In: Focus on Multiple Sclerosis Research ISBN 1-59033-985-1

Editor: Frank Columbus, p. 163-195 © 2004 Nova Biomedical Books.

Chapter 10

A META-ANALYSIS OF HUMAN HERPES VIRUS 6

AND CHLAMYDOPHILA PNEUNONIAE

IN MULTIPLE SCLEROSIS

J. Gutiérrez, M. García, F. Fernández School of Medicine, Granada, Spain; M. Guerrero, University Hospital, Granada

O. Fernández, V.E. Fernández, G. Luque University Hospital; Málaga

J. D. de Luna University of Granada

ABSTRACT

Background: An increasing number of studies have related two infectious agents,

human herpes virus type 6 (HHV-6) and Chlamydophila pneumoniae, with multiple

sclerosis (MS). The situation, however, remains unclear.

Aim: To undertake a meta-analysis of studies published until year 2002.

Methods: A search across electronic databases using key words relating relapsing

remitting MS and infectious agents supposedly involved. Strict selection criteria were

applied.

Results: Sixteen articles fulfilled the methodological selection criteria. Statistical

analysis showed that HHV-6 was related with MS by detection of DNA in serum and

antibodies in serum and cerebro-spinal fluid, but not with DNA in cerebrospinal fluid or

white cells. C. pneumoniae was related with MS by detection of DNA and antibodies and

recovery of bacteria in cerebrospinal fluid, but not with antibodies in serum.

Conclusion: No study was found with a sufficient number of patients and samples,

prospective, and comparative versus healthy controls and patients with other neurological

diseases, which employed a combination of several microbiological techniques in the

same subjects and samples, and then correlated the results with disease activity.

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INTRODUCTION

Multiple sclerosis (MS) is a chronic neurological disease, more common in young adults

from Europe and North America. It is characterised by a demyelinating inflammatory process

of the central nervous system (CNS) which affects the brain, optic nerves and spinal cord. Its

etiology is unknown and its pathogenesis is very likely an autoimmune mechanism. It has two

clinical forms: relapsing remitting (RR-MS) and secondary progressive (SP-MS).

Epidemiological studies have shown the existence of an environmental factor, necessary for

disease onset in genetically susceptible persons. This factor intervenes during childhood,

before the age of 15 years, probably in the form of a non-apparent or banal infection.

For some time it has been speculated that MS could be caused by an infectious agent.

However, it is only recently that different studies have repeatedly reported involvement of the

human herpes virus type 6 (HHV-6) and a bacteria, Chlamydophila pneumoniae, in the

etiopathogenesis of MS. Researchers have not yet come to any definitive conclusion and

controversy still surrounds the validity of the results. We therefore undertook this study

which, if it confirms the association, could have important diagnostic and therapeutic

implications.

This chapter is divided into four sections: first, we revise the aetiopathogenesis of MS

and its possible association with viral or bacterial infections; the second and third section is

an update on the basic microbiological aspects of HHV-6 and C. Pneumoniae, with a

comment on the limitations of laboratory detection techniques, as well as a historical note on

its possible relation with MS; finally, we provide the results of a meta-analysis of studies

published from 1965-2002 in patients with the relapsing-remitting (RR) form of MS (RR-

MS) in whom different laboratory techniques were used to detect these two agents. The meta-

analysis of the published data is an original contribution which may provide new information

or else show up some observation not previously considered. The study was undertaken with

reference to MS-RR patients because they form the greater proportion of all those MS

patients who attend a neurology service and also because they have initial stages of the

disease, where any possible aetiopathogenic association is more likely to be seen.

INFECTION AND MULTIPLE SCLEROSIS

The supposed relation between an infectious agent and MS was already suggested in

1884 by Pierre Marie, a French neurologist, disciple and successor of Charcot in the chair of

Paris University. He even considered the possibility of a future vaccine against the disease

[1]. However, demonstration that an infectious agent is related with a human disease requires

a series of conditions, established by Koch, a 19th Century German microbiologist. These

criteria can be summarised as follows:

1. the infectious agent occurs in all cases of the disease in question and under

circumstance which can justify the pathological changes seen in the patient.

2. it does not exist in other diseases as a fortuitous, non-pathogenic infectious agent.

3. after complete isolation from the human body and repeated pure culture it can again

cause the disease.

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If these conditions are fulfilled, then the occurrence of the infectious agent in the disease

is not accidental, and therefore the only relation which can be considered between this and the

disease is that of a causative agent.

These conditions have not been wholly fulfilled in such diseases as diphtheria and other

bacterial infections in humans. However, after discovery of viruses, Koch’s postulates

became outdated and even, in certain cases, obsolete. First, because of the limited sensitivity

of isolation media, viruses are almost never isolated in all cases of a disease. Second,

pathogenic viruses such as the polio virus or the herpes virus are often recovered from

persons who are not ill. Third, viruses grow in live cells and can not therefore grow in pure

culture, as was defined by Koch. Finally, when they are introduced into experimental animals

they quite often produce different diseases.

It was for these reasons that Rivers highlighted the importance of immunological studies

to demonstrate a primary immune response during the infection to ensure that the virus was

not present fortuitously in the patients or was recovered accidentally in experimental animals.

In certain neurological diseases, however, it is not possible to demonstrate this activation of

the immune system. This is why Gibbs and Johnson, in 1974, proposed the following

alternative criteria to relate a virus with slow infections: 1) there should be consistency in

transmission of the disease to experimental animals or some consistency in virus recovery

from cell cultures, and this transmission and recovery should be confirmed by more than one

laboratory; 2) either the serial transmission of the pathological clinical process should be

fulfilled using filtered material and serial dilutions to establish replication of the agent or else

the recoverable agent should be demonstrated consistently in diseased tissue by electron

microscopy, immunofluorescence or other techniques, and it should be shown in appropriate

cells to explain the lesions; 3) parallel tissue studies from healthy persons or persons with

other diseases should be undertaken to establish that the agent is not a saprophyte or

contaminant [2].

Several different lines of investigation support the hypothesis that MS may be related

with a possible infection: 1) epidemiological studies have indicated that there is an exposure

factor in infancy in the genesis of the disease [3,4]; 2) experimental animal studies (Theiler’s

virus, mouse hepatitis virus) have shown that these viruses produce diseases with long

incubation periods, oscillating courses with remissions and relapses, with different pathogenic

mechanisms of myelin destruction [5]; 3) studies in MS patients have, although not

consistently, shown a relation with some viruses or diverse micro-organisms [6]. (Table 1)

The pathogenesis of the supposed infection should be able to explain the triggering of the

disease, the appearance of bouts and remissions during the first years of the disease, and the

development of the clinical form known as progressive secondary MS, in which there is

neurological worsening independently of the bouts after a certain time. Consideration should

also be given to the role of drugs currently used to treat MS, including corticoids, beta

interferon, Glatiramer acetate (copolymer) and immunosuppressive agents, all with proven,

albeit partial, efficacy.

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Table 1. Presumptively isolated viruses in MS (Modified from Johnson, 1994)

Isolated virus Method Year

Rabies Encephalitis in mouse by inoculation of brain or blood 1946/1964

Herpes simplex Cytopathic changes in cultures inoculated with brain homogenized.

Isolation in CSF during first relapse.

1964

1989

Carp Agent Decreased in polymorphonuclear cells in rats inoculated with MS

tissues

1972

Parainfluenza Cell co-cultures of brain tissue with other cells 1972

Measles Cytopathic changes in monkey kidney cells inoculated with brain 1972

Simian virus-5 Syncitia formation in MRC5 cultures inoculated 1978

Chimpanzee CMV Inoculation to newborn chimpanzee with cerebral cells 1979

Coronavirus Inoculation to rats with fresh non-frozen brain 1980

SMON virus Cytopathic changes in MRC5 cultures inoculated with CSF 1982

Flavivirus Inoculation of rats with blood 1982

HTLV-1 Identification of RNA in T cells of the CSF 1986

LM7 (retrovirus) Founded in CSF leptomeningeal cell lines 1989

HHV-6 Differential detection by PCR in brain plaques 1996

It is possible that an agent or agents may trigger the disease in an immunogenetic

substrate with a particular predisposition and later the same agent or another agent or agents

may be responsible for the development of the disease. Two theories have recently been

proposed, known as “Hit-Hit” and “Hit-Run” which attempt to explain this phenomenon.[7]

According to the former, one or more persistent infections in the CNS could produce multiple

insults (Hit-Hit) over time, with the corresponding direct or indirect damage to the myelin or

the oligodendrocytes, or both; in this case microbiological studies could possibly detect a

relation between the infectious agent and MS. Alternatively, the second theory proposes that

demyelinization is the result of an autoreactive immune response produced by a viral or

peripheral microbial infection which then disappears (Hit-Run). This situation is therefore

more difficult to relate aetiologically with the disease.

To determine, therefore, whether a particular agent, such HHV-6 or C. pneumoniae, is

truly responsible for MS the following criteria should be taken into account to establish a

cause-effect relationship: 1) the cause should always precede the effect (temporal relation); 2)

this causal association appears in patients and not in control subjects (high relative risk, which

indicates a strong association); 3) in the presence of a high exposure to the agent there is also

a high risk of disease (biological gradient); 4) repeated studies by different research groups in

different areas provide the same results (they are consistent); 5) there is a plausible

demonstrable pathogenic explanation according to the present state of scientific knowledge;

6) there is just one disease for just one cause (biological specificity); 6) there must be

experimental evidence, with an animal model which can serve as a reference; and 7) there is

an established cause-effect relationship for a similar exposure (biological analogy) [8].

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Virus and Demyelinating Diseases of the Central Nervous System

The role of viruses in the production of demyelinization of the CNS has long been

known. Two diseases exist in humans which are the prototype of this phenomenon:

progressive multifocal leukoencephalopathy produced by the JC virus (JCV) and subacute

sclerosing panencephalitis secondary to the measles virus. Both diseases are very uncommon

in usual clinical practice and have a very remote temporal relation between the time of

infection and the onset of symptoms, and are therefore considered to be latent in the tissue of

the CNS (brain or spinal cord, or both) by means of mechanisms of viral persistence not yet

clearly defined. Reactivation in the case of JCV is closely related with severe functional

failure of the immune system. This explains why immunosuppressed patients of whatever

origin (AIDS, neoplasms, etc.) more often present this disease. There are more than ten

different classes of different virus which can induce lesions of CNS white matter, both in

humans and in animals [5]. (Table 2)

Table 2. Viruses that have been Associated with Demyelination

(Modified from Stohlman, 2001)

Virus Family Host

Measles Virus Paramyxoviridae Man

JC Virus Papovaviridae Man

HTLV-1 Lentiviridae Man

AIDS Virus (HIV) Lentiviridae Man

SmallpoxVaccine Virus Poxviridae Man

Herpes Simplex Virus (HSV) Herpesviridae Man

Mouse Hepatitis Virus Coronaviridae Mouse

Theiler’s Virus Picornaviridae Mouse

Semliki Forest Virus Alphaviridae Mouse

Sindbis Virus Alphaviridae Mouse

Canine Distemper Virus Paramyxoviridae Dog

Visna Virus Lentiviridae Sheep

Two experimental mouse models of virus-induced demyelinization exist. Both models

are produced by RNA virus belonging to different families, Picornaviridae for the virus of

Theiler’s encephalomyelitis (TEMV) and Coronaviridae for the murine hepatitis virus

(MHV). These two demyelinating infections produced by animal viruses shed light on the

interaction between infectious agent, genetic substrate and immune system regulation to

condition the course of the disease.

The preferred mouse strain to induce Theiler’s encephalomyelitis (TEM) is called SJL,

which is also the most widely used in studies of experimental allergic encephalomyelitis

(EAE), another type of demyelinating disease induced in laboratory animals by immunization

with antigens obtained from their CNS. However, MHV requires inoculation in C57/BL6 or

BALB/c mice, but not in the SJL mouse which is resistant to this infection, as it lacks the cell

receptor for TEMV.

The TEMV is a relatively common natural pathogen in laboratory mice but spontaneous

infection of the CNS is rare. Experimental induction of the disease therefore requires

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inoculation of the virus into the mouse brain in young, preferably female animals which are

more susceptible. Four viral strains can induce the disease, two of which are very

neuropathogenic (GDVII and FA), which produce a monophasic fatal disease, and another

two attenuated strains (BeAn and DA), which work biphasically, first producing an acute

encephalomyelitis with invasion of the neurons, followed by temporal disappearance of the

virus from the CNS, only to reappear later infecting glial cells (astrocytes and, mainly,

microglia, but scarcely in oligodendrocytes). In those mouse strains which are resistant to

demyelinization, the immune system is able to completely and permanently eliminate the

TEMV.

Initial TEMV encephalomyelitis affects the neurones of the thalamus, hypothalamus,

encephalic trunk and motoneurons of the anterior medullar horn which are the initial

objective of the virus, whereas the white matter, meninges, choroid plexus or the ependyma

are not involved, and therefore barely present demyelinization or inflammation of the

parenchyma during this period of the disease. During this phase, a local inflammatory

reaction is produced in the area of the brain infected by TEMV, and is followed later by a

phase of haematogenous dissemination of the virus (viraemia) with the corresponding

generation of humoral immunity (Ab) and cellular immunity (CD4+, CD8+). From 2-4 weeks

after infection there appears in strains of susceptible mice inflammation and progressive

demyelinization of the spinal cord which correlate with persistence of the virus, mainly in

macrophages. In this disease there is typically myelin destruction which does not require

massive oligodendrocyte infection.

Four mechanisms have been proposed to explain demyelinization by TEMV in the

mouse: 1) infected oligodendrocytes which are eliminated by the virus; 2) destruction

mediated by cytokines released by antiviral inflammatory cells of the CD4+ type (Th1); 3)

continued secretion of IFN-gamma which maintains a persistent activation of macrophages

and microglia; and 4) cytolysis mediated by CD8+ cells of infected oligodendrocytes. The

most likely of these mechanism is the second, because susceptible mice generate a vigorous

antiviral response by means of CD4+ cells and neutralizing antibody which, for reasons not

well unknown, do not eliminate the TEMV from the CNS.

Bacteria and Autoimmunity of the Central Nervous System

Numerous studies over the last decade have suggested that the immune response in MS is

related with a bacterial infection which triggers or worsens the disease [9-12]. Shared or cross

antigenic stimulation has been proposed, i.e. similarity between structures of the CNS and

usual commensal bacteria in humans, especially in the upper respiratory tract. These studies

give great importance to the age at which the supposed infection is acquired (childhood or

adolescence), which may be critical for triggering an aberrant immune response.

The main line of defence against these infections is formed by barriers resistant to the

micro-organisms, such as the skin or mucous membranes which cover the various tracts and

organs (respiratory, digestive, genito-urinary, conjunctival). The second line of defence

depends on recognition of the most common bacterial components by the immune system,

which is composed of different molecules and non-specific cells (complement, leukocytes,

monocytes, macrophages) or specific cells (Ig, T lymphocytes, plasma cells).

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The pathogenic mechanisms of the bacteria are related with its microbiological structure

and its invasion mechanisms, persistence or associated toxicity. The double-lipid layer of

gram-negative bacteria is susceptible to mechanisms which can lyse membranes such as

complement and certain other types of cytotoxic cells; the destruction of other types of

bacteria, however, (gram-positive, mycobacteria) requires the participation of phagocytes.

The immune response against bacteria can result in immunomediated tissue damage via

diverse mechanisms such as massive release of cytokines leading to endotoxic septic shock,

Schwartzman reaction with associated disseminated intravascular coagulation or Koch’s

phenomenon of skin necrosis. Recently, however, other novel aspects of the defence response

generated by bacterial infections have been led to great interest, such as the presence of

superantigens or heat shock proteins.

Bacterial superantigens are epitopes of proteins or glycolipids which escape to the

habitually way of activation, the “immunological synapse”, formed by the major

histocompatibility complex class II antigen presenting cell and the T cell receptor.

Heat shock proteins (HSP) are one of the best preserved molecules in the biosphere.

Their synthesis increases to protect, by stabilization of proteins, prokaryote or eukaryote cells

during periods of stress produced by bacterial infection (especially with intracellular

pathogens such as C. pneumoniae), inflammation and other harmful agents such as trauma,

burns, heat stroke, etc. Due to these functions, they are prominent objectives of the normal

and altered immune system (autoimmunity), as they may confuse the response against HSP of

the micro organism with the body’s own HSP (cross reaction).

Finally, according to the “hygiene hypothesis”, numerous epidemiological studies link

the apparent increase in allergic and autoimmune diseases (including MS) in the developed

world with reduced exposure to usual infectious agents in our environment, seen by the low

rate of childhood infections thanks to advances in hygiene and health over the last century.

According to this theory, bacterial and viral infections early in life direct the immune system

towards maturation of Th1 lymphocytes and counteracting Th2 cells, which favour allergic

processes.[13] However, other studies of EAE show just the opposite, i.e. if the laboratory

environment is protected from infectious agents, autoimmune diseases do not develop.

Celullar and Molecular Pathogenesis of Autoimmunity in Multiple Sclerosis

The central role of the phenomenon of autoimmune mediation in MS is supported by

immunological data from the study of the acute lesions of MS. These lesions have T helper

cells (CD4 +) and an anomalous expression of MHC Class II antigens (macrophages and

astrocytes). There is also activation of B cells, shown by the presence of immunoglobulins

synthesised in the CNS (intrathecal synthesis), which lead to the characteristic finding of

oligoclonal bands in cerebro-spinal fluid (CSF).

This autoimmune pathogenetic model is reproducible in EAE. It can be induced in

experimental animals by injecting homogenates of spinal cord or purified myelin proteins and

which is associated with focal demyelinization with a T cell infiltrate, very similar to what is

seen in MS patients. This disorder in the guinea pig can lead to recurrent demyelinating

disease, chronic relapsing experimental allergic encephalomyelitis (CREAE). The main

antigens in EAE are the myelin basic protein (MBP) and the myelin oligodendrocyte

glycoprotein (MOG) [14-16].

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A hereditary predisposition coupled with an unknown environmental factor establish or

maintain autoreactive T cells, which after a latent period may be activated by a systemic or

local factor (viral infection, puerperium, etc.) by means of molecular mimicry (epitopes

shared by the myelin and the possible infectious agents) or by stimulation via viral or

bacterial superantigens.

Molecular mimicry has been shown, as epitopes exist shared by myelin (MBP, CNPase)

and possible infectious agents (coronavirus 229E, Mycobacterium leprae, HHV-6, syncytial

virus) which could trigger the immunological response. Moreover, recent studies have shown

that the conceptual model of molecular mimicry is flexible, as antigen recognition is much

less specific than previously believed and it is not based solely on complete homology of

peptide sequences. Specific MBP derived from MS patients are not necessary for cross-

reactivity in experimental T cell models, but a concomitant increase in the expression of HLA

costimulation and adhesion molecules is necessary for autoimmune phenomena to occur [17-

19].

Activation mechanisms by superantigens involve bacterial or viral proteins able to bind to

the HLA protein of the antigen presenting molecule outside the antigen binding cleft and

activate specific polyclonal reactive V-beta T cells against myelin antigens. Furthermore,

superantigens do not require to be degraded prior to binding with HLA molecules, they have

no HLA restriction to being presented, but the different HLA-DR haplotypes vary in their

ability to bind and present some superantigens. The best studied superantigens involved in the

pathogenesis of MS and the appearance of bouts are the staphylococcal enterotoxins SEB and

TSST-1 [20,21].

Once activated, these T cells selectively pass the blood-brain barrier and on re-exposure

to their autoantigen start a Th1 type inflammatory response. A phenomenon of epitope

amplification has recently been described. Initially, T cells recognise an antigen epitope, but

with time these cells also recognise other epitopes of the same antigen, and even epitopes of

other antigens, and become activated [22-27].

Once inside the CNS, the activated T lymphocyte could find an antigen-presenting cell

(macrophage or microglia) which expresses on its surface the antigen responsible for MS in

the context of an HLA class II molecule and costimulating molecules. The antigen is the least

known factor, and further, it should be borne in mind that more than one antigen may exist

able to trigger the immune response and that the antigen or antigens initiating the disease may

not necessarily be the same antigens which perpetuate it (epitope amplification). The myelin

proteins of the CNS involved in T cell autoreactivity include MBP, MOG, MAG (myelin

associated glycoprotein), PLP (proteolipid protein), B-crystalline, transaldolase,

phosphodiesterases, and other non-myelin proteins such as HSP, astrocyte antigens (S100

protein), some endothelial antigens and nuclear factors [28,29].

After the trimolecular complex has been formed (T cell receptor, the antigen and the

HLA class II molecule), the T cells, which are of the CD4 type 1 helper (Th1) phenotype

produce proinflammatory cytokines (interferon-gamma, TNF-alfa, IL-1, IL-2, IL-12) and

chemokines, which induce clonal proliferation of T cells and attract and activate macrophages

and microglia, thereby initiating inflammation. Type 2 T helper lymphocytes (Th2) release

anti-inflammatory cytokines (IL-4, IL-6, IL-10, TGF) which tend to reduce the

proinflammatory state of the immune system, but which also induce proliferation of B cells

and the resulting elaboration of antibodies by these B cells. The balance between the different

cytokines and their concentrations largely determines the direction of the immune reaction of

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the whole process. Suppressor T cells (CD8+) and T cells expressing the natural killer

receptor produce a reduction in the proliferation of T helper lymphocytes (antiergotypical

response) and also inhibit their activation (anti-idiotypical response), thereby contributing to

counter-regulation of the inflammation [30,31].

Despite the presence of inflammation in MS, the pathogenic role of the inflammatory

response is unclear. Some studies support both the concept that the inflammatory response is

a prerequisite for demyelinization and that it may occur independently. Moreover, the

abundance of inflammation in inactive cases together with recent observations of the local

production of neurotrophic factors by the leukocytes may suggest an important role for

inflammation in the repair of MS lesions. Inflammatory infiltrates in the CNS may therefore

have a neuroprotective effect which could limit the success of non-specific immunotherapy

[32-35].

The pathogenic model of MS based on T cell mediated cellular immunity is currently the

most accepted, although recent immunopathologic data suggest a heterogeneous cause. This

would give rise to a specific immunopathogenic disease spectrum for each patient and for the

different stages of disease development. The pathogenic mechanisms reported are

demyelinization mediated by T lymphocytes and to a variable degree by antibodies (patterns I

and II), oligodendropathy (dying-back) secondary to anomalous expression of myelin proteins

and with apoptosis of the oligodendrocytes (pattern III) and the gradual loss of the

oligodendrocytes combined with demyelinization (pattern IV) [36-38].

HUMAN HERPES VIRUS TYPE 6 AND MULTIPLE SCLEROSIS

Microbiological Aspects

Human herpes virus type 6 is a virus which belongs to the Herpesviridae family, well

known for producing different types of diseases in humans and animals. In humans, there are

various subfamilies and species: subfamily alfa-herpesvirinae with herpes simplex virus

(serotype 1 y 2) and varicella-zoster virus; subfamily beta-herpesvirinae with human

citomegalic virus and human herpes virus 6 and 7; and subfamily gamma-herpesvirinae with

Epstein Barr virus and human herpes virus 8.

They are characterized by presenting a double strand of DNA surrounded by an

icosahedral capsid. Outside the capsid is an envelope with the typical glycoproteins derived

from the nuclear membrane or from the Golgi apparatus. Between capsid and envelope there

is the tegument, which is composed of proteins. Their size varies from 100-200 nm in

diameter [39].

HHV-6 enters the cellule by three different ways: viral receptors, endocytosis or cellular

fusion, and then goes to the nucleus where it replicates. The process of the viral genome

transcription is made by RNA polymerases in three sequential phases, each one depending of

the previous phase of genes transcription and translation. The first phase is denominated early

immediate and begins after viral introduction, it is when the primary proteins needed for

nucleic acids and other proteins are produced. The second or early phase is characterized by

transcription of early viral genes with synthesis of specific viral proteins like thimidinkinase

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or DNA polymerase, that could be important therapeutic targets. In the third o late phase are

synthesized most of the late viral genes and structural viral proteins. Proteins of the early

immediate phase are present along all the viral cycle.

When the virus finish its biological cycle it leaves the host producing cytolysis. In some

cases, cellular infection is latent, with o without cell genome integration; the viral cycle is

then stopped in the early immediate phase and characteristically there are none o few signs of

infection. In another situations, HHV-6 can transform the infected host cell by different

mechanisms, including neoplastic changes. When viral latency is established in cells, there is

a low viral antigen expression with less synthesis of antibodies against it by the host immune

system. Diverse viral and cellular interacting factors determine if viral infection produces

cytolysis, latent persistent infection or cellular transformation.

Isolation of HHV-6 was first done in 1986 by Salahuddin from the blood of six patients

with lymphoproliferative disorders. It was then called the human lymphotropic B cell virus or

HLBV [40]. There are two variants, A and B, very similar genetically though they differ in

cell tropism and pathogenicity. The HHV-6 is able to infect different types of cells including

CD4 lymphocytes, CD8 cells, natural killer cells, oligodendrocytes and microglia. The

pathogenesis of the virus is probably related with different mechanisms: the cytopathic

destructor effect of the infected cell, cytokine induction (inflammatory mediators),

immunosuppressive effects and effects on other viruses (transactivation).

In contrast with other human pathogenic viruses, when a herpes virus infects a subject it

can produce clinical symptoms or not, but it persist along all the life of a person with the virus

remaining quartered latently in host cells (i. e. in the ganglia of the sensitive dorsal roots of

the nerves). Thus, they can produce early infection in infancy with a long period of latency

(many years), and which can reactivate in conditions of low immune defence (AIDS,

transplants). This persistency produces latent o transforming infections with cellular

immortalization.

The epidemiology of infection by HHV-6 differs according to the variant: HHV-6B is

endemic and is acquired in early childhood (seroconversion between the ages of six months

and two years), and is easily recovered from the saliva of infected children, being the most

frequent agent of roseola infantum. The other variant, HHV-6A, however, is not usually

detected until the person reaches adulthood and is associated with reactivations of the virus.

Historical Considerations

The possible relation between HHV-6 and MS first appeared in 1993 when the group of

Luppi, haematologists from the Italian university of Modena, published a study detecting viral

DNA in saliva samples and peripheral blood mononuclear cells from two patients with

lymphoproliferative disorders and one with MS [41]. Just a few months later this group

published a study with serum samples from 126 MS patients and 500 healthy controls

analysing the presence of antibodies against the virus with immunofluorescence. They also

analysed the detection of viral DNA in peripheral blood mononuclear cells using PCR in 31

patients and 24 healthy subjects. The results showed a significantly higher rate of anti-HHV-6

antibodies in patients than controls. PCR was positive in one patient and one control and

Southern blot analysis showed that the MS patient had an unexpectedly high level of viral

sequences compared with the healthy person [42].

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In January 1994, a group of German researchers from Berlin University reported their

experience in patients with different neurological diseases (21 with MS, 19 with facial palsy

and seven with Guillain-Barré syndrome) and compared the results with a control group of 16

persons. They studied simultaneous samples of serum and CSF by PCR to detect HHV-6,

completing the study with ELISA for detection of antiviral antibodies. They found that 3/21

patients with MS (14%) had viral DNA in CSF but not in the corresponding serum sample,

whereas the measurements in all the patients with facial palsy or Guillain-Barré syndrome, as

well as the control persons, were negative. The antibody study showed that the serum titres

against HHV-6 were significantly increased in patients with MS compared to patients with

the other two neurological diseases [43].

In August 1995, the prestigious Proceedings of the National Academy of Sciences,

published the crucial article of Challoner. In this paper with material from 86 brains from MS

patients and controls, they could demonstrate that a fragment of 341 nucleotides identical in a

99,4% to the main binding gene of the HHV-6 DNA, was present in more than 70% of the

patients and controls, being, then, a common virus in human brain. By sequencing the DNA,

they established that in 36/37 (97,2%) of the cases and controls the HHV-6 variant B group 2

was detected. Other herpetic viruses, retroviruses and the measles virus were poorly detected.

They also performed techniques immunocytochemistry with monoclonal antibodies against

the protein of the virus 101K and against the binding protein of the DNA p41, and have found

that nuclear staining of oligodendrocytes was present in MS cases but not in controls, and in

MS cases it was observed around plaques more frequently than in uninvolved white matter.

MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to

plaques, although neurons expressing HHV-6 were also found in certain controls. Because of

this, the authors concluded that it might be an association between the HHV-6 and the

pathogenesis of MS.[44]

Nevertheless sooner than one year later, Sanders et al. performed a similar study,

analysing 5 viruses of the Herpesviridae family (herpes simplex, varicella-zoster, Epstein-

Barr virus, cytomegalovirus and HHV-6), because they considered than all of them are

capable of persistence and latentcy inside the CNS. They investigated the presence with PCR

of the viral DNA in different forms in active and inactive demyelinating plaques, normal

white matter and grey matter of the brains of MS patients, and compared results with brain of

patients with Alzheimer disease, Parkinson disease or not neurological diseases. They found

HHV-6 DNA present in 57% of the MS patients brains against 43% of the controls, as well as

32% in active demyelinating plaque against 17% of inactive plaques. They also found greater

positivity for other herpetic viruses (herpes simplex and VZV) in MS against controls, but

none of these data were statistically significant, so they concluded the article assesing that the

possible association was unclear[45].

In December 1997, a group of investigators from the American National Institutes of

Health, published in the journal Nature the results of a serological study, in which they

showed a significant increase of type IgM antibody rate in serum against the early Ag

(p41/38) of HHV-6 as well as the presence of the virical genome with PCR, in 15 of 50 MS

patients against to none of the 47 controls with other neurological diseases[46].

J. Gutiérrez, M. García, F. Fernández, O. Fernández, V.E. Fernández, et al.

174

Recent Investigations

The situation remained in an unstable equilibrium between opponents and about the

participation of the HHV-6 in MS until 2000, when important works from north American

investigators were published, adding more interest to the controversy. Soldan et al. analysed

the cellular immune response to HHV-6 (the strains 6A-U1102- and 6B-Z29) and against

HHV-7 (H7SB) comparing the linfoproliferative response of 18 MS patients and 21 healthy

controls. They found that 71% and 31% of controls had proliferative response against the

variant HHV-6B and HHV-6A respectively, while 67% and 78% of the patients had a

response to the same variants, resulting significative the difference respect to the variant

HHV-6A, but not against the variant HHV-6B or HHV-7[47].

These same investigators published some months later the tisular distribution of HHV-6

in MS patients and healthy controls. They studied the presence of the virus in different types

of corporal fluids including serum, saliva, urine and peripheral blood lymphocytes. They

found that there was a significant increment of the frequency of DNA in MS patients serum

and also they detected viral sequences in a subgroup of them. Besides, the virus variant

implicated was the HHV-6A that they considered to be the specifically neurotropic, so they

concluded that it might contribute to the process of the disease[48].

Another study performed by Knox et al. with CNS samples from 11 MS patients

autopsies demonstrated that existed actively HHV-6 infected cells in the73% patients, being

the virus located preferably in those zones with active demyelination. In the same article,

these authors communicated the results of the technique of culture of HHV-6 in blood

samples from 41 MS patients and 61 healthy controls. They found that 54% of the patients

presented active HHV-6 infection against to none of the controls, and that those patients with

virus demonstrated in blood, were significant younger and with less time of evolution than the

others[49].

The most important clinical studies recently published[50-70] are summarized in the

following table (Table 3). A critical evaluation is to be done to these papers in regard of some

limitations: 1) HHV-6 is a virus that is acquired early in the childhood (B variant) and

remains since then in the organism (lymphocytes); serological studies in adults show

reactivation instead of primary infection; 2) it is not clear the implication of the different

variants with the disease (although recent studies show that it is A variant) and actual routine

serological techniques do not allow to distinguish between them; 4) cellular culture

techniques (isolation of the virus) and PCR are not standardized, so, different laboratories

might obtain different results depending on the methods they use; 5) the kind of samples

collected and the techniques used are not homogeneous: sometimes only antibodies (Ab) or

DNA are studied, and no cultures were performed; or in another investigations, serum,

PBMCs o CSF are examined separately; 6) sometimes the selection of patients has not been

well defined (RR-MS? or SP-MS?) or there has not been well characterized the time of

sample extraction (was the patient in a relapse?, was there activity by MRI?) and there has not

been correlated with CSF parameters.

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Table 3. Clinical studies recently published about HHV-6 and MS

Authors Population Samples Techniques Results

Merelli[50] 56 MS PBMC, CNS

autopsies

PCR (+)

Nielsen[51] 189 ME /190 OC Serum Ig (-)

Ablashi[52] 21 MS / 35 FCS

20 OND / 28 OC

B, PBMC Ig, PCR, culture ↑IgG, IgM. DNA in MS and

CFS

Alvarez[53] 102 MS / 102 OC PBMC PCR ↑ VHH6, but nor other viruses

Blumberg[54] 8 MS / 25 OND

/6 OC

CNS

autopsies

PCR, IHQ ↑ VHH6 in oligodendrocytes

fron MS plaques

Ferrante[55] 22 MS / 16 OC PBMC, PCR (-)

Hay[56] MS, OC PBMC PCR (-)

Kim[57] 34 MS/ 6 ITM

2 ON / 20 OC

PBMC PCR 7+/27- MS; 2+/4- MIT; 0/0

OC (A variant)

Knox[58] 99 MS/ 80 OC PBMC PCR (+)

Ferrante[59] 30 MS PBMC PCR ?

Locatelli[60] 47 MS/ 30 ON B, CSF PCR (+)

Alvarez-

Lafuente[61]

102 MS / 102 OC B, PBMC PCR (+)

Caselli[62] MS, OND, OC B Ig (+) prevalence: 87% vs 43%,

with ↑ titles

Cirone[63] 22 MS / 16 OC T cell, B,

CSF

Ig, PCR,

lymphoproliferation

assay

(-)

Gutierrez[64] 41 + 27 MS / 31

OND

B, CSF Ig, PCR ?

Rodríguez

Carnero[65]

23MS / 23 OC CSF PCR (-)

Tejada-Simon[66] MS, OC B, CSF Ig, PCR (+)

Al-Shammari[67] 24 MS /13 OND

/20 OC

B PCR (-)

Cermelli[68] 13 MS / 13 OND /

12 OC

CNS

autopsies

PCR (+)

Chapenko[69] 26 MS / 21 OND /

150 OC

PBMC, B PCR, Ig (+)

Goodman[70] 5 MS CNS

autopsies

PCR / IHQ (+)

CSF: cerebrospinal fluid.

B: blood (serum or plasma).

CNS: central nervous system.

Ig: immunoglobulin (antibodies).

IHQ: immunohistochemistry .

ITM: idiopathic transverse myelitis.

MS: multiple sclerosis.

MS?: suspected multiple sclerosis.

ON: optic neuritis.

OND: other neurological diseases.

CFS: chronique fatigue syndrome.

OC: non-neurological control.

PCR: polymerase chain reaction.

PBMC: peripheral mononuclear blood cells.

RNA: ribonucleic acid.

: negative results (no association).

+: positive results (association).

?: non conclusive results.

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CLAMYDOPHILA PNEUMONIAE AND MULTIPLE SCLEROSIS

Microbiological Aspects

The Chlamydiaceae family included the genders Chlamydia and Chlamydophila, being of

clinical interest in humans the species Chlamydia trachomatis, Chlamydophila psitaci and

Chlamydophila pneumoniae; the last one was initially nominated as the TWAR agent. They

have DNA and RNA genetic material, having a cellular wall and ribosomes similar to

gramnegative bacteria. They are obliged intracellular micro organisms because they are not

able to synthesize energetic compounds, so they utilise the biochemical machine of the cell

they infect. Because of this characteristic, in order to recuperate it in the laboratory, special

cells susceptible of infection are needed[71-74].

They have a complex and characteristic replicative cycle, existing two forms that

participate in it: the extracellular elemental body (EB) and intracellular reticular body (RB).

The EB has a size between 0,2 and 0,4 μm able to adapt itself well in the extra cellular

medium because they do not have metabolic and replicative activity. The EB constitute the

infective form of the Cp that is transmitted from one person to another because it is very

resistant to environmental factors and is capable to adhere to susceptible epithelia type cells,

in which penetrates by endocytosis. In less than eight hours after having entered the infected

cell, the EB reorganized itself into RB adapted for survive in the intracellular medium and

multiply by binary division. The RB has a diameter between 0,6 and 1,2 μm, obtains the

nutrients from the cytoplasm of the infected cell by porines, it is fragile and susceptible to

external agents, it multiply itself by binary division being able to generate several replicants

that are inside the membrane of the inclusion body and can occupate most of the infected cell

being visible with the optic microscope (inclusion bodies). After 24 hours, the reticular bodies

condensate and form EB that still are inside the intracellular inclusion. When the intracellular

inclusion breaks out, the cycle begins again infecting adjacent or distant cells.

The step from EB to RB and the contrary requires a cellular cycle, that in case of Cp lasts

2-4 days. In some environmental conditions and depending of the infectious agent, the cycle

can be temporally or permanently stopped, in the RB phase, that now is denominated

persistent body (PB): they are bigger RB and they divide slowly, by lower number of porines.

In this situation less bacterial antigens are expressed, so we can consider these changes as

adaptation of the bacteria to the host to avoid the immune system. The PB have been

observed both in-vivo in places accesibles to the immune system to avoid it, and in-vitro

when there is penicillin or absence of sulphured aminoacids in the medium. All this has

diagnostic and therapeutic repercussions: immunodiagnostic false negatives for less bacterial

antigens; cellular culture false negative for less PB feasibility; and worse response to

antibiotic treatment for the lost of porines.

Cp antigens are complex and partially known, among them outstand two groups:

glycolipids and proteins. The LPS, central oligosacaride with specific unions 1-8 of the

octulopiranoside acid, lacks of O polisacaride and A lipid and is not very toxic. Among the

proteic antigens outstands the major outer membrane protein (MOMP) (gene omp) and outer

membrane complex (Omc). There are also secretive proteins from inclusion bodies. Infected

cells express LPS and secretive proteins; the EB have structural proteins (MOMP and Omc),

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principally. All these antigens are important, because the specific Ab response against Cp the

in blood or CSF of infected patients will allow us detect infections by serological studies.

Epidemiological data of this microorganism are basically from studies of the antibodies

detection and show that primary infections occur in the late childhood with a peak incidence

in young adults, although the risk persist all life long. The seroprevalence rate, that is the

percentage of adults with IgG against Cp, reaches the 40% in different parts of the world. The

possibility of secondary infections (reinfections) exists all the life.

There are outbreaks of pneumonia and other respiratory infections in persons who live

together very closely (military quarters) and where colonized aerosols with EB are the

transmission vectors.; it starts in the high respiratory tract (nasal mucosa, sinus and

oropharynx) persisting in many persons as an asymptomatic opportunist parasite all life long.

Nevertheless, in some cases, the microorganism is transported to far away places (perhaps by

macrophages) as there are data that show replication in arteries and joints sinovial

membranes. Cp external membrane protein can generate a cross over immune response with

human proteins (autoimmunity).

The diagnosis of this infection is difficult, even now a days, because we do not have a

standardized laboratory method, there are no routinely cellular culture media in hospitals and

there have not been developed commercial genetic test (PCR) for studies. So, routine

diagnosis is based on retrospective demonstration of antibodies, commonly by indirect

immunofluorescence, between determinations of serum (or CSF) taken at the beginning of the

supposed infection and in convalescent phase.

Historical Considerations

The journal Medical Hypotheses in 1993 published an article of Perlmutter and Darvish

titled “Posible relationship of Cp with MS”, speculating that this bacteria could be implicated

in the pathogenesisi of this disease[75].

In 1996, some investigators from the Haartman Institute of the University of Helsinki

(Finland), published a paper signed by Koskiniemi in European Neurology journal about CNS

infections associated to Chlamydia pneumoniae, old denomination of the bacteria known

today as Chlamydophila. In this epidemiological study, after analysing the CNS infections in

a population of 3 million inhabitants with serological techniques (Ab detection), 263 cases of

neuroinfection were found, 15 of which were due to contact with Cp or recent reactivation. Of

this later group of patients, in 9 cases no other infectious associated agent was found and a

high morbidity rate was observed, including one death[76].

No more than 2 years later, Neurology, official journal of the American Academy of

Neurology, published an article signed by Sriram, neurologist in the University of Vanderbilt

of Nashville (TN, USA) and 2 pathologists of the same center[77]. This communication

presented the intriguing case of a patient diagnosed of MS with a rapidly progressive course

and refractory to diverse treatments. It was a 24 years old patient seen in 1996 in the MS

clinic of that hospital; the symptoms had started on January 26th of the same year when he felt

numbness in the left arm and leg, showing a progression in five days to complete a Brown-

Séquard sydrome with urine retention. The magnetic resonance (MRI) showed cervical

hyperintense lesions in T2 associated with periventricular lesions in both cerebral

hemispheres. CSF conventional microbiological cultures did not show any bacterial growth.

J. Gutiérrez, M. García, F. Fernández, O. Fernández, V.E. Fernández, et al.

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Weakness in both left members partially improved after i.v. metilprednisole pulse. After a

stabilisation period of one month and a half, he presented double vision when looking to the

right and ataxia. He then received another i.v. corticoids pulse with partial improving. In the

first week of April, he worsened again with ataxic gait, optic neuritis, diplopia and

blepharospasm, with a severe disability needing one aid for walking. At that moment, because

of the neurological deterioration, he started treatment with interferon, associated with

plasmapheresis and azahtioprine, with a slight improvement of vision and gait.

In the last week of June 1996, this patient had a new relapse with ataxic gait, double

vision, severe paraparesia and important weakness in left arm, associated in the following

days with disartria and disphagia, and he was treated with cyclophosphamide. A new lumbar

puncture was done showing a CSF with moderate pleocitosis (35 leucocytes/mm3; 90%

lymphocytes), mild protein elevation (154 mg/dL) and positive oligoclonal bands; special

cultures were performed and serological determinations (IgG and IgM) and PCR both in

blood and CSF in order to detect Cp infection. When high titers of IgG against Cp were found

as well as isolation of the micro organism in CSF, cyclophosphamide treatment as stopped

and antimicrobial therapy was established. After that, all the neurological functions improved,

only remaining mild ataxia in members, nistagmus in lateral extreme gaze and bilateral

pyramidal signs, being able to return to work again in May 1997.

When next year (1999) this same author published in Annals of Neurology the positive

results of the Cp investigation in a short series of 37 MS patients with a control group, the

controversy of the possible implication of this infecting agent in the CNS demyelinization

was already served[78].

Recent Invetigations

After the initial publication of Sriram’s case near five years ago, more than 50 papers

have appeared about the same subject; some of them are original work trying to prove the

discovery done by this investigator, even with the same samples; others, only making a

critical review more or less justified. In the next table we summarize the discoveries of the

more relevant clinical studies[79-93]. (Table 4)

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Table 4. Clinical Studies about C. Pneumoniae and MS

Authors Population Samples Techniques Results

Boman[79] 48 MS / 51 OND CSF PCR, cultures,

Ig

-

Ke[80] /

Hammerschlag[81]

25 MS / 16 OND Autopsy PCR, culture -

Treib[82] 22 MS CSF & B Ig, PCR Ig 8+/22– ; PCR

2+/8-

Morré[83] 10 MS / 10 controls;

18 MS / 30 OND

Autopsy;

CSF

PCR -

Pucci[84] 24 MS / 7 OND CSF & PBMC PCR -

Gutiérrez[85] 31 MS / 116 OC B Ig ? anti-MOMP

Gieffers[86] 58 MS / 47 OND /

10 OC

CSF & PBMC PCR, culture ? Cp in CNS of

MS & OND

Krametter[87] 94 MS / 63 OND CSF & B Ig ? intratecal

syntesis Ig

Saiz[88] 5 MS? / 15 MS / 20

OND

CSF PCR -

Sotgiu[89] 32 MS / 30 OND CSF PCR, Ig, BO ? PCR+

Hao[90] 66 MS / 25 OND CSF & B PCR, culture,

Ig

?

Kaufman[91] 30 MS / 22 OND

or OC

CSF PCR ?

Munger[92] 141 MS?/ 62407 OC B Ig +

Chatzipanagitou [93] 70 MS/ 30 OND CSF PCR, Ig

culture

-

B: blood (serum or plasma).

CSF: cerebrospinal fluid.

CNS: central nervous system.

Ig: immunoglobulin (antibodies).

IHQ: immunohistochemistry.

MS: multiple sclerosis.

MS?: suspected multiple sclerosis.

OB: oligoclonal bands.

OC: non-neurological controls.

OND: other neurological diseases.

PCR: polymerase chain reaction.

PBMC: peripheral mononuclear blood cells.

RNA: ribonucleic acid.

-: negative results (no association).

+: positive results (association).

?: non conclusive results.

Besides the already mentioned clinical studies with MS patients, we have to mention

some publications with animal experimentation or molecular biology that suggest the

possibility that Cp could be involved in autoimmunity processes of the CNS[94-97]. (Table

5). Also it has recently been reported the first epidemiological case-control study linking the

bacteria with MS. It had been performed in the Harvard School of Public Health by

determination of IgG against Cp in serum, and supports a relationship with MS, specially in

the progression of the disease[92].

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Table 5. Animal Experimental or Molecular Studies about Cp and MS

Author Type of study Results

Lenz[94] EAE Cross reaction MBP with Cp’s Ag

Yamaguchi[95] Cellular Cp induce macrophage differentiation

Wizel[96] Cellular Cp induce CD8+ CTL and IFN gamma

Du[97] EAE Cp is neurothropic and worsen EAE

Ag: antigens.

CD8+ CTL: CD8+ lymphocytes.

EAE: experimental allergic encephalomyelitis.

IFN: interferon.

MBP: myelin basic protein.

When evaluating such a different number of publications, we have to bear in mind some

facts: 1) Cp is an intracellular bacteria whose pathogenesis, and though, response to host

immune system response, does not follow common mechanisms as other infectious agents; 2)

Cp is difficult to recuperate and identify; direct microbiological identification (cellular culture

and PCR) are not standardized, existing important differences regarding sensitivity and

specificity between the different laboratories; 3) the samples and techniques used have not

been homogeneous: some of them only use CSF, others only blood; sometimes only Ab or

DNA separately are determined; or some samples have been frozen at –20ºC (being not

suitable for these studies); 4) in some cases the selection of the patients has not been defined

(RR-MS? or SP-MS?) or the extraction of the sample was not well characterized (was on a

relapse? was there activity in MRI?) or it has not been correlated with CSF parameters; and 5)

it is remarkable the lack of basic science research correlating Cp infection and demyelination,

as well as collaborative studies to standardized methods between different laboratories.

META-ANALYSIS OF HUMAN HERPES VIRUS-6 AND

CHLAMYDOPHILA PNEUMONIAE STUDIES IN MULTIPLE SCLEROSIS

In order to find the articles, two electronic data base were used (Medline and Excerpta

Medica) with only one key word (“multiple sclerosis”), to reduce the possible errors

associated to the first screening. We found initially 24.462 publications until year 2002, from

which we decided to reject those with no “abstract” and those published in foreign languages

difficult to translate (i. e. chinese, japanese, turkish, etc). After selection of those studies

concerning to HHV-6 and Cp, a strict exclusion criteria were applied based on the

epidemiological guides on MS[98], so that only those studies describing subjects with RR-

MS fulfilling Poser criteria[99] and a define control group were used. Finally, 16 publication

were selected that related HHV-6 or Cp with MS[46,78,100-113] (Tables 6-13). An internal

quality system was used to verify the results of the afore mentioned method, and consisted in

a careful revision of every publication with a tracking of the bibliography to avoid lost papers.

The meta-analysis had a qualitative component with epidemiological description of the

articles, being the individual studies the subjects of the research; and another quantitative

component that referred to a statistical stratification of the results obtained for each laboratory

probe, in each publication and altogether with the others, statistical signification of the

A Meta-Analysis of Human Herpes Virus 6and Chlamydophila Pneunoniae…

181

analysis, confident interval at 95%, odds ratio and the weight of the publication. The used

method in order to obtain the combined values is that of Dersimonian and Laird[114]. There

was no relationship between RR-MS and the infectious agent when the confident interval

obtained had the unity[115].

RESULTS OF META-ANALYSIS

Antibodies Anti-Human Herpes Type 6 in Serum and CSF

In table 6 we show the publications about the presence of Ab against HHV-6 in serum

and CSF. We underline that in the study of Soldan et al the IgG numerical results have not

been reflected, so this data has not been considered; and that Taus et al employed a small

number of controls. The number of RR-MS patients in both studies was similar. We analyzed

only the CSF results in the study of Ongrádi et al because they do not give numerical data in

serum.

Table 6. Anti HHV-6 Antibodies in Serum and Cerebrospinal Fluid

Studies in serum RR-MS nº Controls Positive Results (%)

Soldan 22 OND: 31

AD: 21

ND: 14

IgM:

RR-MS:11/22 (50)

OND:6/31 (19)

AD:3/21 (14)

ND:0/14 (0)

Taus 23 OND: 8 IgG:

RR-MS:16/23 (69)

OND: 2/8 (25)

Studies in CSF RR-MS nº Controls Positive Results (%)

Ongrádi 6 OND : 6 IgG :

RR-MS:5/6 (83)

OND:1/6 (17)

IgM:

RR-MS:3/6 (50)

OND:0/6 (0)

OND: others neurological diseases.

AD: autoimmunity diseases.

ND: normal donor.

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182

Human Herpes Virus Type 6 DNA in Serum, CSF and White Cells

In table 7 we show the studies about the presence of DNA in serum, CSF ans white cells.

Two studies, Álvarez-Lafuente et al and Berti et al, employed a higher number of cases than

Golberg et al, although this later publication have higher number of controls. None of the

studies found DNA in controls sera, but they were found in two studies with RR-MS patients.

No positive results were obtained in the CSF of cases and controls. In peripheral blood white

cells stands out Tomsone et al paper because of the high number of controls.

Table 7. HHV-6 DNA Detection in Serum, CSF and White Cells

Studies in serum RR-MS nº Controls Positive Results (%)

Berti 149 OND: 19

OIND:15

ND:36

RR-MS:36/149 (24)

Controls:0/70 (0)

Goldberg 13 OND: 16

ND: 14

RR-MS: 0/13 (0)

Controles: 0/30 (0)

Álvarez-Lafuente 103 ND: 46 RR-MS:15/103 (15)

ND: 0/46 (0)

Studies in CSF RR-MS nº Controls Positive Results (%)

Martin 26 OND :39 RR-MS: 0/26 (0)

OND: 0/39 (0)

Goldberg 5 OND : 14 RR-MS: 0/5 (0)

OND : 0/14 (0)

Taus 25 OND : 9 RR-MS : 0/25 (0)

OND : 0/9 (0)

Studies in white cells RR-MS nº Controls Positive Results (%)

Taus 14 OND : 9 RR-MS : 2/14 (14)

OND : 0/9 (0)

Mayne 32 OND : 7

ND: 17

PCR 1:

RR-MS : 1/32 (3)

ND: 2/17 (12)

OND: 1/7 (14)

PCR 2:

RR-MS: 6/26 (23)

ND: 4/17 (23)

OND: 1/7 (14)

Rotola 20

ND: 30

RR-MS: 8/20 (40)

ND: 11/30 (36)

Tomsone 35

OND: 21

ND: 150

RR-MS: 26/35 (74)

OND: 6/21 (28)

ND: 43/150 (28)

Álvarez-Lafuente 103 ND: 46 RR-MS : 55/103 (53)

ND :14/46 (30)

OND: others neurological diseases.

OIND: other inflammatory neurological diseases.

ND: normal donor.

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183

Relationship between Laboratory Techniques that Detect Infection by HHV-

6 and RR-MS Subjects

In tables 8 and 9 we show the statistical relationships between the presence of Ab and

virus DNA and RR-MS. The presence of Ab in serum has moderate relationship with

RRMM, as Soldan et el and Taus et al stated. Ab in CSF show a strong relationship between

having RR-MS, because even if the levels of IgM in Ongradi et al publication are not adjusted

to criteria, those of IgG do, with a higher odd ratio that increases the total one. The presence

of DNA in serum indicates strong relationship with RR-MS. Goldberg et al and Alvarez-

Lafuente et al papers do not satisfied the confident interval; nevertheless, Berti et al paper,

does satisfied it, with an odd ration that elevates the total over the unit. The detection of DNA

in serum is not related with RR-MS, as none of the three studies fulfilled the requirements of

the confident interval. The presence of DNA in blood white cells neither is related with RR-

MS, as only in two of the six studies (Tomsone et al and Alvarez-Lafuente et al. ) the

confident interval does not include the unit and, even having higher odds ratio and weights,

they do not reach the established criteria.

Table 8. Analysis of the Relationship between RR-MS Patients

and anti-HHV-6 Antibodies in Serum and CSF

Studies in serum OR I.C. 95% Weight(%)

Soldan 6.33 2.12-18.88 73.7

Taus 6.86 1.10-42.76 26.3

Total 6.47 2.53-16.52

Studies in CSF OR I.C. 95% Weight (%)

Ongrádi (IgG) 25.00 1.20-520.76 54.1

Ongrádi (IgM) 12.00 0.44-325.08 45.9

Total 17.85 1.91-166.75

Table 9. Analysis of the Relationship between RR-MS Patients and HHV-6 DNA

in Serum, CSF and White Cells

Studies in serum OR I.C. 95% Weight (%)

Goldberg 2.17 0.04-114.99 20.2

Álvarez-Lafuente 15.85 0.93-271.32 39.4

Berti 44.60 2.69-738.55 40.4

Total 16.11 2.71-95.88

Studies in CSF OR I.C. 95% Weight (%)

Martin 1.93 0.04-100.69 34.0

Golberg 2.64 0.05-149.97 32.6

Taus 0.36 0.01-19.53 33.4

Total 1.22 0.12-12.25

Studies in white cells OR I.C.95% Weight (%)

Taus 3.00 0.12-74.88 5.7

Mayne (PCR 1) 0.23 0.02-2.32 9.2

Rotola 1.15 0.36-3.68 19.4

Mayne (PCR 2) 1.14 0.30-4.37 17.3

Tomsone 7.19 3.14-16.45 23.6

Álvarez-Lafuente 2.62 1.25-5.48 24.8

Total 1.97 0.85-4.61

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184

Antibodies anti-Chlamydophila Pneumoniae in Serum and CSF

In table 10 are shown the studies with the presence of Ab in serum and CSF against Cp.

In Yao et al paper the results were obtained by 2 different laboratory techniques, and in this

way will be analyzed later.

Table 10.-Antibodies anti-Chlamydophila Pneumoniae in Serum and CSF

Study in serum RR-MS (nº patients) Controls (nº patients) Positive Results (%)

Derfuss 35

OND : 25

OIND : 35

IgG:

RR-MS :16/35 (46)

OIND: 16/35 (46)

OND: 16/25 (64)

Study in CSF RR-MS nº Controls positive Results (%)

Derfuss

35

OND : 26

OIND: 35

IgG:

RR-MS: 9/35 (26)

OIND: 2/35 (6)

OND: 1/26 (4)

Yao 6

OND : 14 RR-MS:

Inmunoblot 5/5 (100)

Adsortion 5/6 (83)

OND:

Inmunoblot: 2/14 (14)

Absortion: 0/9 (0)

OND: other neurological diseases.

OIND: other inflammatory neurological diseases.

Culture and Detection of DNA in CSF of Chlamydophila Pneumoniae

There is only a single study about Cp recuperation in CSF referred specifically to RR-MS

patients. (Table 11) As Sriram et al (2002) use 3 different primers in the DNA detection, we

have considered the results for each one. The study of Layh-Schmitt et al outstands because

they include a great number of controls.

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185

Table11. Culture and Detection of DNA in Chlamydophila Pneumoniae CSF

Culture Study RR-MS nº patients Controls nº patient Positive Results (%)

Sriram 2001 17 OND : 13 RR-MS: 17/17 (100)

OND: 2/13 (15)

DNA Studies RR-MS nº Controls Positive Results (%)

Layh-Schmitt 2 series :

1. 8 RR-MS

2. 14MSRR

OND: 56 1st serie:

RR-MS: 4/8 (50)

2nd serie:

RR-MS: 1/14 (7)

OND: 0/56 (0)

Derfuss 10 OIND : 5

OND : 5

RR-MS : 0/10 (0)

OIND: 0/5 (0)

OND : 0/5 (0)

Sriram 2002 10 OND : 14 RR-MS

PCR 1: 6/10 (60)

PCR 2 : 5/10 (50)

PCR 3 : 7/10 (70)

OND

PCR 1 : 1/14 (7)

PCR 2 : 0/14 (0)

PCR 3 : 2/12 (16)

Sriram1999 17 OND : 27 RR-MS: 17/17 (100)

OND: 5/27 (18)

Sriram 2001 17 OND : 27 RR-MS : 8/17 (47)

OND : 3/27 (11)

OND: other neurological diseases.

OIND: other inflammatory neurological diseases.

PCR: Polymerase Chain Reaction.

Relationship between Laboratory Probes that Detect Infection by

Chlamydophila Pneumoniae and RR-MS Subjects

In tables 12 and 13 we show the statistical relationships that can be established with RR-

MS. Ab anti-Cp in serum did not have strong relationship with RR-MS as the only study we

had did not reached the criteria for the confident interval. Ab in CSF had big relationship with

the fact of having RR-MS as the 3 studies reviewed have a confident interval higher than the

unit. Cp recuperation in CSF is related with RR-MS as the only study available fulfil the

criteria for the confident interval. The DNA in CSF has a strong relationship with RR-MS,

although only one paper (Derfuss et al) is not adjusted to the criteria for the confident

interval.

J. Gutiérrez, M. García, F. Fernández, O. Fernández, V.E. Fernández, et al.

186

Table 12. Analysis of the Relationship between

Antibodies Anti-Chlamydophila Pneumoniae in Serum and CSF in Patients with RR-MS

Study in Serum OR I.C. 95%

Derfuss 0.74 0.32-1.70

Estudios en CSF OR I.C. 95% Weight (%)

Derfuss 6.69 1.67-26.76 59.7

Yao (Inmun) 60.00 2.28-1578.57 21.5

Yao(Adsorc) 180.00 3.08-10535.93 18.4

Total 21.37 2.88-158.61

Table 13. Culture and Detection of DNA in Chlamydophila Pneumoniae CSF

Culture Study OR I.C. 95%

Sriram 2001 7.11 1.54-32.91

DNA Study OR I.C. 95% Weight (%)

Sriram 2001 192.50 7.93-4673.07 10.9

Layh-Schmitt 33.24 1.73-639.63 12.6

Derfuss 1.00 0.02-55.27 6.9

Sriram 2002 (PCR 1) 19.50 1.78-213.96 19.1

Sriram 2002 (PCR 2) 28.00 1.29-610.01 11.7

Sriram 2002 (PCR 3) 11.67 1.53-89.12 26.3

Sriram 1999 149.60 7.63-2931.34 12.5

Total 25.61 8.89-73.84

DISCUSSION AND CONCLUSIONS

Infections by HHV-6 and Cp are highly prevalent in humans[116]. They penetrate trough

high respiratory mucosa and then can spread through all the organism. The pathogenesis of

the infection by the first infectious agent is better known that of the second one. After primary

infection, HHV-6 quarters in peripheral blood mononuclear cells (PBMC) and macrophages,

and from there it can have more or less important biological reactivations. These reactivations

can be accompanied of the presence of extra cellular virus and/or clinical symptoms. In our

meta-analysis the detection of DNA in CSF and PBMC has not been related with the presence

of RR-MS.

It could be due to a lack of sensitivity of the tests for the CSF as well as the qualitative

character of the assays (authors do not refer the limit of sensitivity of them). That is why new

studies are required to resolve these technical problems.

In the case of the infection by Cp, similar biological phenomenon as the ones already

described for HHV-6 are supposed [73,117]. In just only one study it was not related the

presence of de antibodies with RR-MS. We think it was because the presence of these

antibodies was not quantified and because of the use of not very well characterised antigens.

On the basis of the results obtained, we find that infection by HHV-6 is related with

subjects with RR-MS through the presence of DNA in serum and antibodies in serum and

CSF; but not through the detection of DNA in CSF and white cells in blood. In the case of the

A Meta-Analysis of Human Herpes Virus 6and Chlamydophila Pneunoniae…

187

infection by Cp, it is related with subjects with RR-MS by the presence of DNA, antibodies

and the recuperation of the bacteria in CSF, but not by the detection of antibodies in serum.

In the reviewed literature there is no one article with enough number of patients and

samples, prospective, controlled, using the combination of multiple microbiological

techniques in a same case, evaluating if the MS patient in in an active or inactive phase of the

disease and that analyses serum and CSF with standarized techniques at the same time.

If it could be demonstrated that HHV-6 or Cp are really related with MS, we could: 1)

better diagnose the disease with a biological marker; 2) predict the course of the disease and

establish a prognosis; 3) treat patients with antimicrobials and monitor their effect; 4) state the

possibility of a preventive treatment with the elaboration of a vaccine.

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ACKNOWLEDGMENTS

This study was supported by grants from the Consejerías de Educación y Ciencia

(Investigaction Group "Estudio de los agentes infecciosos relacionados con procesos clínicos

de causa desconocida") and de Salud (Proyect "Posible papel del virus del herpes humano

tipo 6 y Chlamydophila pneumoniae como desencadenante de las recidivas clínicas de los

sujetos con esclerosis múltiple") de la Junta de Andalucía.