Altered interferon-gamma response in patients with Q-fever fatigue syndrome

Q Fever Fatigue Syndrome (QFS) is a well documented state of prolonged fatigue,
following acute Q fever. Up to 20% of patients that are diagnosed with acute Q
fever will develop QFS, leading to a substantial burden for the affected
patients. To date, no diagnostic test is available to diagnose QFS directly.
The diagnosis is based on clinical criteria. Serology is used to exclude
chronic Q fever and to confirm a past Q fever infection, but cannot
differentiate between past infection and QFS. Serology can also become negative
several years after the infection while the patients are still suffering from
QFS. Recently our group developed a C. burnetii-specific whole-blood IFNγ
production assay, which is a promising diagnostic tool for C. burnetii
infection, with similar performance and practical advantages over serology. In
addition, a high IFNγ/IL-2 ratio appeared to be indicative of chronic Q-fever,
and may be a useful diagnostic marker for chronic Q-fever and treatment
monitoring. Based on these results, our group determined the value of these
tests in QFS patients. It appeared that IFNγ production assays were able to
differentiate QFS patients from healthy seropositive controls, and IFNγ/IL-2
ratio (adding IL-2 production assays) was able to differentiate QFS patients
from chronic Q-fever patients. To further investigate the value of this test,
we aim to evaluate C. burnetii-specific whole-blood IFNγ production assays (and
C. burnetii-specific whole-blood IL-2 production assays) in QFS patients that
previously participated in the Qure study that have either recovered from their
complaints, or are still experiencing complaints. This enables us to
investigate the value of IFNγ and IL-2 production as biologic parameters of
recovery. Using the subscale fatigue of the Checklist Individual Strength (CIS)
to assess at time of diagnosis and after treatment, we can specifically
investigate the correlation between level of fatigue, i.e. recovered versus
non-recovered, and IFNγ and IL-2 production.

This study will investigate the value of C. burnetii-specific whole-blood IFNγ
and IL-2 production assays in QFS patients that have either recovered, or are
still substantially fatigued. This will show us if these assays can serve as
potential biologic parameters in the recovery of QFS, and will help us
understand if immunopathologic mechanisms play a role in the complaints that
are seen in QFS.

We will determine the value of C. burnetii-specific whole-blood IFNγ and IL-2
production assays in QFS patients that have either recovered, or are still
substantially fatigued. Severe fatigue (and therefore no recovery) is defined
as a score >= 35 on the subscale fatigue severity of the CIS. Recovery is
defined as a score < 35 on the subscale fatigue severity of the CIS + clinical
significant change, compared to baseline score on the subscale fatigue severity
of the CIS.
After informed consent is obtained, blood will be drawn in 1 Heparine tube of
5ml and 1 Serum tube of 3ml. Whole blood will be divided over 3 0.5ml tubes,
each tube will then be stimulated with either C. burnetii Nine Mile RSA 493
Phase I (CbNM), Phytohaemagglutinin (PHA) as a positive control, or Roswell
Park Memorial Institute medium (RPMI) as a negative control. Samples will be
incubated for 24 hours at 37 *C, after which supernatants will be collected and
stored at -20 *C until analysis. IFNγ and IL-2 production will be measured by
means of ELISA (IFN γ: Pelikine compact, Sanquin, Amsterdam, the Netherlands.)
and multiplex beads assay (Merck Millipore, Billerica, MA, USA), according to
the manufacturer*s instructions. Additionally, levels of CXCL 8/9/10/11 will be
measured in unstimulated serum samples by means of multiplex beads assay
(Bio-Rad, Haryana, India).

The duration of this study is 1 year. Patients will be recruited among previous
participants of the Qure study, performed in the Radboudumc, Nijmegen. In this
study, the effect of three different treatment regimes, i.e. cognitive
behavioral therapy (CBT), doxycycline and placebo, was determined. We aim to
recruit only those who received CBT or a placebo, since tetracyclines have the
potential to influence T-cell activation and could therefore interfere with
IFNγ and IL-2 production assays. Before and after treatment, patients were
asked to fill out the CIS (subscale on fatigue) questionnaire. The outcome of
this questionnaire can be used as an indicator of recovery in our study.
However, since the post-treatment results are most likely dated in several
cases, patients will be asked to fill out this questionnaire once more,
indicating whether or not a change in recovery has occurred. Within the next
few months (which is still in the one-year follow-up period of the Qure study),
patients will receive notification from the coordinating investigator on the
outcome of this study. At that time, we aim to inform patients about our study
by sending them additional information in a return envelope, providing the
option to recline from being contacted for participation in our study. If no
recline has been received after two weeks, patients will be asked by telephone
if they are willing to participate in our study.

Burden: One time collection of blood (1 Heparine tube of 5ml and 1 Serum tube
of 3ml) + filling out CIS questionnaire (subscale on fatigue).

Risk: No risks other than local hematoma and vasovagal collapse are related to
venous puncture.

Benefit: There will be no direct benefits for the subjects enrolled in this
study.