California
Association
for
Medical Laboratory Technology
Distance Learning Program
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Chlamydiae
And Their Role In Human Disease Course
Number: DL-982 © California Association
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Chlamydiae And Their Role In Human Disease
OBJECTIVES
Upon completion of this course the participant will
be able to:
1. Discuss the principal characteristics of chlamydiae, including their intracellular
life cycle and their classification.
2. Contrast the classic chlamydiae species with the newly described Para- chlamydiaceae.
3. Outline the pathogenesis of chlamydial infections.
4. Discuss human diseases caused by chlamydiae, including emerging chlamydial
infections.
5. Describe the possible role of chlamydia in chronic diseases.
6. Summarize current diagnostic methods and treatment options.
INTRODUCTION
Long considered a unique group of intracellular bacteria containing
a few pathogenic species, the chlamydiae have recently been shown through molecular
studies to represent a highly diverse group of ubiquitous organisms. In addition
to well known human pathogens there is an abundance of environmental chlamydiae
symbiotic in free-living amoebae and in other hosts. These symbionts are obligate
intracellular parasites. Phenotypic comparison of newly described chlamydial
groups suggests that all have descended from a common ancestor that replicated
intracellularly within eukaryotic host cells. The minor phenotypic differences
observed among chlamydial groups depend on small genomic differences.
The divergence of environmental and pathogenic chlamydiae
is thought to have taken place about 700 million years ago. The common ancestor
of diverse chlamydial groups was already adapted to intracellular survival in
early eukaryotic cells and contained many virulence factors found in modern
pathogenic chlamydiae (1). Recent molecular studies of environmental chlamydiae
have identified three families: Simkaniaceae, Parachlamydiaceae, and Waddliaceae.
CLASSIFICATION OF CHLAMYDIAE
OLD CLASSIFICATION:
Order Chlamydiales
Family Chlamydiaceae
Genus Chlamydia
Species:
C. psittaci
C. trachomatis
C. pneumoniae
C. pecorum
In 1999 a paper by Everett, Bush, and Anderson introduced a reclassification of chlamydiae. The genus Chlamydia was replaced with the genera Chlamydia and Chlamydophila, with a total of nine species. This classification has not been accepted universally. Ongoing molecular studies have uncovered additional chlamydial groups resulting in further changes in chlamydial classification.
REVISED CLASSIFICATION OF CHLAMYDIAE
Order Chlamydiales
Family Chlamydiaceae
Genus Chlamydophila
Species:
C. abortus
C. psittaci
C. felis
C. caviae
C. pecorum
C. pneumoniae
Genus Chlamydia
Species:
C. trachomatis
C. suis
C. muridarum
Family Parachlamydiaceae
Genus Parachlamydia
Species:
P. acanthamoebae
Genus Neochlamydia
Species:
N. hartmanellae
Family Waddliaceae
Genus Waddlia
Species:
W. chondrophila
Family Simkaniaceae
Genus Simkania
Species:
S. negevensis
Additional species of parachlamydiae have been described; a number of these infect various arthropods.
PRINCIPAL CHARACTERISTICS OF CHLAMYDIAE AND THEIR REPLICATION CYCLE
THE CLASSIC CHLAMYDIAE: CHLAMYDIA AND CHLAMYDOPHILA SPECIES
Role in human disease
Chlamydiae are responsible for a wide range of diseases in humans,
including lymphogranuloma venereum, pelvic inflammatory disease, conjunctivitis,
urethritis, cervicitis, pneumonia, psittacosis, and possibly atherosclerosis.
Genetic organization of chlamydiae
The genome of Chlamydia trachomatis was sequenced in
1998. It is of interest that sets of genes for peptidoglycan synthesis and for
ATP biosynthetic pathways were identified in the C. trachomatis genome,
despite the lack of peptidoglycan in chlamydial cells and their inability to
generate ATP. In addition to the chromosome, chlamydiae commonly possess extrachromosomal
genetic elements (plasmids). The presence of 4 to 10 plasmids per elementary
body (extracellular chlamydial form) has been reported for various strains of
chlamydiae. These plasmids may play a role in the virulence of chlamydiae. Studies
in mice using plasmid-cured C. muridarum demonstrated the ability of
these mutants to infect the murine genital tract, but failure to cause disease
in the oviduct. If plasmid-cured strains of human C. trachomatis strains
have similar characteristics, they have the potential to serve as vaccines to
prevent human disease (2).
Metabolism
Although chlamydiae possess a number of enzymes, they have a restricted
metabolic capacity. Chlamydiae lack cytochromes and therefore their metabolic
reactions do not generate energy (ATP). These organisms are energy parasites
that use ATP produced by their host cells for their own requirements. Energy-rich
metabolic intermediates from host cells are required in order to complete the
chlamydial replication cycle.
Developmental cycle and cell structure
The chlamydiae are nonmotile, Gram-negative, obligate intracellular
bacteria that exhibit an intracellular and an extracellular form, and undergo
a biphasic developmental cycle. All known species of chlamydiae have a common
lipopolysaccharide that differs from the lipopolysaccharide of other bacteria.
This molecule is present in the outer membrane of the cell envelope in both
developmental forms of chlamydiae. Highly antigenic polysaccharide epitopes
are present in the lipopolysaccharide layer.
Extracellular forms of chlamydiae are known as elementary bodies.
This developmental form is hardy, spore-like, infectious, and metabolically
inert. The DNA of elementary bodies is condensed into an eccentrically placed
nucleoid. The elementary body is, generally, spherical and 0.2 to 0.3 micrometers
in diameter. When studied with an electron microscope, an elementary body has
granular cytoplasm reflecting the presence of 70S ribosomes. The cell envelope
is double layered, resembling the cell envelope of Gram-negative bacteria. An
important component of the outer cell layer is a protein, known as the major
outer membrane protein (MOMP). This protein constitutes approximately 60% of
the total protein mass of the elementary body cell wall. MOMP functions as a
membrane channel that is permeable to ATP. Since antibodies to MOMP block cellular
infection with chlamydiae, it is probable that antibody binding to MOMP prevents
the uptake of host cell ATP by the intracellular pathogens. MOMP is also of
major importance in the immunologic diagnosis of chlamydial infections because
the MOMP layer contains strain-specific antigenic sites of chlamydial serotypes.
Intracellular developmental forms are called reticulate bodies.
These are larger than elementary bodies and contain fibrillar DNA plus a high
concentration of ribosomes. The cell envelope appears less complex than that
of the elementary bodies. The reticulate body is the metabolically active replicating
form that does not survive well outside the host cell and appears adapted to
an intracellular environment.
Replication of chlamydiae
Chlamydiae are able to infect a diverse range of both nonphagocytic and phagocytic
cultured cells including insect cells, epithelial cells, endothelial cells,
macrophages and monocyte-derived cell lines. The initial attachment of elementary
body and host cell is mediated by electrostatic interactions with heparan sulfate
molecules on the host cell surface. Specific protein receptors on the host cell
surface are probably involved. Such receptors have not been identified definitively.
Apparently the processes involved in attachment and uptake may differ among
species of chlamydiae and even among variants of the same species. Following
attachment, the elementary body enters the host cell by a process similar to
endocytosis. The entry of the elementary body into the host cell is facilitated
by a reorganization of the cell surface microvilli induced by the attachment
of the microorganism to the host cell receptors (3). Once inside a host cell,
the elementary body reorganizes into a reticulate body within a membrane-bound
vacuole known as an inclusion. The inclusion membrane does not fuse with the
host cell’s lysosomal membrane. The reticulate body replicates by binary fission,
remaining within the inclusion membrane for the duration of the intracellular
growth cycle, and forming characteristic intracellular inclusions that can be
observed by light microscopy. The inclusion membrane is derived from the cytoplasmic
membrane of the host. After a period of exponential growth, the reticulate bodies
reconvert to elementary bodies. This process generally takes 24 to 72 hours
and takes place entirely within the cytoplasm of the infected cell. During the
transformation of reticulate bodies to elementary bodies a number of late-phase
proteins are synthesized, including chlamydial outer membrane complex proteins
and histone-like proteins that are part of the chlamydial chromosome. Elementary
bodies are released into the extracellular environment by the fusion of the
membrane of the inclusion with that of the host cell or upon host cell lysis.
The elementary bodies can then initiate a new cycle of infection.
THE NEWLY DESCRIBED CHLAMYDIAE: THE PARACHLAMYDIACEAE
Molecular studies have demonstrated a huge diversity of chlamydiae
from environmental and clinical sources. Chlamydiae that naturally infect free-living
amoebae have been placed in a separate family, the Parachlamydiaceae, based
on the chlamydia-like cycle of replication and on the 80% to 90% homology of
ribosomal RNA genes. These organisms are endosymbionts of amoebae and are generally
not destroyed by their hosts. Because intra-amoebal growth could increase the
virulence of intracellular bacteria, the parachlamydiae may be pathogenic. Furthermore,
the amoebae could play an important role as reservoirs or vectors of chlamydial
infections. Other parachlamydiae, such as Neochlamydia species and
unclassified species, have been isolated from humans, cats, Australian marsupials,
reptiles, fishes, as well as from various environmental samples. New members
of parachlamydiae infecting invertebrates have recently been characterized.
These include Fritschea and Rhabdochlamydia that infect insects,
presenting a possibility that there are insect vectors of chlamydial infections.
Replication of parachlamydiae
The life cycle of Parachlamydia acanthamoebae in amoebae has been studied
by electron microscopy. Two stages, intracellular and extracellular, are part
of the life cycle. Three morphological forms have been observed: the infective
extracellular elementary bodies and crescent bodies, and the intracellular replicating
reticulate bodies. Infection of amoebae takes place by phagocytosis of elementary
or crescent bodies. Within 8 hours after infection, elementary and crescent
bodies differentiate into the reticulate form. The reticulate bodies divide
by binary fission and are able to invade the amoebal cytoplasm. Multiplication
takes place mainly in the vacuoles and rarely in amoebal cytoplasm. In the vacuoles,
the reticulate bodies condense into elementary and/or crescent bodies, which
are released after amoebal lysis or are expelled within vesicles. A new cycle
of infection can then be initiated by the elementary or crescent bodies. The
presence of crescent bodies is associated with prolonged incubation time. This
developmental form has been observed only in parachlamydiae and could be used
as an important taxonomic feature for this group of microorganisms.
PATHOGENESIS OF CHLAMYDIAL INFECTIONS
Chlamydia trachomatis infections are among the most common
notifiable diseases in USA. Infection with Chlamydophila pneumoniae
is also extremely common: serological surveys indicate a nearly universal occurrence
of infection with this organism. The extremely high prevalence of infections
caused by C. trachomatis and C. pneumoniae reflects the successful
adaptation of these bacteria to persistence in their human hosts. The infected
host’s immune response may fail to eliminate these intracellular bacteria, leading
to clinical persistence of chlamydiae. Similarly, the immune response does not
prevent re-infection with these organisms.
The initial response of the host to chlamydial infection is acute inflammation.
Repeated infection by chlamydiae increases the severity of the inflammatory
response and promotes chronic inflammation that may result in tissue damage
and scarring. The damage may be mediated by immune cells directed against host
tissues. Immune reactivity such as delayed hypersensitivity to chlamydial antigens
or an autoimmune response may be involved. An alternate hypothesis is that host
tissue damage is mediated by inflammation caused by the pathogen (4). According
to this model of chlamydial pathogenesis, chlamydiae infect endothelial or epithelial
cells. Damaged host cells secrete chemokines and growth factors, such as IL-11,
IL-8, IL-12, IL-6, and GM-CSF. These factors induce the appearance of clinical
signs, which include redness, edema, and a mucopurulent discharge. Secreted
cytokines attract and activate neutrophils, macrophages, and immunologically
reactive cells. Activated cells produce their own array of cytokines and growth
factors. These factors promote the inflammatory response, cellular infiltration,
and migration of activated immune cells to lymphoid follicles. Eventually, follicle
necrosis, tissue damage and scarring may occur.
HUMAN DISEASES CAUSED BY CHLAMYDIAE
Infections with Chlamydia trachomatis
C. trachomatis strains infect the eye and the genital
tract. The strains are tissue selective rather than tissue specific. Genital
strains are occasionally found in the eye, while ocular strains are sometimes
isolated from the genital tract. The strains are further subdivided into serotypes
or serovars on the basis of binding affinity for monoclonal antibodies.
Genital infections
Lymphogranuloma venereum; serovars L1, L2, L2a, and L3
The lymphogranuloma strains of chlamydiae are noted for their
ability to invade lymphatic tissue.
Lymphogranuloma venereum is a sexually transmitted disease found
more frequently in the tropical and sub-tropical parts of the world, although
some cases of lymphogranuloma venereum are reported in the Unites States each
year.
The incubation period for this disease ranges from 3 to 12 days.
The primary lesion is a 5 to 8 mm soft, red, painless erosion or ulcer, which
heals spontaneously in a few days. The secondary stage begins 2 to 6 weeks later
and is characterized by the presence of swollen, tender inguinal lymph nodes
which may drain spontaneously. These symptoms may be accompanied by fever, chills,
and malaise. If this condition is not treated, genital ulcers, proctitis, and
other complications may develop.
Lymphogranuloma strains of C. trachomatis are susceptible
to antibiotics. Commonly prescribed medications include tetracycline, doxycycline,
and erythromycin.
Other chlamydial genital infections: serovars D to K, Da, Ia, Ja
These chlamydial strains are the most common causes of urethritis
and mucopurulent cervicitis in females and nongonococcal urethritis in males.
The tissue tropism of these strains is restricted to mucosal epithelial cells.
The same chlamydial strains may also infect neonates causing conjunctivitis
and pneumonia.
Chlamydial genital infections are the most frequently reported
infections in the United States. The age group with the highest prevalence of
chlamydial infection is under 25 years of age. Although the infection is frequently
subclinical and asymptomatic, several important complications may occur. Complications
of chlamydial cervicitis may include pelvic inflammatory disease, ectopic pregnancy,
chronic pelvic pain, and infertility. Chlamydial urethritis in men may lead
to inflammation of the prostate gland, the seminal duct, infertility, and to
Reiter’s syndrome, which includes a triad of symptoms: conjunctivitis, polyarthritis,
and genital inflammation.
Cervicitis is frequently asymptomatic, but some
patients may complain of an abnormal vaginal discharge and occasional vaginal
bleeding. Recommended antibiotic treatment for chlamydial cervicitis includes
oral azithromycin or doxycycline.
Urethritis in men may result from either infectious
or non-infectious causes. Symptoms, if present, include discharge of purulent
material and difficult and/or painful urination. Asymptomatic infections are
common. The most common microbial pathogens that cause urethritis are Neisseria
gonorrhea and C. trachomatis. Chlamydiae are responsible for approximately
15% to 55% of all cases of non-gonococcal urethritis. The same antibiotics that
are used to treat chlamydial cervicitis, azithromycin and doxycycline, are highly
effective in the treatment of chlamydial urethritis.
Chlamydial infections of infants
C. trachomatis infection of neonates results from perinatal
exposure to the mother’s infected cervix. Ocular prophylaxis with silver nitrate
or antibiotic ointments does not prevent eye infection caused by perinatal transmission
of C. trachomatis from mother to infant. The best method for preventing
neonatal chlamydial infections is diagnosis and treatment of pregnant women.
Initial C. trachomatis perinatal infection involves the
mucous membranes of the eye, oropharynx, urogenital tract, and rectum. Chlamydial
infection of these areas of the body may be asymptomatic, or symptoms of disease,
such as conjunctivitis, may develop 5 to 12 days after birth. In general, chlamydial
etiology of infection should be considered for all infants under 30 days of
age who have conjunctivitis, particularly if the mother has a history of untreated
chlamydial infection. Similarly, all infants who are less than three months
of age and develop pneumonia should be tested for C. trachomatis. Characteristic
signs of chlamydial pneumonia in infants include a repetitive, staccato cough,
and chest X-ray findings typical of a chlamydial infection. Generally, fever
is absent and wheezing is rarely observed. When perinatal infections of the
nasopharynx, the urogenital tract, and the rectum occur they may persist for
as long as one year.
Treatment of chlamydial perinatal infections
Topical antibiotic therapy for chlamydial perinatal infections
is generally not effective. Oral erythromycin or ethylsuccinate have been used
for both perinatal chlamydial conjunctivitis and infant pneumonia. The effectiveness
of a single course of erythromycin treatment is approximately 80%.
Chlamydial ocular infections: trachoma (serovars A, B, Ba, and C)
Trachoma occurs worldwide, most often in rural settings in developing
countries. It is primarily a disease of poverty. Although rare in the United
States, trachoma may be found in any geographic area where people live under
crowded conditions, have limited water supply, poor hygiene, and deficient sanitation.
Children are affected most frequently. Serious complications resulting from
trachoma, such as blindness, generally don’t become apparent until later in
life. The disease is transmitted through direct contact with infected tissues
or with secretions from infected eyes, nose, or throat, or from contaminated
towels and clothes. Infection can also be spread by flies.
Trachoma has been recognized as a cause of blindness for centuries.
It has been known in Egypt for more than 3,500 years. Its contagious nature
was recognized, but the identity of the infecting agent was unknown. The fact
that trachoma was a transmissible infectious disease was well known: numerous
First World War conscripts evaded military service by infecting their own eyes
with discharges from trachoma patients. The causative agent of trachoma was
visualized in 1907, when Halberstaedler and von Prowazek described the presence
of inclusion bodies within infected cells. In 1957 T’ang and his coworkers were
able to culture the infectious agent from infected human eyes in yolk sacs of
chicken embryos. In 1966 Moulder described the structure and metabolism of these
disease agents, clearly demonstrating that they were intracellular bacteria
with a distinctive developmental cycle.
Clinical features of trachoma
Severity of trachoma ranges from asymptomatic to mild to severe.
In endemic areas repeat infections occur. Symptoms of acute disease as well
as signs of a chronic infection may be present simultaneously. In an initial
infection, if symptoms develop, they usually appear within 5 to 10 days. These
symptoms include conjunctival infection with an irritated red eye and some mucopurulent
discharge. Other symptoms include swollen eyelids and turned-in eyelashes. When
the cornea is involved it appears cloudy; there is accompanying pain and photophobia.
When sufficient conjunctival scarring accumulates, the upper eyelid may turn
inward so that eyelashes rub against the globe. This is known as trichiasis
and it is intensely irritating. In addition to being painful, trichiasis injures
the cornea. Scarring of the cornea results in impaired vision.
Prevention and treatment
The spread of trachoma can be prevented through improved sanitation
and hygiene, and not sharing items such as towels and clothing.
Early treatment with antibiotics, such as erythromycin, azithromycin,
or doxycycline can prevent long-term complications. In some cases eyelid surgery
may be required to prevent long-term scarring.
Eradication of trachoma as a major cause of blindness
The World Health Organization (WHO) has set a target date of the
year 2020 for eliminating trachoma as a major cause of blindness. In order to
accomplish this goal, intervention with risk factors at individual and community
level would have to take place in affected villages and neighboring communities.
In 2001 WHO published a procedure for rapid, low cost identification
of communities likely to be at risk for trachoma that leads to blindness. This
procedure is known as the Trachoma Rapid Assessment and it involves the following
steps:
a. Areas that have endemic trachoma are identified.
b. Field visits are implemented to areas of highest risk within
the endemic area.
c. Field visits include the selection of at least 15 households
and at least 50 children between the ages of 1 and 9. Specimens from active
cases of disease are collected and submitted for laboratory testing.
d. Areas are ranked to prioritize the need for control measures.
Infections with Chlamydophila pneumoniae
Respiratory infections with C. pneumoniae are extremely
common. It has been suggested that such infections occur at least once in the
lifetime of every human being. Antibody to C. pneumoniae can be demonstrated
in the serum of 40% to 75% of persons tested. Typically infection begins in
the upper respiratory tract. A mild illness develops with fever and a nonproductive
cough, or the infection may remain asymptomatic. If left untreated, or inadequately
treated, the infection may progress to bronchitis, sinusitis, otitis media,
and pneumonia. Untreated C. pneumoniae infections may become chronic.
On the basis of serologic studies, these infections have been associated with
a number of chronic illnesses, such as myocarditis, aseptic meningitis, asthma,
chronic fatigue syndrome, multiple sclerosis, and Alzheimer’s disease. Confirmation
of C. pneumoniae infection is difficult, because the organisms are
fastidious and difficult to isolate from clinical specimens. Generally, diagnosis
relies on immunologic or molecular tests.
Epidemiology of infection
Transmission of infection occurs by the respiratory
route, similar to viral respiratory infections. Most infections occur in schools,
dormitories, military barracks, or within households. Commonly, infections occur
in late childhood with peak incidence between 10 and 20 years of age. New infections
or re-infections are acquired throughout life, in spite of a rising antibody
titer to the pathogen. Infections appear to be more common in men than in women.
Pathogenesis and immune response
C. pneumoniae infects epithelial and endothelial cells,
as well as macrophages and neutrophils. The phagocytic cells are able to serve
as hosts for the pathogen and disseminate the organism throughout the body.
The developing antibody response is not able to eliminate the infecting organism.
Gamma interferon and activated CD8 T cells offer some protection against the
infection.
Treatment
C. pneumoniae is susceptible to a number of antibiotics,
including erythromycin, tetracycline, doxycycline, azithromycin, clarithromycin,
and some fluoroquinolones. It is recommended that treatment be given for at
least 2 to 3 weeks.
Infections with Chlamydophila psittaci
C. psittaci
is primarily an avian pathogen, infecting psittacine birds such as parrots,
cockatiels, and parakeets as well as pigeons, turkeys, ducks, ostriches, and
wild birds. The infectious agent is found in droppings of sick birds and in
dust contaminated by infected droppings. The bacteria can remain infectious
in the environment for many months. Human infection occurs through inhalation
of bacteria shed in bird feces, in dust contaminated by droppings, and in secretions
from infected birds. Sheep, goats, cattle, and reptiles may also become infected
but the transmission of the pathogen from these animals to human cases has not
been documented.
C. psittaci antigenic variants
There are several distinct antigenic variants of C. psittaci:
A, B, C, D, E, and F. The strains can be identified by monoclonal antibodies
that recognize antigenic sites on the major outer membrane protein. These strains,
or serovars, are endemic in different avian species. Recently an additional
serovar, E/B, isolated from turkeys, ducks, and pigeons, has been identified.
Identification of C. psittaci variants is useful in epidemiological
studies.
Human infection with C. psittaci (psittacosis)
Psittacosis is quite rare, with fewer than 50 confirmed cases reported in the
United States each year. Persons at risk include bird owners, pet shop employees,
veterinarians, and poultry workers. Psittacosis has an incubation period of
1 to 4 weeks. The infection may be asymptomatic or there may be fever and chills,
muscle ache, headache, fatigue, a dry cough, shortness of breath, blood-tinged
sputum, and severe pneumonia. Occasionally complications such as endocarditis
and hepatitis as well as neurological problems may occur. The case fatality
rate for untreated psittacosis is 15% to 20%.
Treatment
C. psittaci is susceptible to a number of antibiotics, including tetracycline,
doxycycline, erythromycin, azithromycin, and rifampin.
EMERGING CHLAMYDIAL INFECTIONS
The newly recognized Parachlamydiaceae are only distantly related
to the classic Chlamydia and Clamydophila species. Some of
these new chlamydiae are possible human and animal pathogens.
There is considerable evidence supporting the role of Parachlamydia
acanthamoeba as an emerging respiratory pathogen. A number of serological
studies have demonstrated antibodies to Parachlamydia in patients suffering
from pneumonia. In several cases these patients were immunocompromised. Although
free-living amoebae are hosts for P. acanthamoebae, this bacterium
is also able to enter and to multiply within human macrophages. The results
of various epidemiological studies suggest that exposure to Parachlamydiaceae
may lead to bronchitis, community-acquired pneumonia, and aspiration pneumonia.
Neochlamydia
The pathogenic potential of Neochlamydia hartmanellae remains to be determined.
Neochlamydia has been recovered from free-living amoebae (Acanthamoeba) isolated
from a contact lens of a patient with keratitis. The role of Neochlamydia is
not clear since Acanthamoeba keratitis is a well-established clinical syndrome.
Simkania negevensis
Simkania is found in free-living amoebae, using the amoebae
as an environmental reservoir. This bacterium has a worldwide distribution.
It has been associated with bronchiolitis in infants and with lower respiratory
tract infections in adults. S. negevensis DNA has recently been demonstrated
in human arterial biopsy specimens.
Infections in other vertebrates
Waddlia chondrophila is a newly described agent of bovine
abortion. Parachlamydia salmonis is the probable etiologic agent of
gill epitheliocystis in salmon. Parachlamydiaceae have been implicated as ocular
pathogens of cats.
ROLE OF CHLAMYDIAE IN CHRONIC DISEASE
Many chronic diseases are associated with inflammation. Various
infectious agents have been investigated as possible causes of this inflammation.
Chlamydia species and C. pneumoniae are among the infectious
agents suspected of contributing to the inflammatory process associated with
chronic illness. On the basis of serological evidence C. pneumoniae
has been linked to a number of chronic conditions. Some of these, previously
listed in the section on C. pneumoniae infections, are asthma, chronic
fatigue syndrome, multiple sclerosis, and Alzheimer’s disease. In addition to
these conditions, a recent study demonstrated that male patients with lung cancer
have a higher antibody titer to C. pneumoniae than the control group.
Cardiovascular disease, including atherosclerosis, is one of the chronic diseases
most thoroughly investigated for a link to C. pneumoniae infection.
Inflammation of blood vessels plays an essential role in both initiation and
progression of atherosclerosis, and chronic infection with C. pneumoniae
may be a contributing factor to this inflammation. The presence of C. pneumoniae
was shown in some atherosclerotic plaques of coronary arteries that were studied
with electron microscopy and immunoperoxidase staining. Attempts to isolate
the organisms were negative. A number of serological studies demonstrated an
association between chronic C. pneumoniae infection and cardiovascular
disease but other studies failed to show an increased risk of an adverse outcome
in cardiac patients seropositive for C. pneumoniae.
LABORATORY DIAGNOSIS OF CHLAMYDIAL INFECTIONS
Methods used for identification of chlamydiae in clinical specimens fall into
three categories: 1) culture; 2) immunological tests including immunofluorescence;
and 3) molecular techniques.
1) Cell culture
Culture of chlamydiae has long been considered the gold standard for identification
of chlamydiae. This is the only method that demonstrates the presence of viable
microorganisms and allows determination of antibiotic sensitivity. Cultivation
of chlamydiae is highly specific but not as sensitive as some of the other diagnostic
methods. The procedure is not available in all laboratories and some chlamydial
species are very difficult to grow in cell culture.
Successful isolation of chlamydiae relies on the use of enriched sucrose phosphate
transport medium and strict maintenance of cold storage of clinical specimens
during transport. Various cell lines are used since each species or strain shows
a relative specificity for a given cell type. Examples of cell lines that may
be used are McCoy cells, HeLa 229 cells, and L434 mouse fibroblast cells. The
specimen is centrifuged onto the cell monolayer to aid cellular infection and
increase yield. Cultures are incubated in the presence of cycloheximide which
inhibits host protein synthesis. Bacterial and fungal overgrowth is prevented
by adding gentamicin, vancomycin, and amphotericin. The cultures are incubated
for several days and sometimes for as long as 14 days. Determining whether the
culture is positive or negative requires staining with iodine or Giemsa stain
or the use of labeled polyclonal or monoclonal antibody. One or more blind passages
in which apparently negative cultures are homogenized and inoculated to fresh
cell cultures are sometimes required. Identification of one inclusion is sufficient
to record a positive result.
2) Immunological tests
Complement fixation was the first serological test used
for detecting serum antibodies to the chlamydial polysaccharide antigens in
the lipopolysaccharide layer. These antigens are not strain-specific. In addition,
the complement fixation test has low sensitivity for ocular infections. Immunofluorescence
tests are widely used in the diagnosis of chlamydial infections and are tests
of choice for diagnosis of some of these infections.
The microimmunofluorescence technique developed by Wang and Grayston
was the first method used to identify C. trachomatis serovars. This
test can detect antichlamydial antibodies in serum or tears. Serial dilutions
of the sample are placed on glass slides on which antigens of different C.
trachomatis serovars have been fixed. Following incubation, the slides
are probed with fluorescein-labeled anti-human immunoglobulin. Separate tests
can detect the presence of immunoglobulin A, immunoglobulin M, and immunoglobulin
G.
Other immunofluorescence assays used for diagnosis of chlamydial
infections include direct and indirect fluorescent antibody tests and immunohistochemical
assays.
Enzyme immunoassay
Enzyme-linked immunosorbent assays (EIA) are designed to detect
antigen or antibody by producing an enzyme-triggered color change. In chlamydial
infections enzyme immunoassay usually refers to an antigen detection test with
antibody used to detect chlamydial antigen contained in the specimen. There
are many commercial C. trachomatis enzyme immunoassays available. Most
of these detect the chlamydial lipopolysaccharide.
3) Molecular tests
Hybridization assays Early direct hybridization probe tests used
radiolabeled C. trachomatis DNA and autoradiography requiring an exposure
time of at least 36 hours. Commercial applications of this technique are now
available. These commercial assays offer significant technical improvements
in performance of hybridization probe tests.
Polymerase chain reaction (PCR) tests
PCR is a technique for amplifying DNA, and all assays based on it are part of
the group of nucleic acid amplification tests. In detection of C. trachomatis
a number of different nucleic acid sequences have been used as targets. These
include the chlamydial cryptic plasmid (pCT), genes coding for MOMP, and genes
coding for 16S rRNA. The PCR assay directed at plasmid genes is thought to be
both sensitive and specific for the diagnosis of C. trachomatis infection. A
commercial PCR kit, Amplicor Chlamydia, targets a sequence within the cryptic
plasmid CT. Another commercial kit, the Gen-Probe APTIMA assay detects and amplifies
C. trachomatis ribosomal RNA in cervical specimens. Recently a rapid
PCR assay has been developed that detects and amplifies DNA that codes for 23S
RNA. This assay is able to identify four members of the Chlamydiaceae family:
C. trachomatis, C. psittaci, C. pneumoniae and C.
pecorum.
Additional molecular techniques used for the diagnosis of C. trachomatis
infections are the ligase chain reaction, the strand replacement assay, and
the transcription-mediated amplification. Nucleic acid amplification tests are
probably the most sensitive of all chlamydial diagnostic tests. The introduction
of these tests represents a major advance in the diagnosis of chlamydial infections.
Laboratory diagnosis of infections with newly described chlamydiae
Processing of clinical samples
An adequate specimen must contain infected host cells because of the obligate
intracellular nature of environmental chlamydiae. For mucosal specimens, swabbing
with cotton swabs may be sufficient for the recovery of a sufficient number
of infected host cells. Scraping of mucosal surfaces will increase cell yield
but may also result in bleeding. Transport media developed for rickettsia, such
as the 2-sucrose phosphate medium or the sucrose-glutamate phosphate medium,
may be used for chlamydial clinical samples. Bacterial overgrowth may be suppressed
by the addition of gentamicin and vancomycin to the transport medium. Common
viral transport media should be avoided because they contain antibiotics that
inhibit the growth of Parachlamydiaceae. Amphotericin B should not be added
if the specimen will be tested in amoebal co-culture, since most free-living
amoebae are susceptible to amphotericin B. The use of penicillin should, ideally,
be avoided to prevent the induction of persistent non- multiplying aberrant
bacterial forms. Microbiological specimens should be stored at 4 to 8 degrees
C and processed as soon as possible. After storage for 24 hours the specimens
should be frozen at -70 degrees C.
Cell culture
Historically chlamydiae had been grown in embryonated eggs but
this culture method was relatively rapidly replaced by cell cultures. Embryonated
eggs today are used mostly for production of large quantities of chlamydial
antigens or for cultivation of fastidious strains. Simkania negevensis
and Waddlia spp. have been cultured in a variety of mammalian cell
lines. These include Vero cells, HeLa, Hep-2, human macrophages and other cell
lines. Vero cells are currently used for cultivation of Simkania and
Waddlia species. Centrifugation of the clinical sample before applying
it to the cell monolayer increases the growth yield of the organisms in cell
culture. The culture protocol includes RPMI 1640 medium with 10% calf serum,
the addition of antibiotics, and no cycloheximide. Cytopathic effect develops
as soon as 36 hours after inoculation of clinical samples or after several days,
depending on the species of parachlamydiae.
Parachlamydia acanthamoebae is difficult to grow in mammalian cell lines.
Instead, amoebic co-culture can be used.
Amoebic co-culture
A wide variety of free-living amoebae are able to sustain the growth of new
chlamydiae, but not all strains of a free-living amoeba are susceptible to infection
with these agents. The use of more than one strain of amoeba or the use of several
amoebic species is recommended to increase the rate of isolation of parachlamydiae
from clinical or environmental samples. Amoebic co-culture may be done by inoculating
the clinical sample into a cell culture system where mammalian cells are replaced
by cultured free-living amoebae. An alternative method is the addition of an
enrichment for amoebae to the clinical sample. If parachlamydiae are present
within the amoebae, they will be liberated from their amoebic hosts by lysis.
The released bacteria can then be grown with another strain of free-living amoebae.
Detection of bacteria that grow in either cell culture or amoebic
culture may be done by different staining methods. Since chlamydiae give inconsistent
results with Gram stain, Giemsa or Gimenez stains or immunofluorescence can
be used. In situ fluorescent hybridization technique is also suitable
for demonstration of chlamydiae. When stained by the Gimenez method, the bacteria
appear reddish (fuchsin) against a green background (malachite green).
Serological methods
An enzyme-linked immunosorbent assay (ELISA) has been developed
for the diagnosis of Simkania negevensis infection. This ELISA detects
immunoglobulin A and immunoglobulin G but not immunoglobulin M.
Immunofluorescent techniques have been successfully used in identification of
parachlamydiae in a number of studies. Western blotting technique has been used
to confirm the results of immunofluorescence tests.
Molecular techniques
Nucleic acid amplification by the polymerase chain reaction (PCR) has been successfully
applied to identification of the newly described chlamydiae. PCR techniques
that target ribosomal genes have been most useful for identification of this
group of organisms. For the new chlamydiae sequencing of the 16S rRNA gene appears
to be the best method for strain identification. Primers have also been developed
for 23S rRNA genes. Other genes of parachlamydiae have been used as targets
for amplification by PCR.
Laboratory diagnosis of infections with Chlamydia and Chlamydophila
C. trachomatis genital and neonatal infections:
Cell culture
Direct immunofluorescence
Enzyme immunoassays
Nucleic acid hybridization tests
Nucleic acid amplification tests: these have the highest sensitivity of all
assays listed.
C. pneumoniae respiratory infections
Use of culture for the detection of C. pneumoniae is
difficult because of problems associated with growing C. pneumoniae
in cell culture.
Nucleic acid amplification tests are used in research and, occasionally,
in clinical laboratories.
Serological tests may be used, but complement fixation test does not distinguish
Chlamydophila from Chlamydia.
The microimmunofluorescence test is useful for diagnosis of C. pneumoniae
infections.
Psittacosis
Cell culture
Serology
Lymphogranuloma venereum
This disease is frequently diagnosed on the basis of lymph node
biopsy findings and clinical symptoms. Laboratory tests that could aid in diagnosis
are indirect immunofluorescence and serological tests.
Trachoma
The diagnosis is generally based on clinical symptoms. Diagnostic laboratory
tests include:
Direct microscopy of conjunctival scrapings: the slides are stained
with Giemsa stain or with acridine orange and iodine and examined for inclusion
bodies.
Cell culture Enzyme immunoassays for detection of chlamydial antigen
Serological tests for chlamydial antibody in tears and serum
Nucleic acid hybridization tests
Nucleic acid amplification tests
SUMMARY
Chlamydiae represent a diverse group of intracellular bacteria
widely distributed in the environment. Some chlamydiae are pathogenic in humans
and in other vertebrates whereas others are symbiotic in free-living amoebae.
These diverse chlamydial groups share a common ancestor. Chlamydial classification
is currently undergoing a change. On the basis of molecular studies the chlamydiae
that cause pneumonia and psittacosis are now placed in a new genus Chlamydophila.
A new family and several new genera have been created for environmental chlamydiae.
This classification is not universally accepted at this time.
Chlamydia and Chlamydophila are responsible for a wide range
of human diseases. These diseases include trachoma, lymphogranuloma venereum,
genital infections, neonatal infections, pneumonia, and psittacosis. Chlamydial
genital infections are the most frequently reported infections in the United
States.
The chlamydiae are Gram-negative, obligate intracellular bacteria with a limited
metabolic capacity. They are not able to generate energy and therefore depend
on host cells for their energy requirements. Chlamydiae undergo a biphasic developmental
cycle and have an extracellular and an intracellular phase of growth. The extracellular
forms of chlamydiae are called elementary bodies. These are spherical, spore-like,
infectious, and metabolically inert. The intracellular forms are called reticulate
bodies. They are larger than elementary bodies, are metabolically active, and
are adapted to an intracellular environment.
Chlamydiae are able to infect a wide range of host cells. Replication
of chlamydiae takes place within membrane-bound vacuoles in the cytoplasm of
infected cells. Within the vacuoles, the elementary bodies reorganize into reticulate
bodies which multiply by binary fission. After 24 to 72 hours the reticulate
bodies transform into elementary bodies, which are released from the host cells
and are able to initiate a new cycle of infection.
The recently described environmental chlamydiae have been placed
in a newly created family, the Parachlamydiaceae. These organisms are endosymbionts
of free-living amoebae. The replication cycle of these bacteria is similar to
that of classical chlamydiae with the exception that two infective extracellular
forms are present. The second form is called the crescent body.
Infections with C. trachomatis and C. pneumoniae
are extremely common and have a tendency toward persistence. Re-infections are
also common and tend to increase the severity of the host’s inflammatory response
to the pathogen. Chronic inflammation frequently results in tissue damage and
scarring. C. trachomatis infects the eye and the genital tract depending
on the tissue preference of the infecting strain. Genital infections include
cervicitis, urethritis, and lymphogranuloma venereum. Complications that may
develop as a consequence of the initial infection include pelvic inflammatory
disease, ectopic pregnancy, infertility, and prostatitis. The same strains that
cause genital infections may be involved in neonatal infections. Other strains
of C. trachomatis cause trachoma, an ocular infection that is a major
public health problem in developing countries.
Members of the genus Chlamydophila infect the respiratory
tract. C. pneumoniae causes pneumonia and C. psittaci is the
agent of psittacosis. Some of the newly discovered environmental chlamydiae
are also implicated in human disease. Chronic infections with chlamydiae have
been linked to other diseases such as atherosclerosis.
Chlamydial infections can be treated with antibiotics such as erythromycin,
azithromycin, or doxycycline.
Diagnosis of chlamydial infections is based on
clinical symptoms and on laboratory diagnostic assays. These assays include
cell culture, serological tests, various immunofluorescent assays, and a number
of molecular techniques that involve amplification of chlamydial DNA.
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