Mycoplasmas, rickettsiae, and chlamydiae are classified as true bacteria, but they are extremely
small, and for various reasons cannot be cultured by ordinary bacteriologic methods. The viruses
are the smallest of all microorganisms and are classified separately. The techniques that have developed
over many years for propagating and studying viruses have provided an understanding of their
nature and pathogenicity. The electron microscope, together with elegantly precise biochemical,
physical, molecular, and immunologic procedures, has revealed the structure of viruses and their
role in disease at the cellular level. Prions are proteinaceous infectious particles that cause so-called
slow viral infections because they take many years to develop. Prions are smaller in size than viruses
and are believed to contain no nucleic acids. The means by which such agents can cause disease remains
unknown, but ongoing molecular studies may unravel the answer.
In this exercise we shall review the nature and pathogenicity of these microorganisms.
||To learn the role of mycoplasmas, rickettsiae, chlamydiae, viruses, and prions in disease and to
review some laboratory procedures for recognizing them
||Audiovisual or reading materials illustrating each group
Diagram of the electron microscope
Students will not perform laboratory procedures, but should come to class prepared by assigned
reading to discuss the laboratory diagnosis of diseases caused by these agents.
Following is a brief summary of each group.
The mycoplasmas, previously called “pleuropneumonia-like organisms” (PPLO), were first
known as etiologic agents of bovine pleuropneumonia. Several species are now recognized,
including three that are agents of human infectious disease.
is the causative organism of “primary atypical pneumonia.”
The term implies that the disease is unlike bacterial pneumonias and does not represent a
secondary infection by an opportunistic invader, but has a single primary agent. Clinically,
mycoplasmal pneumonia resembles an influenza-like illness.
Mycoplasma hominis may be found on healthy mucous membranes, but is also associated
with some cases of postpartum fever, pyelonephritis, wound infection, and arthritis.
are strains of mycoplasma that produce very tiny colonies and were,
for this reason, once called “T-mycoplasmas.”They have been renamed in recognition of their
unique possession of the enzyme urease. These mycoplasmas, like M. hominis
, are normally
found on mucosal surfaces, but have sometimes been associated with urogenital and neonatal
infections and female infertility.
Mycoplasmas are extremely pleomorphic (varied in size and shape). They are very
thin and plastic because they lack cell walls. For this reason, unlike other bacteria, they can pass through bacterial filters, they do not stain with ordinary dyes, and they are resistant to
antimicrobial agents (such as penicillin) that act by interfering with cell wall synthesis.
These organisms can be cultivated on enriched culture media, but on agar media their
colonies can be clearly seen only with magnifying lenses. They do not heap on the surface,
but extend into and through the agar from the point of inoculation.
Specimens for laboratory diagnosis include sputum, urethral or cervical discharge,
synovial fluid, or any material from the site of suspected infection. Cultures require 3 to 10
days of incubation at 35°C. Serological methods are also available for detecting mycoplasmal
antibodies in the patient’s serum.
The rickettsiae are very small bacteria that survive only when growing and multiplying intracellularly
in living cells. In this respect they are like viruses; that is, they are obligate parasites.
They have a cell wall similar to that of other bacteria, which can be stained with special
stains so that their morphology can be studied with the light microscope.
Certain arthropods, such as ticks, mites, or lice, are the natural reservoirs of rickettsiae.
They are transmitted to humans by the bite of such insects, by rubbing infected insect
feces into skin (for example, after a bite), or by inhaling aerosols contaminated by infected
insects. The most important rickettsial pathogens are Rickettsia prowazekii
typhus), Rickettsia rickettsii
(Rocky Mountain spotted fever), Rickettsia akari
and Coxiella burnetii
species are classified in the same family as rickettsiae. They are recently
recognized agents of several diseases, especially in Japan and the United States. Some
ehrlichiae are transmitted by ticks. Ehrlichiae sennetsu
, common in Japan, produces a disease
resembling infectious mononucleosis. Ehrlichia chaffeensis
, a tick-borne disease in the
United States, produces symptoms similar to Rocky Mountain spotted fever, but without
Following is a list of the major groups of the rickettsial family and the diseases they cause.
- Typhus group
- Epidemic typhus
- Murine typhus
- Scrub typhus (tsutsugamushi fever)
- Spotted fever group
- Rocky Mountain spotted fever
- Boutonneuse fever
- Coxiella (The genus Coxiella is undergoing reclassification and may be removed from the rickettsial family.)
- Q fever
- Sennetsu fever (Japan)
In the laboratory, rickettsiae can be propagated only in cell culture or in intact animals, such
as chick embryos, mice, and guinea pigs. They are identified by their growth characteristics, by the type of injury they create in cells or animals, and by serological means. Serological diagnosis
of rickettsial diseases can also be made by identifying patients’ serum antibodies.
The chlamydiae are intermediate in size between rickettsiae and the largest viruses, which
they were once thought to be. They are now recognized as true bacteria because of the structure
and composition of their cell walls (the term chlamydia
means “thick-walled”) and because
their basic reproductive mechanism is of the bacterial type. They are nonmotile, coccoid
organisms that, like the rickettsiae, are obligate parasites. Their intracellular life is
characterized by a unique developmental cycle. When first taken up by a parasitized cell, the
chlamydial organism becomes enveloped within a membranous vacuole. This “elementary
body” then reorganizes and enlarges, becoming what is called a “reticulate body.”The latter,
still within its vacuole, then begins to divide repeatedly by binary fission, producing a mass
of small particles termed an “inclusion body” (see colorplate 40
). Eventually the particles are
freed from the cell, and each of the new small particles (again called elementary bodies) may
then infect another cell, beginning the cycle again.
Three chlamydial species are responsible for human disease. Chlamydia psittaci
causes ornithosis, or psittacosis (“parrot” fever), a pneumonia transmitted to humans usually
by certain pet birds. Chlamydia trachomatis
currently is the most common bacterial agent of
sexually transmitted disease; the infection often is referred to as nongonococcal urethritis. In
addition, this species causes a less common sexually transmitted disease, lymphogranuloma
venereum; infant pneumonitis; and trachoma, a severe eye disease that can lead to blindness. Chlamydia pneumoniae
produces a variety of respiratory diseases, especially in young adults.
Because of difficulties growing it, the organism was identified only during the 1980s.
Undoubtedly it has been causing disease for many years, if not for centuries.
and Chlamydia pneumoniae
are almost always diagnosed by serological
means. Cell culture methods are available for growing Chlamydia psittaci
, but isolating this organism
in culture is hazardous and performed only in laboratories with specialized containment
Cell culture methods are also available for isolating Chlamydia trachomatis
, but they
are cumbersome, performed only in specialized laboratories, and generally reserved for cases
of suspected child abuse. The development of nucleic acid probe and amplification assays has
greatly aided diagnosis of this common sexually transmitted disease pathogen. In addition to
genital specimens, eye, urine, and infant respiratory specimens may be tested, depending on
the system used.
Viruses are infectious agents that reproduce only within intact living cells. They are so small
and simple in structure, and so limited in almost all activity, that they challenge our definitions
of life and of living organisms. The smallest are comparable in size to a large molecule.
Structurally, they are not true cells but subunits, containing only an essential nucleic acid
wrapped in a protein coat, or capsid
. The electron microscope reveals that they have various
shapes, some being merely globular, others rodlike, and some with a head and tailpiece resembling
a tadpole. When viruses are purified, their crystalline forms may have distinctive
patterns. An intact, noncrystallized virus particle is called a virion
There are many ways to classify viruses: on the basis of their chemical composition,
morphology, and similar measurable properties. From the clinical point of view, it seems
practical to classify them on the basis of the type of disease they produce. This, in turn, is
based on their differing affinities for particular types of host cells or tissues. Thus, we speak
viruses as those that have a specific affinity for cells of the nervous system. Dermotropic
viruses affect the epithelial cells of the skin, and viscerotropic
viruses parasitize internal
organs, notably the liver. Enteric viruses
are so-called because they enter the body
through the gastrointestinal tract. Their primary disease effects are exerted elsewhere, however,
when they disseminate from this site of initial entry. The term arbovirus
is used for those
viruses that exist in arthropod reservoirs and are transmitted to humans by their biting insect
hosts (i.e., they are arthropodborne
). Still other viruses, such as the human immunodeficiency
virus, have effects on multiple body systems. In table 30.1, some important viruses are
grouped in a clinical and epidemiological classification that reflects either their route of transmission
or the type of disease they cause in humans.
|Table 30.1 Clinical and Epidemiological Classification of Some Clinically Important Viruses
A variety of methods may be used for the laboratory diagnosis of viral infections. These include
isolation of the virus in cell culture; direct examination of clinical material to detect
viral particles, antigens, or nucleic acids; cytohistological (cellular) evidence of infection; and
serological assays to assess an individual’s antibody response to infection. No single laboratory
approach is completely reliable in diagnosing all
viral infections. Therefore, the use of
any one or a combination of these methods may be needed to establish a specific viral etiology
of disease. The choice of method may be determined by several factors, including knowledge of the pathogenesis of the suspected viral agent, the stage of the illness, and the availability
of various laboratory methods for the particular viral infection suspected.
|Figure 30.1 Cell culture of adenovirus. The uninoculated cells on the left form an even monolayer (one cell thick) in the culture tube. Once the cells are infected with virus (right), they undergo a characteristic cytopathic effect, becoming enlarged, granular in appearance, and aggregated into irregular clusters.
Viruses are obligate, intracellular parasites that require metabolically active cells for their
replication. Most can be cultivated in mammalian cell cultures, embryonated chicken eggs,
or laboratory animals, such as mice. In many clinical laboratories, cell culture has supplanted
the other systems for isolating most viruses. Unfortunately, a single, universal cell culture
suitable for the recovery of all viruses is not available. Because of this, several different cell
culture lines are used to optimize recovery of the viral agents most common in human disease.
These include Rhesus monkey kidney cells, rabbit kidney cells, human embryonic lung
cells (called WI-38 cells), and human epidermoid carcinoma cells of the larynx or lung, called
HEp-2 or A549 cells, respectively. These cell lines are cultivated in glass or plastic tubes or
flasks using specially formulated cell culture media. The cells adhere to the glass surface and
produce a confluent, single layer of growth known as a cell monolayer
(see fig. 30.1).
The ability of a virus to infect a particular cell line depends on the presence of
specific receptor sites on the cell membrane to which the virus can attach. Attachment is followed
by virus entry into the cell. The presence or absence of certain receptor sites on the
cell membrane surface determines the susceptibility or sensitivity of that particular cell line
to viral infection.
Once a virus infects a mammalian cell, it may induce certain morphologic
changes in the typical appearance of the cells, known as a cytopathic effect or CPE (see fig.
30.1). Some types of CPE caused by different viruses include generalized cell rounding, syncytia
formation (fusion of cells), and plaque formation (lysis of cells). Importantly, the type
of cell line infected and resultant CPE produced are extremely useful in providing the identity
of the particular virus isolated. The CPE may take from 1 to 25 days to develop, depending
on the virus isolated.
Certain groups of viruses, such as the influenza and parainfluenza viruses, may not
produce CPE when they infect cell cultures, and thus, cell monolayers infected with them appear
normal morphologically. A unique property of these viruses, however, is their ability to produce hemagglutinins, which are proteins projecting from the envelopes of the viruses and
present in the membranes of infected cells. Hemagglutinins have the ability to adhere to erythrocytes
in a process known as hemadsorption, which is used to screen certain cell cultures
for the presence of influenza and parainfluenza viruses. This test is performed by overlaying
the cell monolayer with a suspension of guinea pig erythrocytes, then examining for the presence
of hemadsorption after 30 minutes. Adherence of the guinea pig erythrocytes to the cell
monolayer is regarded as a positive test. Influenza and parainfluenza viruses are the most commonly
isolated hemadsorbing viruses, but mumps virus also gives a positive reaction.
Despite the availability of a large number of different cell culture lines, a number
of clinically important viruses cannot be grown using these conventional methods. The
Epstein-Barr virus (the cause of infectious mononucleosis) and human immunodeficiency
virus (the cause of AIDS) require human white blood cells for growth. Other viruses, such
as some coxsackie A viruses, rabies virus, and arboviruses are best isolated in mice. Because
of the highly specialized nature of these procedures, such methods are generally performed
only in reference laboratories. In addition, some viruses (e.g., hepatitis viruses and rotavirus)
cannot be cultivated at all. Alternative procedures such as electron microscopy, antigen detection
assays, or serology are used for the diagnosis of these viral infections.
Direct Specimen Assays
Immunologic assays, such as immunofluorescence and enzyme immunoassay, are used to detect
viral antigens, and nucleic acid amplification techniques are used to detect viral nucleic
acids directly in patient specimens. Antigen detection assays are available for
a number of different viruses including respiratory syncytial virus, herpes simplex virus, influenza
A and B viruses, rotavirus, and adenovirus. Currently, nucleic acid amplification assays
are limited to the detection of human papillomavirus although assays for quantifying
blood levels of viruses such as HIV are available. If viral products are detected, the laboratory
diagnosis of infection is established and the need to perform viral culture is eliminated.
Results are often available within 10 to 60 minutes.
The earliest nonculture laboratory method used for viral diagnosis was screening for characteristic
changes in infected human cells and tissues. Examination of cell smears or tissue sections
stained with special tissue stains may reveal characteristic viral inclusion bodies that represent
“footprints” of viral replication and are suggestive of certain viral infections. However,
the diagnostic value of such an approach is limited because sensitivity is low (50 to 70%)
compared with other available methods. The major application of this method is for the diagnosis
of infections caused by viruses such as molluscum contagiosum (the cause of genital
warts), which are not culturable. However, a gene amplification method is now commercially
available for detecting these viruses in clinical samples.
Electron microscopy is a powerful tool for the study of viral morphology and size but is of
limited availability in most diagnostic laboratories. Direct electron microscopy also requires
specimens containing high titers (≥107
per ml) of viral particles. The major diagnostic application
of electron microscopy is for the detection of certain nonculturable viruses, particularly
those that cause gastroenteritis (e.g., caliciviruses, astroviruses, and rotavirus).
Serological tests to identify patient’s antibodies are described in more detail in Serological Identification
of Patients’ Antibodies.
A variety of serological tests, however, are available for the diagnosis of many viral infections.
These involve the examination of two serum specimens (acute and convalescent sera spaced
at least 2–4 weeks apart) to detect a significant change in antibody titer. Serology is extremely
useful for the diagnosis of infections caused by the various hepatitis viruses.
Prion is a shorthand term for proteinaceous infectious particles. They are smaller in size than
viruses and are believed to contain neither DNA nor RNA. Prions cause slow neurodegenerative
diseases known as spongiform encephalopathies. They are classified as slow viral infections
because 20 to 30 years following exposure to the agent may elapse before symptoms
of infection develop in the patient. Creutzfeldt-Jakob disease and kuru are examples of human
In recent years, prions have attracted international scientific and public attention
due to the outbreak of bovine spongiform encephalopathy, also known as “mad cow disease,”
in Great Britain and some other European countries. Mad cow disease causes infection primarily
in cattle and sheep, but human infections can result from eating infected animal meat.
Prions are highly resistant to destruction and are not inactivated by thorough cooking of infected
animal products. No treatments are available for diseases caused by prions and the diseases
are universally fatal, with death usually occurring within one year of the onset of symptomatic
The diagnosis of prion infection is problematic. Currently, there is no clinical laboratory
method available to establish the diagnosis. Instead, diagnosis is based on clinical suspicion
confirmed by demonstrating characteristic spongiform changes (spongelike holes) in
histological sections of brain tissue, usually postmortem. Recent evidence indicates that these
spongiform changes may be seen also in more readily accessible tonsillar tissue.