This study maybe explains why Valtrex works with 30% of kid (hhv6/pneumonia)

[Guest guest]

(this post was on another group....very interesting Chlamydia

Pneumonia and HHV6 tied to Autism)

Journal of Neuroscience Research

Evidence for Mycoplasma ssp., Chlamydia pneunomiae and Human Herpes

Virus-6 Co-Infections in the Blood of Patients with Autistic Spectrum

Disorders

Garth L. Nicolson1 Gan1 L. Nicolson1 and Joerg

Haier1,2

1The Institute for Molecular Medicine, Huntington Beach, California,

USA,, 2Department of Surgery, University Hospital, Munster, Germany

Correspondence: Prof. Garth L. Nicolson, Office of the President,

The

Institute for Molecular Medicine, 16371 Gothard Street H, Huntington

Beach, California 92647. Tel: 714-596-6636; Fax: 714-596-3791;

Email:

gnicolson@... <mailto:gnicolson@...> ; Website:

www.immed.org

Running Title: Infections in Autistic Spectrum Disorders

ABSTRACT. We examined the blood of 48 patients from Central and

Southern California diagnosed with Autistic Spectrum Disorders (ASD)

using forensic polymerase chain reaction and found that a large

subset

(28/48 or 58.3%) of patients showed evidence of Mycoplasma spp.

infections compared to two of 45 (4.7%) age-matched control subjects

(Odds Ratio=13.8, p<0.001). Since ASD patients have a high

prevalence of

one or more Mycoplasma species and sometimes show evidence of

infections

with Chlamydia pneumoniae, we examined ASD patients for other

infections. Also, the presence of one or more systemic infections may

predispose ASD patients to other infections, thus we examined the

prevalence of C. pneumoniae (4/48 or 8.3% positive, Odds Ratio=5.6,

p<0.01) and Human Herpes Virus-6 (HHV-6, 14/48 or 29.2%, Odds

Ratio=4.5,

p<0.01) co-infections in ASD patients. We found that

Mycoplasma-positive and –negative ASD patients had similar

percentages of C. pneumoniae and HHV-6 infections, suggesting that

such

infections occur independently in ASD patients. Control subjects also

had low rates of C. pneumoniae (1/48 or 2.1%) and HHV-6 (4/48 or

8.3%)

infections, and there were no co-infections in control subjects. The

results indicate that a large subset of ASD patients show evidence of

bacterial and/or viral infections (Odds Ratio=16.5, p<0.001). The

significance of these infections in ASD is discussed in terms of

appropriate treatment.

Key Words: Autism, Infection, HHV-6 virus, Chlamydia pneumoniae,

Mycoplasma species,

INTRODUCTION Autism was first identified in 1943 (Kanner, 1943),

and

these patients generally suffer from an inability to properly

communicate, form relationships with others and respond

appropriately to

their environments. Autism patients often display repetitive actions

and develop troublesome fixations with specific objects, and they are

often sensitive to certain sounds, tastes and smells. Autism

patients

do not all share the same signs and symptoms but tend to share

certain

social, communication, motor and sensory problems that affect their

behavior in predictable ways (Berney, 2000). Autism and related

disorders have been recently placed into a multi-disorder category

called Autistic Spectrum Disorders (ASD), which includes autism,

Attention Deficit Disorder (ADD) Attention Deficit Hyperactivity

Disorder (ADHD) and other disorders (Keen and Ward, 2004). The

criteria

for diagnosis of ASD are, in general terms, the presence of a triad

of

impairments in social interaction, communication and imagination

(Wing

et al., 2002). These signs and symptoms are thought to be due to

abnormalities in brain function or structure and are thought to have

a

genetic basis (Folstein and Rosen-Sheidley, 2001; Weenstra-

Vanderweele

et al., 2003). The incidence of ASD is currently estimated at 1 in

1,000 children, and in genetically predisposed families the disorder

is

~100-times higher in incidence than in the general population

(Folstein

and Rosen-Sheidley, 2002). The concordance rate in monozygotic

twins is

70-90%, whereas in dizygotic twins the rate is close to 0%,

suggesting a

strong genetic component (Weenstra-Vanderweele et al., 2003). In

some

patients there are also a number of other less specific chronic signs

and symptoms. Among these are fatigue, headaches, gastrointestinal

and

vision problems and occasional intermittent low-grade fevers and

other

signs and symptoms that are generally excluded in the diagnosis of

ASD.

These suggest that a subset of ASD patients may suffer from

bacterial or

viral infections (Takahashi et al., 2001). There are several reasons

for this, including the nonrandom or clustered appearance of ASD,

sometimes in immediate family members or particular regions, the

presence of certain signs and symptoms associated with infection, the

cyclic course of the illness and in some cases its response to

anti-microbial therapies. Although no single underlying cause has

been

established for ASD, there is growing awareness that ASD can have an

infectious nature that may be a cofactor for the illness or appears

as

an opportunistic infection(s) that can aggravate patient morbidity

(Takahashi et al., 2001; Libbey et al., 2005; Yamashita et al.,

2003).

Identifying systemic infections, such as those produced by Mycoplasma

species (Huang et al., 1998; Nicolson et al., 2000; 2003a,b. 2005;

Nijs

et al., 2002) Chlamydia pneumoniae (Chia and Chia, 1999; Nicolson et

al., 2003a, and Human Herpes Virus-6 (HHV-6) (Braun et al., 1997;

Campadelli-Fiume et al., 1999; Nicolson et al., 2003a,, is likely

to

be important in determining the treatment strategies for many ASD

patients. These infections can penetrate the CNS and are associated

with other neurological diseases (Nicolson et al., 2002). In

addition,

heavy metal, chemical and environmental exposures also appear to be

important in ASD (Colborn, 2004; son et al., 2004; Eppright et

al.,

1996). Here we examined ASD patients to see if a subset of patients

show

evidence of infection with Mycoplasma spp., C. pneumoniae or HHV-6.

Since these infections can cause neurological signs and symptoms

(Baseman and Tully, 1997; Nasralla et al., 1999; 2000; Nicolson et

al.,

2003a), they may be important in ASD. Previously we found that

children

of Mycoplasma-positive Gulf War veterans were over 18-times more

likely

to come down with Mycoplasma fermentans than the general population,

and

there was a high incidence of ASD in their children (Nicolson et al.,

2003c). In addition, examination of a group of autism patients from

civilian families revealed that there was a high incidence of

mycoplasmal infections, including M. fermentans, M. pneumoniae and M.

hominis (Nicolson et al., 2005b). Since mycoplasmal infections can

often be found as co-infections with C. pneumoniae or HHV-6

(Nicolson et

al., 2003a,b, 2005a), we examined ASD patients to see if they had

evidence of co-infections of Mycoplasma spp., C. pneumoniae and HHV-

6.

MATERIALS AND METHODS

Patients

All ASD patients (N=48) were from families in contact with patient

support groups and were referred from Central and Southern California

physicians after diagnosis with ASD according to the International

Classification of Diseases (ICD-10) and the Diagnostic and

Statistical

Manual of Mental Disorders (DSM-IV). All patients were assessed by

the

Autism Diagnostic Interview-Revised (ADI-R) (Lord et al., 1997) and

Childhood Autism Rating Scale (CARS) (Van Bourgondien et al., 1992;

Pilowsky et al., 1998). Patients also underwent a medical history,

completed a sign/symptom illness survey and had routine laboratory

tests. Additionally, all parents were questioned about medication

use

during the three months prior to the study, and patients had to be

free

of antibiotic treatment for two months prior to blood collection. Of

the 48 patients, 42 were diagnosed with autism and six with Attention

Deficit Disorder. Control subjects were from families recruited for

unrelated studies (N=45), and they had to be free of any disease for

at

least three months prior to data collection, and 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 (Nicolson et al.,

2003a,b,c, 2005a; Nijs et al., 2002). Samples were shipped with wet

ice

by overnight air courier to the Institute for Molecular Medicine for

analysis. All blood samples were blinded. Whole blood (50 ml) 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 ml) 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

Polymerase Chain Reaction (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 was performed in a total

volume of 50 ml PCR buffer (10 mM Tris-HCl, 50 mM KCl, pH 9)

containing

0.1% Triton X-100, 200 mm each of dATP, dTTP, dGTP, dCTP, 100 pmol of

each primer, and 0.5-1 mg 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 (Nicolson et al., 2003a,b,c, 2005a). 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 ml/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.

Chlamydia pneumoniae Detection by Forensic PCR.

PCR detection of Chlamydia (Chlamydophila) pneumoniae was done as

described above for various Mycoplasma species, except that the

conditions and primers differ (Nicolson et al., 2003a,b, 2005a). 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 b-actin mRNA. The presence of

amplifications inhibitors were evaluated by spiking negative

samples.

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.

HHV-6 Detection by Forensic PCR

PCR detection of HHV-6A was done as described above, except that the

conditions and primers differ and plasma was used for polynucleotide

isolation to detect active infections (Nicolson et al., 2003a,. 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. The efficiency of the PCR

process was monitored by amplification of b-actin mRNA. The

efficiency

of the PCR process was monitored by amplification of b-actin mRNA.

The

presence of amplification inhibitors were evaluated by spiking

negative

samples. 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 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 (Nasralla et al., 1999). Membranes were

pre-hybridized 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

0.5-2

days at –70°C (Nicolson et al., 2003a,b, 2005).

The sensitivity and specificity of the PCR method for detection were

determined by examining serial dilutions of purified DNA from the

microorganisms themselves in blood samples. Control DNA samples were

provided by the American Type Culture Collection (Manasses, VA). The

primers produced the expected amplification product size in all test

species, which was confirmed by hybridization using the appropriate

32P-labeled internal probe (Nasralla et al., 1999). Amounts as low

as a

few fg of purified DNA were detectable for all species with the

specific

internal probes. There was no cross-reactivity between the internal

probes of one species and the PCR product from another species

(Nasralla

et al., 2000; Nicolson et al., 2003a,b,c). The techniques used have

been validated in various studies (for example, Berg et al., 1996;

Bernet et al., 1995).

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. Odds Ratios were

calculated using logistic regression (Logit method) Statistica 5.5

(Statsoft, Tulsa, OK). In some cases Pearson Chi-Square test was

performed to compare prevalence data between patients and control

subjects.

RESULTS

Patients and Control Subjects

ASD patients and control subjects were approximately similar in age

(control subjects mean age = 8.4; ASD patients: mean age = 7.9). ASD

patients differed significantly according to sex distribution (p<

0.05);

75% of the patients were male, whereas 25% of the patients were

female.

Similarly, 62.2% of control subjects were male, while 37.8% were

female.

Patients were from Central and Southern California and resided in

approximately equally in rural and urban environments (Table 1).

Bacterial and Viral Infections in ASD Patients

Using PCR we examined ASD patients' blood for the presence of

bacterial and viral infections. Evidence for Mycoplasma spp.

infections was found in 28/48 or 58.3% of ASD patients and 2/45

(4.7%)

age-matched control subjects (Odds Ratio=13.8, p<0.001). C.

pneumoniae

infections were found in 4/48 or 8.3% of ASD patients and in 1/45 or

2.1% of control subjects (Odds Ratio=5.6, p< 0.01). We also examined

the incidence of HHV-6 infections in ASD patients and found that

14/48

or 29.2% of ASD patients were positive compared to 4/45 (8.8%)

positives

in age-matched control subjects (Odds Ratio=4.5, p<0.01). We did not

find any multiple co-infections in control subjects (Table 2). The

rate

of positive results in control subjects was similar to previous

studies

(Nasralla et al., 2000; Nicolson et al., 2003a,b,c, 2005). The

differences between infections in ASD patients and control subjects

were

highly significant (Odds Ratio=16.5, p< 0.001). Significant

differences

were not found in the prevalence of infections in urban and rural

patients, in male or female patients or between autism and other ASD

diagnoses.

Multiple Co-Infections in ASD Patients

We studied multiple infections in patients by examining whether

patients

who were positive (or negative) for one type of infection also tested

positive for other infections. Eight of 14 patients with HHV-6

positive

results (57.1%) were also positive for mycoplasmal infections,

whereas

of the 6 out of 14 HHV-6-negative patients 50% were mycoplasma-

positive.

C. pneumoniae infections were observed in two of four

mycoplasma-positive ASD patients and two of four mycoplasma-negative

patients. Thus we did not find a preference for particular multiple

infections. Multiple mycoplasmal infections were found in 12 of 48

or

25% of ASD patients; only M. fermentans plus other species were

found.

We examined 45 control subjects who did not show clinical signs and

symptoms and found that only two were positive for a single

mycoplasma

species (Mycoplasma pneumoniae) (Table 2). Differences between ASD

patients and control subjects were highly significant (Table 2).

DISCUSSION

Previously we found that chronic infections in Gulf War veterans

diagnosed with Gulf War Illness could also be found in symptomatic

family members, including their children (Nicolson et al., 2003c).

In

the families chosen for study chronic illnesses were not reported

until

after the veteran in the family returned from the Gulf War.

Interestingly, common diagnoses of illness in the children of Gulf

War

veterans with mycoplasmal infections included ASD-like illnesses,

among

others, and we found the same infection, primarily M. fermentans, in

both the sick adults and children in these families. This suggested

that the M. fermentans was likely passed from the veterans to their

children (Nicolson et al., 2003c). Although preliminary and not

carefully analyzed or studied further, this result suggested that

infections might be present in ASD patients. Therefore, we examined

a

small group of ASD patients (28 autism patients, age range 3-12) in

Central California for evidence of mycoplasmal infections, and we

found

that slightly over one-half were positive for one of four species of

Mycoplasma (Nicolson et al., 2005b). In contrast to the children in

military families where primarily one species of Mycoplasma was found

(usually M. fermentans), the majority of ASD patients in Central

California were found to have single or multiple mycoplasmal

infections

involving M. pneumoniae, M. fermentans, M. hominis or M.

genitalium. We

found similar results in the present study, but in addition to

infections with Mycoplasma spp., we also examined two other commonly

found infections in chronically ill patients, C. pneumoniae and HHV-6

(Nicolson et al., 2003a,. The results suggested that infections

are a

common feature in ASD. Consistent with this hypothesis is the

finding

that autism occurs at greater prevalence during periods of more

frequent

hospitalizations for bronchitis or pneumonia (Tanoue et al., 1988),

and

maternal viral infections during the second trimester of pregnancy

are

associated with increased risk of autism in their offspring

(Ciaranello

and Ciaranello, 1995; Wilkerson et al., 2002). Infections are

thought

to play important roles in a variety of neurodevelopmental diseases,

including ASD (Horning et al., 1999; Libbey et al., 2005; Nicolson et

al., 2002). Such infections could be involved in the etiology of the

disease, or more likely they could cause co-morbid states (Nicolson

et

al., 2003a,b, 2005).

We found higher prevalence of Mycoplasma spp. (Odds Ratio=13.8), C.

pneumoniae (Odds Ratio=5.6) and HHV-6 (Odds Ratio=4.5) among children

diagnosed with ASD compared to age-matched control subjects. The PCR

techniques used in the present study have been validated in other

studies (Nicolson et al., 2003a,b,c, 2005). There are some

similarities

between the environmental exposures of Gulf War veterans and children

with ASD. Both groups were given multiple vaccines prior to their

illnesses, and heavy metals and chemicals have been found in both

groups

(Boyd, 2004; Buttram, 2004; son et al., 2004; Eppright et al.,

1996; Geier and Geier, 2004), but these findings are not universal

( and Garrod, 1978). There are reports of clinical

improvement

with treatment for these environmental exposures (reviewed by Kidd,

2002).

There were some limitations in the present study, including sample

size.

Although all of the patients in the study were ASD patients, almost

all

(42/48) had a diagnosis of autism. Removal of the other six patients

from the analysis, however, did not change the results or

conclusions.

Other factors, such as geography, family socioeconomic status,

vaccination records and educational level were not analyzed.

The infections found in ASD patients in the present and previous

studies

(Libbey et al., 2003; Nicolson et al., 2003c; 2005; Takahashi et al.,

2001; Yamashita et al., 2003) could have originated from vaccines or

from opportunistic infections in immune suppressed children.

Bacterial

contamination has been found in commercial vaccines, and in one

study 6%

of commercial vaccines were contaminated with mycoplasmas (Thornton,

1986). Thus the appearance of infections in children diagnosed with

ASD

may eventually be linked to the multiple vaccines received during

childhood either as a source or from opportunistic infections in

immune

suppressed recipients of multiple vaccines. Although the etiology of

ASD is currently unknown and thought to involve both genetic and

environmental factors (Libbey et al., 2005; Lipkin and Hornig, 2003),

the infections found in ASD patients should be considered along with

other factors in the management of these disorders (Kidd, 2002).

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Table 1. Patient demographic data.

N

Mean age (SD)

Range

Males (%)

Females

(%)

Patients

48

8.4 (2.8)

3-14

36 (75)

12 (25)

Controls

45

7.9 (3.3)

4-11

28 (62.2)

17 (37.8)

Rural patients

18

8.1 (2.9)

3-14

14 (77.7)

4 (22.3)

Urban patients

30

8.6 (3.2)

4-14

22 (73.3)

8 (26.7)

TABLE 2. Prevalence and Odds Ratio analysis of infections in ADS

patients and control

subjects.

Type of infection

ASD Patients

N = 48 (%)

Control Subjects

N = 45 (%)

Odds Ratio, p or Chi2

HHV-6

14 (29.2)

4 (8.3)

4.5, p< 0.01

C. Pneumoniae

4 (8.3)

1 (2.1)

5.6, p< 0.01

Mycoplasma spp.

28 (58.3)

2 (4.7)

13.8, p< 0.001

M. pneumoniae

16

2

9.2, p< 0.001

M. fermentans

17

0

14.8, p< 0.001

M. hominis

5

0

11.8, p< 0.01

M. penetrans

1

0

6.6, p< 0.01

Single mycoplasmal infection

16 (33.3)

2 (4.7)

13.8, p< 0.001

Multiple mycoplasmal infections

12 (25.0)

0 (0)

Chi2 = 11.7, p< 0.001

M. fermentans +M. pneumoniae

7

0

Chi2 = 4.7, p< 0.01

M. fermentans +M. hominis

2

0

Chi2 = 1.9, p< 0.3

M. pneumoniae +M. hominis

1

0

Chi2 = 1.4, p< 0.2

M. fermentans +M. hominis + M. pneumoniae

2

0

Chi2 = 1.9, p<0.2

Mycoplasma + HHV-6

8 (16.7)

0 (0)

Chi2 = 4.4, p< 0.01

Mycoplasma + C. pneumoniae

2 (4.2)

0 (0)

Chi2 = 2.1, p< 0.19

C. pneumoniae + HHV-6

1 (2.1)

0 (0)

Chi2 = 1.6, p< 0.3

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