Journal of Chronic Fatigue Syndrome 2003; 11(2): 7-19.
Evidence for Bacterial (Mycoplasma, Chlamydia) and Viral (HHV-6)
Co-Infections in Chronic Fatigue Syndrome Patients

Garth L. Nicolson,1 PhD, Marwan Y. Nasralla,2 PhD, Kenny De Meirleir,3 MD,
PhD, Robert Gan,2 MB, PhD and Joerg Haier,4 MD, PhD

1The Institute for Molecular Medicine, Huntington Beach, California, USA,,
2International Molecular Diagnostics, Inc., Huntington Beach, California,
USA, 3Department of Internal Medicine, Free University of Brussels,
Brussels, Belgium and 4Department of Surgery, University Hospital, Munster,
Germany

Correspondence: Prof. Garth L. Nicolson, Office of the President, The
Institute for Molecular Medicine, 15162 Triton Lane, Huntington Beach,
California 92649. Tel: 714-903-2900; Fax: 714-379-2082;
Email: gnicolson@immed.org; Website: www.immed.org

ABSTRACT. Using the blood of 100 CFS patients and forensic polymerase chain
reaction we have found that a majority of Chronic Fatigue Syndrome (CFS)
patients show evidence of multiple, systemic bacterial and viral infections
(OR = 18.0, 95% CL 8.5-37.9, P < 0.001) that could play an important role in
CFS morbidity. CFS patients had a high prevalence (51%) of one of four
Mycoplasma species (OR = 13.8, 95% CL 5.8-32.9, P < 0.001) and often showed
evidence of co-infections with different Mycoplasma species, Chlamydia
pneumoniae (OR = 8.6, 95% CL 1.0-71.1, P < 0.01) and/or active Human Herpes
Virus-6 (HHV-6) (OR = 4.5, 95% CL 2.0-10.2, P < 0.001). We found that 8% of
the CFS patients showed evidence of C. pneumoniae and 31% of active HHV-6
infections. Since the presence of one or more chronic systemic infections
may predispose patients to other infections, we examined the prevalence of
C. pneumoniae and active HHV-6 infections in Mycoplasma-positive and
–negative patients. The incidence of C. pneumoniae or HHV-6 was similar in
Mycoplasma-positive and -negative patients, suggesting that such infections
occur independently in CFS patients. Also, the incidence of C. pneumoniae in
active HHV-6-positive and –negative patients was similar. Control subjects
(N=100) had low rates of mycoplasmal (6%), active HHV-6 (9%) or chlamydial
(1%) infections, and there were no co-infections in control subjects.
Differences in bacterial and/or viral infections in CFS patients compared to
control subjects were significant. The results indicate that a relatively
large subset of CFS patients show evidence of bacterial and viral
co-infections.

INTRODUCTION

Chronic illnesses like Chronic Fatigue Syndrome (CFS) are usually complex,
heterogeneous and involve multiple, nonspecific, overlapping signs and
symptoms (1, 2). Such illnesses are usually difficult to diagnose and treat
(3-5). CFS for the most part does not have effective therapies, and
therefore patients often do not completely recover from their illness, even
with therapy (3). CFS patients can be subdivided into clinically relevant
subcategories that may represent different disease states or co-morbid
conditions or illnesses (6). Identifying systemic infections, such as those
produced by Mycoplasma species (4-9), Chlamydia pneumoniae (10) and Human
Herpes Virus-6 (HHV-6) (11-13), is likely to be important in determining the
treatment strategies for many CFS patients.

Although no single underlying cause has been established for CFS, there is
growing awareness that CFS can have an infectious nature that is either
causative for the illness, a cofactor for the illness or appears as an
opportunistic infection(s) that aggravate patient morbidity (14). There are
several reasons for this (15), including the nonrandom or clustered
appearance of CFS, sometimes in immediate family members (16, 17), the
presence of certain signs and symptoms associated with infection, the often
cyclic course of the illness and its response to anti-microbial therapies
(4, 5, 14).
Here we examined CFS patients to see if a subset of patients had more than
one type of chronic bacterial or viral infection. We were particularly
interested in assessing whether patients with one type of infection were
more likely to show evidence of additional infections.

MATERIALS AND METHODS

Patients
All patients were from North America (Canada and the United States, n=100)
and underwent a medical history, completed a sign/symptom illness survey and
had routine laboratory tests. If necessary, medical records were also
reviewed to determine if patients suffered from organic or psychiatric
illnesses that could explain their symptoms. When positive results were
found in any of the evaluations that met the Fukuda et al. (2) exclusionary
criteria, the patients were not included in the study. Additionally, all
subjects were questioned about medication use during the three months prior
to the study, and they had to be free of antibiotic treatment for two months
prior to blood collection. Control subjects (N=100) had to be free of
disease for at least three months prior to data collection, and they had to
be free of antibiotic treatment for three months prior to blood collection.

Blood Collection
Blood was collected in EDTA-containing tubes and immediately brought to ice
bath temperature as described previously (18-20). Samples were shipped with
wet ice by air courier to the Institute for Molecular Medicine and
International Molecular Diagnostics, Inc. for analysis. All blood samples
were blinded. Whole blood (50 µl) was used for preparation of DNA using
Chelex (Biorad, Hercules, USA) as follows. Blood cells were lysed with
nano-pure water (1.3 ml) at room temperature for 30 min. After
centrifugation at 13 000 x g for 2 min, the supernatants were discarded.
Chelex solution (200 µl) was added, and the samples were incubated at 56°C
and at 100°C for 15 minutes each. Aliquots from the centrifuged samples were
used immediately for PCR or flash frozen and stored at –70°C until use.
Multiple aliquots were used for experiments on all patient samples.

Detection of Mycoplasma by Forensic PCR.
Amplification of the target gene sequences (18-20) was performed in a total
volume of 50 µl PCR buffer (10 mM Tris-HCl, 50 mM KCl, pH 9) containing 0.1%
Triton X-100, 200 ?m each of dATP, dTTP, dGTP, dCTP, 100 pmol of each
primer, and 0.5-1 µg of chromosomal DNA. Purified mycoplasmal DNA (0.5-1 ng
of DNA) was used as a positive control for amplification. Additional primer
sets were used to confirm the species specificity of the reaction. The
amplification was carried out for 40 cycles with denaturing at 94?C and
annealing at 60°C (genus-specific primers and M. penetrans) or 55°C (M.
pneumoniae, M. hominis, M. fermentans). Extension temperature was 72°C in
all cases. Finally, product extension was performed at 72°C for 10 min.
Negative and positive controls were present in each experiment. The
amplified samples were run on a 1% agarose gel containing 5 µl/100 ml of
ethidium bromide in TAE buffer (0.04 M Tris-Acetate, 0.001 M EDTA, pH 8.0).
After denaturing and neutralization, Southern blotting was performed as
described below (18-20).

Chlaymdia pneumoniae Detection by Forensic PCR.
PCR detection of Chlaymdia pneumoniae was done as described above for
various Mycoplasma species, except that the conditions and primers differ.
PCR was carried out using the C. pneumoniae-specific primers:
5’-TGACAACGTTAGAAATACAGC-3’ (upstream) and downstream
5’-CGCCTCTCTCTCCTATAAAT-3’. Additional primer sets were used to confirm the
species specificity of the reaction. The DNA was amplified for 30 cycles
using standard cycle parameters, and the product evaluated by agarose-gel
electrophoresis. The efficiency of the PCR process was monitored by
amplification of ?-actin mRNA. The presence of amplifications inhibitors
will be evaluated by spiking negative samples with 2 µl of DNA from stock.
C. pneumoniae-specific oligonucleotides in the PCR product were identified
by Southern Blot and dot-blot hybridization using a 21-mer internal probe:
(5’-CGTTGAGTCAACGACTTAAGG-3’) 3’ end-labelled with digoxigenin–UTP or
32P-labeled probe.

Active HHV-6 Detection by Forensic PCR.
PCR detection of active HHV-6A was done as described above, except that
blood plasma was used instead of whole blood and the conditions and primers
differed. PCR reactions were carried out using the following HHV-6A-specific
primers:
5’-GCGTTTTCAGTGTGTAGTTCGGCAG-3’ (upstream) and downstream
5’-TGGCCGCATTTCGTACAGATACGGAGG-3’. The nucleotides were amplified for 30
cycles using standard cycle parameters, and the product evaluated by
agarose-gel electrophoresis. Additional primer sets were used to confirm the
specificity of the reaction. The efficiency of the PCR process was monitored
by amplification of ?-actin mRNA. The presence of amplification inhibitors
was evaluated by spiking negative samples with 2 µl of DNA from stock.
HHV-6A-specific oligonucleotides in the PCR product were identified by
Southern Blot and dot-blot hybridization using a 21-mer internal probe:
(5’-ATCCGAAACAACTGTCTGACTGGCA-3’) 3’ end-labelled with digoxigenin–UTP or
32P-labeled probe.

Southern Blot Confirmation
The amplified samples were run on a 1% agarose gel containing 5 ml/100 ml of
ethidium bromide in TAE buffer (0.04 M Tris-Acetate, 0.001 M EDTA, pH 8.0).
After denaturating and neutralization, Southern blotting was performed as
follows. The PCR product was transferred to a Nytran membrane. After
transfer, UV cross-linking was performed. Membranes were prehybridized with
hybridization buffer consisting of 1x Denhardt’s solution and 1 mg/ml salmon
sperm DNA as blocking reagent. Membranes were then hybridized with
digoxigenin–UTP or 32P-labeled internal probe (107 cpm per bag). After
hybrization and washing to remove unbounded probe, the membranes were
examined (digoxigenin-UTP-labeled probe) or exposed to autoradiography film
(32P-labeled probe) for 1- 2 days at –70°C.

Statistics
Subjects’ demographic characteristics were assessed using descriptive
statistics and students’ t-tests (independent samples test, t-test for
equality of means, 2-tailed). The 95% confidence interval was chosen for
minimal significance.

RESULTS

Patients and Control Subjects
Patients and control subjects were approximately similar in age
characteristics (control subjects mean age = 34.6; CFS patients: mean age =
39.7). CFS patients differed significantly according to sex distribution (P<
0.05); 72% of the patients were female, while 28% of the patients were male.
Similarly, 69% of control subjects were female, while 31% were male (Table
1). All CFS patients fulfilled current international CDC case definition for
Chronic Fatigue Syndrome (2).

Chronic Infections in CFS Patients
Chronic infections were not found in 29% of CFS patients and 88% of control
subjects (Table 2). When we examined CFS patients’ blood for the presence of
chronic infections using forensic PCR, evidence for Mycoplasma species
infections were found in 51% of CFS patients and 7% of control subjects
(Odds Ratio = 13.8, 95% CL = 5.8-32.9, P<0.001). Evidence for C. pneumoniae
infections were found in 8% of CFS patients and in 1% of control subjects
(Odds Ratio = 8.6, 95% CL = 1.0-71.1, P < 0.01), and evidence for active
HHV-6 infections were found in 31% of CFS patients and 9% of control
subjects (Odds Ratio = 4.5, 95% CL = 2.0-10.2, P < 0.001). We did not find
any multiple co-infections in control subjects. The differences between
chronic infections in CFS patients and control subjects were significant
(Odds Ratio = 18.0, 8.5-37.9, P < 0.001) (Table 2).

Using species-specific primers and PCR the incidence of various Mycoplasma
species in the blood of CFS patients was examined. M. pneumoniae infections
were observed in 29 of 51 Mycoplasma-positive CFS patients (Odds Ratio =
13.2, 95% CL = 3.8-45.4, P < 0.001), M. fermentans infections occurred in 22
patients (Odds Ratio = 13.8, 95% CL = 3.1-61.1, P < 0.001) and M. hominis in
16 patients (Odds Ratio = 18.8, 95% CL = 2.4-147.0, P < 0.001), whereas M.
penetrans infections were found at lower (8 of 51 Mycoplasma-positive
patients) incidence (Odds Ratio = 8.6, 95% CL = 1.0-71.1, P < 0.01) (Table
2). We examined 100 control subjects who did not show clinical signs and
symptoms and found that 7 were positive for a single species of Mycoplasma
(Table 2). Differences between CFS patients and control subjects were highly
significant (Odds Ratio = 18.0, 95% CL = 8.5-37.9, P < 0.001).

Multiple mycoplasmal Co-Infections in CFS Patients
Single infections with one of the four Mycoplasmas that were tested were
observed in 29 of the 51 (56.9%) Mycoplasma-positive patients (Table 2). In
the seven control subjects that were positive for mycoplasmal infections we
found two controls that were positive for M. fermentans, three for M.
pneumoniae and one for M. hominis and one for M. penetrans but these were
found only as single infections. Similar to a previous study (19), the most
commonly observed infection was M. pneumoniae (29 of 51 Mycoplasma-positive
patients), followed by M. fermentans in 22 patients, M. hominis in 16
patients and M. penetrans in 8 patients. Multiple mycoplasmal infections
were detected in 22 of the 51 Mycoplasma-positive patients (43.1% of the
Mycoplasma-positive patients), whereas single infections were found in 29/51
(56.9% of the Mycoplasma-positive patients). The few control or healthy
subjects that showed evidence for mycoplasmal infections only had single
species infections (Chi2 = 24.7, P < 0.001). Similar to our previous results
(25), we have not found patients positive for all four of the tested
Mycoplasma species. In previous studies on North American (19) and European
(6) CFS patients with multiple mycoplasmal infections, all patients showed
combinations of M. pneumoniae and/or M. fermentans (with or without other
species). The combination of M. hominis and M. penetrans was not seen.
Similar results were found here where the most commonly found combination of
Mycoplasma species were M. fermentans plus M. pneumoniae, M. fermentans plus
M. hominis or M. hominis plus M. pneumoniae. The most common triple
infection found was M. fermentans plus M. hominis plus M. pneumoniae found
in two patients (Table 2).

Co-Infections with HHV-6 in CFS Patients
Similar to others, we found evidence of (active) HHV-6 infections in the
plasma in approximately 31% of patients with CFS (Odds Ratio = 4.5, 95% CL =
2.0-10.2, P < 0.001). This finding is similar but somewhat lower than
previously reported for CFS patients in other studies (11-13). When we
examined the incidence of HHV-6 infections in Mycoplasma-positive and
–negative patients, we found that there was no preference for active HHV-6
infections in Mycoplasma-infected patients (Table 2). Evidence for active
HHV-6 infections was found by examination of blood plasma, and we found that
31.4% of Mycoplasma-positive CFS patients also had HHV-6. Similarly, in
Mycoplasma-negative patients evidence of active HHV-6 infections was found
in 29.4% of patients (Table 2). There was also no preference for particular
Mycoplasma species in HHV-6 co-infections (data not shown). In control
subjects without evidence of signs or symptoms we found evidence for active
HHV-6 infections in 9 of 100 subjects. None of these HHV-6-positive control
subjects showed other infections (Table 2).

Co-Infections with C. pneumoniae in CFS Patients
Chlamydia pneumoniae infections were found in 8% of CFS patients and one
control subject out of 100 that also did not have mycoplasmal or HHV-6
infections (Odds Ratio = 8.6, 95% CL = 1.0-71.1, P < 0.01) (Table 2). This
finding is similar but somewhat lower than previously reported for CFS
patients (10). When we examined the incidence of C. pneumoniae infections in
Mycoplasma-positive and –negative patients, we found that there was no
preference for multiple infections, nor was there a preference for
particular Mycoplasma species in C. pneumoniae Mycoplasma co-infections. In
Mycoplasma-positive or –negative patients C. pneumoniae infections were
found in 4 patients in each group. Similarly, in HHV-6-positive patients C.
pneumoniae infections were found in 3 of 31 patients (9.7%), whereas in
HHV-6-negative patients C. pneumoniae infections were found in 5 of 69
patients (7.3%) (Table 2). Thus there appeared to be no preference for
particular combinations of co-infections in CFS patients.

DISCUSSION

Chronic infections appear to be a rather common feature of CFS. Many
patients report that their CFS signs and symptoms slowly evolved after acute
infections, chemical exposures, multiple vaccinations, severe trauma or
other conditions that are associated with immune suppression and
opportunistic infections. Previously we studied North American and European
CFS patients and found that most showed evidence of mycoplasmal infections
(6, 19, 20). Others who studied CFS patients also found evidence of
widespread mycoplasmal infections (7-9). When we examined the incidence of
particular mycoplasmal infections in North American CFS patients, we found
that most patients had multiple infections (two or more species of
Mycoplasma), which were for the most part combinations of M. fermentans and
other Mycoplasma species (19). In our study on the prevalence of multiple
mycoplasmal co-infections in CFS patients we found that double or triple
infections occurred only when one of the species was M. pneumoniae and/or M.
fermentans (19). In a study on European CFS patients a slightly different
picture was found (6). Over two-thirds (68.6%) of 261 consecutive patients
seen at a CFS clinic in Belgium were found to show evidence of Mycoplasmas
in their blood. In contrast to North American patients, however, the most
common species found was M. hominis, and there was a lower overall rate of
multiple mycoplasmal co-infections in the European CFS patients. This could
indicate differences in demography and exposures between North American and
Belgian CFS patients. We also found that more than 50% of North American
patients with rheumatoid arthritis had mycoplasmal infections, and in the
majority of these patients multiple mycoplasmal co-infections infections
were found (18).

Infections of the class Mollicutes can invade a variety of tissues but they
can also present as superficial infections (21, 22). Mycoplasmas are found
commonly in the oral cavity, urogenital tract and as symbiotic gut flora,
but some species can cause acute and chronic illnesses when they penetrate
into the blood vascular system and systemically colonize organs and tissues
(4, 5, 22-23). For example, M. penetrans, M. fermentans, M. pneumoniae, M.
hominis and M. pirum can enter a variety of tissues and cells and cause
systemic signs and symptoms. Mycoplasmas have also been shown to have a
complex relationship with the immune system. They are very effective at
evading host immune responses, and Mycoplasmas can cause changes in cytokine
production (24, 25). In addition to CFS, Mycoplasmas are thought to
contribute to patients’ morbidity in rheumatoid arthritis (22, 26), systemic
lupus erythematosis (27), demyelinating and axonal neuropathies (28),
HIV-AIDS (29, 30) and chronic respiratory conditions (31-33).

In CFS patients we found that multiple co-infections involving Mycoplasmas
were common (19 and the results here), and such mycoplasmal co-infections
also occurred with C. pneumoniae and HHV-6. mycoplasmal infections have been
reported as co-infections with other microorganisms (29, 30). In some cases
synergism with other infectious agents has been seen (34).

Certain types of non-mycoplasmal infections are commonly found in CFS
patients. One of the most common viral infections found is HHV-6 (11-13).
HHV-6 appears to play a role in several chronic illnesses (12, 13). Although
several studies have associated HHV-6 with CFS (13, 35-38), there are also
reports that could not find an association with CFS (39, 40). Although HHV-6
infections are commonly found in children, in adults such infections are
considered latent but can be reactivated in certain illness states. In CFS
patients HHV-6 is frequently reactivated and appears in blood leukocytes and
blood plasma. In contrast, in control subjects HHV-6 may be present as a
latent infection, and although some active HHV-6 may be found in controls,
it is generally found at lower levels compared to CFS patients (11-13).
After peripheral blood mononuclear cells from CFS patients were cultured,
specific HHV-6 glycoprotiens could be found in the culture medium using
monoclonal antibodies. Also, HHV-6 genes could be found using nested PCR
(17). The peripheral leukocytes from a majority of CFS patients showed
active HHV-6 infections. Most of these patients test positive using
antibodies reactive with the HHV-6A variant (11-13).

The use of PCR techniques for detection of microorganism infections in
patients has been questioned in studies where different methods were used in
different laboratories without validation. The PCR tests that we used are
very sensitive and highly specific. These tests are a dramatic improvement
on the relatively insensitive serum antibody tests that are routinely used
to assay for systemic infections. For example, in the determination of
mycoplasmal infections we used primer sets for various genes found in
specific species (18-20). Since some primers that have been used to detect
Mycoplasma species are capable of possible cross-reactions with
Mycoplasma-related organisms (41), we used multiple unique primer set and
conditions that detect only specific Mycoplasma species. As in a previous
study (20), we examined the reliability of the methods by performing
multiple assays, and the results were completely reproducible. The
sensitivity of Mycoplasma detection by the described method was assessed by
the detection of control Mycoplasma and by internal Southern blot
hybridization using Mycoplasma-specific probes. Using serial dilutions of
Mycoplasma DNA the method was able to detect as low as a few fg of DNA (20).
In other experiments, Mycoplasma was added to control blood samples at
various concentrations. We were able to detect specific products down to a
few ccu/ml blood. We have used a specific DNA isolation procedure to avoid
inhibitors, contaminating nucleases and protein complexing. With the use of
specific Southern hybridization the procedure can result in specific test
results of high sensitivity and can detect a few microorganisms in a
clinical sample (19, 20).

The multiple co-infections in CFS probably play an important role in
determining the severity of systemic signs and symptoms found in CFS
patients (19, 23, 42). Since CFS patients that previously tested positive
for mycoplasmal infections have benefited from therapies directed at their
chronic infections (5, 22, 23), we consider it important that such
infections be carefully considered in the clinical management of CFS.

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