23

The spirochetes
Amy M. Woron and A. Christian Whelen

CHAPTER OUTLINE KEY TERMS

Leptospira, 534 Chancre Rapid plasma reagin (RPR) test

General characteristics, 534 Endemic relapsing fever Relapsing fever
Virulence factors and pathogenicity, 534 Endemic syphilis Spirochetes
Infections caused by leptospires, 534 Epidemic relapsing fever Syphilis
Epidemiology, 535 Erythema migrans (EM) Venereal Disease Research
Laboratory diagnosis, 535 Gummas Laboratory (VDRL) test
Antimicrobial susceptibility, 536 Jarisch-Herxheimer reaction Weil disease
Borrelia, 536 Leptospirosis Yaws
Borrelia recurrentis and similar borreliae, 536 Lyme borreliosis Zoonoses
Borrelia burgdorferi sensu lato, 537 Pinta
Treponemes, 538
General characteristics, 538 Case in point
Clinically signicant species, 538
Treponema pallidum subsp. pallidum 538 A 29-year-old male patient arrived at a local medical clinic
Other treponemal diseases, 541 in Los Angeles complaining of diarrhea, fever, chills, muscle
aches, and headaches. He had returned 2 days earlier after
Bibliography, 542
competing in the Eco-Challenge in Malaysian Borneo. During
the competition, he completed various events, including
OBJECTIVES mountain biking, caving, climbing, jungle trekking, swim-
ming, and kayaking in freshwater and salt water. He was still
After reading and studying this chapter, you should be able to: recovering from multiple abrasions from the jungle trekking
1. Describe the general characteristics of the genera of spirochetes. and mountain biking. While kayaking on the Segama River,
2. Discuss the risk factors associated with relapsing fever infection. his kayak capsized, and he inadvertently swallowed several
3. Describe the pathogenesis and clinical manifestations of the borrelioses. mouthfuls of river water. His two teammates took doxycycline
as malaria prophylaxis before and during the race. Neither of
4. Compare the causative agents and arthropod vectors of relapsing
fever and Lyme borreliosis. them became ill.
5. Describe the laboratory diagnosis of relapsing fever and how it differs Issues to consider
from the diagnosis of other spirochetal diseases in the United States.
After reading the patient’s case history, consider:
6. Justify the use of the two-tiered approach to laboratory diagnosis of
Lyme borreliosis. • Risk factors for acquiring infectious disease for the patient
7. Compare the four human pathogens of the genus Treponema • Spirochete agents that cause inuenza-like illness and meth-
ods to identify or rule out those agents
8. Describe the clinical manifestations of the primary, secondary, and
• Effective prophylaxis, if available, for inuenza-like illness
tertiary stages of syphilis.
caused by spirochetes
9. Discuss the epidemiology of leptospirosis in the United States.
• Empiric therapy options
10. Evaluate the tests used to diagnose Treponema pallidum infections in the
clinical laboratory.
533
534 PART 2 23 The spirochetes

The class Spirochaetia contains four orders: Brachyspirales,

Brevinematales, Leptospirales, and Spirochaetales. The order
Leptospirales contains the family Leptospiraceae, and the
order Spirochaetales contains four families: Borreliaceae,
Sphaerochaetaceae, Spirochaetaceae, and Treponemataceae.
The family Leptospiraceae contains the genus Leptospira,
the family Borreliaceae contains the genus Borrelia, and the
family Treponemataceae contains the genus Treponema.
These three genera include the causative agents of important
human diseases, such as syphilis (sexually transmitted); lep-
tospirosis, a zoonoses (transmitted from animals to humans);
and Lyme borreliosis (or Lyme disease) and relapsing fever,
both of which are vector-borne diseases. There has been a
recent insurgence in primary and secondary syphilis as well
as the severe disease congenital syphilis in the United States.
Leptospirosis is likely a prevalent, underreported disease
found worldwide.
Spirochetes are slender, exuous, helically shaped, uni-
cellular bacteria ranging in size from 0.1 to 0.5 µm wide and
from 5 to 20 µm long, with one or more complete turns in the
helix. They differ from other bacteria in that they have a ex- Fig. 23.1 Dark-eld image of Leptospira interrogans serotype Sejroe Wolf 3705.
ible cell wall around which several brils are wound. These The tight coils and bent ends are characteristic of this organism (×1000). (Courtesy
brils, termed periplasmic agella (also known as axial brils, State Laboratories Division, Hawaii Department of Health.)
axial laments, endoagella, and periplasmic brils), are respon-
sible for motility. A multilayered outer sheath resembling
the outer membrane of gram-negative bacteria surrounds In contrast with both Treponema and Borrelia organisms, the
completely the protoplasmic cylinder (the cytoplasmic and spirals are very close together, so the organism may appear
nuclear regions are enclosed by the cytoplasmic membrane– to be a chain of cocci. One or both ends of the organism have
cell wall complex and periplasmic agella). The spirochetes hooks rather than tapering off. Their motion is rapid transla-
exhibit various types of motion in liquid media. tional (back and forth) and rotational.
Spirochetes are free living or survive in association with Electron microscopy reveals a long axial lament covered
animal and human hosts as normal biota or pathogens. In by a very ne sheath, like treponemes and borreliae. All spe-
addition, they can use carbohydrates, amino acids, long- cies have two periplasmic agella. The organisms cannot
chain fatty acids, or long-chain fatty alcohols as carbon and be readily stained, but they can be impregnated with silver
energy sources. Metabolism can be anaerobic, facultatively and visualized. Unstained cells are not visible by bright-eld
anaerobic, or aerobic, depending on the species. Treponema microscopy but are visible by dark-eld, phase-contrast, and
spp. reproduce via transverse ssion, whereas Leptospira and immunouorescent microscopy. Leptospires are obligate
Borrelia divide by the more common binary ssion. aerobes.

Virulence factors and pathogenicity

Leptospira
General characteristics
Historically, pathogenic leptospires were identied as
Leptospira interrogans, and environmental saprophytes were
named Leptospira biexa. Currently, the leptospires are divided
into serovars (serotypes). More than 200 different serovars
of L. interrogans sensu lato are recognized. Antigenically
related serovars are placed into serogroups, which have no
taxonomic standing but are useful for epidemiologic studies.
Genetic typing (16 S rRNA gene) based on nucleic acid simi-
larities has identied 23 genomospecies that include all of the
Leptospira serovars. Although genetic typing is taxonomically
correct, scientists and health care providers continue to prefer
serogroup-based nomenclature. Identication requires deter-
mining serovars by serotyping and determining species by
nucleic acid sequencing or matrix-assisted laser desorption/
ionization–time-of-ight (MALDI-TOF) mass spectrometry.
Organisms of the genus Leptospira are tightly coiled, thin,
exible spirochetes, 0.1 µm wide and 5 to 15 µm long (Fig. 23.1).
Leptospiral disease in the United States is caused by more
than 20 different serovars, the most common of which are
Icterohaemorrhagiae, Australis, and Canicola. Some sero-
vars of L. interrogans sensu lato and L. biexa sensu lato are
pathogenic for a wide range of wild and domestic animals
and humans, but the mechanisms of pathogenicity are not
well understood. Factors that may play a role in pathoge-
nicity include reduced phagocytosis in the host, a soluble
hemolysin produced by some virulent strains, cell-medi-
ated sensitivity to leptospiral antigen by the host, and small
amounts of endotoxins produced by some strains. The clin-
ical ndings in animals with leptospirosis suggest the pres-
ence of endotoxemia.
Infections caused by leptospires
Zoonotic leptospires contaminate water or mud when shed
in the urine of infected animals and often enter the human
host through small breaks in the skin or intact mucosa. The
initial sites of multiplication are unknown. Nonspecic
host defenses do not stop multiplication of leptospires, and

leptospiremia occurs during acute illness. Late manifesta-
tions of the disease can be caused by the host’s immunologic
response to the infection.
The incubation period of leptospirosis is usually 10 to
12 days but ranges from 3 to 30 days. Individuals often do
not seek treatment because the majority of cases are sub-
clinical or very mild. The clinical presentation is usually
abrupt, with nonspecic, inuenza-like symptoms, such
as fever, chills, headache, severe myalgia, and malaise. The
subsequent course is protean, frequently biphasic, and can
result in hepatic, renal, and central nervous system (CNS)
involvement. The major renal lesion is an interstitial nephri-
tis with associated glomerular swelling and hyperplasia that
does not affect the glomeruli. The most characteristic phys-
ical nding is conjunctival suffusion, but this is seen in less
than 50% of patients. Severe systemic disease (Weil disease)
occurs in about 5% to 10% of cases and includes renal fail-
ure, hepatic failure, and intravascular disease and can result
in death. The duration of the illness ranges from less than
1 week to 3 weeks. Late manifestations can be caused by the
host’s immunologic response to the infection. In patients with
a leptospiral bacteremia, immunoglobulin M (IgM) antibod-
ies are detected within 1 week after onset of disease and can
persist in high titers for many months. Immunoglobulin G
(IgG) antibodies are usually detectable 1 month or more after
infection. Convalescent serum contains protective antibodies.
Epidemiology
Leptospira can be free living or associated with colonization
of kidneys in animals. The most important hosts are small
mammals. Worldwide, leptospirosis is considered the most
common zoonosis, causing an estimated over 1 million cases
annually. In 2013, leptospirosis was reinstated as a notiable
disease in the United States. The Centers for Disease Control
and Prevention (CDC) estimates that 100 to 150 cases of lepto-
spirosis occur annually in the United States, mainly in Puerto
Rico, followed by Hawaii. Since most cases are believed to be
asymptomatic, cases likely remain unrecognized nationwide
and go unreported.
Leptospirosis is a zoonosis primarily associated with occu-
pational or recreational exposure. Working with animals or
in rat-infested surroundings poses hazards for veterinarians,
dairy workers, swine handlers, slaughterhouse workers, min-
ers, sewer workers, and sh and poultry processors. In the
United States, most cases of leptospirosis result from recre-
ational exposures.
In the natural host, leptospires live in the lumen of renal
tubules and are excreted in urine. Dogs, rats, and other
rodents are the principal animal reservoirs. Hosts acquire
infections directly by contact with the urine of carriers or
indirectly by contact with bodies of water contaminated with
the urine of carriers. Leptospires can survive in neutral or
slightly alkaline waters for months. Protective clothing (boots
and gloves) should be worn in situations involving possi-
ble occupational exposure to leptospires. Control measures
include rodent elimination and drainage of contaminated
waters. Vaccination of dogs and livestock has been effective
in preventing disease but not the initial infection and lepto-
spiruria. Short-term prophylaxis consisting of weekly doxy-
cycline therapy may be appropriate in high-risk groups with
expected occupational exposure.
Leptospira 535
Case check 23.1
Leptospires are present in water and mud contaminated by the
urine of reservoir animals. The Case in Point describes significant
and repeated exposure risk that should be reported to the primary
health care provider on presentation. Otherwise, the initial clinical
impression might resemble influenza, especially if presentation
occurs during periods of high influenza activity.
Laboratory diagnosis
Specimen collection and handling
Leptospiremia occurs during the acute phase (rst week) of
the disease, before symptoms are present. Toward the end
of the rst week as symptoms appear (rst 4 to 6 days of ill-
ness), blood or cerebrospinal uid (CSF) should be collected.
Optimal recovery occurs if fresh specimens are inoculated
directly into laboratory media. Urine can also be collected, but
the yield is much higher after the rst week of illness, and
shedding can occur intermittently for weeks. Ideal submission
is whole blood and serum during the rst week of acute illness
and serum with optional urine during convalescent illness.
Microscopic examination
Although direct demonstration of leptospires in clinical spec-
imens during the rst week of the disease by special stains,
dark-eld microscopy, or phase-contrast microscopy is pos-
sible, it is not recommended. Direct demonstration is suc-
cessful in only a small percentage of cases, and false-positive
results may be reported because of the presence of artifacts,
especially in urine.
Isolation and identication
Isolation of leptospires is accomplished by direct inoculation
of one to two drops of freshly drawn blood or CSF into labo-
ratory media, such as Fletcher semi-solid medium, Stuart liq-
uid medium, or Ellinghausen-McCullough-Johnson-Harris
(EMJH) semi-solid medium, and incubation of the media
in the dark at room temperature. Several dilutions of urine
should be used (undiluted, 1:10, and 1:100) and/or ltered
(0.45 µm) to minimize the effects of inhibitory substances.
Tubes are examined weekly for evidence of growth, such
as turbidity, haze, or a ring of growth. A drop taken from a
few millimeters below the surface is examined by dark-eld
microscopy for tightly coiled, rapidly motile spirochetes with
hooked ends. Serotypes have historically been identied by
microscopic agglutination testing using sera of dened reac-
tivity; however, 16 S ribosomal nucleic acid sequencing and
MALDI-TOF mass spectrometry have been shown to provide
accurate species identication.
Polymerase chain reaction (PCR) testing for leptospire
DNA in blood, plasma, serum, urine, CSF, and tissue is
as sensitive or more so than culture methods. PCR has the
advantage of providing a diagnosis in the acute stage, when
treatment is most benecial. Real-time PCR can also quantify
the infection burden.
536 PART 2 23 The spirochetes

Serologic tests
Most cases of leptospire infection are diagnosed by serology.
In patients with leptospiral bacteremia, IgM antibodies are
detected within 1 week after onset of disease and may per-
sist in high titers for many months. One month or more after
the onset of illness, IgG antibodies can be detected in some
patients. A visually read IgM enzyme-linked immunosorbent
assay (ImmunoDOT Leptospira IgM; GenBio, San Diego, CA)
has been approved by the U.S. Food and Drug Administration
(FDA) and has demonstrated good performance in cases of
acute leptospirosis. A macroscopic slide agglutination test for
rapid screening and the gold standard microscopic agglutina-
tion test are available for detection of leptospiral antibodies,
but both require maintenance of dened serotypes in culture,
so their use is typically limited to conrmatory laboratories.
Acute and convalescent samples are required to demonstrate
a fourfold rise in titer to conrm a diagnosis. Fig. 23.2 Appearance of Borrelia recurrentis (arrows) in blood (Giemsa stain,
×850).
Antimicrobial susceptibility
Susceptibility testing of leptospires is not normally per-
formed in the clinical laboratory; leptospires have been leptospires and treponemes, borreliae stain easily and can be
shown to be susceptible in vitro to streptomycin, tetracycline, visualized by bright-eld microscopy. Electron microscopy
doxycycline, and the macrolide antimicrobials. For severe shows the same general features as are seen with the trepo-
disease, intravenous penicillin and ceftriaxone are equally nemes—long, periplasmic agella (15 to 20 per cell) coated
effective. Doxycycline appears to shorten the course of the with sheaths of protoplasm and periplasm.
illness in adults and reduce the incidence of convalescent
leptospiruria.
Borrelia recurrentis and similar borreliae
Case check 23.2
Virulence factors
At least two deaths occurred in 2009, when confusion with
As the disease name suggests, relapsing fever is character-
pandemic influenza delayed appropriate antimicrobial therapy in
patients with severe leptospirosis. The Case in Point describes two ized by acute febrile episodes that subside spontaneously but
teammates who were taking doxycyline for malaria prophylaxis, tend to recur over a period of weeks. Borrelia spp. responsi-
which is also effective against many bacterial agents, including ble for this disease rst evade complement by acquiring and
Leptospira. Adherence to this preventive medicine regimen likely displaying suppressive complement regulators, C4b-binding
contributed to disease avoidance in these individuals. protein, and factor H. The relapses are potentiated by anti-
genic variation; borreliae systematically change their surface
antigens, thereby rendering specic antibody production
ineffective in completely clearing the organisms.
Borrelia
General characteristics Clinical manifestations
The genus Borrelia comprises several species of spiro- After an incubation period of 2 to 15 days, massive spirochete-
chetes that are morphologically similar but have different mia develops and remains at varying levels of severity during
pathogenic properties and host ranges. Some species cause the entire course of relapsing fever. The infection is accompa-
relapsing fever, whereas several other species cause Lyme nied by sudden high temperature, rigors, severe headache,
borreliosis. All pathogenic Borrelia are arthropod-borne. myalgia, arthralgia, and weakness. The febrile period lasts
Over 20 species are categorized as relapsing fever Borrelia that about 3 to 7 days and ends abruptly with the development of
include Borrelia recurrentis, Borrelia hermsii, and Borrelia dut- an adequate immune response. The disease recurs several days
tonii. The B. burgdorferi sensu lato complex, sometimes simply to weeks later, following a less severe but similar course. The
called B. burgdorferi, causes a spectrum of syndromes known febrile periods worsen during the spirochetemia and wane as
as Lyme borreliosis or Lyme disease. Over 20 species belong the immune response clears the bacteria from circulation.
to this group and include B. burgdorferi sensu stricto (often
referred to as B. burgdorferi), Borrelia afzelii, Borrelia bavariensis,
and Borrelia bissettiae
Borreliae are highly exible organisms ranging in thick-
Epidemiology
ness from 0.2 to 0.5 µm and in length from 3 to 20 µm. The spi- Relapsing fever can be tickborne (endemic relapsing fever)
rals range in number from 3 to 10 per cell and are much less or louseborne (epidemic relapsing fever). Tickborne borreliae
tightly coiled than those of the leptospires (Fig. 23.2). Unlike are transmitted by a large variety of soft ticks of the genus

Ornithodoros except for Borrelia miyamotoi, which is believed
to be transmitted by the larval hard tick Ixodes scapularis.
Tickborne borreliae are widely distributed throughout the
world, although specic species of borreliae tend to be limited
geographically by their vector. Transmission to a vertebrate
host takes place via infected saliva during tick attachment.
Louseborne fever, due to B. recurrentis, is transmitted
via the body louse, Pediculus humanus, and humans are the
only reservoir. Borreliae infect the hemolymph of the louse.
Unlike tickborne disease, transmission of the louseborne dis-
ease occurs when infected lice are crushed and scratched into
skin, rather than through the bite of an infected arthropod.
Relapsing fever is best prevented by controlling exposure to
the arthropod vectors. For tickborne relapsing fever, limiting
exposure to ticks includes wearing protective clothing, rodent
control, and the use of insect repellents. For louseborne
relapsing fever, control is best achieved by good personal
and public hygiene, especially improvements in measures to
avoid overcrowding and in delousing.
Laboratory diagnosis
Microscopic examination
Diagnosis of borreliosis is readily made by observing Giemsa-
or Wright-stained smears of peripheral blood taken during
the febrile period collected in ethylenediaminetetraacetic
acid (EDTA). Relapsing fever is the only spirochetal disease
in which the organisms are visible in blood with bright-eld
microscopy. The appearance of the spirochete among the red
blood cells is characteristic (see Fig. 23.2).
Isolation and identication
Whole blood collected in EDTA can also be used for cul-
ture and PCR testing, although citrate-treated blood might
be superior. Borreliae can be recovered by using Barbour-
Stoenner-Kelly medium, but it is rarely attempted. B. recur-
rentis, Borrelia hermsii, Borrelia parkeri, Borrelia turicatae, and
Borrelia hispanica have been successfully cultivated. Antigenic
variation in the spirochetes that cause relapsing fever makes
the serodiagnosis of their diseases difcult and impractical
with the possible exception of B. miyamotoi. Because the bor-
relia can be readily detected microscopically and by PCR,
serologic methods have limited use.
Antimicrobial susceptibility
Borreliae are susceptible to many antimicrobial agents; how-
ever, tetracyclines are the drugs of choice because they reduce
the relapse rate and rid the CNS of spirochetes. Up to 39% of
patients treated with antimicrobial agents experience fever,
chills, headache, and myalgia believed to be caused by the
sudden release of endotoxin from the spirochetes, a condition
referred to as Jarisch-Herxheimer reaction.
Borrelia burgdorferi sensu lato
Virulence factors
Bacterial spread may occur depending on the organism’s
ability to bind plasminogen and urokinase-type plasminogen
Leptospira 537
activator to its surface. This binding can convert plasminogen
to plasmin, which is a potent protease and could facilitate tis-
sue invasion. Binding factor H allows complement evasion
and immune system suppression and might explain, in part,
why IgM antibody titer does not peak for 3 to 6 weeks. In
vitro, the organism can stimulate proinammatory cytokines,
such as tumor necrosis factor and interferons, which can be
important in controlling disease but may also contribute to
inammatory manifestations as untreated disease progresses.
Clinical manifestations
Lyme borreliosis is a complex disease that can generally be
divided into three stages. Early infection includes two stages,
the rst of which is localized (stage 1). About 60% to 80%
of patients exhibit erythema migrans (EM), the classic skin
lesion that is normally found at the site of the tick bite, within
1 week of the tick bite. It begins as a red macule and expands
to form large annular erythema with partial central clearing,
sometimes described as having a target (“bull’s eye”) appear-
ance. EM often occurs alone but can be accompanied by fever,
fatigue, headache, mild stiff neck, arthralgia, or myalgia. Stage
2 is disseminated early and produces widely variable symp-
toms that include secondary skin lesions, migratory joint and
bone pain, alarming neurologic and cardiac disease, spleno-
megaly, and severe malaise and fatigue. Late manifestations,
or late persistent infections (stage 3), occur mainly in the car-
diac, musculoskeletal, and neurologic systems. Arthritis is the
most common symptom, occurring weeks to years later.
Epidemiology
Lyme borreliosis is the number one vector-borne disease in
the Northern Hemisphere. Organisms are transmitted via
the bite of infected Ixodes ticks, so most cases occur from
June to September, when more people are involved in out-
door activities and ticks are more active. Lyme disease was
rst described after an outbreak among children in Lyme,
Connecticut, in 1975. A total of 23,453 conrmed and 11,492
probable cases were reported in the United States in 2019. The
CDC estimates as many as 300,000 Americans contract Lyme
borreliosis annually.
At least three genospecies of B. burgdorferi sensu lato cause
Lyme borreliosis. B. burgdorferi, B. garinii, and B. afzelii are the
most common causes of Lyme borreliosis in the United States
and Europe. Protective clothing and repellents should be
used in areas in which tick exposure is intense. Attached ticks
should be removed immediately because pathogen transmis-
sion is associated with the length of attachment.
Laboratory diagnosis
Specimen collection and handling
The most common and productive specimen for the labora-
tory diagnosis of B. burgdorferi sensu lato infection is serum
for serology. Other tests have too many limitations (e.g., poly-
merase chain reaction) or have been inadequately validated
(e.g., urine and CSF antigen). Large-volume plasma cultures
have been shown to be positive in about 50% of adult patients
538 PART 2 23 The spirochetes

with erythema migrans. The paucity of bacteria present in

clinical samples renders direct microscopy insensitive.

Serologic tests
Diagnosis follows a two-tier approach in which the rst step
is a sensitive immunouorescence antibody (IFA), chemilu-
minescence immunoassay (CLIA), or enzyme immunoassay
(EIA) screening that detects IgM and IgG. If the test result
is negative, no further testing is needed. Reactive or equivo-
cal results are conrmed with separate IgM and IgG Western
immunoblots. If symptoms were present for 30 days or less,
the patient is tested for IgM only. Immunoblot conrma-
tion of IgM antibody presence includes reactivity for two of
the three following bands: 24, 39, and 41 kilodaltons (kDa).
Conrmation of IgG antibody presence is acceptable when 5
of the 10 scored bands are present: 18, 21, 28, 30, 39, 41, 45, 58,
66, and 93 kDa. Because of the low sensitivity of the immuno-
blot, two-tiered testing is insensitive, particularly in patients
with early disease.
To improve sensitivity, in 2019, the FDA cleared several
serologic tests that use EIA and CLIA rather than immuno-
blot in a modied two-test approach. These assays detect
antibodies to the proteins C6 and VlsE found in B. burgdorferi.
If serologic test results are negative and symptoms are consis-
tent with Lyme borreliosis, a convalescent serum should be
obtained and tested.
Antimicrobial susceptibility
Early diagnosis and antimicrobial treatment are important for
preventing neurologic, cardiac, and joint abnormalities that
can occur late in the disease. Doxycycline is the recommended
therapy, but macrolides and β-lactams have also been effec-
tive in treating early stages of Lyme disease without compli-
cations. For refractile or late stages, prolonged treatment with
ceftriaxone has been effective. Antimicrobial susceptibility
testing is not warranted.
Treponemes
General characteristics
Pathogenic treponemes are thin, spiral organisms about 0.1
to 0.2 µm thick and 6 to 20 µm in length. They are difcult to
visualize with a bright-eld microscope because they are very
thin, but they can be seen easily on dark-eld microscopy.
The spirals are regular and angular, with 4 to 14 spirals per
organism (Fig. 23.3). Three periplasmic agella are inserted
into each end of the cell. The ends are pointed and covered
with a sheath. The cells exhibit graceful exuous movements
in liquid.
Clinically signicant species
The genus Treponema comprises four microorganisms that are
pathogenic for humans: T. pallidum subsp. pallidum, the caus-
ative agent of syphilis; T. pallidum subsp. endemicum, the caus-
ative agent of endemic syphilis; T. pallidum subsp. pertenue,
the causative agent of yaws; and Treponema carateum, the
Fig. 23.3 Scanning electron micrograph of Treponema pallidum. Two treponemes
are shown adjacent to an erythrocyte (Nichols strain, ×2500).
causative agent of pinta. The four pathogenic strains exhibit a
high degree of DNA homology (>99%) and shared antigens.
At least six nonpathogenic species have been identied in the
normal microbiota, and they are particularly prominent in the
oral cavity. Some of these species, notably Treponema denticola,
have been linked to the polymicrobial infections gingivitis
and periodontitis.
Treponema pallidum subsp. pallidum
Virulence factors
Treponema pallidum subsp. pallidum can cross intact mucous
membranes and the placenta, disseminate throughout the
body, and infect almost any organ system. It has also been
postulated that antigenic variation of cell surface proteins
contributes to the organism’s ability to evade host immune
response and establish persistent infection.
Clinical manifestations
Treponema pallidum subsp. pallidum causes syphilis. The word
syphilis comes from a poem written in 1530 that described a
mythical shepherd named Syphilus, who was aficted with
the disease as punishment for cursing the gods. The poem
represented the compendium of knowledge at the time about
the disease.
Treponema pallidum subsp. pallidum transmission nor-
mally occurs during direct sexual contact with an individual
who has an active primary or secondary syphilitic lesion.
Consequently, the genital organs—the vagina and cervix in
females and the penis in males—are the usual sites of inocula-
tion. Syphilis can also be acquired by nongenital contact with
a lesion (e.g., on the lip) or transplacental transmission to a
fetus, resulting in congenital syphilis. After bacterial inva-
sion through a break in the epidermis or penetration through
intact mucous membranes, the natural course of syphilis can
be divided into primary, secondary, and tertiary stages based

on clinical manifestations. Co-infection with human immu-
nodeciency virus (HIV) can result in variation of the natural
course of the disease. Furthermore, ulcers caused by syphilis
might contribute to the efciency of HIV transmission in pop-
ulations with high rates of both infections. Syphilis has a wide
variety of clinical manifestations, which gave rise to the name
the “great imitator.”
Primary stage of syphilis
The three stages of syphilis are primary, secondary, and ter-
tiary. For reporting and surveillance purposes, the CDC uses
the case denition categories primary; secondary; early,
nonprimary nonsecondary; and unknown duration or late
latent. After inoculation, the spirochetes multiply rapidly and
disseminate to local lymph nodes and other organs via the
bloodstream. The primary mucocutaneous lesion develops 10
to 90 days after infection and is a result of an inammatory
response to the infection at the site of inoculation. The lesion,
known as a chancre, is typically a single erythematous lesion
that is nontender but rm, with a clean surface and raised bor-
der. The lesion is teeming with treponemes and is extremely
infectious. Because the chancre is commonly found on the
cervix or vaginal wall and is nontender, the lesion might not
be obvious in women. The lesion can also be found in the
anal canal of both sexes and remain undetected. The penis is
the usual location of chancres in men. No systemic signs or
symptoms are evident in the primary stages of the disease.
Secondary stage of syphilis
Approximately 4 to 10 weeks after development of the pri-
mary lesion, due to spirochete dissemination, the patient
may experience secondary disease, with clinical symptoms of
fever, sore throat, generalized lymphadenopathy, headache,
lesions of the mucous membranes, and rash. The rash can
present as macular, papular, follicular, papulosquamous, or
pustular and is unusual in that it can also occur on the palms
and soles. All secondary lesions of the skin and mucous mem-
branes are highly infectious. The secondary stage can last
for several weeks and can relapse. It might also be mild and
go unnoticed by the patient. Relapses of secondary syphilis
occur in about 25% of untreated patients.
Tertiary stage of syphilis
Following the secondary stage, patients enter latent syphi-
lis, when clinical manifestations are absent, and the patient
is not infectious. Latency within 1 year of infection is
referred to as early latent syphilis, whereas latency greater
than 1 year is late latent syphilis. Approximately one third
of untreated patients exhibit biological cure, losing sero-
logic reactivity. Another one third remain latent for life
but have reactive sera. The remaining one third ultimately
develop tertiary or late syphilis, generally decades later.
Symptoms of tertiary syphilis include the development of
granulomatous lesions (gummas) in skin, bones, and the
liver (benign tertiary syphilis), degenerative changes in the
CNS (neurosyphilis), and syphilitic cardiovascular lesions,
particularly aortitis, aneurysms, and aortic valve insuf-
ciency. Patients in the tertiary stage are usually not infec-
tious. In the United States and most developed countries,
the tertiary stage of disease is not often seen because most
patients are adequately treated with antimicrobial agents
before the tertiary stage is reached.
Treponemes 539
Congenital syphilis
Treponemes can be transmitted from an infected mother
to her fetus by crossing the placenta. Congenital syphilis
affects many body systems and is therefore severe and muti-
lating. Early-onset congenital syphilis (onset before 2 years
of age) resembles secondary syphilis in adults. It is charac-
terized by mucocutaneous lesions, osteochondritis, anemia,
hepatosplenomegaly, and CNS involvement, and it occurs
when mothers have early syphilis during pregnancy. Late-
onset congenital syphilis, corresponding to tertiary syphi-
lis in adults, results following pregnancies when mothers
have chronic, untreated infections. Symptoms of late-onset
congenital syphilis occur after 2 years of age but generally
are not apparent until the second decade of life. Symptoms
include interstitial keratitis, bone and tooth deformities, cra-
nial nerve VIII deafness, neurosyphilis, and other tertiary
manifestations.
Epidemiology
Under natural conditions, Treponema pallidum subsp. pallidum
is an exclusively human pathogen. Syphilis was rst recog-
nized in Europe at the end of the 15th century, when it reached
epidemic proportions. Two theories have been proposed con-
cerning the introduction of syphilis into Europe. One theory
suggests that Christopher Columbus’s crew brought the dis-
ease from the West Indies back to Europe. The second theory
suggests that the disease was endemic in Africa and trans-
ported to Europe via the migration of armies and civilians.
The venereal transmission of syphilis was not recognized
until the 18th century. The causative agent of syphilis was not
discovered until 1905.
The incidence of syphilis in the United States decreased
through the 1990s, and the fewest cases (31,618) since
reporting began in 1941 were reached in 2000. However,
since 2000, the incidence of the disease has been increasing
almost every year, including a 16.4% increase from 2018 to
2020. In 2020, 133,945 cases of all stages of syphilis were
reported, which included 41,655 cases of primary and sec-
ondary syphilis. From 2016 to 2022, cases of primary and
secondary syphilis per 100,000 increased from 15.5 to 20.8
in males and 1.9 to 4.7 in females. Men who have sex with
men account for the majority of reported primary and sec-
ondary syphilis cases (56.7% in 2019); however, heterosex-
ual syphilis is also increasing. It is up 58.2% among women
from 2018 to 2020. An increase in the rate among women
corresponds to an increase in the rate of congenital syph-
ilis. The rate of congenital syphilis increased from 8.4 per
100,000 live births in 2012 to 57.3 per 100,000 live births in
2020.
High-risk sexual behavior and co-infection with HIV con-
tinue to complicate syphilis control efforts. Educating peo-
ple about sexually transmitted diseases (STDs), including
providing information about the proper use of barrier con-
traceptives, reporting each case of syphilis to public health
authorities for contact investigation, and treating all sexual
contacts of persons infected with syphilis are cornerstones of
syphilis control efforts. Serologic screening of high-risk pop-
ulations should be performed, and to avoid congenital syphi-
lis, pregnant women should undergo serologic testing during
early and late pregnancy.
540 PART 2 23 The spirochetes
Laboratory diagnosis
Specimen collection and handling
Lesions of primary and secondary syphilis typically contain
large numbers of spirochetes. The surface of primary or sec-
ondary lesions is cleansed with saline and gently abraded
with dry, sterile gauze; induction of bleeding should be
avoided. Serous transudate is placed onto a slide, diluting it
with nonbactericidal saline if the preparation is thick. A cov-
erslip is added, and the slide is transported immediately to a
laboratory, where dark-eld microscopy is performed. Oral
lesions should not be examined because the numerous non-
pathogenic spirochetes present in these specimens will lead
to misinterpretation. Culture methods are not available, and
dark-eld microscopy equipment and expertise are uncom-
mon, so serology is the normal basis of diagnosis. Nucleic
acid amplication tests are not commonly used, and no FDA-
cleared commercial assay is available. Rabbit inoculation tests
are considered the gold standard, but they are not practical
for routine diagnosis.
Microscopic examination
The organisms are too thin to be observed by bright-eld
microscopy, so spirochetes are illuminated against a dark
background. Dark-eld microscopy requires considerable
skill and experience; however, demonstration of motile trepo-
nemes in material from the chancre is diagnostic for primary
syphilis.
Serologic tests
Serology is the primary method used for the laboratory diag-
nosis of syphilis. Because of the antigenic similarity between
the four Treponema strains pathogenic for humans, serologic
assays cannot distinguish them. Two major types of serologic
tests exist: nontreponemal and treponemal. Both have lower
sensitivities in the primary stage of syphilis, but approach
100% in the secondary stage. Treponemal tests retain a very
high sensitivity in the tertiary stage as well, whereas nontrepo-
nemal tests have a lower sensitivity. A co-infection with HIV
can result in false-negative serologic test results. Comparisons
between CSF and serum antibody responses can be helpful in
cases of neurosyphilis. With congenital syphilis, comparing
antibody responses in the mother’s serum and infant’s serum
can aid in the diagnosis.
The nontreponemal tests detect reaginic antibodies that
develop against lipids released from damaged cells. Although
they are biologically nonspecic and known to react with
organisms of other diseases and conditions (causing
false-positive reactions), the nontreponemal tests are excel-
lent screening tests. The antigen used is a precisely dened
cardiolipin-lecithin-cholesterol complex made from bovine
hearts. Nontreponemal tests have a lower specicity than
treponemal tests. Because a nontreponemal antigen is used
in these assays, they are prone to false-positive reactions. All
reactive results should be conrmed with a treponemal anti-
gen test.
The two nontreponemal tests widely used today are the
Venereal Disease Research Laboratory (VDRL) test and the
rapid plasma reagin (RPR) test. These tests are inexpensive to
perform, demonstrate rising and falling reagin titers, and cor-
relate with the clinical status of the patient. For the VDRL test,
cardiolipin antigen is mixed with the patient’s serum or CSF.
Flocculation is a positive reaction and is observed micro-
scopically. The RPR test is more commonly used; carbon par-
ticles are used to visually enhance the reaction and is read
on a white card macroscopically. When mixed with a posi-
tive serum on a disposable card, the black charcoal particles
clump together with the cardiolipin-antibody complexes. The
occulation is easily observed without a microscope. Reactive
or weakly reactive sera should undergo titration and be tested
with a treponemal test. Recently automated RPR testing has
gained FDA clearance. A third nontreponemal antigen test
occasionally used is the toluidine red unheated serum test. It
is similar to the RPR assay, except toluidine red is substituted
for charcoal.
The treponemal tests detect antibodies specic for trepo-
nemal antigens. Historically, they have been used to conrm
positive nontreponemal test results, although some labora-
tories use reverse sequence syphilis screening. In this strat-
egy, automated treponemal test–positive serum is retested
with a nontreponemal assay and a second treponemal assay.
This strategy resulted in higher numbers of false-positive
results in ve laboratories studied from 2006 to 2010, so
the CDC continues to recommend the original approach.
Treponemal tests are also helpful in the detection of late-stage
infections because the titers remain high and usually do not
drop in response to therapy, as with nontreponemal tests.
Consequently, treponemal tests are not useful in following
therapy or detecting reinfection.
The treponemal antigens used are spirochetes derived from
rabbit testicular lesions. Two commonly used treponemal
antigen test methods are the Treponema pallidum–particulate
agglutination (TP-PA) test (Fujirebio America, Faireld, NJ)
and EIAs. The TP-PA test uses gelatin particles sensitized with
T. pallidum antigens. Agglutination indicates the presence of
antitreponemal antibodies. EIA kits are simple to use, commer-
cially available, and comparable with other treponemal tests.
They also have the advantage of being semi-automated. The
majority use recombinant T. pallidum antigens and detect IgG,
IgM, or both. The uorescent treponemal antibody absorption
(FTA-ABS) assay uses a uorescent-labeled antihuman anti-
body that detects patient antitreponemal antibodies bound to
treponema afxed to a commercially prepared slide. Because
of subjectivity in reading the samples and the use of expensive
uorescent microscopy, the FTA-ABS test has become less fre-
quently used, and agglutination assays and EIAs are favored.
Neurosyphilis is diagnosed based on clinical and labora-
tory ndings. The VDRL test on CSF has high specicity but
low sensitivity. Because of plasma IgG entering the CSF, the
FTA-ABS assay on CSF has low specicity but high sensitivity
for neurosyphilis. Therefore a negative FTA-ABS assay result
is a strong indicator against neurosyphilis, but a negative
VDRL test result cannot rule out neurosyphilis.
Rapid immunochromatographic, point-of-care assays
using whole blood collected via nger stick have demon-
strated sensitivities and specicities like those of Treponema
antigen tests. Reactive samples need a nontreponemal anti-
gen titer test. The assays employ recombinant T. pallidum anti-
gens to detect IgG and IgM. They are useful in STD diagnosis
and antenatal clinics. Immunoblot assays are commercially
available; some use fractionated T. pallidum proteins, and
others use recombinant bacterial proteins. The assays can
differentiate between IgG and IgM if a separate anti–human
immunoglobulin antibody is used.
 Treponemes 541

Antimicrobial susceptibility POINTS TO REMEMBER

Antimicrobial susceptibility testing of T. pallidum is not avail-
• Spirochetes are slender, exuous, helically shaped, motile
able. Penicillin is the drug of choice for treating patients with
bacteria.
syphilis. It is the only proven therapy that has been widely used
• Leptospires are most likely to enter the human host through
for patients with neurosyphilis, congenital syphilis, and syphilis
small breaks in the skin or intact mucosa.
during pregnancy. Resistant strains have not developed. Long-
• The incubation period of leptospirosis is usually 10 to 12
acting penicillin, such as benzathine penicillin, is preferred.
days but ranges from 3 to 30 days. The onset of clinical
Alternative regimens for patients who are allergic to penicillin
illness is generally abrupt, with nonspecic, inuenza-like
and not pregnant include doxycycline and tetracycline. A typ-
constitutional symptoms, such as fever, chills, headache,
ical Jarisch-Herxheimer reaction and exacerbation of cutaneous
severe myalgia, and malaise.
lesions can occur within hours following treatment.
• The pathogenic borreliae are commonly arthropod-borne (by
a tick or louse) and cause relapsing fever and Lyme borrelio-
Other treponemal diseases sis (disease).
• B. recurrentis and similar species cause relapsing fever. The
Three nonvenereal treponemal diseases—yaws, pinta, and
relapses are caused by immune evasion, including antigenic
endemic syphilis—occur in different geographic locations.
variation. During a single infection, borreliae systematically
These treponematoses are found in developing countries
change their surface antigens.
where hygiene is poor, little clothing is worn, and direct skin
• During the febrile period, diagnosis of relapsing fever is
contact is common because of overcrowding. All three dis-
readily made by Giemsa or Wright staining of blood smears.
eases have primary and secondary stages, but tertiary man-
Relapsing fever is the only spirochetal disease in which the
ifestations are not always seen. All diseases respond well to
organisms are visible in blood with bright-eld microscopy.
penicillin or tetracycline. These infections are rarely transmit-
• Laboratory diagnosis of Lyme borreliosis caused by
ted by sexual contact, and congenital infections do not occur.
B. burgdorferi sensu lato is accomplished by two-tiered
serology. Initial reactive or equivocal EIA results are con-
rmed with an immunoblot or secondary EIA.
Yaws • Serologic assays are the most common method for diag-
nosing syphilis. However, these assays cannot distinguish
Yaws is a spirochetal disease caused by T. pertenue. It is endemic
among the four pathogenic treponemes.
in the humid, tropical belt, the tropical regions of Africa, parts
• Among adults, treponemes are transmitted by close, intimate
of South America, India, Indonesia, and many of the Pacic
contact. Treponemes can cross the placenta and be transmit-
Islands. It is not seen in the United States. The course of yaws
ted from an infected mother to her fetus. Congenital syphilis
resembles that of syphilis, but the early-stage lesions are ele-
affects many body systems and is therefore severe and muti-
vated, granulomatous nodules (hyperkeratoric papilloma).
lating. All pregnant women should have serologic testing for
syphilis early in pregnancy.

Endemic syphilis
Endemic syphilis (“bejel”) is caused by T. pallidum subsp. LEARNING ASSESSMENT QUESTIONS
endemicum and resembles yaws in clinical manifestations. It
is characterized by ulcerative lesions on the oropharyngeal 1. What are the general characteristics of spirochetes?
mucosa with split papules in the corners of the mouth. It is a. Slender (0.1 to 0.5 µm wide)
found in the Middle East and in the arid, hot areas of the world. b. Helix-shaped with one or more complete turns in the helix
The primary and secondary lesions are usually papules that c. Flexible cell wall around which several brils are wound
often go unnoticed. They can progress to gummas of the skin, d. All of the above
bones, and nasopharynx. Dark-eld microscopy is not useful 2. Which risk factor is not associated with Borrelia spp.
because of normal oral spirochetal biota. Poor hygienic condi- endemic relapsing fever?
tions are important in perpetuating these infections. Endemic a. Geographic location
syphilis is transmitted by direct contact or sharing contami- b. Louse infestation
nated eating utensils. c. Outdoor exposure
d. History of tick bites
3. Which tickborne spirochete is associated with a rash or skin
lesion?
Pinta a. Borrelia burgdorferi sensu lato
Pinta, caused by T. pallidum subsp. carateum, is found in the b. Treponema pallidum subsp. carateum
tropical regions of Central America and South America. It is c. Borrelia recurrentis
acquired by person-to-person contact and is rarely transmit- d. Leptospira interrogans sensu lato
ted through sexual intercourse. Lesions begin as scaling, pain- 4. What is the signicance on infectious disease transmission
less papules and are followed by an erythematous rash that of nding partially engorged ticks attached to skin?
becomes hypopigmented with time. The tertiary stage pres- a. Pathogen transmission is more probable the longer the
ents with hyperchromic, hypochromic, achromic, or dyschro- vector is attached.
mic plaques. It is unique among the pathogenic treponemes b. Pathogen transmission does not occur unless fully
in being limited to the skin. engorged.
542 PART 2 23 The spirochetes

c. More than one tick is required for disease transmission.

d. There is no signicance. BIBLIOGRAPHY
5. What is the typical test of choice for the laboratory diagno-
Aguero-Rosenfeld, M. E., & Stanek, G. (2019). Borrelia. In K. C. Carroll,
sis of relapsing fever? et al. (Eds.), Manual of clinical microbiology (12th ed., p. 1066).
a. Serology Washington, DC: ASM Press.
b. Dark-eld microscopy Centers for Disease Control and Prevention. (2011). Discordant results
c. Peripheral blood smear stained with Giemsa stain from reverse sequence syphilis screening—ve laboratories, United
States, 2006–2010. MMWR. Morbidity and Mortality Weekly Report,
d. Culture in Ellinghausen-McCullough-Johnson-Harris 60, 133. Available at: http://www.cdc.gov/mmwr/preview/
(EMJH) media mmwrhtml/mm6005a1.htm?s_cid=mm6005a1_w. (Accessed 1 May
6. A nonreactive result is obtained on a nontreponemal assay 2022).
performed on serum from a patient suspected of having Centers for Disease Control and Prevention. (2019). Lyme disease: Lyme
disease data tables. Last updated May 5, 2021. Available at: https://
syphilis. What should be the next step?
www.cdc.gov/lyme/stats/tables.html. (Accessed 29 April 2022).
a. Repeat the nontreponemal assay to conrm the nonreac- Centers for Disease Control and Prevention. (2021). Lyme disease: how
tive result. many people get Lyme disease? Available at: https://www.cdc.gov/
b. Perform a treponemal assay to conrm the nonreactive lyme/stats/humancases.html. (Accessed 1 May 2022).
result. Centers for Disease Control and Prevention. (2021). Sexually transmitted
infections treatment guidelines, 2021. Available at: https://www.cdc.
c. Request a second specimen in 1 week. gov/std/treatment-guidelines/default.htm. (Accessed 1 May
d. Report the result as nonreactive. 2022).
7. Which stage of syphilis is characterized by neurologic Centers for Disease Control and Prevention. (2021). Leptospirosis:
symptoms and a reactive treponemal antigen test? healthcare workers. Available at: https://www.cdc.gov/leptospiro-
sis/health_care_workers/index.html. (Accessed 1 May 2022).
a. Primary
Centers for Disease Control and Prevention. (2022). Sexually transmitted
b. Secondary disease surveillance, 2020. Available at: https://www.cdc.gov/std/
c. Tertiary statistics/2020/tables.htm. (Accessed 28 April 2022).
d. Latent Katz, A. R., et al. (2011). Leptospirosis in Hawaii, USA, 1999–2008.
8. Where are the most cases of leptospiroses contracted within Emerging Infectious Diseases, 17, 221. Available at: http://wwwnc.
cdc.gov/eid/article/17/2/10-1109_article.htm. (Accessed 1 May
the United States and its territories? 2022).
a. Florida Levett, P. N., & Galloway, R. L. (2019). Leptospira. In K. C. Carroll, et al.
b. California (Eds.), Manual of clinical microbiology (12th ed., p. 1058).
c. Virginia Washington, DC: ASM Press.
Lo, Y. -C., et al. (2011). Severe leptospirosis similar to pandemic (H1N1)
d. Puerto Rico
2009, Florida and Missouri, USA. Emerging Infectious Diseases, 17,
9. Leptospira are typically transmitted from natural hosts, 1145. Available at: http://wwwnc.cdc.gov/eid/arti-
dogs, and rodents to humans via which type of contact? cle/17/6/10-0980_article.htm. (Accessed 1 May 2022).
a. Animal bites Meri, T., et al. (2006). Relapsing fever spirochetes Borrelia recurrentis
b. Insect vectors and B. duttonii acquire complement regulators C4b-binding protein
and factor H. Infection and Immunity, 74, 4157.
c. Contact with animal feces Seña, A. C., et al. (2019). Treponema and Brachyspira, human host–associ-
d. Contact with water contaminated with urine ated spirochetes. In K. C. Carroll, et al. (Eds.), Manual of clinical
10. Which of the following are a treponemal antigen serologic microbiology (12th ed., p. 1083). Washington, DC: ASM Press.
test for syphilis? Select all that apply Sejvar, J. B., et al. (2003). Leptospirosis in “Eco-Challenge” athletes,
Malaysian Borneo, 2000. Emerging Infectious Diseases, 9, 702.
a. VDRL
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b. FTA cle.htm. (Accessed 1 May 2022).
c. RPR
d. TPPA
 24
Chlamydia, Rickettsia, and similar organisms
Donald C. Lehman

10. Compare the characteristics and pathogenesis of Ehrlichia spp. and

CHAPTER OUTLINE
Anaplasma phagocytophilum
11. Compare the characteristics of the Rickettsia and Coxiella and the
Chlamydiaceae, 544
diseases they cause.
General characteristics, 544
12. Justify the use of serologic assays to diagnose ehrlichiosis and
Chlamydia trachomatis, 544
anaplasmosis.
Chlamydia pneumoniae, 547
Chlamydia psittaci, 548
Rickettsiaceae and similar organisms, 552 KEY TERMS
Rickettsia, 552
Orientia, 554 Brill-Zinsser disease Lymphogranuloma venereum
Anaplasmataceae, 555 Bubo (LGV)
Coxiella, 556 Elementary body (EB) Morulae
Bibliography, 557 Human granulocytic Pelvic inammatory disease (PID)
anaplasmosis (HGA) Reiter syndrome
Human monocytic ehrlichiosis Reticulate body (RB)
OBJECTIVES
(HME) Trachoma
After reading and studying this chapter, you should be able to:
1. List the members of the family Chlamydiaceae. Case in point
2. Discuss the unique growth cycle of Chlamydia, describing elementary
A 7-day-old female neonate was brought by her grandmother
and reticulate bodies.
to the emergency department of a large city hospital. She had
3. Describe how the Chlamydia and Rickettsia differ from other bacteria been discharged 3 days after birth, with the last nursing note
and viruses.
indicating that the child was “fussy.” The newborn arrived at
4. Discuss the most important human diseases caused by the the emergency department with a temperature of 39° C, loss
Chlamydia, Rickettsia, and similar microorganisms. of appetite, profuse yellow discharge from the right eye, and
5. Describe the modes of transmission for each species of Chlamydia, general irritability. Medical history revealed the mother to be a
Rickettsia, and similar microorganisms. 17-year-old intravenous drug abuser with no prenatal care, who
6. Compare the epidemiology and pathogenesis of the serovars of had a vaginal delivery in the parking lot of a local hospital. Eye
Chlamydia trachomatis discharge and cell scrapings were cultured. Routine bacterial
7. Evaluate the available assays for the laboratory diagnosis of cultures were negative; however, a rapid nucleic acid amplica-
C. trachomatis and C. pneumoniae infections. tion test (NAAT) was diagnostic.
8. Discuss the problems with serologic cross-reactivity among the
rickettsial species. Issues to consider
9. For the following human rickettsial diseases, compare the causative After reading the patient’s case history, consider:
agents and mode of transmission to humans: • The various organisms that can be recovered from exudative
• Louseborne typhus material from newborns
• Rocky Mountain spotted fever • The clinical infections and disease spectrum associated with
• Scrub typhus these organisms

543
544 PART 2 24 Chlamydia, Rickettsia, and similar organisms
• How these organisms are transmitted and the risk factors
associated with the diseases produced
• The appropriate methods of laboratory diagnosis
This chapter covers obligate intracellular organisms that are
either extremely difcult to culture or are nonculturable.
Molecular biology assays are used to detect the more com-
monly seen human pathogens among this group. Their very
small size and obligate intracellular parasitism are major
characteristics that differentiate the organisms of the genera
Chlamydia, Rickettsia, Orientia, Anaplasma, and Ehrlichia from
other bacterial species.
The genus Chlamydia is the only genus in the family
Chlamydiaceae. A proposal to add the genus Chlamydophila
was ultimately rejected. The creation of a second genus was
somewhat controversial. Therefore readers may nd both tax-
onomic classications in published literature. Members of the
family share characteristics (Table 24.1) and have a unique life
cycle. Within the genus Chlamydia, four species were previ-
ously recognized: C. pecorum, C. pneumoniae, C. psittaci, and C.
trachomatis. Based on analysis of 16 S and 23 S ribosomal ribo-
nucleic acid (rRNA) gene sequences, the taxonomic classica-
tion was revised. The genus Chlamydia consists of the human
pathogens C. trachomatis, C. pneumoniae, and C. psittaci. Other
named species of Chlamydia exist, but they are rarely isolated
from humans.
The term rickettsiae can specically refer to the genus
Rickettsia, or it can refer to a group of organisms included in
the order Rickettsiales. There has been signicant reorgani-
zation in the order Rickettsiales in recent years. The order
includes the families Rickettsiaceae and Anaplasmataceae.
The family Rickettsiaceae includes the genera Rickettsia
and Orientia. The family Anaplasmataceae includes the
genera Ehrlichia, Anaplasma, Neorickettsia, and Wolbachia. As
a result of this reorganization, Coxiella has been removed
from the family Rickettsiaceae and placed into the family
Coxiellaceae
Chlamydiaceae
General characteristics
Currently, there are about 15 Chlamydia species. As shown
in Table 24.2, initial differentiation of the Chlamydia spp.
associated with human disease was based on selected char-
acteristics of the growth cycle, susceptibility to sulfa drugs,
accumulation of glycogen in inclusions, and deoxyribonu-
cleic acid (DNA) relatedness. Table 24.2 also lists additional
properties of the Chlamydiaceae species that have helped fur-
ther differentiate the three human species based on natural
host, major diseases, and number of antigenic variants (i.e.,
serovars).
Chlamydiae are decient in energy metabolism and are
therefore obligate intracellular parasites. Historically, it
was believed that the chlamydiae were strictly energy par-
asites. However, gene sequencing identied the presence
of enzymes for the metabolism of glucose 6-phosphate to
pyruvate via glycolysis, allowing the bacteria to generate
adenosine triphosphate (ATP) via substrate-level phos-
phorylation. The bacteria depend on the phosphorylated
sugar, d-glucose 6-phosphate, from the host cell. Although
chlamydiae do take up ATP from the host cell, they are able
to produce their own ATP from d-glucose 6-phosphate also
taken from the host cell. The tricarboxylic acid (Krebs) cycle
is incomplete in all Chlamydiaceae, and they are unable
to synthesize most amino acids, cofactors, and purine and
pyrimidine nucleotides.
Their unique growth cycle involves two distinct forms,
an elementary body (EB), which is infectious, and a reticu-
late body (RB), which is noninfectious. The EB has sporelike
features in that they are resistant to environmental physical
stress. It was believed that EBs are inert; however, recent
studies have demonstrated some metabolic activity without
cellular division. The growth cycle (Fig. 24.1) begins when
the small EB infects the host cell by inducing energy-requir-
ing active phagocytosis where they remain within mem-
brane-bound phagosome. The bacteria prevent interaction of
the phagosome with endosomes.
In vivo, host cells are primarily the nonciliated, columnar,
or transitional epithelial cells that line the conjunctiva, respi-
ratory tract, urogenital tract, and rectum. During the next
8 hours after cellular penetration, they organize into larger,
less dense RBs, which divert the host cell’s synthesizing func-
tions to their metabolic needs and begin to multiply by binary
ssion. About 24 hours after infection, the dividing organisms
begin reorganizing into infective EBs. At about 30 hours, mul-
tiplication ceases, and by 35 to 40 hours, the disrupted host
cell dies, releasing new EBs (Fig. 24.2) that can infect other
host cells, continuing the cycle.
The EB has an outer membrane like that of many gram-
negative bacteria. The most prominent component of this
membrane is the major outer membrane protein (MOMP).
The MOMP is a transmembrane protein that contains both
species-specic and subspecies-specic epitopes that can be
dened by monoclonal antibodies. The Chlamydia outer mem-
brane also contains lipopolysaccharide (LPS). This extractable
LPS is the primary antigen detectable in genus-specic sero-
logic assays.
Chlamydia trachomatis
C. trachomatis has been divided into two biovars: trachoma
and lymphogranuloma venereum. In addition, characteriza-
tion of the MOMP has separated C. trachomatis into 20 serovar-
iants, or serovars (Table 24.3). The trachoma biovar includes
serovars A to K. Serovars A, B, Ba, and C are associated with
the severe eye infection trachoma, whereas serovars D to K,
Da, Ia, and Ja are associated with inclusion conjunctivitis, a
milder eye infection, and urogenital infections. Serovars L1,
L2, L2a, L2b, and L3 are associated with lymphogranuloma
venereum (LGV), an invasive urogenital tract disease. The
serovars L are referred to as the biotype LGV, while serovars
A to K are called the trachoma biovar.
Most Chlamydia species carry a plasmid. Analysis of the
genus-wide plasmid produced three lineages and indicates
that all three evolved from a common ancestor. The plasmids’
high level of conservation across the genus implies a strong
evolutionary selection for their retention. The plasmid found
in C. trachomatis is important for virulence and establishing
persistent infections, but its role in pathogenesis is unknown.
NAATs, such as polymerase chain reaction (PCR), are cur-
rently the most commonly used methods to diagnose these
infections.
 Chlamydiaceae 545

Table 24.1 Comparative properties of microorganisms

Typical bacteria Chlamydiae Rickettsiae Mycoplasmas Viruses

DNA and RNA + + + + −
Obligate intracellular parasites − + + − +
Peptidoglycan in cell wall + + − − −
Growth on nonliving medium + − − + −
Contain ribosomes + + + + −
Sensitivity to antimicrobial agents + + + + −
Sensitivity to interferon − + − − +
Binary ssion (replication) + + + + −
+, Characteristic is present; −, characteristic is absent.

Table 24.2 Initial differentiation of Chlamydia species

Properties Chlamydia trachomatis Chlamydia pneumoniae Chlamydia psittaci
Inclusion morphology Round, vacuolar Round, dense Variable shape, dense
Glycogen in inclusions + − −
Elementary body morphology Round Pear-shaped Round
Sulfa drug sensitivity + − −
DNA relatedness (against C. pneumoniae) 10% 100% 10%
Natural hosts Humans Humans Birds, lower animals
Major human diseases Sexually transmitted Pneumonia Pneumonia
diseases Pharyngitis FUO
Trachoma Bronchitis
Lymphogranuloma
venereum
+, Characteristic is present; −, characteristic is absent; FUO, fever of unknown origin.

Clinical infections Lymphogranuloma venereum

Trachoma
C. trachomatis causes the chronic eye infection trachoma
(Fig. 24.3). Although cataracts is the leading cause of blind-
ness worldwide, an estimated 1.2 million people currently
have blindness caused by trachoma. An estimated 200
million people live in trachoma-endemic areas. In 1996, the
World Health Organization began the Alliance for the Global
Elimination of Trachoma by the year 2020 (GET2020). Despite
widespread success, trachoma is still the number-one cause of
preventable blindness in the world.
Trachoma is associated with serotypes A, B, Ba, and C.
These serovars are most frequently found near the equator
and in climates with high temperature and high humidity;
they are not commonly seen in the United States. These sero-
vars produce a chronic infection that, if left untreated, gener-
ally results in blindness in adults. Prevention and treatment
include antimicrobial therapy, facial cleanliness, environ-
mental improvement, and a simple surgical procedure on the
eyelid. Trachoma is a chronic disease that begins as follicular
conjunctivitis. The chronic inammation causes the eyelid to
turn inward, which results in continual abrasion to the cor-
nea from the eyelashes. The condition results in scarring and
ulceration of the cornea. This can result in secondary bacterial
infection and blindness.
C. trachomatis serovars L1, L2, L2a, L2b, and L3 cause LGV, a
sexually transmitted disease (STD); these serovars are more
invasive than the others. Patients with LGV present with
inguinal and anorectal symptoms (Fig. 24.4). Following an
incubation period of 1 to 4 weeks, patients develop a small
papule or lesion at the site of infection (e.g., penis, vaginal
wall, cervix). This is followed by swelling of the regional
lymph nodes. The serovars causing LGV can survive inside
mononuclear cells, and the bacteria enter the lymph nodes
and produce a strong inammatory response that often
results in uctuant bubo formation and subsequent rupture
of the lymph node. Proctitis is common in women because of
lymphatic spread of bacteria from the vagina or cervix. Men
can develop proctitis as a result of anal-receptive intercourse
or lymphatic spread from the urethra. The LGV serovars have
also been linked to Parinaud oculoglandular conjunctivitis.
Because LGV is no longer a reportable disease, its preva-
lence in the United States is unknown; however, it is believed
to be uncommon. LGV is usually seen in immigrants and
travelers returning from countries in which the disease is
endemic, typically the tropics and subtropics. Studies have
shown that men who have sex with men and individuals
who are infected with the human immunodeciency virus are
disproportionately affected with LGV.
546 PART 2 24 Chlamydia, Rickettsia, and similar organisms

Elementary body 0 hour

8 hours
Release

Reticulate
body
Phagocytosis
Reorganization
to reticulate bodies and
synthetic diversion

35 to 40 hours
Multiplication
Multiplication
cessation
Continued multiplication
and reorganization into
elementary
bodies

24 hours
30 hours

Fig. 24.1 Life cycle of Chlamydia

Table 24.3 Human diseases caused by Chlamydiaceae species

Species Serovarsa Disease Host
Chlamydia A, B, Ba, C Trachoma Humans
trachomatis D, Da, E, F, Inclusion conjunctivitis Humans
G, H, I, Ia, (adult and newborn)
J, Ja, K Nongonococcal urethritis
Cervicitis
Salpingitis
Pelvic inammatory
disease
Endometritis
Acute urethral syndrome
Proctitis
Epididymitis
Pneumonia of newborns
Perihepatitis (Fitz-Hugh–
Curtis syndrome)
Fig. 24.2 Elementary bodies and cells in a Chlamydia trachomatis–positive direct
L1, L2, Lymphogranuloma
specimen (×400). (Courtesy Syva Microtrak, Palo Alto, CA.)
L2a, L2b, venereum
L3
Chlamydia 1 Pneumonia, bronchitis Humans
Other urogenital diseases pneumo- Pharyngitis
niae Inuenza-like febrile
C. trachomatis causes urogenital infections in both women and
illness
men. Serovars D to K are associated with these clinical infections,
which can be persistent and subclinical as well as acute. The same Chlamydia 10 Psittacosis Birds
serovars can produce conjunctivitis in both males and females. psittaci Endocarditis
Abortion
Typical clinical manifestations of urogenital infection include
cervicitis, endometritis, salpingitis, proctitis in women and
a
Predominant serovars associated with disease.

Fig. 24.3 Conjunctival scarring and hyperendemic blindness caused by Chlamydia
trachomatis in ocular infections.
Fig. 24.4 Inguinal swelling and lymphatic drainage caused by Chlamydia tracho-
matis serovars L1, L2a, L2b, or L3 (i.e., lymphogranuloma venereum).
nongonococcal urethritis (NGU), epididymitis, prostatitis, and
proctitis in men. Pelvic inammatory disease (PID) and peri-
hepatitis are not uncommon complications in women. Between
45% and 68% of female partners of men with Chlamydia-positive
NGU yield chlamydial isolates from the cervix. Approximately
50% of current male partners of women with a cervical chlamyd-
ial infection are also infected. Most infected women (up to 80%)
and some men (approximately 25%) remain asymptomatic,
which facilitates spread of bacteria through unprotected sex-
ual contact. Salpingitis can lead to scarring and dysfunction of
the oviductal transport system, resulting in infertility or ectopic
pregnancy. In the United States, this is a signicant cause of ste-
rility. Reiter syndrome (urethritis, conjunctivitis, polyarthritis,
and mucocutaneous lesions), also known as reactive arthritis, is
believed to be caused by C. trachomatis
C. trachomatis is the most common sexually transmitted
bacterial pathogen and the most common notiable disease
in the United States. In 2020, a total of 1,579,885 cases were
reported, a decrease of 13% compared to 2019. This follows an
increase of 19.4% from 2014 to 2018. More worrisome is that
many infections remain undiagnosed, and the Centers for
Disease Control and Prevention (CDC) estimates that 2 mil-
lion to 3 million new cases occur annually in the United States.
It was reported in 2020 that 84% of all reported chlamydia
Chlamydiaceae 547
Table 24.4 Inclusion conjunctivitis in the neonate caused by
Chlamydia trachomatis
Characteristic Comments
Incubation period 4–5 days
Signs Edematous eyelids
Discharge Copious, yellow
Course Untreated, weeks to months
Complications Corneal panus formation, conjunctival
scarring
cases in females were among those 15 to 29 years of age. For
males, 71% of cases occurred in the same age group. Genital
warts, caused by human papillomavirus, is a more common
STD in the United States.
Chlamydial infection in the newborn
Because of the high incidence of chlamydia infection in
women of childbearing age, health professionals have sug-
gested routine screening of women who are pregnant for col-
onization. Newborns can be infected with C. trachomatis while
traveling through an infected birth canal. Chlamydial infec-
tion in a newborn delivered by cesarean section is rare, and
infection from seronegative mothers has not been reported.
Newborns with chlamydial infection can experience con-
junctivitis, nasopharyngeal infections, and pneumonia.
Table 24.4 shows selected features associated with neonatal
inclusion conjunctivitis. The portal of entry is ocular or aspi-
ration, with oropharynx colonization being a necessary event
before infection. Infants born in the United States receive pro-
phylactic eye drops, generally erythromycin, to prevent eye
infections by C. trachomatis and Neisseria gonorrhoeae
Clinically, it is believed that pneumonia in infants younger
than 6 months of age is associated with C. trachomatis, unless
proven otherwise. This pneumonia also can occur as a mixed
infection with gonococcus and cytomegalovirus and other
viruses. The incubation period for C. trachomatis pneumo-
nia is variable, but symptoms generally appear 2 to 3 weeks
after birth. Studies have suggested that some cases of sudden
infant death syndrome might be caused by C. trachomatis
Case check 24.1
In the Case in Point, the neonate presented with conjunctivitis and
symptoms of pneumonia. The signs and symptoms along with the
neonate’s history are suggestive of C. trachomatis infection.
Chlamydia pneumoniae
Chlamydia pneumoniae, formerly known as Chlamydia sp. strain
TWAR, was originally identied in 1965 from a conjunctival
culture of a child (TW) enrolled in a Taiwan trachoma vac-
cine study. In 1983 at the University of Washington, a simi-
lar organism was isolated in HeLa cells from a pharyngeal
specimen of a college student (AR). To date, only a single C.
pneumoniae serovar has been found.
C. pneumoniae is recognized as an important respiratory
pathogen thought to be transmitted in human secretions. It is
known to be a cause of sinusitis, pharyngitis, acute respiratory
548 PART 2 24 Chlamydia, Rickettsia, and similar organisms

Table 24.5 Summary of key epidemiologic and clinical features of Chlamydia pneumoniae infections
Epidemiologic
Almost no antibody detectable before 5 years of age
Antibodies present in >50% of adults
Attack rate highest between 6 and ≈25 years of age, often
focusing on college-age students
No seasonal incidence; epidemics have been reported
every 4–6 years
Reinfection common
Clinical
Estimated to account for approximately 6%–10% of outpatient and hospitalized
pneumonia; 90% of infections are asymptomatic or mildly symptomatic
Biphasic illness—prolonged sore throat, crouplike hoarseness, followed by
lower respiratory tract (ulike) symptoms
Pneumonia and bronchitis, rarely accompanied by sinusitis
Fever relatively uncommon
Chest radiograph shows isolated pneumonitis
One in nine infections results in pneumonia.
Sarcoidosis, cardiovascular relationships (?)

Table 24.6 Evaluating for Chlamydia pneumoniae

Population or situation Evaluation methods Comments
Pneumonias requiring hospitalization C. pneumoniae–specic IgM and 12% antibody prevalence
(age 6–20 years) IgG: acute and convalescent, use
Pharyngitis in college students MIF IgM, single visit 9% antibody prevalence
Retrospective, undiagnosed outbreaks in young CF or MIF, IgG specic
adults, college students, or military trainees
Serious pneumonia, undiagnosed; clinically C. pneumoniae–specic IgM and If negative, rather than perform cultures for similar
presents like Mycoplasma pneumoniae IgG by MIF respiratory pathogens (i.e., Mycoplasma pneumo-
niae), reevaluate the patient to determine whether
additional testing is necessary
CF, Complement xation; IgG, immunoglobulin G; IgM, immunoglobulin M; MIF, microimmunouorescence.

disease, bronchitis, and pneumonia. It also has been isolated
from patients with otitis media with effusion, pneumonia
with pleural effusion, and aseptic pharyngitis. C. pneumoniae
can cause a mild atypical pneumonia resembling pneumonia
caused by Mycoplasma pneumoniae. Probably 90% of infections
are asymptomatic or mildly symptomatic, causing a chronic
cough and malaise. Hospitalization is rare. Unlike viral respi-
ratory diseases, there seems to be no seasonal incidence.
Reinfection with C. pneumoniae appears to be common and
can be milder or more severe than the initial infection. The
epidemiologic and clinical features of C. pneumoniae are listed
in Table 24.5. The mode of transmission, incubation period,
and infectiousness of C. pneumoniae infections are still largely
unknown. No animal reservoir or vector is known. Table 24.6
summarizes situations and/or populations at risk that would
benet from the detection of C. pneumoniae, usually by sero-
logic methods.
The prevalence of C. pneumoniae infection is controversial,
but infections are thought to be common, with an estimated
200,000 cases per year in the United States. C. pneumoniae is
regarded as the third most common cause of infectious respi-
ratory disease. It accounts for up to 10% to 15% of community-
acquired cases of pneumonia. However, a meta-analysis of
63 research articles published between 1995 and 2019 indi-
cated that C. pneumoniae caused a low number of commu-
nity-acquired pneumonia cases. In PCR studies, the rate of
identication of C. pneumoniae as a possible pathogen ranged
from 0% to 8.95%. Supporting the fact that many infections
are asymptomatic or mild, antibodies were demonstrated in
50% to 70% of adults in some populations. It is thought that
the attack rate is highest between 6 and 20 years of age, with a
particular emphasis in college-age students. Outbreaks have
been reported in schools, prisons, and the military.
The clinical picture in college-age students, although it
may vary, has a biphasic clinical course. C. pneumoniae infec-
tion results in prolonged sore throat (5–7 days) and hoarse-
ness, followed by ulike lower respiratory tract symptoms
(8–15 days). Because of its striking clinical similarity to bac-
terial pharyngitis, the result of a streptococcal antigen test
often is thought to produce false-negative results. The sec-
ond phase of the biphasic illness often results in pneumonia
(approximately one in nine infections) and bronchitis but is
rarely accompanied by sinusitis. Fever is relatively uncom-
mon, and x-rays show isolated pneumonitis.
C. pneumoniae has been implicated as a possible factor in
asthma and cardiovascular disease. The organism has been
detected in atherosclerotic tissue by culture and NAATs; how-
ever, its possible pathogenic role remains under investigation.
Association of this organism with other vascular diseases, such
as abdominal aortic aneurysm, has also been considered.
Chlamydia psittaci
Chlamydia psittaci is the cause of psittacosis (psittakos is the
Greek word for parrot) among psittacine birds, also known
as ornithosis or parrot fever. Many avian species can harbor
C. psittaci. DNA sequencing led to the naming of new spe-
cies that were formerly considered C. psittaci. They include
Chlamydia felis, Chlamydia caviae, and Chlamydia abortus
Inhalation of C. psittaci bacteria following contact with
poultry is the greatest risk for infection. Person-to-person
spread seems unlikely. The bacteria enter the respiratory tract
and can spread to the reticuloendothelial cells of the liver
and spleen. A lymphocytic inammatory response occurs in
the alveolar and interstitial spaces that can produce edema,
necrosis, and bleeding in the lungs. Human infection can
 Chlamydiaceae 549

Table 24.7 Appropriate Chlamydia trachomatis assays for selected patient populations
Assay Patient population
Prenatal Newborn Clinicsa Legal applicability Test of
Low High Eye Throat Plasma Low High (rape or child abuse?) cure
risk risk risk risk
Culture A/B A B A — A/B B Yes Yes
Nonculture, nonamplied
DFA B B A B — B B No No
EIA B B B B — B B No No
OIA — — — — — B B No No
Nonculture, amplied
PCR, SDA, IUO IUO IUO IUO — A A Yes No
TMA
Serology
CF B, LGV B, LGV — — — B, LGV B, LGV No No
EIA B B — — B B B NA No
MIF NA B — — A B B No No
(IgM)
A, Most useful, stands alone; B, probable, but needs verication or complementary assay recognizing different Chlamydia trachomatis macromolecules, i.e., lipopolysac-
charide (enzyme immunoassay [EIA]) versus major outer membrane protein (direct uorescent antibody [DFA]) or competition assay for DNA probes; CFI, complement
xation; IgM, immunoglobulin M; IUO, investigational use only; LGV, lymphogranuloma venereum; MIF, microimmunouorescence; NA, not available; OIA, optical immuno-
assay; PCR, polymerase chain reaction; SDA, strand displacement amplication; TMA, transcription-mediated amplication.
a
A low-risk population is dened as one with a less than 5% incidence, such as in an obstetrics-gynecology or family practice patient group (e.g., birth control, annual gyne-
cologic examination). A high-risk population is dened as one with a more than 10% incidence, such as those in sexually transmitted disease clinics, those in university or
college student health centers, and emergency department patients.

be asymptomatic or mild, or it can present as a severe, even
life-threatening pneumonia. Symptoms include fever, chills,
muscle aches, and severe headache. In the United States, fewer
than 50 cases of C. psittaci are reported annually, although the
number of cases might be underreported because infection
can be mild. Most cases are associated with occupational
exposure by breeding pet birds and working in poultry pro-
cessing plants, particularly turkey processing plants.
Laboratory diagnosis of chlamydial diseases
There are numerous methods for the laboratory diagnosis of
C. trachomatis that differ in sensitivity, specicity, negative
predictive value (NPV), and positive predictive value (PPV).
Table 24.7 identies the situations in which the tests may be
most applicable and identies the population groups at great-
est risk. Table 24.8 provides the predictive values for isolation,
detection, and identication methods. The most appropriate
tests or combinations of assays used depend on the following
factors:
• Knowledge of the population at risk
• Capability and facilities available for testing
• Cost of assays
• Ability to batch specimen types
• Experience of the laboratory scientist
Prevalence in the population to be tested is an important
criterion in determining which method or combination of
methods should be used. For any assay, the PPV increases
(assuming optimal technical conditions) when the prevalence
of the disease in the population is high. The type of specimen
selected for laboratory processing depends on the symptoms
of the patient and the clinical presentation. Regardless of the
source, however, the specimen should consist of infected epi-
thelial cells and not exudate. First morning voided urine for
men and vaginal swab specimens for women are excellent
for detecting infection. Urine is an acceptable alternative for
women.
Dacron, cotton, and calcium alginate swabs can be used,
but it should be noted that toxicity has been associated
with different lots of each, which is a concern if culture is
attempted. Furthermore, it is important to remember that
swabs with plastic or metal shafts are superior to those with
wooden shafts, which are toxic to cells. Table 24.9 lists the
optimal specimens for detection of Chlamydia spp. in patients
with a variety of clinical manifestations. Specimens for cul-
ture must be placed into a transport medium, such as sucrose
phosphate glutamate buffer, and transported to the labora-
tory at 4° C or below within 24 hours. Because molecular biol-
ogy assays are widely available, cultures are rarely attempted
in clinical laboratories.
Specimens collected for the detection of C. pneumoniae
include nasopharyngeal and throat swabs, sputum, bronchial
lavage uid, and tracheal aspirates (see Table 24.9). C. psittaci
is most often diagnosed based on the clinical presentation,
history of bird exposure, and serologic assays.
Direct microscopic examination
Direct specimen examination by cytologic methods pri-
marily involves trachoma and inclusion conjunctivitis
(Fig. 24.5). This method is technically demanding and inu-
enced by the quality of the specimen and expertise of the lab-
oratory scientist. Although this method is difcult to use with
550 PART 2 24 Chlamydia, Rickettsia, and similar organisms

Table 24.8 Detection capabilities of various methods for Chlamydia trachomatis

SENSa SPECa PPV a NPV a Specimen site False Reported Comments
(%) (%) (%) (%) Cervical- Rectal Urine Eye ± cross-reactivity
urethral
Culture 50–85 100 73–98 90–100 + + + + False None Labor-intensive
gold standard for
specicity
Nonculture, nonamplied
DFA 75–85 92–98 73–98 95–99 + + − + False Staphylococci Screen only;
± experience in FA
needed
EIA 72–95 90–99 45–92 95–99 + − LA LA False Streptococci, Verify with com-
± GC, plementary assay
Acinetobacter
Nonculture, amplied
PCR, 85–95 99–100 85–95 100 + − + False None reported No verication
SDA, necessary
TMA
a
Range—low to high prevalence as described in the text.
DFA, Direct uorescent antibody; EIA, enzyme immunoassay; FA, uorescent antibody; LA, limited availability; NPV, negative predictive value; PCR, polymerase chain reac-
tion; PPV, positive predictive value; SDA, strand displacement amplication; SENS, sensitivity; SPEC, specicity; TMA, transcription-mediated amplication.

Table 24.9 Appropriate specimens for detection of Chlamydial infections

Clinical manifestation, site of Specimen site, type Comments
infection
Inclusion conjunctivitis and trachoma Conjunctival swab, scraping with spatula Specimen collection in neonates is difcult
Urethritis Urethral swab In males, insert swab >4 cm; do not use
discharge
Epididymitis Epididymis aspirate
Cervicitis Endocervical swab Remove exudate rst
Salpingitis Fallopian tube (lumen) or biopsy
Sexually transmitted disease, result Rectal, vaginal swabs May be used for supplemental information and
clarication in clarifying previous isolates or diagnostic
dilemmas
Sexually transmitted disease, male Urine Noninvasive diagnostic procedure;
sex partner EIA antigen detection is 80% accurate, PCR is
98% accurate
Lymphogranuloma venereum Bubo or lymph node aspirate
Infant pneumonia Throat swab, nasopharyngeal aspirate, or lung
tissue
Chlamydia pneumoniae pneumonia Throat washing, throat swab, sputum, bron- Tissue culture isolation and direct immunou-
or pharyngitis chial lavage uid, lung tissue orescence are relatively new and need further
evaluation
Psittacosis Sputum, bronchial lavage uid, pleural uid,
lung tissue
EIA, Enzyme immunoassay; PCR, polymerase chain reaction.

large numbers of specimens, it does offer rapidity in selected
cases, particularly in detecting ocular infection in newborns.
Direct uorescent antibody (DFA) testing should not be used
routinely for genital tract specimens. It is, however, appropri-
ate for rapid diagnosis of inclusion conjunctivitis in infants and
sometimes adults. The assay requires an experienced micros-
copist and is labor-intensive. The sensitivity is 75% to 85%, and
cross-reactivity to other bacteria has been reported. The assay
was developed in the 1980s and uses a species-specic epitope
on the MOMP. Direct specimen examination offers one addi-
tional important advantage—it allows immediate quality con-
trol of the specimen, revealing whether columnar epithelial cells
are present. Fig. 24.6 shows inclusion bodies demonstrated by
direct examination of cytologic stains of endocervical smears.
An indirect uorescent antibody method has been reported for
detecting C. pneumoniae in respiratory secretions; the antibody

reacts with the MOMP (Fig. 24.7). This same antibody can be
used to identify infected cell culture monolayers.
Immunoassays
Direct enzyme immunoassays (EIAs) use monoclonal or
polyclonal antibodies directed against the chlamydial LPS.
Several commercial kits are available for C. trachomatis. They
can screen large numbers of specimens, obtain objective
results, obtain results in 3 to 5 hours, and use various spec-
imen types. However, none of them equals the sensitivity
of culture or NAATs, and many are prone to false-positive
results. Because of these limitations, the CDC considers EIAs
substandard for the detection of C. trachomatis, and they are
not recommended for diagnosis.
Cell culture
Until the development of NAATs, chlamydial cell culture
was considered the gold standard for detecting C. trachoma-
tis infection; however, the usefulness of cell culture has been
limited because of the inherent technical complexity, time and
specimen handling requirements, expense, biosafety hazard
Fig. 24.5 Inclusion body from an ocular swab from a 7-day-old newborn who was
discharged but then readmitted with fever, weight loss, lack of eating, and “fussi-
ness.” At 3 days after delivery, Neisseria gonorrhoeae was isolated from the ocular
discharge, although the patient had been given silver nitrate eye drops. Eye cultures
conrmed the presence of Chlamydia trachomatis (Giemsa stain, ×600).
A B
Fig. 24.6 Cytologic examination of endocervical specimens demonstrating inclusion bodies consistent with Chlamydia trachomatis (Papanicolaou stain; A, ×600;
B, ×600).
Chlamydiaceae 551
concerns, and labile nature of the organisms. Even under
the most stringent and optimal conditions, isolation of chla-
mydiae is only approximately 80% sensitive. C. pneumoniae
can also be recovered in cell cultures. Isolation of C. psittaci in
culture, although diagnostic, is difcult, dangerous, and not
routinely used or recommended.
Fluorescein-labeled monoclonal antibodies can be used
to detect the chlamydial inclusions found associated with
infected cell lines. Several uorescent antibodies are avail-
able commercially. Some laboratories use species-specic
monoclonal antibodies that bind to the MOMP, whereas
others prefer the family-specic antibody, which binds
to an LPS component. Monoclonal antibodies against the
MOMP are reported to offer the brightest uorescence,
with consistent bacterial morphology and less nonspe-
cic staining than monoclonal antibodies against the LPS.
Iodine or Giemsa stain can be used, but these methods are
less sensitive and specic and are no longer recommended
(see Fig. 24.7).
Nucleic acid hybridization and amplication assays
Because of high sensitivity and specicity, the NAATs are the
preferred method for the diagnosis of C. trachomatis infection.
NAATs detect 30% to 50% more C. trachomatis infections than
culture or earlier nonculture assays. NAATs offer several
advantages, including U.S. Food and Drug Administration
(FDA) approval to detect C. trachomatis in endocervical swabs
from women, urethral swabs from men, and urine from men
and women. These assays can have the added advantage of
detecting two STDs in one sample—gonorrhea and C. tracho-
matis infection. The sensitivity, specicity, NPV, and PPV are
higher than those reported for EIAs and cultures. Results can
be obtained quickly, and testing is less technically demand-
ing than culture. However, currently no NAAT has been
approved for use on conjunctival, oropharyngeal, or rectal
specimens. The assays also cannot distinguish LGV strains
from other C. trachomatis serotypes (A to K).
Five NAATs are currently licensed for use in the United
States for the detection of C. trachomatis in clinical specimens:
the PCR-based Cobas CT/NG (Roche Molecular Systems,
Indianapolis, IN); the GenXpert CT/NG (Cepheid, Sunnyvale,
CA); the Abbott RealTime m2000 CT/NG (Abbott Molecular,
Des Plaines, IL); the APTIMA Combo 2 CT/GC transcrip-
tion-mediated amplication assay (Hologic, San Diego, CA);
552 PART 2 24 Chlamydia, Rickettsia, and similar organisms
Fig. 24.7 Chlamydia pneumoniae detection from a direct sputum smear using a u-
orescent-labeled monoclonal antibody, highlighting cytoplasmic inclusion (×400).
(Courtesy DAKO Reagents, Carpinteria, CA.)
and the BD ProbeTec ET strand displacement amplication
(BD Diagnostic Systems, Sparks, MD). Although commercial
tests differ in their amplication methods and target nucleic
acid sequences, the increased sensitivity of NAATs is ascribed
to their ability to produce positive signals from as little as
a single copy of the target DNA and do not require viable
organisms. The assays target sequences on the plasmid, and
all ve have some degree of automation to facilitate testing
and can provide results in the same day. Two NAATs have
FDA approval for detecting C. pneumoniae; unfortunately, nei-
ther method has been thoroughly evaluated for diagnosing
infections. Currently, no commercially available NAATs are
approved for diagnosing C. psittaci infections.
Case check 24.2
In the Case in Point, the diagnosis of C. trachomatis infection was
confirmed by a NAAT. These assays are generally rapid and highly
sensitive and specific. Currently, five FDA-approved commercial
NAATs are available; in-house tests can be used if they have been
properly validated.
Antibody detection
Serologic assays have little value in the detection of C. tra-
chomatis infections. Many individuals have chlamydial anti-
bodies from previous infections, and because chlamydial
infections tend to be chronic, it is difcult to demonstrate
the traditional fourfold rise in C. trachomatis antibody titer
between acute and convalescent stage specimens. Serologic
testing of uncomplicated genital infections and screening of
asymptomatic individuals is not recommended. Infections by
the LGV strains are more invasive and therefore more likely to
produce a detectable systemic antibody titer. Serologic testing
in these patients can support a clinical diagnosis. Detection of
high levels of IgM directed against C. trachomatis in neonates
can be diagnostic of pneumonia.
Also because of high antibody levels in adults, serologic
testing has limited value in diagnosing C. pneumoniae infec-
tions. However, human psittacosis diagnosis is often made
by demonstrating a fourfold rise in antibody titer. A single
antibody titer greater than 1:32 is suggestive of acute illness
in a symptomatic patient during an outbreak of psittacosis.
The increase in the levels of antibodies is usually not demon-
strable until the acute illness is over, however, and it is often
weak or absent if appropriate antimicrobial therapy is given.
The microimmunouorescence (MIF) assay has been
the most widely used serologic test for the chlamydiae.
Demonstration of high IgG titers can help the diagnosis of
LGV, epididymitis, ectopic pregnancy, and human psittaco-
sis. Commercial reagents for these assays are available. The
MIF assay is considered the method of choice for detecting
antibodies to C. trachomatis. EIA tests are also commercially
available, but they have not been sufciently evaluated to rec-
ommend their routine use.
Rickettsiaceae and similar organisms
The genera Rickettsia and Orientia are the only two genera
in the family Rickettsiaceae. Most members of the rickettsial
group are arthropod-borne, obligate intracellular pathogens
that can grow only in the cytoplasm of host cells. These bac-
teria are extremely well adapted to their arthropod hosts. The
primary hosts usually have minimal or no disease from their
rickettsial infection. The arthropod host allows rickettsiae
to persist in nature in two ways. First, rickettsiae are passed
through new generations of arthropods by transovarial trans-
mission. Because of this mechanism, arthropods are not only
vectors for rickettsioses but also reservoirs. Second, arthropods
directly inoculate new hosts with rickettsiae during feeding.
An exception to this pattern occurs with Rickettsia prowazekii.
In this case, the arthropod vector, the body louse, can die of
the rickettsial infection, and humans act as a natural reservoir.
Rickettsia
Rickettsiae are short, nonmotile, gram-negative bacilli about
0.8 to 2.0 µm × 0.3 to 0.5 µm in size. The members of the genus
Rickettsia have not been grown in cell-free media but have been
grown in the yolk sacs of embryonated eggs and several cell
lines. They lack enzymes for sugar metabolism and amnio
acid, nucleotide, and lipid synthesis. However, they can pro-
duce small amounts of ATP via the tricarboxylic acid pathway.
Nutritional building blocks (e.g., amino acids) and ATP using
an ATP/ADP translocase are acquired from host cells.
At least 30 species of Rickettsia are recognized, and most
are divided into three groups according to the types of clini-
cal infections they produce and phylogenetic clustering. The
typhus group contains only two species: R. prowazekii and R.
typhi. The spotted fever group includes several species gen-
erally recognized as human pathogens, such as R. rickettsii,
R. parkeri, R. philipii, R. conorii, and R. africae. The transitional
group contains R. akari, R. australis, and R. felis. Because the
infective aerosol dose is low, R. rickettsii, R. prowazekii, R. typhi,
and R. conorii are considered potential bioterrorism agents.
Following the bite of the arthropod vector, the rickettsiae are
phagocytosed into endothelial cells (cells that line blood ves-
sels), where they escape from the phagosome and replicate in
the cytoplasm of the host cell. Replication in the nucleus also
occurs. The rickettsiae pass directly through the plasma mem-
branes of infected cells into adjacent cells without causing dam-
age to the host cells. The rickettsiae are spread hematogenously
throughout the host and induce vasculitis in internal organs,
including the brain, heart, lungs, and kidneys.

Spotted fever group
Rocky Mountain spotted fever
The most severe of the rickettsial infections, Rocky Mountain
spotted fever (RMSF) is caused by R. rickettsii. It was rst
described in the western United States during the latter part of
the 19th century. It was not until the early 1900s that research-
ers demonstrated the infectious nature of the disease, when
they infected laboratory animals with the blood of infected
patients. The nature of the agent was a mystery because no
bacteria were apparent on direct examination or on culture.
However, researchers discounted a viral cause because the
agent was nonlterable. The organism was rst seen using
light microscopy in 1916. Since 2010, the CDC has used the
category spotted fever rickettsiosis (SFR), which includes RMSF,
Rickettsia parkeri rickettsiosis, Pacic Coast tick fever (R. phil-
ipii), and rickettsialpox (R. akari). The number of SFR cases in
the United States has increased in the past two decades, and
between 4500 and 6200 cases of RMSF have been reported
annually since 2012.
RMSF is a zoonosis, and humans typically acquire the
infection by tick bites. Ticks are the principal vectors and
reservoirs for R. rickettsii. The most common tick vectors
are Dermacentor variabilis (Fig. 24.8) in the southeastern
United States and Dermacentor andersoni in the western part
of the country. Other tick species, however, can be vectors.
Ticks transmit the organism into humans via saliva, which
is passed into the host when the tick feeds. After an incuba-
tion period of approximately 7 days, the patient experiences
ulike symptoms for approximately 1 week. The symptoms
include fever, headache, myalgia, nausea, vomiting, and rash.
The rash, which may be hard to distinguish in individuals of
color, begins as erythematous patches on the ankles and wrists
during the rst week of symptoms. The rash can extend to the
palms of the hands and soles of the feet but normally does not
affect the face. The maculopapular patches eventually consol-
idate into larger areas of ecchymoses.
Once disseminated, the organisms cause vasculitis in
the blood vessels of the lungs, brain, and heart, leading to
Fig. 24.8 Dorsal view of Dermacentor variabilis, the American dog tick, a vector for
Rocky Mountain spotted fever (×20,000). (Courtesy Janice Carr, Centers for Disease
Control and Prevention, Atlanta, GA.)
Rickettsiaceae and similar organisms 553
pneumonitis, central nervous system (CNS) manifestations,
and myocarditis. The patient experiences symptoms second-
ary to vasculitis, including decreased blood volume, hypo-
tension, and disseminated intravascular coagulation. The
mortality rate for untreated or incorrectly treated patients
can be as high as 25%, although correct antimicrobial ther-
apy with tetracycline or chloramphenicol lowers the rate to
3% to 6%.
Boutonneuse fever
Boutonneuse fever, also known as Mediterranean spotted fever,
is caused by R. conorii and occurs in France, Spain, and Italy.
R. conorii also causes Kenya tick typhus, South African tick
fever, and Indian tick typhus. Like the agent for RMSF, this
rickettsia is tickborne, and its reservoirs include ticks and dogs.
Boutonneuse fever clinically resembles RMSF. The rash
involves the palms of the hands and soles of the feet, just as in
RMSF. The rash of boutonneuse fever, however, also involves
the face. In contrast with RMSF, this disease is characterized
by the presence of taches noires (black spots) at the primary
site of infection. Taches noires lesions are caused by the intro-
duction of R. conorii into the skin of a nonimmune person. As
the organism spreads to the blood vessels in the dermis, dam-
age occurs to the endothelium. Edema secondary to increased
vascular permeability reduces blood ow to the area and
results in local necrosis.
Typhus group
The typhus group of rickettsiae includes the species R. typhi
(which causes eaborne typhus, also referred to as endemic
typhus and murine typhus) and R. prowazekii (which causes
louseborne or epidemic typhus). Brill-Zinsser disease is a
recrudescent disease caused by reactivation of R. prowazekii
years after epidemic typhus. Generally, the typhus rickettsiae
differ from the other rickettsial groups in that they cause cell
lysis, thereby releasing the rickettsiae. Other rickettsiae pass
directly through an uninjured cell.
Fleaborne typhus
The arthropod vector for R. typhi is the oriental rat ea
Xenopsylla cheopis, and the rat (Rattus exulans) is the primary
reservoir. The cat ea, Ctenocephalides felis, can also harbor the
organism. Because this ea infests many domestic and wild
animals, it may be an important factor in the persistence of
infection in humans. Rickettsia felis, described in 1990, also
causes eaborne typhus.
The rickettsiae survive in nature, to a lesser extent, by
transovarial transmission. When a ea feeds on an infected
host, the rickettsiae enter the ea’s midgut, where they rep-
licate in the epithelial cells. They are eventually released into
the gut lumen. Humans become infected when eas defecate
on the surface of the skin while feeding. The human host
reacts to the bite by scratching the site, allowing direct inocu-
lation of the infected feces into abrasions. R. typhi can also be
transmitted to humans directly from the ea bite itself.
In the 1940s, approximately 5000 cases of murine typhus
were reported annually in the United States. Rigid control
measures have reduced that number to fewer than 100 cases
annually. The disease essentially occurs only in southern
Texas and southern California. However, it continues to be a
problem in warmer climates of the world in areas where rats
and their eas are present in urban settings. As is the case
554 PART 2 24 Chlamydia, Rickettsia, and similar organisms
with RMSF, the clinical course of endemic typhus includes
fever, headache, and rash. Unlike RMSF, endemic typhus does
not always produce a rash; only about 50% of those infected
will have a rash. When the rash is present, however, it usu-
ally occurs on the trunk and extremities. Rash on the palms of
the hands occurs rarely. Complications are rare, and recovery
usually occurs without incident. Gastrointestinal symptoms,
including anorexia, nausea, vomiting, and abdominal pain,
are often present. The fatality rate is about 4% with treatment.
Louseborne typhus
The vectors for louseborne typhus include the human louse
(Pediculus humanus; Fig. 24.9), squirrel ea (Orchopeas how-
ardii), and squirrel louse (Neohaematopinus sciuriopteri). The
reservoirs are primarily humans and ying squirrels located
in the eastern United States. The louse often dies because of
rickettsemia, unlike vectors of other rickettsiae.
Louseborne typhus is still found commonly in areas of
Africa and Central and South America where unsanitary
conditions promote the presence of body lice. As seen during
World War II, epidemic louseborne typhus can recur even in
developed countries when sanitation is disrupted. More than
20,000 cases were documented during the 1980s, with the vast
majority originating in Africa. Louseborne typhus resembles
the other rickettsioses.
Lice are infected with R. prowazekii when feeding on infected
humans. The organisms invade the cells lining the gut of the
louse. They actively divide and eventually lyse the host cells,
spilling the organisms into the gut lumen. When the louse
feeds on another human, it defecates, and the infected feces
are scratched into skin, just as in murine typhus. Following
an incubation period of 1 to 3 days, the disease progression is
like that of RMSF, including involvement of the palms of the
hands and soles of the feet with the rash. Unlike the case with
RMSF, the face may also be affected by rash. Patients have
high fever, severe headache, and myalgia. The mortality rates
for untreated patients can approach 40%, although mortality
rates in treated patients are very low.
Brill-Zinsser disease, also called recrudescent typhus, is
seen in patients who previously had louseborne typhus.
R. prowazekii lies dormant in the lymph tissue of the human
host until the infection is reactivated. Brill-Zinsser disease is a
milder disease compared with louseborne typhus, and death
is rare. Patients with latent infections constitute an important
reservoir for the organism.
Fig. 24.9 The female head louse, Pediculus humanus, which is a vector for
Rickettsia prowazekii, the agent of epidemic typhus (×40). (Courtesy Dr. Dennis D.
Juranek, Centers for Disease Control and Prevention, Atlanta, GA.)
Transitional group
Rickettsialpox
Rickettsialpox is caused by R. akari, whose reservoir is
the common house mouse, and the vector, the mouse mite
Liponyssoides sanguineus. Rickettsialpox occurs in Korea and
Ukraine and in the eastern United States, including the cities
of New York, Boston, and Philadelphia. The infections occur
in crowded urban areas where rodents and their mites exist.
Rickettsialpox has similarities to RMSF but is a milder
infection and has a biphasic presentation. The rickettsial
organism enters the human host following a mite (chigger)
bite. The incubation period is about 7 days, after which the
rst phase presents as a papule at the site of inoculation.
The papule progresses to a pustule and then to an indurated
eschar. During this state, the bacteria spread hematogenously.
This is followed by the second phase, when the patient
becomes febrile. The patient also experiences headache, nau-
sea, chills, and a papulovesicular rash. Unlike RMSF, the rash
of rickettsialpox appears on the face, trunk, and extremities
and does not involve the palms of the hands or soles of the
feet. Rickettsialpox symptoms resolve without medical atten-
tion in 2 to 3 weeks.
Orientia
Scrub typhus is a disease that occurs in India, Pakistan,
Myanmar, eastern Russia, Asia, and Australia. The causative
agent is Orientia (formerly Rickettsia) tsutsugamushi. The vec-
tor is the Leptotrombidium deliensis mite, and the reservoir is
the rat. Because mites only feed once during their life, they
are not considered important reservoirs for human disease.
Mites are also reservoirs because the bacteria are transmitted
transovarially. The transmission of O. tsutsugamushi to the
human host is followed by an incubation period of approxi-
mately 2 weeks. A tache noire, like that of boutonneuse fever,
often forms at the site of inoculation. The normal rickett-
sial symptoms of fever, severe headache, and rash are also
present. The macular to papular rash starts on the trunk and
spreads to the extremities but is present on less than 50% of
the patients. Unlike the case with RMSF, the rash does not
involve the palms of the hands and soles of the feet, and the
face is also not involved. Systemic lymphadenopathy, heart
failure, and CNS involvement can be present. Without treat-
ment, mortality approaches 30%.
Laboratory diagnosis of rickettsial diseases
Because of their infectious nature, isolation of the rickett-
siae is not recommended and should be attempted only by
biosafety level 3 laboratories. If culture is attempted, blood
should be collected as early in the disease course as possible.
Methods for immunohistochemical testing of tissue speci-
mens have been established for several rickettsial diseases.
Monoclonal antibodies directed against the spotted fever or
typhus group have been used, but no antibody is commer-
cially available. NAATs are particularly useful for eschar
specimens. Only NAATs and immunohistochemical assays
can provide a diagnosis in the acute stage, when treatment
is critical. Unfortunately, neither method is readily available.
Typically, serologic assays are the only laboratory
tests performed for the diagnosis of rickettsial diseases.
Unfortunately, these methods can conrm a diagnosis only

in convalescent specimens and offer little help in diagnosing
acute infections that could guide antimicrobial therapy. An
acute blood sample should be taken as early as possible, and
a second sample should be taken 1 to 2 weeks later. If the
second sample does not detect a fourfold or greater increase
in titer, a third sample is recommended 4 weeks after onset
of symptoms.
The immunouorescent antibody (IFA) test is considered
the gold standard method for antibody detection. Because of
cross-reactivity among members of the same groups (spotted
fever and typhus), generally only group-specic antibody
testing is available. Antibodies to certain rickettsial species
are known to cross-react with bacteria in the genus Proteus.
This gave rise to the Weil-Felix agglutination test. Because
this assay does not use rickettsial antigens, it is nonspecic
and rarely used in the United States. However, because of
its low cost, it is used in some other countries. An aggluti-
nation test using latex beads coated with rickettsial antigens
is commercially available for the diagnosis of RMSF (Panbio,
Baltimore, MD) as are EIAs.
Anaplasmataceae
The family Anaplasmataceae contains several genera of
importance to human and veterinary medicine. Anaplasma
and Ehrlichia are the most signicant for human disease.
Members of the family are small, nonmotile, obligate intracel-
lular bacteria. They replicate within membrane-derived vacu-
oles within the cytoplasm. Most members of the family grow
in vertebrate hosts and one invertebrate vector. In vertebrate
hosts, they reside in cells of hematopoietic origin.
Ehrlichia
Ehrlichiosis was rst noted in France in the 1930s in dogs.
Postmortem examination revealed rickettsial-like inclusions
in the monocytes of the dead animals. These rickettsiae were
named Rickettsia canis. They were obligately intracellular,
arthropod-borne coccobacilli. They differ from members of
the rickettsiae in that they multiply in the phagosomes of host
leukocytes and in other cells derived from the bone marrow,
not in the cytoplasm of endothelial cells.
Because these organisms grew within host cell vacuoles,
they were reclassied into a new genus, Ehrlichia, in 1945. The
ehrlichiae have a developmental cycle like that of the chla-
mydiae. There are two forms: the denser, infective EB, and the
RB that replicates in the phagosome and prevents phagolyso-
some formation. As the cells divide within the phagosome,
they develop into morulae (Latin for “mulberry”; Fig. 24.10).
Morulae are round to oval clusters of bacteria 1 to 3 µm in
diameter that stain blue in Wright’s and Giemsa stains. As the
host cell ruptures, the morulae break into many individual
EBs, which continue the infective cycle.
Ehrlichia chaffeensis causes E. chaffeensis ehrlichiosis, for-
merly called human monocytic ehrlichiosis because the
bacteria primarily reside in mononuclear cells such as
monocytes. Most cases occur in the United States, but some
cases are reported in Europe, Africa, and South and Central
America. In the United States, about 1600 case are reported
annually, and most cases are found in the southeastern and
south-central states as well as in the Mid-Atlantic states. In
2019, four states, Missouri, Arkansas, New York, and North
Carolina, accounted for over one half of all reported cases of
Rickettsiaceae and similar organisms 555
Fig. 24.10 Anaplasma morula (arrow) in an infected white blood cell (×1000).
ehrlichiosis. Most cases occur in the summer months June
and July, which is expected for a tickborne disease. E. chaf-
feensis causes the most cases, but Ehrlichia ewingii, the second
most common, produces a disease indistinguishable from E.
chaffeensis. Currently, no serologic test can distinguish these
agents.
The seroprevalence of E. chaffeensis ranges from 1.3% to
12.5% in Arkansas and Tennessee, two states with a high inci-
dence of infection. This indicates that cases may be under-
reported. Natural hosts of the organism include dogs, deer,
and humans, with the lone star tick (Amblyomma americanum)
being the primary vector. This tick also transmits E. ewingii.
E. muris eauclairensis is the third most common cause of ehrli-
chiosis in the United States, with most cases occuring in
Minnesota and Wisconsin, and is transmitted by the black-
legged tick (Ixodes sccapularis)
Many patients with E. chaffeensis ehrlichiosis experience
asymptomatic infection. The organism has an incubation
period of 5 to 10 days. Patients often experience fever, head-
ache, malaise, myalgia, and nausea. Vomiting, diarrhea,
cough, and joint pain are occasionally present. A petechial,
macular, or maculopapular rash is sometimes present (57%)
in pediatric patients infected with E. chaffeensis; however,
adults rarely experience a rash (<30%). Patients may also have
evidence of leukopenia and neutropenia, thrombocytopenia,
and elevated liver enzyme levels. Patients can experience
severe complications, including a toxic shock–like syndrome
presentation, CNS involvement, and acute respiratory dis-
tress syndrome. Mortality rates are approximately 2% to 3%.
Direct staining (Giemsa or Wright) of peripheral blood
smears or buffy coats for morulae can be used for diagnosing
E. chaffeensis infections; however, this method is not very sen-
sitive (29%). The bacteria are primarily found in monocytes,
and the percentage of cells infected is less than 10%. Antigen
detection in tissues, such as bone marrow, liver, and spleen,
has been described. Again, the sensitivity is low (40%), and
cross-reaction with other species has been noted. This leaves
NAATs as a frequently used method for direct detection of E.
chaffeensis from whole blood. The use of real-time PCR and
multiplex PCR has been described. The bacteria can be iso-
lated from peripheral blood in cell culture, but this method
is rarely used. Most cases of E. chaffeensis ehrlichiosis are
diagnosed retrospectively by serologic testing demonstrating
a fourfold increase in IgG antibody. The IFA test is the most
widely used method. Signicant antibody cross-reactivity
has been seen among Ehrlichia spp. and between Ehrlichia and
Anaplasma
556 PART 2 24 Chlamydia, Rickettsia, and similar organisms
Anaplasma
Anaplasma phagocytophilum, formerly known as Ehrlichia
phagocytophilum, causes the disease human granulocytic
anaplasmosis (HGA). The incubation period for HGA is 1 to
5 days. The symptoms closely resemble those of E. chaffeensis
ehrlichiosis and include fever, headache, malaise, and myal-
gia. Less than 10% of infected individuals have a rash.
The number of HGA cases is probably underreported.
Annually, about 4000 cases are reported in the United
States, with most cases occurring in the upper Northeast
region. In 2018, the highest rates per 100,000 people were
in Vermont (391), Maine (355), New Hampshire (158), and
Rhode Island (141). Signicant infection rates also occur in
Minnesota (88) and Wisconsin (63). Natural hosts include
deer, rodents, horses, cattle, and humans. Tick vectors
include I. scapularus in the northeast and Ixodes pacicus
along the west coast. The geographic range of anaplasmosis
is increasing corresponding to the blacklegged tick’s
expanding range.
Typical laboratory test results include elevated transami-
nase levels, thrombocytopenia, and leukopenia with lympho-
penia. As with E. chaffeensis ehrlichiosis, staining of peripheral
blood buffy coats can be used to diagnose HGA. The moru-
lae are found in granulocytes (see Fig. 24.10), and the sensi-
tivity is about 60%. The percentage of granulocytes infected
can reach 40%. Diagnosis can also be made by using direct
antigen detection, NAATs, and isolation in cell cultures. IFA
serologic kits are available for the detection of antibodies to
A. phagocytophilum
Coxiella
Coxiella burnetii is the only species in the genus. This organism
differs in several ways from many members of the families
Rickettsiaceae and Anaplasmataceae. For example, C. burnetii
has been grown in cell-free media and should be considered
a facultative intracellular parasite. The bacterium does not
transport ATP across its plasma membrane, and it develops
within the phagolysosomes of infected cells. The acidic envi-
ronment activates its metabolic enzymes. The bacteria exist in
two forms. The large-cell variant is rod shaped (0.4–1.5 µm)
and metabolically active inside host cells. Under adverse
conditions, the large-cell variant converts into the denser
small-cell variant (0.22 µm) that survives harsh environmen-
tal conditions. Another distinguishing feature is that C. bur-
netii is generally not transmitted by arthropods, although it is
known to infect more than 12 genera of ticks and other arthro-
pods. The bacteria can infect shes, birds, rodents, livestock,
and other mammals.
C. burnetii is the causative agent of Q (query) fever, a dis-
ease found worldwide. Roughly 160 cases are reported annu-
ally in the Unites States. Q fever is highly contagious and,
as such, is considered a potential bioterrorism agent (see
Chapter 30). Most infections are spread by the inhalation of
dried birthing uids of several animals. Therefore occupa-
tional hazards include ranching or livestock management.
The ingestion of unpasteurized milk is also a recognized
risk factor. Approximately 50% of infected individuals will
remain asymptomatic. In the remaining cases after an incu-
bation period of 2 to 3 weeks, acute Q fever generally has an
abrupt onset of an undifferentiated febrile disease consisting
of high fever that can be accompanied by chills, headaches,
myalgia, arthralgia, cough, and rarely, a rash. Patients may
present with elevated liver enzyme levels, increased erythro-
cytic sedimentation rate, and thrombocytopenia. Because of
the rapid dissemination of the bacteria, several tissues can be
infected. Chronic disease occurs in a small number of patients
and can result in vascular disease, endocarditis, and bone and
joint infections.
Because the symptoms vary among patients and can be
difcult to distinguish from other diseases, it can be a chal-
lenging disease to diagnose. The laboratory diagnosis of Q
fever can be made by direct IFA of infected tissue. However,
except for heart tissue in cases of endocarditis, infected tis-
sue contains low numbers of bacteria. NAATs, such as the
PCR assay, have also been successful in diagnosing acute
and chronic infections; whole blood and buffy coats are often
successful in detecting the organism. However, NAAT sen-
sitivity drops signicantly after antibody production, which
occurs about 2 weeks after infection. C. burnetii is highly
contagious; isolation in cell cultures should be attempted
only in biosafety level 3 facilities. Several serologic assays
have been described for detecting antibodies in acute and
chronic cases. The IFA test is the method of choice. EIA kits
are commercially available and FDA approved and have
sensitivities and specicities comparable with those of the
IFA test.
POINTS TO REMEMBER
• Chlamydiae and rickettsiae are obligate intracellular
organisms.
• Chlamydia trachomatis is the most common sexually transmit-
ted bacterial pathogen, and certain serovars are associated
with trachoma, which can result in blindness.
• The LGV strains of C. trachomatis are more invasive, pro-
ducing a more serious infection and pronounced antibody
response.
• NAATs are better assays for the diagnosis of C. trachomatis
infections compared with cultures.
• C. pneumoniae is a relatively common respiratory tract patho-
gen considered responsible for many cases of community-ac-
quired pneumonia.
• C. psittaci is the cause of psittacosis, also known as parrot fever
or ornithosis. This bacterium produces lower respiratory tract
infections in humans.
• The Rickettsia spp. are important human pathogens responsi-
ble for several diseases, including RMSF, rickettsialpox, and
typhus.
• The Rickettsia, Orientia, Ehrlichia, and Anaplasma are typically
transmitted to humans by arthropod bites.
• Ehrlichia and Anaplasma are intracellular parasites of
white blood cells—mononuclear cells and granulocytes,
respectively.
• Coxiella burnetii is the causative agent of Q fever. Infection is
most often transmitted by inhalation of dried birthing uids.
The ingestion of unpasteurized milk is also a risk factor.
• C. burnetii differs from the ricketsiae by developing within
the phagolysosome of infected cells and is generally not
transmitted by arthropods. C. burnetii, unlike the Rickettsia
spp., has been grown in cell-free media.
 Rickettsiaceae and similar organisms 557

blue inclusions about 2 µm in diameter were found in the

LEARNING ASSESSMENT QUESTIONS neutrophils. What is the most likely diagnosis?
a. Ehrlichia chaffeensis ehrlichiosis
1. Which organisms should be considered as probable causes
b. Human granulocytic anaplasmosis
of neonatal conjunctivitis?
c. Fleaborne typhus
a. Chlamydia trachomatis and Chlamydia pneumoniae
d. Q fever
b. Chlamydia pneumoniae and Chlamydia psittaci
11. For the neonate described in the Case in Point, what other
c. Chlamydia trachomatis and Neisseria gonorrhoeae
clinical conditions could have resulted from infection with
d. Chlamydia pneumoniae and Neisseria gonorrhoeae
the causative organisms?
2. Which Chlamydia trachomatis serotypes are associated with
12. How does lymphogranuloma venereum differ from
lymphogranuloma venereum?
other sexually transmitted diseases caused by Chlamydia
a. A, B, and C
trachomatis?
b. A to K
13. Which types of infections are associated with Chlamydia
c. D to K
pneumoniae?
d. L1, L2, and L3
14. How does Coxiella burnetii differ from the Rickettsia spp.?
3. How does psittacosis or ornithosis present in human
infections?
a. Endocarditis
b. Pneumonia
BIBLIOGRAPHY
c. Nausea and vomiting
d. Tissue abscess Blanton, L. S., & Walker, D. H. (2017). Flea-borne rickettsioses and
4. What is the most common laboratory method used to diag- rickettsiae. American Journal of Tropical Medicine and Hygiene, 96, 53.
nose rickettsial diseases? Blanton, L. S., et al. (2019). Rickettsia and Orienta. In K. C. Carroll,
a. Serology et al. (Eds.), Manual of clinical microbiology (12th ed., p. 1149).
Washington, DC: ASM Press.
b. Growth in cell monolayers Centers for Disease Control and Prevention. (2022). Sexually transmitted
c. Direct immunouorescent assay diseases surveillance 2020: tables—sexually transmitted disease
d. Histologic tissue stain with Giemsa stain surveillance, 2020. Available at: https://www.cdc.gov/std/
5. Which cells do the Ehrlichia typically infect in humans? statistics/2020/tables.htm. (Accessed 1 June 2022).
Centers for Disease Control and Prevention (2021). Rocky Mountain
a. Hematopoietic stem cells
spotted fever. Available at: https://www.cdc.gov/rmsf/stats/index.
b. Reticulocytes html. (Accessed 1 June 2022).
c. Granulocytes Centers for Disease Control and Prevention (2022). Ehrlichiosis.
d. Monocytes Available at: https://www.cdc.gov/ehrlichiosis/index.html.
6. During human infection by the rickettsiae, where do the (Accessed 1 June 2022).
Centers for Disease Control and Prevention (2022). Anaplasmosis.
bacteria usually replicate? Available at: https://www.cdc.gov/anaplasmosis/index.html.
a. Attached to the outside of the plasma membrane (Accessed 1 June 2022).
b. Within the endosome Centers for Disease Control and Prevention (2019). Q fever. Available at:
c. Free in cytoplasm https://www.cdc.gov/qfever/index.html. (Accessed 1 June 2022).
Centers for Disease Control and Prevention. (2014). Recommendations
d. Within a phagolysosome
for the laboratory-based detection of Chlamydia trachomatis and
7. Which stain should be performed on a conjunctival Neisseria gonorrhoeae—2014. MMWR. Recommendations and Reports,
scraping for microscopic examination for the diagnosis of 63, 1.
inclusion conjunctivitis? Fujita, J., & Kinjo, T. (2020). Where is Chlamydophila pneumoniae
a. Gram pneumonia? Respiratory Investigation, 58, 336.
Kersh, G. J., & Bleeker-Rovers, C. P. (2019). Coxiella. In K. C. Carroll,
b. Giemsa et al. (Eds.), Manual of clinical microbiology (12th ed., p. 1180).
c. Machiavello Washington, DC: ASM Press.
d. Fluorescent antibody Mouméne, A., & Meyer, D. F. (2016). Ehrlichia’s molecular tricks to
8. Which time of year do most cases of Rocky Mountain manipulate their host cells. Microbes and Infection, 18, 172.
Pritt, B. S., & Dumler, J. S. (2019). Ehrlichia, Anaplasma, and related
spotted fever occur in the United States?
intracellular bacteria. In K. C. Carroll, et al. (Eds.), Manual of
a. Winter clinical microbiology (12th ed., p. 1163). Washington, DC: ASM Press.
b. Spring Schachter, J., & Chernesky, M. (2019). Chlamydia. In K. C. Carroll, et al.
c. Summer (Eds.), Manual of clinical microbiology (12th ed., p. 1137).
d. Fall Washington, DC: ASM Press.
Sheinman, M. D., & Vinod, J. (2020). Lymphogranuloma venereum
9. Areas of overcrowding and poor personal hygiene are proctocolitis. In StatPearls [Internet]. Treasure Island, FL: StatPearls
known for a high incidence of infections by which organism? Publishing. Available at: https://www.ncbi.nlm.nih.gov/books/
a. Rickettsia prowazekii NBK544283. (Accessed 1 June 2022).
b. Rickettsia coronii Szabo, K. V., et al. (2020). Diversity in chlamydial plasmids. PloS One,
15, e0233298. https://doi.org/10.1371/journal.pone.0233298.
c. Rickettsia akari
World Health Organization. (2022). Trachoma. Available at: https://
d. Rickettsia typhi www.who.int/health-topics/trachoma. (Accessed 1 June 2022).
10. In July, a 28-year-old male patient presents to his primary World Health Organization. (2017). GET 2020: WHO alliance for global
care provider complaining of headache, malaise, and myal- elimination of trachoma by 2020. Available at: https://www.who.int/
gia. He is also found to have a fever, but no rash is present. initiatives/who-alliance-for-the-global-elimination-of-tracho-
ma-by-2020. (Accessed 1 June 2022).
His medical history is unremarkable, and he reports that Yang, C., et al. (2020). Chlamydia trachomatis plasmid gene protein 3 is
he recently returned from a camping trip in Maine. A essential for the establishment of persistent infection and
complete blood count revealed leukopenia, and clumps of associated immunopathology. mBio, 11, e01902
 25
Mycoplasma and Ureaplasma
Donald C. Lehman and Connie R. Mahon

CHAPTER OUTLINE 9. Justify the use of serologic assays for diagnosing M. pneumoniae
infections.
General characteristics, 559 10. Describe two selective media for the detection of the mycoplasmas.
Clinical infections, 560 11. Evaluate the use of different classes of antimicrobial agents to treat
Mycoplasma pneumoniae, 560 mycoplasmal infections.
Genitourinary tract infections associated with the mollicutes, 561 12. Provide recommendations for the proper interpretation and reporting for
Mycoplasma fermentans, 562 Mycoplasma and Ureaplasma
Laboratory diagnosis, 562
Specimen collection and transport, 562
KEY TERMS
Nucleic acid amplication tests, 563
Culture, 563 Cell wall decient Nongonococcal urethritis (NGU)
Serologic diagnosis, 566 L-forms Pelvic inammatory disease (PID)
Antimicrobial susceptibility, 567 Pleuropneumonia-like organism Primary atypical pneumonia
Interpretation of laboratory results, 567 (PPLO) T-strain mycoplasma
Bibliography, 569

OBJECTIVES
After reading and studying this chapter, you should be able to:
1. Describe the general characteristics of the Mycoplasma and
Ureaplasma, and how they differ from other bacterial species.
2. Name the clinical specimens from which the mycoplasma species are
most likely to be isolated.
3. Compare the clinical diseases caused by Mycoplasma pneumoniae,
Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma fermen-
tans, and Ureaplasma urealyticum
4. Evaluate the recovery of M. hominis from a genital specimen.
5. Compare the pneumonia caused by Mycoplasma pneumoniae with
that caused by Streptococcus pneumoniae
6. Discuss the possible roles of M. hominis and U. urealyticum in infec-
tions of low-birth-weight and high-risk neonates.
7. Discuss the clinical manifestations of Mycoplasma spp. in immuno-
compromised patients.
8. Analyze the diagnostic methods appropriate for the detection and
identication of mycoplasmal and ureaplasmal infections including
specimen collection, transport, storage, and appropriate media.
Case in point
A premature male neonate in the neonatal intensive care unit,
who weighed 1.5 lb at birth (low birth weight), developed signs
of meningitis, and a lumbar puncture was performed. The
results of a white blood cell count of cerebrospinal uid (CSF)
were negative. The Gram-stained smear showed “no organisms
seen,” and the result of routine culture at 3 days was reported
as “no growth.” The infant was still symptomatic at this time,
and the pediatric infectious disease physician, after consulta-
tion with the clinical microbiologist, performed another lumbar
puncture and ordered additional cultures. An organism was
recovered by the laboratory.
Issues to consider
After reading the patient's case history, consider:
• The cause of meningeal infections in the given patient
population
• Supporting laboratory ndings and how they help establish
the diagnosis
• Methods for recovery of the suspected causative agent

558
 General characteristics 559

This chapter discusses bacteria within the class Mollicutes, Mycoplasmas are generally slow-growing, highly fastidi-
the smallest self-replicating organisms once thought to be ous, facultative anaerobes requiring complex media contain-
viruses because their small size allowed them to pass through ing nucleic acid precursors, cholesterol, and fatty acids for
lters, hence the name lterable. Members of this class are also growth; important exceptions include aerobic M. pneumoniae
referred to by the common names mollicute or mycoplasma. and the more rapidly growing M. hominis. The acholplasma
This group of bacteria is characterized by permanently lack- do not require cholesterol supplements in laboratory media.
ing a cell wall. They range in size for coccoid forms from The mycoplasmas produce small colonies ranging in size
approximately 0.2 to 0.3 µm in diameter to tapered rods of from about 15 µm to over 300 µm in diameter. Mycoplasma
approximately 1 to 2 µm in length and 0.2 to 0.3 µm in diame- spp. often grow embedded beneath the surface of solid
ter. Seven families, at least 12 genera, and over 200 species of media; therefore transferring colonies with a loop is ineffec-
mollicutes have been described. At least 16 species of molli- tive. On solid media, some species (e.g., M. hominis) form
cutes have been isolated from humans. colonies with slightly raised centers, giving the classic “fried
Mycoplasma and Ureaplasma are two of the four genera in the egg” appearance (Fig. 25.1). In the laboratory, mycoplasmas
family Mycoplasmataceae. Numerous species of Mycoplasma are common and hard to detect contaminants of cell cultures.
and Ureaplasma have been identied in plants and animals;
however, the following species are the most signicant human
pathogens (Table 25.1):
Table 25.2 Human clinical isolates in the class Mollicutes
• Mycoplasma pneumoniae, which causes respiratory disease Feature Mycoplasma Ureaplasma Acholeplasma
• Mycoplasma hominis, associated with urogenital tract
Cell wall + + +
disease
decient
• Ureaplasma urealyticum, associated with urogenital tract
disease Gram stain − − −
Penicillin − − −
The family Acholeplasmataceae contains ve genera, susceptible
including Acholeplasma. Members of this genus do not appear Urease activity − + −
to cause signicant infections in humans.
Lack of cell − − −
wall induced
in hypertonic
solution and
General characteristics by penicillin,
lysozyme, or
The rst Mycoplasma sp. was isolated in the late 1800s from salts
a cow with pleuropneumonia. Later, a mycoplasma was Exists in nature − − +
isolated from humans and was referred to as a pleuropneu- as a free-living
monia-like organism (PPLO) and the Eaton agent, after the organism
researcher who rst isolated it from humans. This human iso- Pleomorphic + + −
late was ultimately named Mycoplasma pneumoniae. Because shape
of the absence of a cell wall, the mycoplasmas are pleomor-
Other shared Smaller than other bacteria; close in size to
phic and are not visible microscopically with the Gram stain. characteristics myxoviruses
In addition, this characteristic makes them resistant to cell
wall–active antibiotics, such as the penicillins and cepha- Smaller genome than other bacteria
losporins. They were originally grouped under the general Lower guanidine-to-cytosine ratio than most
term cell wall–decient bacteria given the absence of a cell bacteria
wall. They are not, however, classied as L-forms, which are Limited metabolic activity (i.e., fastidious)
bacteria that have temporarily lost their cell wall attributable Many mollicutes contain DNase
to environmental conditions. Table 25.2 compares features of
+, Feature present; −, feature absent.
the three genera found in human specimens.

Table 25.1 Divergent ecosystems inhabited by genera of the class Mollicutes

Ecosystem Mycoplasma Ureaplasma Acholeplasma Spiroplasma Anaeroplasma
Soil and grasses − − − − −
Crops and plants − − − + −
Mown hay − − − − −
Water − − + − −
Deciduous trees − − − + −
Humans + + − − +
Cattle + + − − +
+, Present in ecosystem; , rarely associated with ecosystem.
560 PART 2 25 Mycoplasma and Ureaplasma
The mycoplasmas adhere to the epithelium of muco-
sal surfaces in the respiratory and urogenital tracts and are
not eliminated by mucous secretions or urine ow. Fig. 25.2
depicts electron micrographs of ciliated tracheal epithelial
cells before and after M. pneumoniae adherence. M. pneumo-
niae utilizes the P1 adhesion and other proteins to adhere
to host cells of the respiratory tract. P1 is associated with a
polar extension called an attachment organelle or foot. The
organelle is responsible for cytoadherence. This ability of M.
pneumoniae to adhere and attach to hosts’ cells is crucial for
the bacteria to survive and infect. Once attached, the host’s
mucociliary clearance mechanisms are compromised and
prevented from eliminating the organism, damaging the
respiratory epithelial cells. Fig. 25.3 is an electron micrograph
demonstrating the shape of M. pneumoniae and its orientation
of attachment. Although M. pneumoniae mainly inhabits the
surface of the respiratory epithelial cells, it may also invade
tissues and replicate intracellularly.
Mycoplasma spp. indigenous to humans are listed in Table
25.3. The human mollicutes are susceptible to adverse envi-
ronmental conditions, such as heat and drying. Transmission
of mollicutes in humans can occur via direct sexual contact,
Fig. 25.1 Typical large Mycoplasma colony showing “fried egg” appearance.
(Courtesy Bionique Testing Laboratories, Saranac Lake, NY.)
A B
Fig. 25.2 Electron micrographs showing the effect of Mycoplasma pneumoniae on ciliated tracheal cells. A, Infected animal model. B, Uninfected animal
model (×20,000).
from mother to child during delivery or in utero, and by
respiratory secretions or fomites in cases of M. pneumoniae
infections.
Clinical infections
Mycoplasma pneumoniae
M. pneumoniae does not occur as a normal commensal
organism; therefore its isolation is always signicant and
pathognomonic. Infection can result in a relatively com-
mon pneumonia known as primary atypical pneumonia
or walking pneumonia. The clinical manifestations resemble
those caused by Chlamydia pneumoniae. The disease differs
from the typical pneumonia caused by Streptococcus pneumo-
niae in that it is milder and has a higher incidence in young
adults. Although infection is not considered seasonal, more
cases occur in autumn and early winter. Outbreaks also have
been noted when adolescents return to school in the fall.
c
mv
Fig. 25.3 Electron micrograph of Mycoplasma pneumoniae attaching by specic
attachment features to ciliated trachea (×100,000). Arrow indicates attachment
organelle. c, Cilia; m, mycoplasma; mv, microvilli.
 Clinical infections 561

Table 25.3 Mycoplasma species indigenous to humans

Mycoplasma species Usual habitat Reported frequency Colony morphologya
M. salivarium Oropharynx Very common Large, “fried egg”
M. orale Oropharynx Common Small, spherical
M. buccale Oropharynx Uncommon Large, fried egg
M. faucium Oropharynx Uncommon Large, fried egg
M. lipophilum Oropharynx Rare Large, fried egg
M. pneumoniae Oropharynx Common (with disease) Small, spherical, granular
M. fermentans Oropharynx Rare Small, fried egg, spherical
Urogenital tract Uncommon
M. hominis Oropharynx Uncommon Large, fried egg, vesicu-
lated peripheral zone
Urogenital tract Common (9%–50% of women)
M. genitalium Urogenital tract Occasional
Ureaplasma urealyticum Urogenital tract Very common (81% of women; Tiny, spherical, fried egg,
30%–50% of men) granular
Acholeplasma laidlawii Oropharynx Rare Small, fried egg, spherical
a
Relative sizes: large, greater than 100 nm; small, 50 to 100 nm; tiny, less than 50 nm.

M. pneumoniae is extremely susceptible to desiccation because

it lacks a cell wall; hence, transmission is probably through
aerosol droplet spray produced while coughing.
M. pneumoniae causes 4% to 8% of reported pneumonias
in the general population and up to 70% in conned popula-
tions, such as those in military settings, prisoners, and college
students. School-age children and young adults are especially
susceptible to infection. Epidemics are known to occur in
these populations. Historically, clinical disease was uncom-
mon in very young children and older adults. However,
recent outbreaks and endemicity have been reported in older
adults and children younger than 5 years of age. A recent
study found that 40% of 830 Chinese children with communi-
ty-acquired pneumonia tested positive for M. pneumoniae by
polymerase chain reaction (PCR).
Many infections are asymptomatic or very mild. The most
common presentation is tracheobronchitis often accompa-
nied by pharyngitis. About one third of infected patients
demonstrate clinically apparent pneumonia (Fig. 25.4). The
incubation period is usually 2 to 3 weeks, and early symp-
Fig. 25.4 Typical chest x-ray of a patient with a 3-week course of atypical pneumo-
toms are nonspecic, consisting of headache, low-grade fever,
nia. Note nonspecic interstitial pneumonia and a patchy inltrate delineated by a
malaise, and anorexia. Dry cough and earache are accompa- feathery outline.
nying symptoms. Extrapulmonary complications, including
cardiovascular, central nervous system, dermatologic, and
gastrointestinal problems, are rare occurrences. In addition, Genitourinary tract infections associated with
Mycoplasma pneumoniae infection has been associated with the mollicutes
chronic lung disease and bronchial asthma. M. pneumoniae is
not associated with infections of the urogenital tract. It has, Although the mollicutes do not cause vaginitis, M. hominis,
however, been implicated as a co-infection or cofactor in epi- M. genitalium, and U. urealyticum are associated with infec-
demic group A meningococcal meningitis (Neisseria menin- tions of the urogenital tract. M. genitalium and U. urealyticum
gitidis) and infant pneumonitis. Recently, a unique virulence probably play a role in nongonococcal urethritis (NGU).
factor known as community acquired respiratory distress M. hominis and U. urealyticum are frequently isolated from
syndrome (CARDS) toxin has been identied and associated asymptomatic sexually active individuals, making interpre-
in the pathogenesis of M. pneumoniae infections. It appears tation of a positive culture difcult. U. urealyticum has been
that the CARDS toxin helps in the colonization and develop- recovered from more than 60% of healthy sexually active
ment of disease, causing an inammatory response and sub- females. Because these species are opportunistic pathogens,
sequent airway dysfunction. the immune status of the host is an important factor in the
562 PART 2 25 Mycoplasma and Ureaplasma
occurrence and severity of disease. In addition, it has been
reported that among sexually active individuals, the rate of
colonization is directly related to the number of sexual part-
ners. Higher rates of colonization have been noted in adults
of lower socioeconomic status.
M. hominis is found in the lower genitourinary tracts
of approximately 50% of healthy adults but has not been
reported as a cause of NGU. The organism may, however,
invade the upper genitourinary tract and cause salpingitis,
pyelonephritis, pelvic inammatory disease (PID), or post-
partum fever. It might also play a role in bacterial vaginosis.
Ureaplasma parvum (U. urealyticum biovar 1) and U. urealyt-
icum (U. urealyticum biovar 2) do not cause disease in the
female lower genital tract. However, U. urealyticum has been
associated with NGU in men, whereas U. parvum has not,
although recent studies have found that both serovars often
occur together, and horizontal gene transfer between the two
species is common. Therefore maintaining separate serovars
might not be valid. Evidence also does not support the role
of the Ureaplasma spp. causing upper female genitourinary
tract disorders.
While somewhat controversial, the urogenital mollicutes
have also been linked to female infertility. Most females
with infertility suffer from inammation of the oviduct
and nearby tissue. Chlamydia trachomatis and Neisseria gon-
orrhoeae are the most common causes of this condition,
although other infectious agents have been implicated.
The mollicutes can be part of the normal microbiota of
the vagina and cervix. However, they can ascend and col-
onize the endometrium, fallopian tubes, and ovaries and
cause inammation and damage to the ciliated epithelium
of the human fallopian tube. This can lead to scaring and
infertility.
Ureaplasma spp. and M. hominis can be transmitted to neo-
nates during delivery and have been associated with cho-
rioamnionitis, congenital bacteremia, and pneumonia, as
well as development of chronic lung disease in premature
infants. Although it is not a primary cause of chronic lung
disease, U. urealyticum is a common organism isolated from
tracheal aspirates of low-birth-weight infants with respira-
tory disease; 14% of infections were in newborns delivered
by cesarean section, thus indicating that infection occurred
in utero and not during passage through the birth canal.
M. hominis and Ureaplasma spp. have been recovered from
the CSF of certain high-risk newborns, including preterm
and low-birth-weight infants. Table 25.4 summarizes the
known association of genital mollicutes with urogenital and
newborn diseases. It has been recommended that culture
for these organisms be attempted when the CSF specimen
from a newborn with evidence of meningitis is negative for
bacteria on Gram stain and routine bacteriology culture.
However, because healthy neonates can be asymptomati-
cally colonized with mollicutes, routine screening cannot be
justied.
Mycoplasma genitalium, rst isolated in 1980, has been asso-
ciated with NGU, cervicitis, endometriosis, and PID. There
is also evidence linking M. genitalium to some cases of tubal
sterility. Its prevalence is unknown, but PCR assays found M.
genitalium more frequently in urethral samples taken from
men with acute NGU than in those from men without ure-
thritis. It is estimated that M. genitalium could be responsible
for 30% of persistent urethritis in males.
Case check 25.1
In the Case in Point, CSF was culture negative for the more
common causes of neonatal meningitis: group B Streptococcus,
Escherichia coli, and Listeria monocytogenes. Both M. hominis and
U. urealyticum have been isolated from the CSF of low-birth-weight
infants. Because of the absence of a cell wall, these bacteria are
not visible with Gram stain, so the lack of bacteria seen in direct
Gram-stained smear supports the diagnosis.
In immunocompromised individuals, bacteremia and
invasive disease of the joints and respiratory tract caused
by mollicutes species have occurred. U. urealyticum has been
reported to cause chronic inammatory diseases, such as
arthritis and cystitis, in patients with hypogammaglobulin-
emia. Mycoplasma isolates have been intermittently associated
with patients with endocarditis, sternal wound infections,
and arthritis.
Mycoplasma fermentans
Mycoplasma fermentans has been noted as a likely opportunis-
tic respiratory pathogen. It is not known how often M. fermen-
tans occurs in the respiratory tracts of healthy children, but it
has been detected in throats of patients with lower respiratory
tract infection, in some of whom a specic causative agent
was not identied. Other groups of patients from whom M.
fermentans has been recovered include adult patients with
respiratory illness and those with acquired immunode-
ciency syndrome (AIDS). M. fermentans has been isolated
from tissue in patients with and without AIDS who died of
systemic infection. M. fermentans has also been isolated from
synovial uid of patients with rheumatoid arthritis.
Laboratory diagnosis
Because they lack a cell wall, the mollicutes will not be vis-
ible by Gram stain. A DNA uorescent stain (e.g., acridine
orange) can be used, but this is not specic for the mol-
licutes. Laboratory diagnosis is often based on a nucleic
acid amplication test (NAAT), culture, and serology.
Immunochromatographic tests were released commercially
in the 1980s. However, the assays demonstrated low sensitiv-
ity and specicity and are not recommended.
Specimens for culture and nonculture detection of the mol-
licutes include blood, synovial uid, amniotic uid, urine,
prostatic secretions, semen, bronchoalveolar lavage uid,
as well as swabs from the nasopharynx, throat, cervix, and
urethra. If swabs are used, it is important to vigorously rub
the tissue because the mollicutes are strongly cell associated.
Tissue samples may also be submitted for culture.
Specimen collection and transport
The absence of a cell wall makes all mollicutes extremely sen-
sitive to drying and heat. Ideally, specimens should be inocu-
lated at the bedside. If this is not possible, specimens should
be delivered immediately to the laboratory in a transport
 Laboratory diagnosis 563

Table 25.4 Summary of association of genital mollicutes with urogenital and newborn diseases
Disease, target population Mycoplasma Mycoplasma Ureaplasma Comments
hominis genitalium spp.
Nongonococcal urethritis None Weak Stronga Ureaplasma spp. cause some cases, but the
proportion is unknown.
Prostatitis None Weak None An association with a few cases of chronic
disease has been reported; a causal relation is
unproven.
Epididymitis None None Weak Mycoplasma spp. are not an important cause.
Vaginitis and cervicitis None None None M. hominis is often associated with disease, but a
causal relation is unproven.
Pelvic inammatory disease Strong Strong None M. hominis causes some cases, but the propor-
tion is unknown.
Postpartum fever Strong None Weak Recent studies indicate that M. hominis may be a
major cause.
Urinary calculi None None Weak Ureaplasma spp. cause calculi in male rats, but
no convincing evidence exists that they cause
natural human disease.
Pyelonephritis Strong None None M. hominis causes some cases.
Involuntary infertility Weak Weak Weak Ureaplasma spp. are associated with altered
motility of sperm.
Chorioamnionitis Strong None Strong An association exists, but a causal relation is
unproven.
Low birth weight None None Strong An association exists, but a causal relation is
unproven.
Neonatal infections, including Strong None Strong Further clarication is needed, but importance is
sepsis, pneumonia, meningitis growing in a selected prenatal population.
Neonatal period, particularly Strong Weak Strong These ndings need further clarication because
preterm delivery, very low birth most neonatal infections resolve without therapy,
weight; clinical signs compatible but in low socioeconomic groups, the diagnostic
with meningitis (CSF), pneumo- workup of newborns should include CSF and
nia (trachea), sepsis (blood) blood cultures for detection of mycoplasmas. This
includes low-birth-weight and preterm newborns,
in whom traditional CSF cell counts and cultures
would be negative.
CSF, Cerebrospinal uid.
a
U. urealyticum has been implicated in nongonococcal urethritis, while U. parvum has not been implicated.

medium, such as SP4 (sucrose phosphate buffer, Mycoplasma
base, horse serum [20%], and neutral red) or Shepard 10B
broth or 2SP, which are designed for Mycoplasma. Cotton-
tipped swabs and wooden shafts should be avoided because
of possible inhibitory effects. Swabs should be made of
Dacron polyester or calcium alginate with aluminum or plas-
tic shafts, and the swabs should be removed when the sample
is placed in a transport medium. On arrival in the laboratory,
the specimens should be frozen at −70° C if plating within
24 hours is not possible.
asymptomatic carriage and acute infection. Patients with M.
pneumoniae infections can persistently harbor the organism
for various lengths of time after the acute infection. Therefore
it is difcult to interpret a positive PCR result. NAATs are less
valuable for the more rapidly growing mollicutes, M. homi-
nis and Ureaplasma spp., than the more difcult to isolate M.
pneumoniae and M. genitalium. At least four NAATs for molli-
cutes have U.S. Food and Drug Administration approval. The
BioFire Diagnostics (Salt Lake City, UT) FilmArray RP (respi-
ratory panel) detects nucleic acid from 14 respiratory patho-
gens, including M. pneumoniae, in nasopharyngeal swabs.

Nucleic acid amplication tests

Real-time PCR assays are the preferred method for the detec-
tion of many mollicutes. NAATs have the advantage of detect-
ing bacteria earlier in the course of infection than serology, are
more sensitive than serology, and have a rapid turnaround
time. PCR assays can also differentiate Ureaplasma spp. A
drawback to NAATs is that they cannot distinguish between
Culture
Because recovery from culture is difcult (sensitivity approx-
imately 40%), isolation of M. pneumoniae from respiratory
sites is infrequently attempted. Growth may take several
weeks, and technical expertise is necessary. M. hominis and
Ureaplasma spp. are less stringent in their growth requirements
564 PART 2 25 Mycoplasma and Ureaplasma

Table 25.5 Major clinical and corresponding diagnostic manifestations of Mycoplasma pneumoniae
Manifestation Days after onset
5 10 15 20 25 35 40
Headache and malaise +1 +3 +3 +2 +1
Dry cough +2 +4 +4 +1
Chest soreness +3 +3 +1
Fever
With antimicrobial treatment 104° F 104° F 102° F 100° F Absent
Without antimicrobial treatment 104° F 100° F Absent Absent Absent
Chest radiography +2 +3 +2 +2 +1
Mycoplasma culture with or without antimicrobial + + + + + +
treatment
Complement xation (titer) ≤8 8 32 64 256 256 128
Mycoplasma-specic Ig
IgM − + + + + + +
IgG − − + + + +/− −
+ 4, Most severe; + 1, least severe; +, present or positive; −, absent or negative; IgG, immunoglobulin G; IgM, immunoglobulin M.

but require cholesterol for synthesis of plasma membranes.
M. hominis is the only species that will grow on sheep blood
and chocolate agars. Diagnosis of M. pneumoniae infection is
usually established serologically, traditionally with acute-
phase and convalescent sera collected 2 to 3 weeks apart to
demonstrate a fourfold increase in titer. A representation of
classic clinical and corresponding diagnostic manifestations
of M. pneumoniae is shown in Table 25.5. As noted, many of
the early symptoms are nonspecic, and a thorough under-
standing of the disease process is necessary for interpretation
of serum and culture results.
Media
Several media have been developed for the recovery of molli-
cutes, and no single medium is suitable for all species isolated
from humans. Penicillin can be added to minimize bacterial
contamination. M. hominis and Ureaplasma spp. are more rapid
growers and relatively easy to recover compared with the other
mollicutes. SP4 broth and agar are ideal for M. pneumoniae and
M. hominis. M. pneumoniae and M. genitalium require glucose
(their major energy source), M. hominis requires arginine, and
Ureaplasma spp. require urea. Ureaplasma spp. also require media
to have a pH near 6.0 (Shepherd 10B arginine broth) with a buf-
fer to maintain the pH. It is difcult to sustain Ureaplasma spp. in
culture because death occurs rapidly when the urea is depleted,
and the bacteria are sensitive to changes in pH because of urea
utilization. M. hominis and U. urealyticum require cholesterol
for synthesis of plasma membranes and other undetermined
growth factors; fetal calf serum (20% vol/vol) is the traditional
nutrient source. A8 agar can be used as a solid medium to
recover M. hominis and Ureaplasma spp. Because mycoplasmas
do not produce turbidity in broth media, a pH indicator, such as
phenol red, should be added to detect growth.
Recovery of mycoplasma from blood can be performed by
placing uncoagulated blood into mycoplasmal broth media.
A ratio of 1:10 (blood to broth) and 10 mL of blood for adults
is recommended. Sodium polyanethol sulfonate (SPS), an
additive often found in commercial blood culture media, is
inhibitory to mycoplasma. The addition of 1% (weight per
volume) gelatin might help overcome the inhibitory effect
of SPS. Nevertheless, the use of commercial blood culture
media, whether or not used in automated instruments, is not
recommended. Fig. 25.5 presents a schematic representation
of media and methods used in the traditional procedures for
isolation and identication of Mycoplasma spp.
Fluids should be centrifuged and the pellet resuspended in
a small volume of liquid for inoculation of media. It is import-
ant that specimens be diluted in broth up to 10 3 before each
dilution is plated. This helps minimize the inhibitory effects of
antimicrobial agents, antibodies, and other antibacterial fac-
tors that might be present in the specimen.
Commercial culture media and kits for the detection and
recovery of mollicutes have been developed and are available
in the United States and Europe. Such products may detect,
quantify, identify, and determine the antimicrobial suscepti-
bility of genital mycoplasmas from urogenital specimens and
M. pneumoniae from respiratory secretions. These kits are use-
ful in laboratories that infrequently perform cultures for the
mollicutes, but laboratory scientists must be aware of the lim-
itations of these assays and perform internal quality control.
Isolation and identication
Once inoculated, broth media should be placed at 35° C under
atmospheric conditions, whereas solid agar media may be
incubated in an environment of room air enhanced with 5%
to 10% carbon dioxide (CO 2) or in an anaerobic atmosphere of
95% nitrogen gas (N2) with 5% CO2. M. hominis and Ureaplasma
spp. colonies may appear within 2 to 4 days, whereas M.
pneumoniae colonies may take 21 days or longer. Mycoplasma
like colonies are stained with Dienes or methylene blue stain.
Staining is performed by placing a small block of the agar
on a glass slide, covering the colony with the stain, adding a
coverslip, and examining the agar microscopically under low
power. M. hominis has a typical “fried egg” appearance, with
 Laboratory diagnosis 565

Collect respiratory specimen

(e.g., bronchoalveolar lavage, nasopharyngeal swab, throat swab, sputum)

Inoculate transport medium

(SP4* medium minus agar base, plus antimicrobial agents)

Inoculate transport medium

onto SP4 agar and incubate at 35to 37C
Incubate SP4 broth in CO2; perform weekly microscopic
with transport medium observation for small (10 to 100 m),
at 35 to 37C (no CO2) grainy colony with thin “apron,”†
hold for 4 weeks before reporting as negative

+
Color change in liquid phase + GP-RBC-HAD§
(+) (–) (+) (–)

Subculture to SP4 agar Subculture to SP4 agar Mycoplasma pneumoniae Mycoplasma sp.
and follow incubation and at 2 weeks and follow incubation (not M. pneumoniae)
observation procedure for and observation procedure for
SP4 agar SP4 agar

*SP4 is sucrose phosphate buffer, Mycoplasma base, fetal bovine serum (20%), phenol red. Medium stabilizes and
decontaminates specimen. Storage at –70° C for repeated testing is recommended.
†
Thin colony periphery. Examine with stereomicroscope using ×20 to ×60 magnification.
‡
Color change: positive, yellow color with no gross turbidity; negative, red color.
§
Guinea pig red blood cell hemadsorption (GP-RBC-HAD). -Hemolysis test for presumptive identification of
Mycoplasma pneumoniae may be used in lieu of GP-RBC-HAD.
Note: Methylene blue or Dienes stain can be used for detection of Mycoplasma spp. on SP4 agar; plate immunofluorescence
using labeled antibody and nucleic acid amplification can be used for identification.
Fig. 25.5 Flow diagram for Mycoplasma spp. isolation using classic traditional methods.

the periphery staining a light blue and the center dark blue
(Fig. 25.6). Mycoplasma spp. almost universally show a mixed
colony presentation on primary isolation when examined
with a stereomicroscope (Fig. 25.7).
Ureaplasma spp., once called T-strain mycoplasma (T for
“tiny”), form extremely small colonies that are difcult to see
with the naked eye; hence, mycoplasmal cultures on solid media
should always be examined with a stereomicroscope. Fig. 25.8
shows M. hominis and U. urealyticum grown on New York City
agar. Urease activity of Ureaplasma may be detected on solid
agar containing urea and manganese chloride (U9B urease color
test medium). Urease-positive colonies are a dark golden-brown
color because of the deposition of manganese dioxide.
Although uncommon, extragenital M. hominis infections
are emerging. This organism should be considered whenever Fig. 25.6 Dienes stain of Mycoplasma spp. colonies demonstrating typical fried
many polymorphonuclear cells are seen on Gram stain, but egg appearance (×40).
566 PART 2 25 Mycoplasma and Ureaplasma
Fig. 25.7 Typical mixed sizes of Mycoplasma spp. on primary isolation media,
Mycoplasma salivarium (×20). (Courtesy Bionique Testing Laboratories, Saranac
Lake, NY.)
Fig. 25.8 Mixed isolation of Mycoplasma hominis and Ureaplasma urealyticum
showing why U. urealyticum was originally called T-strain mycoplasma, T for “tiny”
(arrow) (×40).
there is no growth on routine bacterial culture. M. hominis
grows well anaerobically and will appear as pinpoint (0.05-
mm), clear, glistening, raised colonies on Columbia colis-
tin–nalidixic acid agar or anaerobic blood agar (Centers for
Disease Control and Prevention formula) in 48 hours. Under
anaerobic conditions, the colonies do not display the “fried
egg” morphology. The anaerobic plate should be examined
by using oblique light. The colonies that do not take up Gram
stain should be subcultured to A7 medium, on which they
demonstrate typical “fried egg” growth and stain positive
with Dienes or methylene blue stain if they are Mycoplasma
spp.
Although not conclusive, growth rate, body site recovered
from, and colony appearance can aid in the identication of
Mycoplasma. Glucose utilization in SP4 broth will cause an
acid shift producing a yellow color, whereas arginine metab-
olism will produce an increase in pH, changing the indicator
to a deeper red color. In 10B broth, urea or arginine utiliza-
tion will increase the pH, changing the pH indicator from
orange to deep red. A slow-growing mycoplasma from a
respiratory specimen producing a yellow color in SP4 broth
is likely M. pneumoniae. Production of an alkaline reaction in
10B broth after overnight incubation of a urogenital speci-
men is suggestive of U. urealyticum, whereas an alkaline shift
in media with arginine within 24 to 72 hours is likely caused
by M. hominis
Case check 25.2
In the Case in Point, an infection caused by U. urealyticum would
produce an alkaline shift in media containing urea in about
24 hours. If the infection was caused by M. hominis, an alkaline
shift would occur in media containing arginine in 24 to 72 hours.
Previously, identication of the mollicutes was often done by
typing methods using monoclonal antibodies and immuno-
uorescence and observation of plate media with a stereomi-
croscope. A direct plate immunouorescent method also can
be used. Fluorescent-labeled, anti–M. pneumoniae antibody
is ooded on colonies on the plate; the plate is then washed
and examined for immunouorescence. The characteristic of
guinea pig red blood cells (0.4% in saline) adhering to colo-
nies of M. pneumoniae and not M. hominis is another standard
assay that helps distinguish the two species. Furthermore,
guinea pig cells do not adhere to large-colony Mycoplasma
spp., which are common inhabitants of the upper respiratory
tract. PCR-based assays are now commonly used because
they are widely available, less subjective, and easier to per-
form. Matrix-assisted laser desorption/ionization–time-
of-ight mass spectrometry was shown to identify most
human mollicute species. However, testing requires 30 mL to
100 mL of culture for the mollicutes, limiting the use of this
method.
Serologic diagnosis
Because of the inherent difculties of cultures and interpre-
tations of a positive PCR assay result, M. pneumoniae has his-
torically been diagnosed by serologic methods. Optimally,
serum samples for serologic testing should be collected at the
onset of symptoms and 2 to 3 weeks later for acute-phase and
convalescent measurements. This delay in testing is an obvi-
ous drawback. However, using a single immunoglobulin M
(IgM) determination is awed. Some individuals, especially
those older than 40 years of age, might not produce IgM, pos-
sibly due to previous exposure. In addition, IgM antibody can
persist for weeks to months.
M. pneumoniae induces the formation of autoantibodies
that bind to a number of antigens on host cells, including the I
antigen found on human red blood cells. Because they agglu-
tinate type O Rh-negative erythrocytes at temperatures below
normal body temperature (optimal is 4° C), anti-I antibodies
are referred to as cold agglutinins. The cold agglutinin anti-
body titer was used for many years as an indicator of primary
atypical pneumonia, but it is insensitive and nonspecic for
M. pneumoniae. Approximately 50% of patients with primary
atypical pneumonia produce a detectable cold agglutinin
antibody titer. This assay is no longer recommended for the
diagnosis of M. pneumoniae infection.
Previously, the most used technique for demonstration of
M. pneumoniae–specic antibodies was the complement x-
ation assay, which was time-consuming and had inherent
technical problems. The assay also suffered from false-posi-
tive results caused by autoantibodies and cross-reactivity to
M. genitalium. Several commercially available enzyme immu-
noassays and immunouorescence assays are now available
for the detection of serum antibodies and, in some cases,

detect IgM or IgG. Table 25.6 highlights selected features of
these immunologic assays and other methods. It is important
to remember that demonstration of a signicant increase in
antibody titer in conjunction with culture isolation is prefera-
ble for a denitive diagnosis. Serologic methods are available
for M. hominis and U. urealyticum but are generally performed
only by reference laboratories and are not recommended for
routine diagnosis.
Antimicrobial susceptibility
The mollicutes are inherently resistant to the β-lactams (pen-
icillins and cephalosporins) as well as sulfonamides, tri-
methoprim, and rifampin because they lack a cell wall. M.
pneumoniae has remained susceptible to the tetracyclines,
newer uoroquinolones, and the macrolides (e.g., erythromy-
cin). However, there have been scattered reports of high-level
macrolide resistance. Roughly 15% of United States isolates
are macrolide resistant. Because of side effects, tetracycline
is used only for the treatment of adults. M. hominis, which
is more resistant than M. pneumoniae, is usually resistant to
erythromycin but susceptible to clindamycin and lincomycin,
whereas U. urealyticum is generally resistant to clindamycin
and lincomycin and susceptible to erythromycin. Both organ-
isms are often susceptible to tetracycline, but high-level resis-
tance is emerging and is common in some geographic areas.
Standard minimal inhibitory concentration methods for
susceptibility testing by broth microdilution and agar dilu-
tion of mycoplasma have been established by the Clinical and
Laboratory Standards Institute. The agar dilution method is
regarded as the reference method; however, because of the
high degree of technical expertise required and the few mol-
licute isolates, this assay is not offered by most hospital lab-
oratories. The broth microdilution is the most used method
to determine the minimal inhibitory concentration. With
antimicrobial resistance increasing, the availability of newer
broad-spectrum antimicrobials, and the emergence of more
Table 25.6 Comparative features of various laboratory methods used to detect Mycoplasma pneumoniae, Mycoplasma hominis, and
Ureaplasma urealyticum
Detection method Mycoplasma pneumoniae
Nonserologic
Culture Traditionally difcult
Polymerase chain Commonly used
reaction
Serologic
Complement xation Traditional assay, but need to demonstrate
fourfold increase between acute-phase and
convalescent sera; >32 single titer might
be suggestive
Immunouorescent Separately measures IgG and IgM
antibody
Enzyme immunoassay Separately or simultaneously measures IgG
and IgM
IgG, Immunoglobulin G; IgM, immunoglobulin M.
Interpretation of laboratory results 567
infections caused by Mycoplasma spp., antimicrobial suscepti-
bility testing methods are becoming more important. Because
of the variable susceptibility pattern of M. hominis, antimicro-
bial susceptibility testing is usually recommended for clini-
cally signicant isolates. These isolates should be forwarded
to a reference laboratory for testing.
Historically, M. pneumoniae had a predictable sensitivity
pattern, so antimicrobial susceptibility testing was not often
warranted. However, with high-level macrolide resistance
increasing, molecular assays were developed to detect muta-
tions in 23 S rRNA that results in macrolide resistance. These
assays can be performed directly on clinical specimens elimi-
nating the need for cultures to isolate the organism.
Interpretation of laboratory results
M. pneumoniae detected by any method from pulmonary or
nonpulmonary specimens should be considered signicant
and a pathogen. The high sensitivity of the PCR assay means
that a positive result must be correlated with the clinical
picture. Interpretation of M. hominis isolation is not as obvi-
ous; differentiation from colonization and infection requires
detailed clinical analysis and potentially repeated cultures.
Isolation from a normally sterile site is signicant.
U. urealyticum is the most difcult to assess clinically. In
urogenital specimens, it has been reported to colonize up
to 70% of men and 45% of women with no apparent infec-
tion. Its isolation is not indicative of pathogenicity, and it is
incumbent on the laboratory to educate the health care pro-
vider. Culture results should include a statement suggesting
its potential for colonization versus pathogenicity. In these
specimens, quantication is important. In sterile specimens,
particularly CSF isolates, it is reasonable to assume that iso-
lation is signicant.
Respiratory specimens received in the laboratory often
provide limited clinical information. Specimens are pro-
cessed and inoculated onto the appropriate media given the
Mycoplasma hominis Ureaplasma urealyticum
Method of choice, but must Method of choice using urease
differentiate infection from detection, but must differentiate
colonization infection from colonization
Occasionally used Occasionally used
Not recommended Not recommended
Not recommended Not recommended
Not recommended Not recommended
568 PART 2 25 Mycoplasma and Ureaplasma

Table 25.7 Laboratory detection of frequent respiratory pathogens

Epidemiologic factors Laboratory methods
Age Organism Disease Season Specimen Source Stain Culture Nonculture
Frequently
Involved
Newborn 1, 3, 4, 7 Pneumonia Year Tracheal suction Gram Routine,
rounda plus myco-
plasmal
Grade 2, 4 Atypical Fall and Sputum, Gram Routine, Mycoplasmal
school pneumonia winter nasopharynx plus myco- serology
plasmal
College 1, 2, 8 Biphasic disease Year Sputum, Nonec B. pertussis Mycoplasmal
student with pharyn- roundb nasopharynx serology
gitis and later,
bronchitis
Adult 2, 4, 5, 6 Pneumonia Year Sputum Gram Routine Mycoplasmal
or atypical rounda serology
pneumonia Bronchoalveolar Gram, Gomori Routine,
lavage specimen methenamine plus fungal
silver, and/
or calcouor
white
1, Chlamydia pneumoniae; 2, Mycoplasma pneumoniae (outbreak); 3, Mycoplasma hominis; 4, viral, e.g., adenovirus, respiratory syncytial virus, inuenza virus (seasonal);
5, other—fungus, Legionella, or Pneumocystis pneumonia; 6, Streptococcus pneumoniae; 7, Streptococcus agalactiae; 8, Bordetella pertussis .
a
Seasonal incidence depends on the pathogen.
b
The greatest seasonal incidence of pertussis is spring and summer, followed by winter.
c
In place of direct stains, nucleic acid amplication tests for C. pneumoniae and/or B. pertussis should be performed.

most likely candidate for the disease, clinical presentation,

age of the patient, and seasonality, recognizing that there LEARNING ASSESSMENT QUESTIONS
is a certain predictability with selected pathogens. Table
25.7 presents laboratory methods used to diagnose infec- 1. From which source did the neonate described in the Case in
tions caused by several pathogens—Mycoplasma, Chlamydia, Point likely acquire the infection?
Legionella, mycobacteria, fungi, and viruses—in various age a. Breast milk
groups. All respiratory specimens should be stored at −80° b. Inhalation after birth
C if storage time is likely to exceed 24 hours. Acute-phase c. Passed through the birth canal
sera should also be stored frozen for subsequent antibody d. Infectious agent crossed the placenta
titer testing. 2. Why was the Gram stain result negative in the Case in Point?
a. Mollicutes do not Gram stain.
b. The Gram stain is too insensitive.
c. Mollicutes do not retain safranin; need to use a different
POINTS TO REMEMBER counterstain.
d. When mollicutes are suspected, the time needed to heat
• The mollicutes are minute organisms characterized by the x the smear must be extended.
lack of a cell wall. Because of the lack of a cell wall, the myco- 3. How does primary atypical pneumonia caused by M. pneu-
plasmas are inherently resistant to the β-lactam antibiotics. moniae differ from pneumonia caused by S. pneumoniae?
• The most clinically signicant species of the a. Has a lower mortality rate
Mycoplasmataceae are M. pneumoniae, M. hominis, M. genita- b. Is not spread person to person
lium, M. fermentans, and Ureaplasma spp., although others are c. Is seen more often in older adults
beginning to be recognized as opportunistic pathogens. d. Is characterized by a bloody, productive cough
• M. pneumoniae is an important cause of community-acquired, 4. Which special stain is used on suspected colonies of
atypical pneumonia. It is not considered part of the normal Mycoplasma?
human microbiota. a. Acridine orange
• M. hominis, M. genitalium, and Ureaplasma spp. are genital b. Calcouor white
mollicutes. c. Dienes
• Because of the difculty to isolate, M. pneumoniae infection d. Silver
is most often diagnosed by serologic methods. M. hominis 5. An amniotic uid is submitted to the microbiology labora-
and Ureaplasma spp. are commonly diagnosed by culture, tory for isolation of mycoplasma. It will be 48 hours before
although PCR technology is also available. M. genitalium and the specimen can be processed. At what temperature should
M. fermentans are generally detected by PCR. the specimen be stored?
 Interpretation of laboratory results 569

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this organism? org/10.1371/journal.ppat.1009621.
Seo, H. Y. (2020). Immunochromatography for the diagnosis of
7. List the four common species of mollicutes associated with
Mycoplasma pneumoniae infection: a systematic review and
the genitourinary tracts of humans. meta-analysis. PLoS One, 15, e0230338.
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Zhu, X., et al. (2016). Epidemiology of Ureaplasma urealyticum and
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