[Emerging Infectious Diseases]                                                         
[Volume 4 No. 3 / July - September 1998]

Special Issue

Infectious Causes of Chronic Inflammatory Diseases and Cancer

Gail H. Cassell
Lilly Research Laboratories, Indianapolis, Indiana, USA

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      Powerful diagnostic technology, plus the realization that
      organisms of otherwise unimpressive virulence can produce
      slowly progressive chronic disease with a wide spectrum of
      clinical manifestations and disease outcomes, has resulted in
      the discovery of new infectious agents and new concepts of
      infectious diseases. The demonstration that final outcome of
      infection is as much determined by the genetic background of
      the patient as by the genetic makeup of the infecting agent is
      indicating that a number of chronic diseases of unknown
      etiology are caused by one or more infectious agents. One
      well-known example is the discovery that stomach ulcers are due
      to Helicobacter pylori. Mycoplasmas may cause chronic lung
      disease in newborns and chronic asthma in adults, and Chlamydia
      pneumoniae, a recently identified common cause of acute
      respiratory infection, has been associated with
      atherosclerosis. A number of infectious agents that cause or
      contribute to neoplastic diseases in humans have been
      documented in the past 6 years. The association and causal role
      of infectious agents in chronic inflammatory diseases and
      cancer have major implications for public health, treatment,
      and prevention.

The belief that infectious agents are a cause of chronic inflammatory
diseases of unknown etiology and of cancer is not new. Approximately 100
years ago, doctors noted a connection between cervical cancer and sexual
promiscuity that transcended mere coincidence (1). By 1911, a connection
between viruses and cancers in animals had become well established (2). As
early as the 1930s, mycoplasmas were proposed as a cause of rheumatoid
arthritis in humans, and shortly thereafter, they were proven to be the
most common cause of naturally occurring chronic arthritis in animals (3).
Proof of causality of cancer and arthritis in humans was more difficult.
When searches for infectious agents in cancer and arthritis found none,
research began to focus on mechanisms of inflammation, tumorogenesis, and
drug discovery. More recently, however, scientists have renewed searches
for infectious agents.

Advances in molecular biology and medical devices have revolutionized our
ability to detect very low numbers of infectious agents in specimens
collected directly from the affected site. HIV has demonstrated the ability
of infectious agents to produce slowly progressive, chronic disease with a
wide spectrum of clinical manifestations and disease outcomes. Increased
understanding of the body's defense mechanisms and the demonstration that
final outcome of infection is as much determined by the genetic background
of the host as by the genetic makeup of the infecting agent suggest that a
number of chronic diseases of unknown etiology may be caused by an
infectious agent.

Recent data suggest a role for one or more infectious agents in the
following chronic diseases: chronic lung diseases (including asthma),
cardiovascular disease, and cancer. Many of the agents implicated are
commonly transmissible and are either treatable with existing antibiotics
or are potentially treatable with antiviral drugs. Thus, proof of causality
in any one of these diseases would have enormous implications for public
health, treatment, and prevention. Few areas of research hold greater
promise of contributing to our understanding of infectious diseases and the
eventual relief of human suffering.

The intent of this paper is not to provide a comprehensive review of
chronic inflammatory diseases of unknown etiology and the agents implicated
but rather to utilize several models to discuss available data and to
illustrate the difficulty in proving causality in chronic inflammatory
diseases. The discussion is based upon the following assumptions. Most
chronic inflammatory diseases are likely multifactorial. Heredity,
environment, and nutrition are critical determinants of disease expression
with heredity being the most important.

Theoretically, chronic inflammatory diseases currently of unknown etiology
could result from three different types of pathogens: 1) those that are
fastidious and previously recognized but because of their fastidiousness or
lack of appreciation of their disease-producing potential are not included
in the differential diagnosis, and 2) infectious agents previously not
recognized that therefore go undetected. Infection with either group can
result in misdiagnosis and lack of treatment. Depending upon the biology of
the organism and intrinsic and extrinsic factors of the host the organism
can persist, resulting in chronic inflammation. The third group of
pathogens would be those that elicit an autoimmune response resulting in
persistent inflammation without the persistence of the inciting agent.
Examples of the first two groups of pathogens will be discussed here using
mycoplasmas to typify the first group and Chlamydia pneumoniae the second.
Finally, recent advances in our understanding of the role of infectious
agents in cancer will also be summarized.

Chronic Lung Diseases

Murine Chronic Respiratory Disease as a Model System

The difficulty in establishing the infectious etiology of a chronic
obstructive lung disease is well illustrated by Mycoplasma pulmonis and
murine chronic respiratory disease. Proof that M. pulmonis can cause this
disease took nearly 50 years and required inoculation of germ-free animals
(4). Chronic bronchopneumonia in rats was first described in 1915 when this
species came into general use for experimental purposes (5). In
approximately 1940, a Mycoplasma, later identified as M. pulmonis, was
recognized as a possible cause (6), but the ubiquity of the organism and
its frequent isolation from healthy as well as diseased rats and mice (even
from trachea and lungs) soon gave it the reputation of being a commensal
with little pathogenic potential. The failure of pure cultures of this
organism to consistently produce disease of the lower respiratory tract
also precluded its acceptance as the etiologic agent. Only in the early
1970s was M. pulmonis alone shown to consistently reproduce all of the
characteristic clinical and pathologic features of the natural respiratory
disease when inoculated into animals maintained under germ-free conditions
(7). Subsequent studies provided explanations for previous difficulties in
reproducing the disease.

The respiratory disease caused by M. pulmonis is slow to begin and
long-lasting. Consequently, the disease has various stages of pathologic
lesions and a lack of uniform lesions, even among animals in the same cages
(due partly to variables that can affect development of the disease in the
lower respiratory tract, such as intracage ammonia produced by bacterial
action on soiled bedding, synergy with murine respiratory viruses and other
bacterial pathogens, and nutritional factors) (7). However, comparison of
animals matched for age, sex, and microbial and environmental factors
indicates that heredity is the most critical determinant of susceptibility,
lesion character, and disease severity. Susceptibility among animal species
and among strains of the same species differ dramatically (8-11).

Intranasal inoculation of M. pulmonis produces markedly different lesions
in F344 rats and in CD-1 mice, even when the dose is comparable on the
basis of lung and body weight. In rats the lesions progress slowly from the
upper respiratory tract distally, with alveolar involvement occurring days
to months following inoculation, whereas in mice, alveolar lesions develop
within hours after infection and are responsible for acute alveolar disease
and death within 3 to 5 days. Depending on their genetic background, mice
that survive the acute disease develop chronic lung disease characterized
by bronchiectasis that persists for up to 18 to 24 months or the lifetime
of the animal.

Studies of naturally occurring and experimentally induced disease indicate
that M. pulmonis also causes a slowly progressing upper genital tract
disease in LEW and F344 rats (18). Pups can become infected in utero, at
the time of birth due to cervical and vaginal infection of the dams, or via
aerosol from dams shortly after birth. Even though the organisms can be
shown to colonize the ciliated epithelium of the upper and lower
respiratory tracts of pups, microscopic lesions are not detectable for 2 to
6 months depending on the strain of rat. Development of obstructive lung
disease can require as long as 12 to 18 months.

Differences in severity and progression of the lung lesions due to M.
pulmonis in LEW and F344 rats are related to differences in the degree of
nonspecific lymphocyte activation in the two strains or an imbalance in
regulation of lymphocyte proliferation in LEW rats (12). M. pulmonis
possesses a potent B cell mitogen, and, in addition, the organism is
chemotactic for B cells (13). Interestingly, LEW rats are also more
susceptible to other chronic inflammatory diseases, including streptococcal
cell-wall induced arthritis, adjuvant-induced arthritis, and allergic
encephalomyelitis (12).

Ureaplasma urealyticum as a Cause of Pneumonia in Newborns and Its
Association with Chronic Lung Disease (CLD) in Premature Infants

Respiratory dysfunction represents the most common life-threatening problem
in premature infants and one of the largest costs of neonatal intensive
care (14). Infants weighing less than 1,000 g at birth are more likely than
those with greater birth weights to die within the first few days of birth
of respiratory-related problems; those who survive are at an increased risk
of CLD (15). Approximately 20% of stillborn babies and infants dying within
72 hours of delivery have histologic evidence of pneumonia (16). Yet the
true incidence of lower respiratory infection acquired either in utero or
at the time of delivery and its contribution to death or development of CLD
are unknown. The cause of lower respiratory disease in newborn babies is a
diagnostic dilemma because pneumonia in early neonatal life is usually
clinically and radiologically indistinguishable from surfactant-deficiency
syndrome (17). Furthermore, meaningful cultures from the lung are not
easily obtained, whereas cultures of the throat, nasopharynx, and blood are
unrevealing or misleading.

Pneumonia

The mycoplasma U. urealyticum, a common commensal of the lower female
genital tract, has recently been shown to cause respiratory disease in
newborn infants. Retrospective (18) and prospective (19-21) studies
indicate an association of U. urealyticum with congenital pneumonia. Case
reports also provide evidence that U. urealyticum is a cause of pneumonia
in newborn infants (22-23). The organism has been isolated from affected
lungs in the absence of chlamydiae, viruses, fungi, and bacteria and in the
presence of chorioamnionitis and funisitis (40) and has been demonstrated
within fetal membranes by immunofluorescence (24) and in lung lesions of
newborns by electron and immunofluorescent microscopy (20). The specific
immunoglobulin (Ig) M response in several cases of pneumonia in newborns
further documents in utero infection (20).

We have found that U. urealyticum is the single most common microorganism
isolated from endotracheal aspirates of infants who weigh </=2,500 g and who
require supplemental oxygen within the first 24 hours after birth (19).
Infants weighing </=1,000 g and from whom U. urealyticum is isolated from the
endotracheal aspirate are twice as likely to die as infants of similar
birth weight but who are uninfected or infected infants >/1,000 g. These
findings support the hypothesis that only a select group of infants, i.e.,
those with very low birth weights, is subject to disease due to U.
urealyticum. This fact may account for the seeming disparities in
conclusions regarding the role of U. urealyticum in neonatal respiratory
disease reached in earlier prospective studies that failed to distinguish
this subpopulation at high risk from the whole (25,26).

That endotracheal isolations of U. urealyticum represent true infection of
the lower respiratory tract is supported by initial isolation of
ureaplasmas in numbers exceeding 1,000 CFUs (and sometimes exceeding 10,000
CFUs) and repeated isolations of the organism from tracheal aspirates for
weeks and even months in some infants that continue to require mechanical
ventilation. That the tracheal isolates are not merely a reflection of
contamination from the nasopharynx is supported by the discrepancy in
isolation rates between the two sites and recovery of U. urealyticum in
pure culture from endotracheal aspirates in more than 85% of the infants
(19). Concomitant recovery of the organism from blood of up to 26% of those
with positive endotracheal aspirates and from cerebrospinal fluid (CSF) of
some infants indicate that in some infants the organism is invasive (19).
Fourteen percent of U. urealyticum endotracheal isolates were from infants
born by cesarean section with intact membranes, indicating that in utero
transmission occurs rather commonly, at least in premature infants.

In a study of 98 infants, respiratory distress syndrome, the need for
assisted ventilation, severe respiratory insufficiency, and death were
significantly more common among those infants <34 weeks gestation from whom
U. urealyticum was recovered from endotracheal aspirates at the time of
delivery than among uninfected infants (27). U. urealyticum was isolated
from 34% of blood cultures and also from four of six CSF samples and in 6
of 11 postmortem brain and lung biopsy pecimens. Eighty-two percent of the
ureaplasma isolates were present in pure culture, and 48% of infants born
by cesarean section with intact membranes had ureaplasmas isolated from one
or more sites.

U. urealyticum can induce ciliostasis and mucosal lesions in human fetal
tracheal organ cultures (20). Furthermore, we have shown that ureaplasmas
isolated from the lungs of human infants with congenital and neonatal
pneumonia produce a histologically similar pneumonia in newborn mice (28).
Even in this mouse model, age is a critical determinant of disease. Newborn
mice are susceptible to colonization of the respiratory tract and
development of pneumonia; 14-day-old mice are resistant.

We have shown that endotracheal inoculation of premature baboons
(well-established models of premature human infants) with U. urealyticum
isolated from human infants results in the development of pathologically
recognizable pulmonary lesions, including acute bronchiolitis with
epithelial ulceration and polymorphonuclear infiltration, which is
distinguishable from hyaline membrane disease (29). U. urealyticum can be
isolated from blood, endotracheal aspirates, and pleural fluid and lung
tissue from some of these animals 6 days after infection.

The available evidence provides a strong argument that U. urealyticum is a
common cause of pneumonia in newborn infants, particularly those born
before 34 weeks of gestation. The organism can be isolated from
endotracheal aspirates in up to 34% of infants weighing <2,500 g;
radiographic evidence of pneumonia is twice as common in these infants as
in U. urealyticum negative infants (30% vs. 16%, p = .03) (30). Many of
these infections develop as a result of in utero exposure. Cases of
ureaplasmal pneumonia occur much less frequently in term infants. These
findings in infants are consistent with the fact that U. urealyticum
infection of the chorioamnion is also much more common before 34 weeks of
gestation. Lack of transplacental passage of immunoglobulin prior to 32
weeks gestation (31) may partially explain these findings. Experience from
mycoplasmal respiratory diseases of animals indicates that preexisting
antibody is protective, whereas antibody in the presence of an established
infection is rarely effective in elimination of the organism (32).

CLD in Premature Infants

Some, but not all, studies (33-36) show an association between isolation of
U. urealyticum from the respiratory tract of newborn infants and the
development of CLD (33). Differing results may be obtained because some
studies do not limit culture isolation to the affected site (the lower
respiratory tract), do not limit their patient population to those at
greatest risk (birth weight <1,000 g); or do not limit culture isolation to
within 12 hours of delivery, i.e., most likely infected in utero. Several
facts suggest that infants who acquire U. urealyticum in utero may be at
greatest risk for development of CLD. Dyke et al. (34) found U. urealyticum
in the gastric aspirates of infants </=1,000 g was associated with a
significantly increased risk of CLD in those infants delivered by cesarean
section but not in those delivered vaginally. This could result from a
longer exposure to U. urealyticum as a result of in utero exposure, or it
may be a reflection of differences in the virulence of those organisms
found only in the cervix versus those that have invasive potential and that
can cause an ascending infection from the vagina into the uterus. Along
these lines it is of interest that a recent study of 49 preterm infants
which included only three infants from whom U. urealyticum was recovered
within 24 hours of birth found no association with development of CLD (35).
The remaining 11 infants were not culturally positive until 48 to 72 hours
after birth suggesting that only the three study infants were infected in
utero. In another recent study reported by Valencia et al. (36) CLD was
found in 26% of U. urealyticum infected infants compared to only 4.7% of
the noncolonized group. However, these results were not statistically
significant possibly because of the small number of patients studied but
also possibly because 22% of the patients included did not have cultures
performed until between 2 days and 3 months postnatal life.

Isolation of U. urealyticum from endotracheal aspirates is not only a risk
factor for development of pneumonia but also of precocious dysplastic
changes (30). Walsh et al. (38) isolated U. urealyticum directly from
pleural fluid and tissue collected by open lung biopsy in four of eight
infants cultured who had CLD. We (19) continued to recover ureaplasmas from
endotracheal aspirates of infants with CLD for months following initial
recovery of the organism from endotracheal aspirates within 12 hours of
birth.

Available evidence creates a cohesive argument that U. urealyticum
infection of the lower respiratory tract is a likely risk factor for, and
not only associated with, CLD. Because U. urealyticum has only recently
been suggested as a cause of pneumonia in newborns, it is not routinely
sought by most hospital laboratories. Furthermore, the organism is not
susceptible to antibiotics used prophylactically in very low birth-weight
infants with evidence of respiratory distress. Consequently, the infection,
i.e., pneumonia, goes undetected and untreated. Due to the difficulties in
diagnosis, most hospital laboratories do not culture for this organism.

The pathophysiology of CLD in premature infants suggests that U.
urealyticum produces undetected and untreated pneumonia and results in an
increased requirement for oxygen and subsequent development of CLD as a
result of oxygen toxicity (33,37) or a synergistic effect between the
ureaplasmas and hyperoxia. It has been proposed that hyperoxia-induced lung
injury contributes to development of CLD by stimulating the proinflammatory
cytokine interleukin (IL)-6 (38). U. urealyticum may also contribute to the
development of CLD by stimulation of proinflammatory cytokines. Infants
from whom ureaplasmas are isolated from endotracheal aspirates within the
first 24 hours of life are more likely to have neutrophils in their
tracheal aspirates on day 2 than are those not colonized (39). Aspirates
from colonized infants are also more likely to have class II cytology than
those from uncolonized patients at day 2 of life. This may explain why
ureaplasma-infected infants respond to dexamethasone therapy (39). These in
vivo findings are consistent with the recent demonstration of U.
urealyticum induction of IL-6 and IL-8 in human neonatal pulmonary
fibroblasts even in the absence of hyperoxia (38). Interestingly, together
ureaplasmas and hyperoxia resulted in greater stimulation of IL-6 and IL-8
than either alone. This is consistent with the synergism previously
demonstrated in vivo between ureaplasmal infection and hyperoxia (37).

Studies in mice also suggest that increased oxygen requirements of very low
birth-weight infants might predispose them to lower respiratory tract
infection or, alternatively, that U. urealyticum infection potentiates
oxygen-induced injury (28,37). Exposure to oxidants is known to enhance
respiratory disease and death due to M. pulmonis respiratory disease in
mice (41).

That U. urealyticum is a cause of pneumonia in newborns can no longer be
questioned. Data provide strong evidence that U. urealyticum can be a
primary cause or a contributing cofactor in development of CLD in humans,
but the data are not definitive. Cohort studies allow follow-up of exposed
persons and thus reduce bias, but the designs of these studies cannot rule
out the possibility that a third factor associated with U. urealyticum is
actually the true cause of CLD. However, a randomized trial of exposure to
infection in humans is not ethical or practical. Although a randomized
trial of antibiotic treatment could provide critical information related to
patient management, it would still not bring us closer to proving
causality. Even if treatment is found to be efficacious, conclusions about
causation will be limited by the fact that the third factor might also be
susceptible to the antibiotic chosen. If it is not found to be efficacious,
it may be because ureaplasma infection in utero or soon after birth results
in irreversible lung damage. Nevertheless, a treatment trial is urgently
needed to determine whether appropriate therapy can reduce the incidence of
illness and death associated with CLD. First, studies are needed to
determine dose and duration of antibiotic therapy and whether currently
available antibiotics will even eliminate the organism.

Mycoplasma pneumoniae and C. pneumoniae as Causes of Chronic Asthma

Asthma, a CLD characterized by airway obstruction, inflammation, and
bronchial hyperresponsiveness to a variety of stimuli, including
infections, is a common illness in both pediatric and adult populations. In
the United States alone, approximately 12 million people have asthma,
resulting in health-care costs of approximately 4.6 billion dollars
annually (42). In children, asthma is the most common reason for hospital
admissions and school absenteeism (43). Yet the etiology and pathogenesis
of this important disease remains poorly defined. Historically, viruses
that commonly infect the respiratory tract have been thought to play a role
in both provoking asthma exacerbations and in altering responses to other
environmental agents that might be involved (44).

M. pneumoniae is a common cause of both upper and lower respiratory
infection in humans; tracheobronchitis is the most common clinical
manifestation (45). Previously thought to cause acute, self-limited disease
primarily in persons between 6 and 21 years of age (45), M. pneumoniae is
now known to be the cause of pneumonia in 20% to 25% of all age groups and
to persist in certain persons for weeks to months, resulting in prolonged
reduced pulmonary clearance and airway hyperresponsiveness (46-47).
Epidemiologic evidence links mycoplasma infection with asthma exacerbation
and possibly with the pathogenesis of asthma (47-50).

While M. pneumoniae has been associated with exacerbations of asthma, its
role in sustaining chronic asthma or in initiating exacerbation is unknown.
However, the proven role of mycoplasmas in similar chronic respiratory
diseases of numerous animal species, including M. pulmonis in rodents,
suggests that careful, systematic studies should be undertaken in humans
(45).

C. pneumoniae, the most recently described Chlamydia species, has been
associated with a wide range of respiratory tract illnesses, from
pharyngitis to pneumonia with empyema (51). C. pneumoniae has been isolated
from 15% to 20% of adults and children with community-acquired pneumonia
(51). On the basis of serologic results only, C. pneumoniae has been
associated with acute exacerbations of asthma in adults (52); on the basis
of nasopharyngeal cultures, it has been associated with asthma in children
(53). In both children and adults, the organism persists for months in the
upper respiratory tract of patients with wheezing (54).

If M. pneumoniae, or for that matter any infectious agent, is a causal
factor in initiating and sustaining asthma in certain persons, the agent
should be present and persistent in the lungs of some persons with stable,
chronic asthma. We have recently undertaken a study to determine if M.
pneumoniae can be detected in the lungs of adults with stable, chronic
asthma versus asymptomatic controls (55). To facilitate interpretation of
results, we also evaluated the presence of other fastidious infectious
agents that have previously been implicated in the pathogenesis of asthma,
including the seven common respiratory viruses (44) and C. pneumoniae
(56,57).

M. pneumoniae was detected by PCR in 10 of 18 asthma patients and 1 of 11
controls (p = 0.02). All patients were culture, EIA, and serologically
negative for M. pneumoniae. All PCR and cultures were negative for C.
pneumoniae and all EIAs for respiratory viruses were negative. Nine persons
with asthma and one control exhibited positive serology for C. pneumoniae
(p = 0.05). For C. pneumoniae, the lack of correlation between serologic
results and culture and PCR was not unexpected. We have seen discordance
between culture and serologic results in patients with community-acquired
pneumonia (58,59), but in these cases more patients were culture positive
than seropositive. Thus, the culture methods we used in the study have
previously been shown to be valid.

Our failure to culture M. pneumoniae might be explained by its extreme
fastidiousness or its low numbers. Culture is the least sensitive of the
methods used in this study for detection of M. pneumoniae. However, the
culture methods we used in this study we also used to evaluate more than
2,000 respiratory specimens during the same period in patients with
radiographically confirmed, community-acquired pneumonia. These methods
have resulted in recovery of M. pneumoniae by culture in up to 17% of
patients (G. Cassell, et al., unpub. obs.; 58,59).

Recent studies indicate that some other mycoplasma species of human origin
may be able to survive intracellularly in chronic infections of cell
cultures (60). Likewise, some strains of M. hyorhinis, the etiologic agent
of chronic respiratory disease of swine, can become so adapted to growth in
the presence of cells that it is no longer cultivable on artificial media
(61). If this occurs in vivo, organisms like M. pneumoniae could be
difficult if not impossible to recover by culture.

In the absence of other known respiratory pathogens in other patient
populations, the presence of M. pneumoniae can be detected longer by PCR
than by either culture or serologic test. Guinea pigs experimentally
infected with M. pneumoniae become chronically infected as detected by PCR
for up to 200 days but are culture negative by 70 days. Also by 70 days,
antibody levels become negative (62). Thus, patients with asthma appear
chronically infected with M. pneumoniae, despite negative culture results,
because they are PCR positive. That positive PCR results truly reflect
involvement of the lower respiratory tract by M. pneumoniae is supported by
the fact that 9 of the 10 M. pneumoniae-positive patients were positive in
the bronchoalveolar lavage (BAL), bronchial biopsies, or bronchial brush
specimens. Furthermore, the organism was detected in the nasopharynx or the
throat of only five of the nine asthma-positive patients, thus indicating
that detection in the lower tract was not merely due to contamination by
organisms from the upper tract during sample collection. More importantly,
a significant number of persons with asthma were positive in the lower
respiratory tract on repeat sampling (2 to 4 months between samples), thus
indicating persistent colonization. By cloning and sequencing the PCR
product in BAL from several representative patients, we demonstrated 100%
sequence homology with M. pneumoniae. Use of multiple primer pairs as well
as confirmation of PCR findings in two different laboratories also attests
to the validity of the PCR results. Our failure to detect M. pneumoniae in
specimens from age-matched control patients as well as in specimens from
100 asymptomatic children using the same PCR methods further verifies the
specificity of our PCR methods and argues that finding M. pneumoniae in
persons with chronic asthma does not merely reflect a carrier state.

We have previously noted the lack of antibody response to M. pneumoniae in
both pediatric and adult populations with community-acquired pneumonia (G.
Cassell et al, unpub. obs.; 58,59,63). Study results indicate that a subset
of infected persons do not mount an antibody response, perhaps due to
genetic differences. Lack of antibody may in fact contribute to the
organism's persistence. The immunomodulatory properties of M. pneumoniae
(12) also could facilitate the organism's persistence.

Recent studies indicate that M. pneumoniae respiratory disease is often
misdiagnosed and inappropriately treated, which would also contribute to
persistence. Admitting physicians chose other pathogens as the most likely
agents in 46% of the cases subsequently documented as M. pneumoniae
infections (64). Even upon correct diagnosis, at least 10% of the patients
did not receive appropriate antibiotics during their hospitalization.

In summary, we have demonstrated that persons with chronic asthma, but not
healthy persons, exhibit evidence of M. pneumoniae colonization of the
lower airways. Like several other investigators (56,57), we found more
persons with asthma than control subjects had serologic evidence of C.
pneumonia infection. Further study is needed to determine if these findings
are an epiphenomenon or, as we expect, a pathogenic mechanism in asthma. If
the latter is correct, greater evaluation of the process involved is needed
to further our understanding of the pathogenesis and treatment of asthma.

Role of C. pneumoniae in Atherosclerosis

Infection was proposed as a cause of atherosclerosis by Sir William Osler
and others at the beginning of the century (65). However, it was not until
the 1970s that experimental infection of germ-free chickens with an avian
herpesvirus was found to produce arterial disease that resembled human
atherosclerosis (66). Associations have since been reported of human
coronary heart disease with certain gram-negative bacteria (i.e.,
Helicobacter pylori and C. pneumoniae) (67,68), with certain herpesviruses
(especially cytomegalovirus) (69), and with clinical markers of chronic
dental infection (70). Rather than an exhaustive evaluation of each of
these purported associations, it seems reasonable to focus on the
respiratory pathogen, C. pneumoniae, for which the evidence seems
strongest.

C. pneumoniae, like M. pneumoniae, is a common cause of community-acquired
pneumonia (70,71). C. pneumoniae infects more than 50% of people at some
point in their lives (51,71). It can often go undiagnosed and improperly
treated because again it is fastidious and diagnostic methods are not
routinely available. Even in the best reference laboratories, diagnosis can
be a challenge (71). It, like M. pneumoniae, is also thought to play a role
in acute asthma and chronic bronchitis (52) as well as to cause
extrapulmonary manifestations (51,71). It, like M. pneumoniae, can also
result in persistent infection following acute respiratory disease (54).

Eighteen seroepidemiologic studies evaluated the association of C.
pneumoniae infection and cardiovascular disease (67). Most found at least
twofold or larger odds ratios; some reported increasing odds ratios with
increasing antibody titers. The general consistency of their findings in a
total of 2,700 cases supports the existence of some real association
between C. pneumoniae and coronary heart disease because the studies were
done in different populations, used different criteria for cases, adjusted
for potential confounders to differing degrees, and were, therefore, prone
to different biases. While diagnosis by serology has its limitations, C.
pneumoniae has been demonstrated by a variety of laboratory techniques
(including culture, PCR, electron microscopy, and immunocytochemistry) in
the atherosclerotic lesions of coronary arteries, carotid arteries, aorta,
smaller cerebral vessels, and larger peripheral arteries (72-78). In the
more than 13 published studies of C. pneumoniae in human pathology samples
(67), chlamydiae were present in 257 (52%) of 495 atheromatous lesions but
in only 6 (5%) of 118 control samples of arterial tissue, yielding a
weighted odds ratio of about 10 (95% confidence interval 5-22). It seems
unlikely that sampling biases can entirely account for this extreme
difference between case and control tissue.

C. pneumoniae, an obligatory intracellular bacterium capable of causing
persistent infection and multiplying in endothelial and smooth muscle cells
and macrophages (79), can also be disseminated by macrophages (80). Hence,
some have argued that macrophages may ingest C. pneumoniae in the lung or
elsewhere before migrating to atheromatous lesions, in which case it may
only be a bystander. However, in two different rabbit models,
atherosclerotic changes develop only after infection with C. pneumoniae
(81,82). The organism by itself induces the production of cytokines (83)
and adhesion molecules (84), and it possesses an endotoxin (85) capable of
modulating the host inflammatory response. Thus, the biologic properties of
C. pneumoniae make it a logical candidate for triggering the chronic
inflammation found in atherosclerosis (82).

Finally, some studies have found rising or elevated levels of antibodies to
C. pneumoniae in some males during the months just preceding a heart attack
(86). Recent studies indicate that antibiotics given during or after a
first heart attack may decrease the risk of a second cardiac problem
(86-88). This finding also raises the possibility that antibiotics may have
a role in the treatment of cardiovascular illnesses; that could be
especially beneficial in developing countries where traditional treatments
like angioplasty are expensive.

Some have proposed additional large-scale antibiotic treatment trials in an
attempt to further prove causality. Several major issues need to be
resolved first. Ideally, one should treat patients with documented C.
pneumoniae infection; however, reliable diagnostic methods and treatment
protocols are lacking (71). Because most available antibiotics are
bacteriostatic, not bacteriocidal, some patients may remain infected up to
11 months after treatment. Even if these issues could be resolved,
antibiotic treatment trials will not prove causality, just as is the case
with U. urealyticum and CLD of prematurity or M. pneumoniae and chronic
asthma. The nonantimicrobial effects may also influence the outcome of such
studies. For example, tetracyclines can inhibit metalloproteinases, which
may contribute to acute coronary syndromes (89). Some macrolides have
antiinflammatory effects (90-93). Moreover, antibiotics are not selective,
thus making it impossible to determine the effects of treatment upon C.
pneumoniae versus other potential culprits, e.g., H. pylori, which is also
susceptible to tetracyclines and macrolides. However, if antibiotic
treatment could reduce atherosclerotic events, the public health
implications could be enormous.

Causal Role of Viruses and Bacteria in Cancer

Early in this century, Peyton Rous (2) established beyond doubt that cancer
can be caused by an infectious agent in chickens. Since then, evidence has
accumulated that other viruses cause cancer in a number of different animal
species (94). A growing body of research suggests that a number of viruses,
bacteria, and parasites cause cancer in humans, thus providing new
possibilities for treatment and prevention of cancer (94). In 1997, the
World Health Organization estimated that up to 84% of cases of some cancers
are attributable to viruses, bacteria, and parasites and that more than 1.5
million (15%) new cases each year could be avoided by preventing the
infectious disease associated with them (95).

H. pylori, found in the stomachs of a third of all adults in the United
States, causes inflammation of the mucous membrane of the stomach (96). In
20% of infected persons, H. pylori induces gastric ulcers (96). Peptic
ulcer disease, a chronic inflammatory condition of the stomach and
duodenum, affects as many as 10% of people in the United States at some
time in their lives. In the early 20th century, pathogenesis was believed
related to stress and dietary factors. Thus treatment focused on bed rest
and bland food. Later, gastric ulcers were believed to be caused by the
injurious effects of digestive secretions. Following the identification of
the histamine receptor that appeared to be the principal mediator of
gastric acid secretion, antagonists of this receptor were used for therapy
for peptic ulcer disease. In 1982, H. pylori was first isolated from the
human stomach, but it was not until one decade later and after Marshall
ingested pure cultures of the organism that causality was accepted by the
medical and scientific community (97).

In 1994, the International Agency for Research on Cancer concluded that
infection of humans with H. pylori is causally associated with the risk of
developing adenocarcinoma of the stomach (98), one of the most common
malignancies in the world, although relatively uncommon in the United
States (24,000 new cases and 14,000 deaths per year). However, also in
1994, a Consensus Panel of the National Institutes of Health (NIH)
concluded that available evidence was insufficient to recommend eradication
of H. pylori for the purpose of preventing gastric cancer (99). The NIH
conclusion was based upon the existence of clear examples of disparity in
the epidemiology of the two diseases. Gastric cancer is more common in
males than in females, whereas the rates of H. pylori infection are not
different for the two genders. Some populations are reported to have a high
rate of H. pylori infection but low rates of gastric cancer. Gastric cancer
occurs in some persons with no evidence of H. pylori infection, and in the
United States, fewer than 1% of H. pylori-infected persons will ever
develop gastric cancer. The strongest evidence that H. pylori is associated
with gastric cancer comes from three prospective studies that indicate that
H. pylori-infected persons have a significantly increased rate of gastric
cancer (96,98).

Only some retrospective serologic studies have shown an association. These
disparities indicate that factors other than H. pylori infection are also
important in gastric cancer risk. It is possible that only some strains of
H. pylori are involved in the carcinogenic process. For example, infection
with H. pylori strains possessing the cagA virulence factor is associated
with an increased risk of developing adenocarcinoma of the stomach
(100,101).

H. pylori is also associated with two less common forms of cancer,
non-Hodgkin lymphoma and mucosa-associated lymphoid tissue lymphomas of the
stomach (96). These types of lymphomas in the stomach only arise in the
setting of H. pylori inflammation. In 70% of H. pylori-infected patients
with lymphoma, treatment with appropriate antibiotics leads to regression
(96). This finding not only suggests a causal role but that treatment of a
bacterial infection can actually result in regression of cancer.

Another landmark study, published in June, 1997, shows that a 12-year
nationwide vaccination program against hepatitis B virus in Taiwan resulted
in a significant reduction in the number of cases of childhood liver cancer
(102). The role of chronic infection with hepatitis B virus in the etiology
of hepatocellular carcinoma is well established (103,104). Yet this is the
first evidence that prevention of a viral infection is also effective
against cancer. The implications are profound. Hepatitis B infection causes
some 316,000 cases of liver cancer (60% of all liver cancer) a year
worldwide (103,104). While hepatitis C causes a further 118,000 cases (22%
of all cases) a year (103,104), some cases result from infections with both
viruses (104).

The infectious origin of carcinoma of the cervix has long been suspected,
because known risk factors for the disease are linked to sexual activity
(105). Recent evidence indicates that human papillomavirus (HPV) types 16
and 18 are definitely carcinogenic in humans (94,105). Types 31 and 33 are
classified as probably carcinogenic (94,105). In the United States, HPVs,
are associated with 82% of the 15,000 cases and 4,600 deaths due to
cervical cancer each year. They are also associated with more than a
million precancerous lesions of varying severity. The combined direct
medical costs due to HPV are approximately 1.3 billion dollars per year in
the United States alone. Thus, effective therapy and vaccines would have a
major impact.

The pathogenic mechanisms by which infectious agents cause cancer have not
been resolved but they appear to be diverse. In cervical cancer, there
seems to be a clear role for HPV-encoded genes in tumor cell growth (106).
In addition to stimulation of cell proliferation, inactivation of tumor
suppressor genes, such as p53 may be a common pathway leading to malignancy
in HPV and hepatitis B virus (106,107). In the case of other viruses and H.
pylori, active oxygen and nitrogenic species generated by inflammatory
cells may cause DNA damage, induce apoptosis, and modulate enzyme
activities and gene expression (94,108).

Future Research Opportunities

The basic biology of agents implicated in chronic diseases and cancer, in
contrast to many other infectious agents, is relatively unknown. With rare
exception, the means by which pathogens suppress, subvert, or evade host
defenses and establish chronic or latent infection have received little
attention. Few areas of basic research compared with microbial latency hold
greater promise of substantially contributing to our understanding of
infectious diseases and the eventual relief of human suffering (109). Given
that the diseases discussed are among the most common in the world, even if
only some cases are proven to be of infectious origin and effective
therapies or vaccines can be developed, the impact on reducing health-care
costs would be substantial. Thus, further research to clarify the etiologic
agents and pathogenic mechanisms involved in chronic diseases and cancer
should be given the highest priority.

To address the potential role of infectious agents in chronic diseases
requires a new research paradigm compared to that which most investigators
and funding agencies in infectious diseases are accustomed. Such an
approach will require high levels of sustained funding of networks of
research groups (ideally at least for 10 years). The approach will require
collaborative research groups that follow a large number of well-defined
patients over long periods. Success will depend on involvement of
researchers highly skilled in clinical and epidemiologic investigation
supported by laboratory personnel with proven expertise in detection of a
wide spectrum of fastidious organisms. No single agent is likely to be the
cause of chronic obstructive lung disease, asthma, or cancer; rather a
number of infectious agents are likely to have this potential, hence the
need for studies of large numbers of patients. Because the infecting agent
may only be present in the very early stages of disease followed by an
inflammatory response, different stages of disease need to be studied. A
critical component of the investigative approach will be the ability to
determine the genetic background and immune response of the patients.

The randomized, controlled clinical trial provides a scientific experiment
that conforms to the standard model of biomedical research and is
undoubtedly the best theoretical approach to evaluating any new therapy
(110,111). Antibiotic treatment trials are commonly used to prove an
infectious etiology. While clinical trials are at their best in evaluating
the efficacy of therapies for acute diseases, clinical trials may not be
the best approach for evaluating the efficacy of therapies for chronic
diseases, most of which are likely to be complex, multifactorial illnesses
in which behavioral and lifestyle factors play an important role. Some of
the difficulties associated with this approach have already been discussed.

Well-defined, relevant animal models will be extremely important in
elucidating the role of infectious agents in chronic inflammatory diseases.
The animal studied should be the most genetically susceptible to the
infecting agent and chronic infection. All too often inappropriate
conclusions are based on use of a single strain of a single species. The
value of using a naturally occurring disease with features that closely
parallel those of the human disease cannot be overestimated.

As we attempt to prove the role of infection in chronic inflammatory
diseases and cancer, the biggest challenge will be convincing peer review
groups who establish research priorities and who facilitate funding
decisions that these are not "fishing expeditions." Likewise, the challenge
will be to convince journal editors that the findings are not merely
coincidental. To make rapid progress we must keep an open mind and accept
the likely possibility that fulfillment of Koch's postulates for infectious
agents involved in chronic inflammatory diseases and cancer may not be
possible.

Dr. Cassell is a recent past president of the American Society for
Microbiology, a member of the National Institutes of Health Director's
Advisory Committee, and a member of the Advisory Council of the National
Institute of Allergy and Infectious Diseases of NIH. She was named to the
original Board of Scientific Councilors of the National Center for
Infectious Diseases, CDC, and is the immediate past chair of the board.

Address for correspondence: Gail H. Cassell, Lilly Research Laboratories,
Lilly Corporate Center, Indianapolis, IN 46285, USA; fax: 317-276-1743;
e-mail: Cassell_Gail_H@Lilly.com.

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Emerging Infectious Diseases
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