHunter, New South Wales Health Pathology, Locked
Bag 1, Hunter Region Mail Centre, 2310, Australia (stephen.
Clinical Infectious Diseases 2013;56(12):17523
© The Author 2013. Published by Oxford University Press
on behalf of the Infectious Diseases Society of America. All
rights reserved. For Permissions, please e-mail: journals.
DOI: 10.1093/cid/cit132
1752 CID 2013:56 (15 June) EDITORIAL COMMENTARY
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this new assay in prevaccination testing?
Yes, because the current skin test and
serological tests may both give false-
negative results, with the result that the
patient is immunized and may develop
an adverse reaction to QVAX. But would
the greater sensitivityof the new assay
result in some normal person not being
vaccinated (due to a false-positive reac-
tion) and then being susceptible to Q
fever in their workplace? Possibly. I have
seen several cases in which Q fever has
occurred in patients who were refused
vaccination on the basis of faulty (false-
positive) pretesting. The answer to this
conundrum is to develop a better Q fever
vaccine that does not require the patient
to be pretested.
Potential conicts of interest. Author certi-
es no potential conicts of interest.
The author has submitted the ICMJE Form
for Disclosure of Potential Conicts of Interest.
Conicts that the editors consider relevant to the
content of the manuscript have been disclosed.
1. Schoffelen T, Joosten LAB, Herremans T, et al.
Specic interferon γdetection for the diagno-
sis of previous Q fever [published online
ahead of print 5 March 2013]. Clin Infect Dis
2013; 56:174251.
2. Penttila IA, Harris RJ, Storm P, Haynes D,
Worswick DA, Marmion BP. Cytokine dys-
regulation in the post-Q-fever fatigue syn-
drome. QJM 1998; 91:54960.
3. Marmion BP, Ormsbee RA, Kyrkou M, et al.
Vaccine prophylaxis of abattoirassociated Q
fever: eight years experience in Australian ab-
attoirs. Epidemiol Infect 1990; 104:27587.
4. Izzo AA, Marmion BP, Worswick DA.
Markers of cell-mediated immunity after vac-
cination with an inactivated whole cell Q fever
vaccine. J Infect Dis 1988; 157:7819.
5. Izzo AA, Marmion BP, Hackstadt T. Analysis
of the cells involved in the lymphoproliferative
response to Coxiella burnetii antigens. Clin
Exp Immunol 1991; 85:98108.
6. Izzo AA, Marmion BP. Variation in interfer-
on-gamma responses to Coxiella burnetii anti-
gens with lymphocytes from vaccination or
naturally infected subjects. Clin Exp Immunol
1993; 94:50715.
EDITORIAL COMMENTARY CID 2013:56 (15 June) 1753
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... Overall, the IGRA identified more individuals as having prior exposure than did the skin test or serology and amongst those individuals who passed the pre-vaccination screen based on IFA and skin test there was a trend for more common local adverse reactions in those with high pre-vaccination IFNg responses (22). Based on these data, IGRA has been proposed as a more suitable tool for prevaccination exposure screening (40,41). Additional arguments that have been raised for using an IGRA for pre-vaccination screening are that in contrast to IFA, the IGRA has an internal negative and positive control, continuous scale readout and is not prone to inter-operator interpretation differences (22). ...
... Additional arguments that have been raised for using an IGRA for pre-vaccination screening are that in contrast to IFA, the IGRA has an internal negative and positive control, continuous scale readout and is not prone to inter-operator interpretation differences (22). And in contrast to the skin test, the IGRA does not require a follow-up visit, is not reactogenic and does not boost immune responses (40,41). Consequently, the Q-detect IGRA was incorporated in a 2016 pilot study to compare detection of prior C. burnetii exposure by IGRA, intradermal skin test and four clinically used serological assays (42). ...
... Moreover, there was poor correspondence between the other clinically used tests. Followup studies in larger groups are now needed to confirm these results and to assess the cut-off with which the IGRA could be implemented for pre-vaccination screening to avoid unnecessarily excluding individuals from vaccination (40). Availability of the new ready-to-use IGRA format will greatly facilitate such studies and real-life implementation of the IGRA in a routine diagnostic setting. ...
Full-text available
For the zoonotic disease Q fever, serological analysis plays a dominant role in the diagnosis of Coxiella burnetii infection and in pre-screening for past exposure prior to vaccination. A number of studies suggest that assessment of C. burnetii-specific T-cell IFNγ responses may be a more sensitive tool to assess past exposure. In this study, we assessed the performance of a whole blood C. burnetii IFNγ release assay in comparison to serological detection in an area of high Q fever incidence in 2014, up to seven years after initial exposure during the Dutch Q fever outbreak 2007-2010. In a cohort of >1500 individuals from the Dutch outbreak village of Herpen, approximately 60% had mounted IFNγ responses to C. burnetii. This proportion was independent of the Coxiella strain used for stimulation and much higher than the proportion of individuals scored sero-positive using the serological gold standard immunofluorescence assay. Moreover, C. burnetii-specific IFNγ responses were found to be more durable than antibody responses in two sub-groups of individuals known to have sero-converted as of 2007 or previously reported to the municipality as notified Q fever cases. A novel ready-to-use version of the IFNγ release assay assessed in a subgroup of pre-exposed individuals in 2021 (10-14 years post exposure) proved again to be more sensitive than serology in detecting past exposure. These data demonstrate that C. burnetii-induced IFNγ release is indeed a more sensitive and durable marker of exposure to C. burnetii than are serological responses. In combination with a simplified assay version suitable for implementation in routine diagnostic settings, this makes the assessment of IFNγ responses a valuable tool for exposure screening to obtain epidemiological data, and to identify previously exposed individuals in pre-vaccination screens.
... The Q-detect test (Innatoss Laboratories BV, Netherlands) has a sensitivity of 93% and specificity of greater than 90% but is not currently available for human clinical diagnostic testing [80]. Advantages of the IGRA assay over the IFA include elimination of inter-assay and inter-laboratory variability [81]. However, the IGRA is more time-consuming, and unlike samples for serological testing, which can be stored frozen, whole blood for measurement of cellular immunity must be fresh (<12 hrs.), which limits its usefulness in certain diagnostic situations [81]. ...
... Advantages of the IGRA assay over the IFA include elimination of inter-assay and inter-laboratory variability [81]. However, the IGRA is more time-consuming, and unlike samples for serological testing, which can be stored frozen, whole blood for measurement of cellular immunity must be fresh (<12 hrs.), which limits its usefulness in certain diagnostic situations [81]. ...
Full-text available
Q fever is a disease caused by the bacterial pathogen Coxiella burnetii. This hardy organism can easily spread long distances in the wind, and only a few infectious aerosolized particles are necessary to cause serious illness. Presentations of Q fever disease can be wide ranging, allowing it to masquerade as other illnesses, highlighting the importance of laboratory testing for diagnosis and treatment. This review summarizes Q fever's epidemiology and clinical presentations and presents classical laboratory diagnostic assays and novel approaches to detecting this troubling disease.
Full-text available
Background: Current practice for diagnosis of Q fever, caused by the intracellular pathogen Coxiella burnetii, relies mainly on serology and, in prevaccination assessment, on skin tests (STs), which both have drawbacks. In this study, C. burnetii-specific interferon γ (IFN-γ) production was used as a new diagnostic tool for previous Q fever, circumventing most of these drawbacks. Our aim was to compare this test to serology and ST. Methods: One thousand five hundred twenty-five individuals from an endemic area with a risk for chronic Q fever were enrolled. IFN-γ production was measured after in vitro stimulation of whole blood with C. burnetii antigens. Various formats using different C. burnetii antigens were tested. Serology and ST were performed in all individuals. Results: In all assay formats, C. burnetii-specific IFN-γ production was higher (P < .0001) in seropositive or ST-positive subjects than in seronegative and ST-negative subjects. Whole blood incubated for 24 hours with C. burnetii Nine Mile showed optimal performance. After excluding subjects with equivocal serology and/or borderline ST results, IFN-γ production was 449 ± 82 pg/mL in the positive individuals (n = 219) but only 21 ± 3 pg/mL in negative subjects (n = 908). Using Bayesian analysis, sensitivity and specificity (87.0% and 90.2%, respectively) were similar to the combination of serology and ST (83.0% and 95.6%, respectively). Agreement with the combination of serology and ST was moderate (84% concordance; κ = 0.542). Conclusions: Specific IFN-γ detection is a novel diagnostic assay for previous C. burnetii infection and shows similar performance and practical advantages over serology and ST. Future studies to investigate the clinical value in practice are warranted.
Full-text available
During the period 1981–8 a clinical trial of a Q fever vaccine (Q-vax; Commonwealth Serum Laboratories, Melbourne) has been conducted in abattoir workers and other at-risk groups in South Australia. Volunteers in four abattoirs and visitors to the abattoirs were given one subcutaneous dose of 30 μg of a formalin-inactivated, highly-purified Coxiella burnetii cells, Henzerling strain, Phase 1 antigenic state, in a volume of 0·5 ml. During the period, over 4000 subjects have been vaccinated and the programme continues in the abattoirs and related groups. ‘Common’ reactions to the vaccine comprised tenderness and erythema, rarely oedema at the inoculation site and sometimes transient headache. Two more serious ‘uncommon’ reactions, immune abscess at the inoculation site, were observed in two subjects, and two others developed small subcutaneous lumps which gradually dispersed without intervention. Protective efficacy of the vaccine appeared to be absolute and to last for 5 years at least. Eight Q fever cases were observed in vaccinees, but all were in persons vaccinated during the incubation period of a natural attack of Q fever before vaccine-induced immunity had had time (≥ 13 days after vaccination) to develop. On the other hand, 97 Q fever cases were detected in persons working in, or visiting the same abattoir environments. Assays for antibody and cellular immunity showed an 80–82% seroconversion after vaccination, mostly IgM antibody to Phase 2 antigen, in the 3 months after vaccination. This fell to about 60%, mostly IgG antibody to Phase 1 antigen, after 20 months. On the other hand, 85–95% of vaccinees developed markers of cell mediated immunity as judged by lymphoproliferative responses with C. burnetii antigens; these rates remained elevated for at least 5 years. The Q fever vaccine, unlike other killed rickettsial vaccines, has the property of stimulating long-lasting T lymphocyte memory and this may account for its unusual protective efficacy as a killed vaccine.
Vaccination with an inactivated, whole cell, Q fever vaccine (Q-vax) induces lasting antibody conversion and a positive delayed-type hypersensitivity (DTH) skin reaction in about 60% of recipients but a long-lasting positive lymphoproliferative or mitogenic response to C. burnetii antigens with peripheral blood mononuclear cells (PBMC) in 85-95% of subjects. Analysis of the lymphoproliferative response to C. burnetii antigens has now been made by fractionation-reconstitution experiments with PBMC from vaccines, from past infections, and from healthy controls. The major contributor to the response in immune subjects proved to be the T lymphocyte. T cells were stimulated by both the phase I and phase II antigens of two prototype strains of C. burnetii and responses were greatly amplified by addition of IL-2. Similar T lymphocyte stimulation profiles were obtained with the 'Priscilla' strain of C. burnetii which represents a different biotype of Coxiella isolated from Q fever endocarditis; Q-vax is therefore likely to protect against endocarditis strains. Fractionation-reconstitution experiments with T and B cells from vaccines and subjects infected in the past, using various antigenic or haptenic fractions from C. burnetii indicate that protein, non-lipopolysaccharide components of the organism are responsible for the mitogenic response of immune T cells. However, the role of the lipopolysaccharide in the protective immunogen has still to be defined.
A clinical trial of Q fever vaccine in four South Australian abattoirs showed apparently complete protection against natural infection; however, only 50%–60% of vaccinees developed complement-fixing or immunofluorescent antibody after vaccination. Cell-mediated immunity to Coxiella burnetii antigens, as measured by an index of lymphoproliferative responses (LSI) of peripheral blood mononuclear cells, was therefore assessed. Eighty-five percent of 13 subjects with “low risk” of exposure to Q fever and with an initially negative LSI converted to a positive LSI after vaccination; conversion was noted nine to 13 days after vaccination, and positive values were obtained for at least 96 d. Only 35% of this group seroconverted. In a “high-risk” group (abattoir workers), higher rates of positive LSI (>95%) and of antibody (50%–70%) were observed after vaccination; >95% of vaccinees in this group, who had been vaccinated five years previously, had positive LSI values.
Previous work in our laboratory has shown that lymphocytes from persons vaccinated with a formalin-inactivated Phase I Q fever vaccine (Q-Vax CSL Ltd) show a mitogenic response to Coxiella burnetii antigens. The mitogenic response is the sum of that from various subsets of CD4+, T helper cells, CD8+ T cells and probably B cells. It does not distinguish between T helper cell responses leading to formation of interferon-gamma (IFN-gamma)--a cytokine responsible for clearing intracellular infection with C. burnetii organisms--and responses of other T cell subsets which may produce disease-enhancing cytokines. The present study analyses (i) the capacity of Q-Vax to induce T cell sensitization which leads to IFN-gamma responses on antigen stimulation, and (ii) the immunomodulatory, (down-regulatory) effects of the Phase I lipopolysaccharide (LPS) of the organism, which interacts with monocyte/macrophages to limit IL-2 production and production of IFN-gamma by sensitized T lymphocytes.
The post-Q-fever fatigue syndrome (QFS) (inappropriate fatigue, myalgia and arthralgia, night sweats, changes in mood and sleep patterns) follows about 20% of laboratory-proven, acute primary Q-fever cases. Cytokine dysregulation resulting from chronic immune stimulation and modulation by persistence of Coxiella burnetii cells or their antigens is hypothesized. We studied cytokine release patterns of peripheral blood mononuclear cells (PBMC) stimulated with various ligands in short-term culture, from 18 patients with active QFS, and 27 controls: six with resolving QFS, five who had had acute primary Q-fever without subsequent QFS, eight healthy Q-fever vaccinees and eight healthy subjects without Q-fever antibody. Conditioned media (CM) from PBMC stimulated in short-term culture with Q-fever antigens, PHA or measles antigen (as an unrelated antigen) were assayed for IL-2, IL-4, IL-5, IL-6, IL-10 and IFN gamma by AgEIA, and for IL-1 and TNF alpha/beta by bioassay. Aberrant cytokine release patterns were observed with PBMC from QFS patients when stimulated with Q-fever antigens: an accentuated release of IL-6 which was significantly [p = 0.01, non-parametric one-way analysis of variance (ANOVA)] in excess of medians for all four control groups. With IL-2, the number of responders in the active QFS group was decreased relative to control groups (Fisher's exact test, p = 0.01) whereas the number of IFN gamma responders was increased (Fisher's exact test, p = 0.0008). Significant correlations were observed between concentrations of IL-6 in CM, total symptom scores, and scores for other key symptoms.