Markers of exposure to spotted fever rickettsiae in patients with chronic illness, including fatigue, in two Australian populations
+ Author Affiliations
- Address correspondence to Dr S. Graves, Australian Rickettsial Reference Laboratory, Barwon Biomedical Research, the Geelong Hospital, Geelong, Victoria, Australia 3220. email: Stephen.Graves@hnehealth.nsw.gov.au
- Received June 23, 2007.
- Revision received October 4, 2007.
Abstract
Background: Some investigators believe that a proportion of chronically unwell patients, many with fatigue, have an underlying rickettsial
disease.
Aim: To investigate the prevalence of markers of rickettsial infection in patients with chronic illnesses.
Design: Observational study.
Methods: A 526 patient
cohort with chronic illnesses from Melbourne, Australia and 400 control
patients from Newcastle, Australia
were assessed using serology, culture and PCR for
the detection of rickettsiae. Rickettsial serology was performed on
another
cohort of 581 chronically unwell patients (and 34
non-fatigued patients from the same practice) from Adelaide, Australia.
Results: Of the Melbourne patient cohort, 14/526 (3%) were real-time PCR positive for rickettsial DNA compared to none of the 400
control patients (P < 0.001). Of these 14 patients, Rickettsia honei strain ‘marmionii’ was detected in 5 and isolated from 2. Rickettsaemia was seasonal, with more in winter (8/145; P < 0.03) and less in spring (0/143; P
< 0.03). Positive rickettsial serology titres of ⩾1:256 were seen in
206 (39%) patients. Of the Adelaide patient cohort,
238/581 (41%) had positive rickettsial antibodies
titres. Of the 34 control sera, 5 (15%) were serologically positive (P < 0.002). Both Melbourne and Adelaide patient cohorts had significantly higher seropositivity than the Newcastle control
cohort (3/399; P < 0.0001).
Conclusions: In patients
with chronic illness, rickettsial DNA in peripheral blood and/or
rickettsial seropositivity may represent exposure
to rickettsiae or underlying rickettsial diseases.
It is not known whether the presence of rickettsiae is causally related
to the patients’ chronic illnesses, or reactivation
of a latent rickettsial infection.
Introduction
Chronic illness can be frustrating for patient and doctor alike. This is only accentuated when no diagnosis can be made.1–3 Often the patient feels as if no one can give them a credible diagnosis and they often seek assistance from ‘alternative
medicine’.
It has been claimed that some patients with chronic illnesses, including those with post-infection fatigue syndrome, have
underlying rickettsial disease, although control group investigations are lacking.4 Although this view requires further evidence, it is well known that an infectious disease may lead to chronic infection and
chronic illness. Such infections include Epstein-Barr Virus (EBV),5 Cytomegalovirus (CMV),6 Parvovirus B-19,7 Barmah Forest Virus,8 Human Herpes type 6 Virus (HHV-6),9 Brucellosis (including Brucella melitenis),10 Lyme disease (Borrelia burgdorferi),11–12 Mycoplasma species (especially M. pneumoniae and M. fermentans)13 and Chlamydia pneumoniae.14 Post-infection fatigue syndrome after Q-fever (Coxiella burnetii) has also been well documented in both adults15–18 and children.19 It has associations with cytokine aberrations,20 specific immune system genes alleles21 and the persistence of C. burnetii DNA.22–24
The microbial agents listed above grow
mainly within an intracellular environment or in the case of
mycoplasmas, on the cell
surface. Rickettsiae are small obligate
intracellular bacteria usually transmitted to a human via the bite of an
arthropod,
often a tick. Rickettsial disease usually has an
acute onset and has major symptoms of headache, rash, fever, myalgia,
arthralgia
and fatigue.25
Most cases of acute rickettsial disease resolve without complication after antibiotic therapy, however, cases of Brill–Zinsser
disease, a recurrent form of epidemic typhus (Rickettsia prowazekii), are well documented decades after the initial infection.26 Cases of persistent scrub typhus (Orientia tsutsugamushi) infections in humans27 and rickettsial spotted fever infections in dogs28 have also been documented. Thus it is reasonable to speculate that spotted fever group (SFG) rickettsia may also be able
to cause a chronic infection or be associated with a chronic illness.
To test the hypothesis that some
chronically unwell patients have underlying rickettsial disease, two
groups of chronically
ill patients were studied. The first cohort was
from Victoria, Australia, under the care of Dr Geoff Kemp was tested for
the
presence of rickettsia, by in vitro
culture, rickettsial DNA via real-time PCR and serology. The second
cohort of patients from South Australia under the care
of Dr John Graham was tested serologically for
rickettsial antibodies but not by rickettsial PCR or culture.
Methods
A patient cohort of 526 was recruited from the practice of Dr Geoff Kemp in Camberwell, Victoria from February 2003 to February
2005 (‘Melbourne cohort’). Patients came from both urban and rural areas. Patients were added to the study ad seriatim
as they presented. Dr Kemp's usual diagnostic tests included
investigations on peripheral blood for rickettsial DNA (by real-time
PCR), viable rickettsia (by culture) and
rickettsial serological tests. The patients of Dr Kemp had actively
sought his advice
due to his expertise in treating chronic conditions
and had thus come from all over Victoria. As such they did not
represent
a typical patient cohort.
A control group of 400 peripheral blood
samples were randomly selected from another medical practice in
Newcastle, New South
Wales and subjected to the same rickettsial
real-time PCR and serological tests. The investigators had no details on
any of
these control patients but it can be assumed that
they were not completely well and had their blood taken for purposes
that
may have included full blood counts, blood
grouping/cross matching, HbAlc or red cell folate testing, as they were
discarded
bloods from a diagnostic haematology laboratory.
SFG rickettsial serological assays were performed using the indirect microimmunofluorescence assay with the antigens R. akari, R. australis, R. conorii, R. honei, R. rickettsii and R. sibirica using previously described methods.29 SFG rickettsial titres of =256 were considered positive.
Rickettsial culture was performed only on the patient group (not controls) using previously described methods.30
DNA was extracted from buffy coat
specimens from the patient and control groups as well as positive
rickettsial cultures with
a DNA extraction kit (Gentra, USA) using the
manufacturer's protocols. Rickettsial specific citrate synthase
real-time PCR
was performed on each DNA extract and rickettsial
cell culture as previously described.31
To rule out any false positive results, positive samples were repeated
on two other occasions for confirmation. Additionally,
UDG was incorporated in all master mixes in order
to prevent carryover contamination from amplified products. Conventional
gel-based PCR for the rickettsial 17 kDa antigen
gene using the primers MTO-1 and MTO-2 was performed on real-time PCR
positive
cultures and buffy coat extracts as previously
described, except with an annealing temperature of 51°C.32 Subsequent sequencing of the 17 kDa antigen gene amplicons was used to identify the aetiological rickettsia in PCR positive
cultures and buffy coat specimens (Newcastle DNA, Newcastle, Australia).
The second cohort of 581 chronically
unwell patients was recruited from the practice of Dr John Graham, a
physician specializing
in chronic illnesses, in Adelaide, South Australia
(‘Adelaide cohort’). These patients, like Dr Kemp's, had rickettsial
serological
testing performed as apart of their routine
diagnostic testing. Testing for rickettsial DNA and culture was not
done. A group
of 34 patients without a chronic condition, from Dr
Graham's practice, was used as controls for serological comparisons.
All
patients had rickettsial serology performed using
the methods described before.
Results
Examination of the ‘Melbourne cohort’ by citrate synthase real-time PCR revealed that 14/526 (3%) had detectable rickettsial
DNA in their peripheral blood, compared to none of the Newcastle control cohort (P < 0.001; chi-squared test; Table 1).
Of the 14 patients positive by rickettsial real-time PCR, only 5 had a
420bp 17 kDa antigen gene product amplified by traditional,
gel-based PCR that was subsequently sequenced (Table 1). A rickettsial isolate was also obtained from 2 of the 14 patients (Table 1),
but neither isolate could be maintained in continuous culture. Each of
the 17 kDa PCR products from the five positive buffy
coat specimens and the two rickettsial isolates
yielded a 400 bp DNA sequence that was 100% homologous with the Rickettsia honei strain ‘marmionii’ 17 kDa antigen gene (GenBank accession number AY37683).33
View this table:
Table 1
Rickettsial DNA positive patients from the ‘Melbourne cohort’; their clinical and laboratory findings
Of the 14 patients positive by real-time PCR, significantly fewer were detected in spring than all other seasons (P < 0.03; Fisher's exact test) and significantly more were detected in winter than all other seasons (P < 0.03; Fisher's exact test) (Figure 1).
Figure 1.
A graph showing the seasonal incidence of rickettsial DNA positive patients from the ‘Melbourne cohort’. P-values are shown above statistically significant seasons.
The summary of the 14 rickettsial DNA positive patients in the ‘Melbourne cohort’ is shown in Table 2. Common clinical features from these 14 patients included arthralgia, fatigue or tiredness and clinical depression. Other
symptoms included myalgia or fibromyalgia, and chronic headache (Table 1).
The most common laboratory abnormality detected was raised inflammatory
markers (erythrocyte sedimentation rate and C-reactive
protein; Table 1).
Females were more commonly afflicted than males (11:3) with most
patients being middle-aged (mean 43 years). The average
length of illnesses was 8 years. Of the 14
patients, 9 were from urban Victoria (Melbourne) and the remaining 5
from rural
areas (Table 2).
View this table:
Table 2
Summary of the 14 patients of the ‘Melbourne cohort’ with rickettsial DNA in their peripheral blood
Positive rickettsial serology titres (=256) were seen in only 5/14 (36%) of the patients who were positive in the rickettsial
real-time PCR assay (Table 1).
Serologically the ‘Melbourne cohort’ had 206/521 (39%) patients with
positive rickettsial titres. When compared to serological
results of the Newcastle control cohort, the latter
contained significantly less patients (3/399; 1%) with positive
rickettsial
titres (P < 0.0001; two sample test for binomial proportions) (Figure 2).
Figure 2.
A graph showing
the distribution of rickettsial serology titres from the control cohort
of Newcastle (light) and chronically
ill patient cohorts of Adelaide (medium) and
Melbourne (dark). Only titres of ⩾256 were considered genuine positives.
A significantly higher proportion of patients in the ‘Adelaide cohort’ (238/581; 41%) had positive rickettsial serology, when
compared to the Newcastle control cohort (3/399; 1%) (P < 0.0001; two sample test for binomial proportions) (Figure 2). The control cohort from the same Adelaide clinic also had significantly fewer patients with positive rickettsial serology
(5/34; 15%) than the ‘Adelaide cohort’ of patients (P < 0.002; two sample test for binomial proportions).
Discussion
We now report that 14 of 526 (3%) chronically unwell patients from a medical practice in Melbourne, Victoria had rickettsial
DNA in their blood. Of the 14 patients, 5 had a R. honei strain ‘marmionii’ DNA sequence amplified, an aetiological agent of Flinders Island Spotted Fever (FISF).33 Rickettsia honei strain ‘marmionii’ was able to be cultivated from a further two patients. As a whole, both the Melbourne and Adelaide cohorts
had a significantly higher proportion of rickettsial seropositivity than the control cohorts.
The 14 cases of rickettsaemia in the current study had similar symptoms to cases of acute rickettsial infection (e.g. myalgia,
arthralgia, headache and lethargy).25
Abnormal laboratory findings (e.g. thrombocytopenia, aberrations in
leukocyte and neutrophil counts, raised C-reactive protein
and liver transaminase levels) were also found in
both rickettsaemic patients within our study and acute rickettsioses.34 The pattern of rickettsial serology in the 14 patients who were rickettsial DNA positive (only 5/14 seropositive) was not
dissimilar to the seven acute rickettsial patients with FISF.33
In the current 14 cases the failure of some patients to seroconvert is
not understood and may have been due to the extremely
low quantity of rickettsial DNA/organisms
circulating in the patient's blood. The Melbourne and Adelaide cohorts’
serology
findings were not dissimilar, with 39 and 41%,
respectively having positive rickettsial serology. Such a high
sero-prevalence
could be explained by Adelaide and surrounding
areas, being endemic for spotted fever illnesses.30,,34 However, endemic rickettsial areas in Victoria are unknown with the exception of Gippsland.35
The significantly higher proportion of chronically unwell patients that
were seropositive for rickettsia suggests that exposure
to rickettsiae may be causally related to their
chronic conditions. It is unclear if the underlying rickettsaemia is
(partially)
responsible for the patients’ chronic ailments or
simply a reactivation of a latent infection caused by immunosuppression
generated from the chronic condition itself or the
treatment thereof. Regarding the later, bacterial diseases such as
tuberculosis,36 syphilis,37 brucellosis,38 Q-fever22–24 and, with most relevance, typhus (Brill–Zinsser disease)26
are known to cause latent infections using yet to be defined
mechanisms. A rickettsial infection would normally be eradicated
from the host, unless there is an aberration in the
host's immune response. Gene expression studies in patients with
chronic
fatigue syndrome revealed an increase in T-cell
activation.39 Although a cytotoxic T-cell response is an important factor for eradicating a rickettsial infections,40
chronic antigenic stimulation of T-cells may be responsible for fatigue
induced as a result of an aberration in the patient's
immune response. It has been postulated that post
Q-fever fatigue syndrome, along with its cytokine dysregulation, may be
caused by a similar chronic antigenic stimulation.20,22–24
The number of rickettsia found in the patients’ blood was very low, being detectable in most cases only by a highly sensitive
and specific rickettsial real-time PCR.31
The amplicons of the citrate synthase real-time PCR assay are very
small (74 base pairs) and cannot be sequenced. The low
sensitivity of the traditional gel-based 17 kDa PCR
may explain why only 5/14 real-time PCR positive patients were positive
with the traditional assay. Hence it was not
possible to determine the rickettsial species present in the other nine
patients.
The two isolates of R. honei strain ‘marmionii’ were initially isolated in Vero cell culture and then grown for another two passages in XTC-2 cells before
they could no longer be passaged in cell culture. This phenomenon has been noted with previous isolates of R. honei strain ‘marmionii’ from acute cases.33 Unfortunately it was not possible to obtain control patient specimens from Melbourne (rather than Newcastle) for the ‘Melbourne
cohort’, although this would have been ideal.
To complement the current study
additional cohort and prospective studies are needed. As apart of these
additional studies
a more comprehensive effort needs to be taken in
examining patient blood for rickettsaemia via real-time PCR. Further
study
of patient immune gene alleles and cytokine
concentration should determine whether immune system dysfunction may be
contributing
to the patients’ chronic illnesses and whether the
persistence of rickettsaemia is causal. The presence of rickettsaemia
and
a high proportion of rickettsial seropositivity
within two cohorts of chronically ill patients suggest that rickettsiae
may
be an underlying factor to some of the patient's
illnesses.
Acknowledgements
We would like to thank Mr Leonard Izzard for undertaking some of the rickettsial serology, Dr Margaret Henry for helping perform
the statistical analyses for this manuscript and Prof. Barrie Marmion for kindly reviewing this manuscript.
