Ebook Textbook of diagnostic microbiology (5th edition): Part 2

(BQ) Part 2 book Textbook of diagnostic microbiology presents the following contents: Laboratory identification of significant isolates, aboratory diagnosis of infectious diseases an organ system approach to diagnostic microbiology.

Virulence Factors and Pathogenicity

Infections Caused by Leptospires

Clinically Significant Species

Borrelia recurrentis and Similar Borreliae Borrelia burgdorferi

■ TREPONEMESGeneral CharacteristicsClinically Significant Species

Treponema pallidum subsp pallidum

Other Treponemal Diseases

CHAPTER OUTLINE

OBJECTIVES

After reading and studying this chapter, you should be able to:

1 Describe the general characteristics of the genera of spirochetes

2 List the risk factors associated with Borrelia spp endemic relapsing

fever infection

3 Describe the pathogenesis and clinical manifestations of Borrelia

spp infection, including high-risk factors

4 Compare the causative agents and arthropod vectors of relapsing

fever and Lyme disease

5 Describe the laboratory diagnosis of relapsing fever and how it

differs from the diagnosis of other spirochete diseases in the United

8 Discuss the epidemiology of leptospirosis in the United States

9 Evaluate the diagnostic tests used to identify Treponema pallidum in the clinical laboratory

10 Describe the two-tiered approach to laboratory diagnosis of Lyme disease

Case in Point

A 29-year-old man arrived at a local medical clinic in Los Angeles

complaining of diarrhea, fever, chills, muscle aches, and

head-aches He had returned 2 days earlier after competing in the

Eco-Challenge in Malaysian Borneo During the competition, he

had completed various events, including mountain biking,

caving, climbing, jungle trekking, swimming, and kayaking in

fresh and salt water He was still recovering from multiple

abra-sions from the jungle trekking and mountain biking While

kaya-king the Segama River, his kayak capsized and he had

inadvertently swallowed several mouthfuls of river water His two

teammates were on doxycycline as malaria prophylaxis before

and during the race Neither of them became ill

Issues to Consider

After reading the patient’s case history, consider:

■ Risk factors for acquiring infectious disease for the

■ Agents that cause influenza-like illness and methods to identify or rule out those agents

■ Effective prophylaxis, if available, for influenza-like illness

■ Empiric therapy options

Key TermsChancre

Endemic relapsing feverEndemic syphilisEpidemic relapsing feverErythema migrans (EM)Gummas

Jarisch-Herxheimer reactionLeptospirosis

Lyme borreliosisPinta

Endemic Relapsing feverRapid plasma reagin (RPR) test

SpirochetesSyphilisVenereal Disease Research Laboratory (VDRL) testWeil disease

YawsZoonoses

are very close together, so the organism may appear to be a chain

of cocci One or both ends of the organism have hooks rather than tapering off Their motion is rapid and rotational Histori-

cally, pathogenic organisms were identified as Leptospira rogans and saprophytes were categorized as Leptospira biflexa More than 200 different serovars (serotypes) of L interrogans

inter-sensu lato have been reported Although genetic typing has lished relatedness based on nucleic acid similarities and is taxo-nomically correct, serogroup-based nomenclature continues to be preferred by scientists and physicians

estab-Electron microscopy reveals a long axial filament covered by

a very fine sheath, similar to treponemes and borreliae All species have two periplasmic flagella The organisms cannot be readily stained, but they can be impregnated with silver and visualized Unstained cells are not visible by bright field micros-copy but are visible by dark field, phase contrast, and immuno-fluorescent microscopy Leptospires are obligately aerobic and can be grown in artificial media such as Fletcher’s semisolid, Stuart liquid, or Ellinghausen-McCullough-Johnson-Harris (EMJH) semisolid media

Virulence Factors and PathogenicityLeptospiral disease in the United States is caused by more than

20 different serovars, the most common of which are

Icterohaem-orrhagiae, Australis, and Canicola Some serovars of L gans sensu lato and L biflexa sensu lato are pathogenic for a wide

interro-range of wild and domestic animals and humans, but mechanisms

of pathogenicity are not well understood Factors that may play

a role in pathogenicity include reduced phagocytosis in the host,

a soluble hemolysin produced by some virulent strains, mediated sensitivity to leptospiral antigen by the host, and small amounts of endotoxins produced by some strains The clinical findings in animals with leptospirosis suggest the presence of endotoxemia

cell-Infections Caused by LeptospiresLeptospires present in water or mud are most likely to enter the human host through small breaks in the skin or intact mucosa The initial sites of multiplication are unknown Nonspecific host defenses do not stop multiplication of leptospires, and leptospi-remia occurs during the acute illness Late manifestations of the disease may 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 The onset of clinical illness is usually abrupt, with nonspecific, influenza-like constitutional symptoms such as fever, chills, headache, severe myalgia, and malaise The subsequent course is protean, frequently biphasic, and often results in hepatic, renal, and central nervous system involvement The major renal lesion is an interstitial nephritis with associated glomerular swelling and hyperplasia that does not affect the glomeruli The most characteristic physical finding

is conjunctival suffusion, but this is seen in less than 50% of

patients Severe systemic disease (Weil disease) includes renal

failure, hepatic failure, and intravascular disease and can result

in death Duration of the illness varies from less than 1 week to

3 weeks Late manifestations can be caused by the host’s nologic response to the infection In patients with a leptospiral bacteremia, immunoglobulin M (IgM) antibodies are detected

immu-The order Spirochaetales contains two families:

Leptospi-raceae and Spirochaetaceae The family LeptospiLeptospi-raceae

contains the genus Leptospira, and the family

Spirochae-taceae contains Borrelia and Treponema These three genera

include the causative agents of important human diseases such

as syphilis, zoonoses (transmitted from animals to humans) such

as leptospirosis, and vector-borne diseases such as Lyme

bor-reliosis or Lyme disease and relapsing fever.

The spirochetes are slender, flexuous, helically shaped,

uni-cellular bacteria ranging 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 flexible cell wall

around which several fibrils are wound These fibrils, termed the

periplasmic flagella (also known as axial fibrils, axial filaments,

endoflagella, and periplasmic fibrils), are responsible for motility

A multilayered outer sheath similar to the outer membrane of

gram-negative bacteria completely surrounds the protoplasmic

cylinder (the cytoplasmic and nuclear regions are enclosed by the

cytoplasmic membrane–cell wall complex and periplasmic

fla-gella) The spirochetes exhibit various types of motion in liquid

media They are free-living, or survive in association with animal

and human hosts as normal biota or pathogens In addition, they

can use carbohydrates, amino acids, long-chain fatty acids, or

long-chain fatty alcohols as carbon and energy sources

Metabo-lism can be anaerobic, facultatively anaerobic, or aerobic,

depending on the species Treponema reproduce via transverse

fission, whereas Leptospira and Borrelia divide by the more

common binary fission

Leptospires

General Characteristics

Organisms of the genus Leptospira are tightly coiled, thin,

flex-ible spirochetes, 0.1 µm wide and 5 to 15 µm long (Figure 23-1)

In contrast to both Treponema and Borrelia organisms, the spirals

FIGURE 23-1 Dark field image of Leptospira

interrogans sero-type Sejroe Wolffi 3705 The tight coils and bent ends are

characteristic of this organism (×1000) (Courtesy State Labo-ratories Division, Hawaii Department of Health.)

Isolation and Identification

Isolation of leptospires is accomplished by direct inoculation of

1 to 2 drops of freshly drawn blood or CSF into laboratory media such as Fletcher, Stuart, or EMJH, and incubating the media in the dark at room temperature Urine can also be cultured and is most productive after the first week of illness Several dilutions should be used (undiluted, 1 : 10, and 1 : 100) and/or filtered (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 field microscopy for tightly coiled, rapidly motile spirochetes, with hooked ends Serotypes have historically been identified by microscopic agglu-tination testing using sera of defined reactivity; however, other methods such as pulsed field gel electrophoresis and 16s rRNA DNA sequencing are also being investigated

in culture, so performance is typically limited to confirmatory laboratories

Antimicrobial SusceptibilitySusceptibility testing of leptospires is not normally performed in the clinical laboratory; leptospires have been shown to be sus-ceptible in vitro to streptomycin, tetracycline, doxycycline, and the macrolide antimicrobials in vitro Although treatment data are too sparse to be definitive, penicillin is considered beneficial and alters the course of the disease if treatment is initiated before the fourth day of illness Doxycycline appears to shorten the course

of the illness in adults and reduce the incidence of convalescent leptospiruria

within 1 week after onset of disease and may 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

Leptospirosis is a zoonoses primarily associated with

occupa-tional or recreaoccupa-tional exposure Working with animals or in

rat-infested surroundings poses hazards for veterinarians, dairy

workers, swine handlers, slaughterhouse workers, miners, sewer

workers, and fish and poultry processors In the United States,

most cases of leptospirosis disease result from recreational

expo-sures In California residents, 59% of leptospirosis cases were

acquired during freshwater recreation from 1982 to 2001; in the

last 5 of those years, the rate was 85% Leptospirosis ceased to

become a nationally notifiable disease in 1995 Leptospirosis is

still reportable in Hawaii, and from 1999 to 2008, Hawaii

aver-aged 20 confirmed cases annually Cases are likely unrecognized

nationwide and also go unreported in Hawaii

In the natural host, leptospires live in the lumen of renal

tubules and are excreted in the 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

car-riers Leptospires can survive in neutral or slightly alkaline

waters for months Protective clothing (boots and gloves) should

be worn in situations involving possible occupational exposure

to leptospires Control measures include rodent elimination and

drainage of contaminated waters Vaccination of dogs and

live-stock has been effective in preventing disease but not the initial

infection and leptospiruria Short-term prophylaxis consisting of

weekly doxycycline may be appropriate in high-risk groups with

expected occupational exposure

Laboratory Diagnosis

Specimen Collection and Handling

During the acute phase (first week) of the disease, blood or

cere-brospinal fluid (CSF) should be collected Optimal recovery

occurs if fresh specimens are inoculated directly into laboratory

media Urine can also be collected, but yield is much higher after

the first week of illness, and shedding can occur intermittently

for weeks

Microscopic Examination

Although direct demonstration of leptospires in clinical

speci-mens during the first week of the disease by special stains, dark

field, or phase contrast microscopy is possible, it is not

recom-mended Direct demonstration is only successful in a small

percentage of cases, and false-positive results may be reported

✓ 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 provider on

presentation Otherwise, the initial clinical impression might resemble

influenza, especially if presentation occurs during periods of high

influ-enza activity.

✓ Case Check 23-2

At least two deaths occurred in 2009, when confusion with pandemic influenza delayed appropriate antimicrobial therapy in patients with severe leptospirosis The Case in Point describes two teammates who were on doxycyline for malaria prophylaxis, which is also effective against many bacterial agents, including Leptospira Adherence to this preventive

medicine likely contributed to disease avoidance in these individuals.

Borreliae General Characteristics

The genus Borrelia comprises several species of spirochetes that

an adequate immune response The disease recurs several days

to weeks later, following a less severe but similar course The spirochetemia worsens during the febrile periods and wanes between recurrences

Epidemiology

Relapsing fever can be tick-borne (endemic relapsing fever) or louse-borne (epidemic relapsing fever) The tick-borne bor-

reliae are transmitted by a large variety of soft ticks of the genus

Ornithodoros Species-specific borreliae often bear the same epithet as their vectors (e.g., Ornithodoros hermsii transmits Borrelia hermsii) Tick-borne borreliae are widely distributed

throughout the Eastern and Western hemispheres, and sion to a vertebrate host takes place via infected saliva during tick attachment

transmis-Louse-borne fever is transmitted via the body louse, Pediculus humanus, and humans are the only reservoir The borreliae infect

the hemolymph of the louse Unlike tickborne disease, sion of the louse-borne disease occurs when infected lice are crushed and scratched into the skin rather than through the bite

transmis-of an infected arthropod Relapsing fever is best prevented by control of exposure to the arthropod vectors For tick-borne relapsing fever, exposure control includes wearing protective clothing, rodent control, and the use of repellents For louse-borne relapsing fever, control is best achieved by good personal and public hygiene, especially improvements in overcrowding and delousing

Laboratory Diagnosis

Microscopic Examination Diagnosis of borreliosis is readily made by observing Giemsa- or Wright-stained blood smears of blood taken during the febrile period Relapsing fever

is the only spirochetal disease in which the organisms are visible

in blood with bright-field microscopy The appearance of the spirochete among the red cells is characteristic (Figure 23-2)

Isolation and Identification Borreliae can be recovered using Kelly medium or animal inoculation (involving suckling

Swiss mice or suckling rats), but it is rarely attempted B rentis, B hermsii, Borrelia parkeri, Borrelia turicatae, and Bor- relia hispanica have been successfully cultivated Antigenic

recur-variation in the spirochetes that cause relapsing fever makes the serodiagnosis of their diseases difficult and impractical

Antimicrobial Susceptibility

Borreliae are susceptible to many antimicrobial agents; however, tetracyclines are the drugs of choice because they reduce the relapse rate and rid the central nervous system of spirochetes Studies indicate that up to 39% of patients treated with antimi-crobial 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

FIGURE 23-2 Appearance of Borrelia recurrentis (arrows) in

blood (Giemsa stain, ×850)

properties and host ranges Most species cause relapsing fever,

with the notable exception of Lyme borreliosis, which is caused

by several species in the Borrelia burgdorferi sensu lato complex

All pathogenic Borrelia are arthropod-borne.

The borreliae are highly flexible organisms varying in

thick-ness from 0.2 to 0.5 µm and in length from 3 to 20 µm The

spirals vary in number from 3 to 10 per organism and are much

less tightly coiled than those of the leptospires (Figure 23-2)

Unlike the leptospires and treponemes, the borreliae stain easily

and can be visualized by bright field microscopy Electron

microscopy shows the same general features as are seen with the

treponemes—long, periplasmic flagella (15 to 20/cell) coated

with sheaths of protoplasm and periplasm The borreliae are

typi-cally cultivated in the clinical laboratory using Kelly medium

Clinically Significant Species

A number of borreliae, including Borrelia recurrentis and

Bor-relia duttonii, cause relapsing fever The complex B burgdorferi

sensu lato causes a spectrum of syndromes known as Lyme

disease

Borrelia recurrentis and

Similar Borreliae

Virulence Factors

As the disease name suggests, relapsing fever is characterized by

acute febrile episodes that subside spontaneously but tend to

recur over a period of weeks Borrelia spp responsible for this

disease first evade complement by acquiring and displaying

sup-pressive complement regulators, C4b-binding protein and factor

H The relapses are potentiated by antigenic variation; the

bor-reliae systematically change their surface antigens, thereby

ren-dering specific antibody production ineffective in completely

clearing the organisms

Clinical Manifestations

After an incubation period of 2 to 15 days, a massive

spiro-chetemia develops and remains at varying levels of severity

during the entire course of relapsing fever The infection is

accompanied by sudden high temperature, rigors, severe

head-ache, muscle pains, and weakness The febrile period lasts

about 3 to 7 days and ends abruptly with the development of

and symptoms are consistent with Lyme disease, 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 and amoxicillin are equally effective in treating early stages of Lyme disease without com-plications For refractile or late stages, prolonged treatment with ceftriaxone has been effective

Treponemes General CharacteristicsPathogenic treponemes are thin, spiral organisms about 0.1 to 0.2 µm in thickness and 6 to 20 µm in length They are difficult

to visualize with a bright field microscope because they are so thin, but they can be seen very easily using dark field microscopy The spirals are regular and angular, with 4 to 14 spirals per organism (Figure 23-3) Three periplasmic flagella are inserted into each end of the cell The ends are pointed and covered with

a sheath The cells are motile, with graceful flexuous movements

in liquid

Clinically Significant 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 pertenue, the causative agent of yaws; T pallidum subsp endemicum, the causative agent

of endemic syphilis; and Treponema carateum, the causative

agent of pinta The four pathogenic strains exhibit a high degree

of DNA homology and shared antigens At least six genic species have been identified in the normal microbiota, and they are particularly prominent in the oral cavity

nonpatho-Treponema pallidum Subsp pallidum

Virulence Factors

Treponema pallidum subsp pallidum has the ability to cross

intact mucous membranes and the placenta, disseminate

Binding factor H allows for complement evasion and immune

system suppression and might explain, in part, why IgM antibody

does not peak for 3 to 6 weeks In vitro, the organism can

stimu-late proinflammatory cytokines such as tumor necrosis factor and

interferons, which can be important in controlling disease but

may also contribute to inflammatory 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 first of which is localized (stage 1) About 60% of patients

exhibit erythema migrans (EM), the classic skin lesion that

is normally found at the site 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

appearance Regional lymphadenopathy is common with minor

constitutional symptoms Stage 2 is early disseminated and

produces widely variable symptoms that include secondary skin

lesions, migratory joint and bone pain, alarming neurologic

and cardiac pathology, splenomegaly, and severe malaise and

fatigue Late manifestations, or late persistent infections (stage

3), focus on the cardiac, musculoskeletal, and neurologic

systems Arthritis is the most common symptom, occurring

weeks to years later

Epidemiology

Organisms are transmitted via the bite of infected Ixodes ticks,

so most cases occur during June through September, when more

people are involved in outdoor activities and ticks are more

active Lyme disease was first described after an outbreak among

children in Lyme, Connecticut, in 1975 A total of 33,097 cases

were reported in the United States in 2011

At least three species of B burgdorferi sensu lato cause Lyme

disease (Lyme borreliosis) B burgdorferi sensu stricto occurs in

North America B garinii and B afzelii have been confirmed in

Asia, and all three species have caused disease in Europe

Protec-tive clothing and repellents should be worn in areas in which tick

exposure is intense Attached ticks should be removed

immedi-ately because pathogen transmission is associated with the length

of attachment

Laboratory Diagnosis

Specimen Collection and Handling The most common

and productive specimen collected for the laboratory diagnosis

of B burgdorferi sensu lato infection is serum for serology Other

tests have too many limitations (e.g., polymerase chain reaction)

or have not been adequately validated (e.g., urine antigen, CD57

lymphocyte)

Serologic Tests Diagnosis follows a two-tiered approach

in which the first step is an immunofluorescent antibody (IFA)

or enzyme immunoassay (EIA) screen Positive or equivocal

results are confirmed with IgM and/or just IgG Western blot,

depending on whether symptoms were present for longer than

30 days (IgG only) Western blot confirmation of IgM antibody

presence includes reactivity for two of the three following

bands—24, 39, and 41 kDa Confirmation of IgG antibody

pres-ence is acceptable when five of the scored bands are present—18, FIGURE 23-3pallidum. Two treponemes are shown adjacent to an erythro- Scanning electron micrograph of Treponema

patients exhibit a biologic cure, losing serologic reactivity Another third remain latent for life but have reactive serology The remaining 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 liver (benign tertiary syphilis), degenerative changes in the central nervous system (neurosyphilis), and syphilitic cardiovas-cular lesions, particularly aortitis, aneurysms, and aortic valve insufficiency 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 ter-tiary stage is reached

Congenital Syphilis Treponemes can be transmitted from

an infected mother to her fetus by crossing the placenta genital syphilis affects many body systems and is therefore severe and mutilating Early-onset congenital syphilis, onset at less than 2 years of age, is characterized by mucocutaneous lesions, osteochondritis, anemia, hepatosplenomegaly, and central nervous system involvement and occurs when mothers have early syphilis during pregnancy Late-onset congenital syphilis 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, eighth nerve deafness, neu-rosyphilis, and other tertiary manifestations

Con-Epidemiology

Treponema pallidum subsp pallidum is an exclusively human

pathogen under natural conditions Syphilis was first recognized

in Europe at the end of the fifteenth century, when it reached epidemic proportions Two theories have been proposed concern-ing the introduction of syphilis to Europe The first theory sug-gests that Christopher Columbus’ crew brought the disease from the West Indies back to Europe The second theory suggests that the disease was endemic in Africa and transported to Europe via the migration of armies and civilians The venereal transmission

of syphilis was not recognized until the eighteenth century The causative agent of syphilis was not discovered until 1905.The incidence of syphilis in the United States dropped through the 1990s, and the fewest cases since reporting began in 1941 (31,618) was reached in 2000 However, since 2000 the disease has increased, peaking at 46,290 cases in 2008 The next 2 years saw only a slight decrease from the peak number of cases at 44,830 in 2009 and 46,042 in 2011 High-risk sexual behavior and coinfection with HIV continue to complicate syphilis control efforts Educating people about sexually transmitted diseases, including the proper use of barrier contraceptives, reporting each case of syphilis to the public health authorities for contact inves-tigation, and treating all sexual contacts of persons infected with syphilis are cornerstones of syphilis control efforts Sero-logic screening of high-risk populations should be performed, and to avoid congenital syphilis, pregnant women should have serologic examinations early and late in their pregnancy

Laboratory Diagnosis

Specimen Collection and Handling Lesions of primary and secondary syphilis typically contain large numbers of

throughout the body, and infect almost any organ system It has

also been postulated that antigenic variation of cell surface

pro-teins 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 afflicted with the

disease as punishment for cursing the gods The poem

repre-sented the compendium of knowledge at the time regarding the

disease

Treponema pallidum subsp pallidum transmission normally

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 inoculation Syphilis can

also be acquired by nongenital contact with a lesion (e.g., on the

lip) or transplacental transmission to a fetus, resulting in

congeni-tal syphilis After bacterial invasion through a break in the

epi-dermis or penetration through intact mucous membranes, the

natural course of syphilis can be divided into primary, secondary,

and tertiary stages based on the clinical manifestations

Coinfec-tion with human immunodeficiency virus (HIV) can result in

variation of the natural course of the disease Furthermore, ulcers

caused by syphilis may contribute to the efficiency of HIV

trans-mission in populations 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 After inoculation, the

spiro-chetes multiply rapidly and disseminate to local lymph nodes and

other organs via the bloodstream The primary lesion develops

10 to 90 days after infection and is a result of an inflammatory

response to the infection at the site of the inoculation The lesion,

known as a chancre, is typically a single erythematous lesion

that is nontender but firm, with a clean surface and raised border

The lesion is teeming with treponemes and is extremely

infec-tious Because the chancre is commonly found on the cervix or

vaginal wall and is nontender, the lesion might not be obvious

The lesion can also be found in the anal canal of both genders

and remain undetected No systemic signs or symptoms are

evident in the primary stages of the disease

Secondary Stage of Syphilis Approximately 2 to 12

weeks after development of the primary lesion, 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 membranes 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

Tertiary Stage of Syphilis After the secondary stage

heals, individuals are not contagious; however, relapses of

sec-ondary syphilis occur in about 25% of untreated patients

Fol-lowing the secondary stage, patients enter latent syphilis, when

clinical manifestations are absent Latency within 1 year of

infec-tion is referred to as early latent, whereas latency greater than 1

year is late latent syphilis Approximately one third of untreated

results Consequently, treponemal tests are also not useful in lowing therapy or detecting reinfection.

fol-The treponemal antigens used are spirochetes derived from rabbit testicular lesions Two commonly used treponemal test

methods are the Treponema pallidum–particulate agglutination

(TP-PA) Test (Fujirebio America, Fairfield, NJ) and EIAs The

TP-PA test uses gelatin particles sensitized with T pallidum

anti-gens Agglutination indicates the presence of anti-treponemal antibodies EIA kits are simple to perform, commercially avail-able, and comparable to other treponemal tests The fluorescent treponemal antibody absorption (FTA-ABS) assay utilizes a fluorescent-labeled anti-human antibody that detects patient anti-treponemal antibodies bound to treponema affixed to a commer-cially prepared slide Because of subjectivity in reading the samples and the use of expensive fluorescent microscopy, the FTA-ABS test has become less frequently used in favor of the EIAs

Antimicrobial Susceptibility

Penicillin is the drug of choice for treating patients with syphilis

It is the only proven therapy that has been widely used for patients with neurosyphilis, congenital syphilis, and syphilis during pregnancy Resistant strains have not developed Long-acting penicillin such as benzathine penicillin is preferred Alter-native regimens for patients who are allergic to penicillin and not pregnant include doxycycline, tetracycline, and chlorampheni-col A typical Jarisch-Herxheimer reaction and exacerbation of cutaneous lesions can occur within hours following treatment.Other Treponemal Diseases

Three nonvenereal treponemal diseases—yaws, pinta, and endemic syphilis—occur in different geographic locations These treponematoses are found in developing countries in which hygiene is poor, little clothing is worn, and direct skin contact is common because of overcrowding All three diseases have primary and secondary stages, but tertiary manifestations are uncommon All diseases respond well to penicillin or tetracy-cline These infections are rarely transmitted by sexual contact, and congenital infections do not occur

spirochetes The surface of primary or secondary lesions is

cleaned with saline and gently abraded with dry, sterile gauze;

bleeding should not be induced Serous transudate is placed onto

a slide, diluting with nonbacteriocidal saline if the preparation is

too thick A coverslip is added and the slide is transported

imme-diately to a laboratory where dark field microscopy is performed

Oral lesions should not be examined because numerous

non-pathogenic spirochetes present in these specimens will lead to

misinterpretation Culture methods are not available and dark

field microscopy equipment and expertise are uncommon, so

serology is the normal basis of diagnosis

Microscopic Examination Organisms are too thin to be

observed by bright field microscopy, so spirochetes are

illumi-nated against a dark background Dark field microscopy requires

considerable skill and experience; however, demonstration of

motile treponemes in material from the chancre is diagnostic for

primary syphilis

Serologic Tests Serology is the primary method used for

the laboratory diagnosis of syphilis Two major types of serologic

tests exist, nontreponemal tests and treponemal tests Both have

lower sensitivities in the primary stage, but approach 100% in

the secondary stage of syphilis The treponemal tests retain a very

high sensitivity in the tertiary stage as well A coinfection with

HIV can result in false-negative serologic test results

Compari-sons between CSF and serum antibody responses can be helpful

in potential cases of neurosyphilis With congenital syphilis,

comparing antibody responses in the mother’s and baby’s serum

can aid diagnosis

The nontreponemal tests detect reaginic antibodies that

develop against lipids released from damaged cells Although

they are biologically nonspecific and known to react with

organ-isms of other diseases and conditions (causing false-positive

reactions), the nontreponemal tests are excellent screening tests

The antigen used is a cardiolipin-lecithin complex made from

bovine hearts

The two nontreponemal tests widely used today are the

Vene-real Disease Research Laboratory (VDRL) and rapid plasma

reagin (RPR) tests These tests are inexpensive to perform,

dem-onstrate rising and falling reagin titers, and correlate with the

clinical status of the patient The VDRL test uses a cardiolipin

antigen that is mixed with the patient’s serum or CSF

Floccula-tion occurs in a positive reacFloccula-tion and is observed microscopically

The RPR test is more commonly used; it uses carbon particles

and is read macroscopically When mixed with a positive serum

on a disposable card, the black charcoal particles clump together

with the cardiolipin-antibody complexes The flocculation is

easily observed without a microscope Reactive or weakly reactive

sera should undergo titration and be tested with treponemal tests

The treponemal tests detect antibodies specific for treponemal

antigens Historically, they have been used to confirm positive

nontreponemal test results, although some laboratories use

reverse sequence syphilis screening In this strategy, automated

treponemal test–positive sera is tested with nontreponemal and a

second treponemal assay This algorithm resulted in higher

numbers of false-positives in five 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

Centers for Disease Control and Prevention: Lyme disease: resources for

clinicians: diagnosis, treatment, and testing, 2013 Available at:

http://www.cdc.gov/lyme/healthcare/clinicians.html Accessed June

30, 2013.

Centers for Disease Control and Prevention: 2010 sexually transmitted

syphilis.htm Accessed September 4, 2012.

Centers for Disease Control and Prevention: Sexually transmitted

dis-eases treatment guidelines, 2010, MMWR 59(RR-12):1, 2010

Avail-able at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6131a3 htm?s_cid=mm6131a3_w Accessed November 14, 2013.

Centers for Disease Control and Prevention: Summary of notifiable

diseases—United States, 2011, MMWR 60(53):1, 2013 Available at:

http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6053a1.htm Accessed November 13, 2013.

Hawaii Department of Health Communicable Disease Division:

Com-municable disease report, September/October 2004 Personal

com-munication for 2004 leptospirosis data.

Kassutto S, Doweiko JP: Syphilis in the HIV era, Emerg Infect Dis

10:1471, 2004 Available at: http://wwwnc.cdc.gov/eid/article/10/8/ 03-1107_article.htm Accessed November 14, 2013.

Katz AR, et al: Leptospirosis in Hawaii, USA, 1999–2008, Emerg Inf

17/2/10-1109_article.htm Accessed November 14, 2013.

Levett PN: Leptospira In Versalovic J, et al, editors: Manual of clinical

microbiology, ed 10, Washington, DC, 2011, ASM Press, p 916.

Lo Y-C, et al: Severe leptospirosis similar to pandemic (H1N1) 2009,

Florida and Missouri, USA, Emerg Infect Dis 17:1145, 2011

Avail-able at: http://wwwnc.cdc.gov/eid/article/17/6/10-0980_article.htm Accessed November 14, 2013.

Meri T, et al: Relapsing fever spirochetes Borrelia recurrentis and

B duttonii acquire complement regulators C4b-binding protein and

factor H, Infect Immun 74:4157, 2006.

Radolf JD, et al: Treponema and Brachyspira, human host–associated spirochetes In Versalovic J, et al, editors: Manual of clinical micro-

biology, ed 10, Washington, DC, 2011, ASM Press, p 941.

Schriefer ME: Borrelia In Versalovic J, et al, editors: Manual of clinical

microbiology, ed 10, Washington, DC, 2011, ASM Press, p 924.

Sejvar JB, et al: Leptospirosis in “Eco-Challenge” athletes, Malaysian

Borneo, 2000, Emerg Infect Dis 9:702, 2003 Available at:

http://wwwnc.cdc.gov/eid/article/9/6/02-0751_article.htm Accessed November 14, 2013.

Steere AC: Borrelia burgdorferi (Lyme disease, Lyme borreliosis) In Mandell GL, et al, editors: Mandell, Douglas, and Bennett’s princi-

ples and practice of infectious diseases, ed 7, Philadelphia, 2010,

Churchill Livingstone Elsevier, p 3071.

Pinta

Pinta, caused by T carateum, is found in the tropical regions of

Central and South America It is acquired by person to person

contact and is rarely transmitted by sexual intercourse Lesions

begin as scaling, painless papules and are followed by an

ery-thematous rash that becomes hypopigmented with time

Points to Remember

■ Spirochetes are slender, flexuous, helically shaped bacteria.

■ Leptospires are most likely to enter the human host through small

breaks in the skin or intact mucosa.

■ The incubation period of leptospirosis is usually 10 to 12 days but

ranges from 3 to 30 days after inoculation The onset of clinical

illness is generally abrupt, with nonspecific, influenza-like

consti-tutional symptoms such as fever, chills, headache, severe myalgia,

and malaise.

■ The pathogenic borreliae commonly are arthropod-borne (by a tick

or louse) and cause relapsing fever and Lyme disease.

are caused by immune evasion, including antigenic variation

During the course of a single infection, borreliae systematically

change their surface antigens.

■ During the febrile period, diagnosis of relapsing fever is readily

made by Giemsa or Wright staining of blood smears Relapsing

fever is the only spirochetal disease in which the organisms are

visible in blood with bright field microscopy.

■ Laboratory diagnosis of Lyme borreliosis caused by B burgdorferi

sensu lato is accomplished by a two-tiered serology Initial positive

or equivocal EIA results are confirmed with Western blot.

■ Treponemes can cross the placenta and be transmitted from an

infected mother to her fetus Congenital syphilis affects many body

systems and is therefore severe and mutilating All pregnant

women should have serologic testing for syphilis early in

pregnancy.

Learning Assessment Questions

1 What are the general characteristics of spirochetes?

2 What risk factors are associated with Borrelia spp endemic

relapsing fever infection?

3 Which tickborne species of Borrelia is associated with a skin rash

or lesion?

4 What is the significance on infectious disease transmission of

finding partially engorged ticks attached to the skin?

5 What is the test of choice for the laboratory diagnosis of

relaps-ing fever borreliosis?

6 Name the four strains of the genus Treponema that are

patho-genic for humans.

7 What are the stages of a Treponema pallidum subsp pallidum

infection? Is the final stage usually seen in developed countries?

8 Where are most cases of leptospiroses contracted within the

United States, and why is this important when considering the

typical incubation period of the infection?

9 Compare the difference(s) between treponemal and mal tests for syphilis.

nontrepone-10 What is the recommended methodology for laboratory diagnosis

of Lyme borreliosis?

CHAPTER

and Similar Organisms

Donald C Lehman and Connie R Mahon *

Anaplasmataceae

Coxiella

CHAPTER OUTLINE

OBJECTIVES

After reading and studying this chapter, you should be able to:

1 List the members of the family Chlamydiaceae

2 Discuss the unique growth cycle of Chlamydia, describing

elementary and reticulate bodies

3 Compare and contrast Chlamydia and Rickettsia and distinguish

them from other bacteria and viruses

4 Discuss the most important human diseases caused by the

Chlamydia, Chlamydophila, and Rickettsia, species and similar

microorganisms

5 Describe the modes of transmission for each species of Chlamydia,

Chlamydophila, Rickettsia, and similar microorganisms

6 Compare the epidemiology and pathogenesis of the serovars of

Chlamydia trachomatis

7 Evaluate the available assays for the laboratory diagnosis of

C trachomatis and Chlamydophila pneumoniae infections

8 Discuss the problems with serologic cross-reactivity among the rickettsial species

9 For the following human rickettsial diseases, link the causative agent and compare the mode of transmission to humans:

A 7-day-old newborn girl was brought by her grandmother to

the emergency department of a large city hospital She had been

discharged 3 days after birth, with the last nursing note

indicat-ing that the child was “fussy.” The child presented to the

emer-gency department with a fever of 39° C, loss of appetite, a

profuse yellow discharge from the right eye, and general

irritabil-ity Medical history revealed the mother to be a 17-year-old

intravenous drug abuser with no prenatal care, who had a

vaginal delivery in the parking lot of a local hospital Eye charge and cell scrapings were cultured Routine bacterial cul-tures were negative; however, a rapid nucleic acid amplification test was diagnostic

dis-Issues to Consider

After reading the patient’s case history, consider:

■ The various organisms that can be recovered from tive material from newborns

exuda-■ The clinical infections and disease spectrum associated with these organisms

■ How these organisms are transmitted and the risk factors associated with the diseases produced

of Chlamydia exist, but they are rarely isolated from humans

The creation of a second genus was somewhat controversial and

is still being debated Therefore, readers may find both taxonomic classifications in published literature

The term rickettsiae can specifically refer to the genus ettsia or it can refer to a group of organisms included in the order

Rick-Rickettsiales There has been significant reorganization in the order Rickettsiales in recent years The order includes the fami-lies Rickettsiaceae and Anaplasmataceae The family Rickettsia-

ceae includes the genera Rickettsia and Orientia The family Anaplasmataceae includes the genera Ehrlichia, Anaplasma, Cowdria, Neorickettsia, and Wolbachia As a result of this reorganization, Coxiella has been removed from the family

Rickettsiaceae

Chlamydiaceae General Characteristics

As shown in Table 24-2, initial differentiation of the Chlamydia

spp was based on selected characteristics of the growth cycle, susceptibility to sulfa drugs, accumulation of glycogen in inclu-sions, and DNA relatedness Table 24-2 also lists additional prop-erties of the Chlamydiaceae species that have helped further differentiate the three human species on the basis of natural host, major diseases, and number of antigenic variants (i.e., serovars)

The genus Chlamydia is in the family Chlamydiaceae;

members of the family share selected characteristics

genus Chlamydia, four species were previously recognized—

C pecorum, C pneumoniae, C psittaci, and C trachomatis All

except C pecorum have been associated with human disease

Based on analysis of 16S and 23S rRNA gene sequences, a

revised taxonomic classification has been accepted The family

Chlamydiaceae now consists of two genera: (1) Chlamydia

to include C trachomatis; and (2) Chlamydophila to include

C pneumoniae, C psittaci, and C pecorum Other named species

Reiter syndromeReticulate body (RB)Trachoma

TABLE 24-1 Comparative Properties of Microorganisms

Characteristic

Organisms

Obligate intracellular parasites − + + − + Peptidoglycan in cell wall + + − − − Growth on nonliving medium + − − + −

Sensitivity to antimicrobial agents + + + + − Sensitivity to interferon − + − − + Binary fission (replication) + + + + −

+, Characteristic is present; −, characteristic is absent.

TABLE 24-2 Initial Differentiation of Chlamydiaceae Species

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 diseases

Trachoma Lymphogranuloma venereum

Pneumonia Pharyngitis Bronchitis

Pneumonia FUO Number of serovars 20 1 10

FUO, Fever of unknown origin; +, characteristic is present; −, characteristic is absent.

separated C trachomatis into 20 serovariants, or serovars (Table24-3) The trachoma biovar includes serovars A through K Serovars A, B, Ba, and C are associated with the severe eye infection trachoma, whereas serovars D through K, Da, Ia, and

Ja are associated with inclusion conjunctivitis, a milder eye tion, and urogenital infections Serovars L1, L2, L2a, L2b, and L3

infec-are associated with lymphogranuloma venereum (LGV), an

Chlamydiae are deficient in energy metabolism and are

there-fore obligate intracellular parasites Their unique growth cycle

involves two distinct forms, an elementary body (EB), which is

infectious, and a reticulate body (RB), which is noninfectious

The growth cycle (Figure 24-1) begins when the small EB infects

the host cell by inducing energy-requiring active phagocytosis

In vivo, host cells are primarily the nonciliated, columnar, or

transitional epithelial cells that line the conjunctiva, respiratory

tract, urogenital tract, and rectum During the next 8 hours, they

organize into larger, less dense RBs, which divert the host cell’s

synthesizing functions to their own metabolic needs and begin to

multiply by binary fission About 24 hours after infection, the

dividing organisms begin reorganizing into infective EBs At

about 30 hours, multiplication ceases, and by 35 to 40 hours, the

disrupted host cell dies, releasing new EBs (Figure 24-2) that can

infect other host cells, continuing the cycle

The EB has an outer membrane similar to that of many

gram-negative bacteria The most prominent component of this

mem-brane is the major outer memmem-brane protein (MOMP) The MOMP

is a transmembrane protein that contains both species-specific

and subspecies-specific epitopes that can be defined by

monoclo-nal antibodies The chlamydial outer membrane also contains

lipopolysaccharide (LPS) This extractable LPS, with

ketode-oxyoctonate, is shared by most members of the family and is the

primary antigen detectable in genus-specific tests and serologic

assays for the chlamydiae

Chlamydia trachomatis

C trachomatis has been divided into three biovars—trachoma,

lymphogranuloma venereum, and mouse pneumonitis (renamed

30 hours

Multiplication cessation

35 to 40 hours

Release

Elementary body

FIGURE 24-2 Elementary bodies and cells in Chlamydia

Microtrak, Palo Alto, CA.)

C trachomatis is unique in that it carries 10 stable plasmids

whose function is currently unknown This unique characteristic

is a major reason for the applications of nucleic acid

amplifica-tion by polymerase chain reacamplifica-tion (PCR) assay and identificaamplifica-tion

by hybridization

Clinical Infections

Trachoma C trachomatis causes the chronic eye infection

trachoma (Figure 24-3), 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 are seen in climates with high temperature and high

humidity; they are not commonly seen in the United States

These serovars produce a chronic infection resulting in scarring

and continual abrasion of the cornea as the eyelid turns

down-ward todown-ward the cornea and, if left untreated, infection generally

ends in blindness in adults The World Health Organization

FIGURE 24-3 Conjunctival scarring and hyperendemic

blind-ness caused by Chlamydia trachomatis in ocular infections FIGURE 24-4 Inguinal swelling and lymphatic drainage caused

by Chlamydia trachomatis serovars L1 , L 2a , L 2b , or L 3 —that is, lymphogranuloma venereum

TABLE 24-3 Human Diseases Caused by Chlamydiaceae Species

D, Da, E, F, G, H, I, Ia, J, Ja, K Inclusion conjunctivitis (adult and newborn)

Nongonococcal urethritis Cervicitis

Salpingitis Pelvic inflammatory disease Endometritis

Acute urethral syndrome Proctitis

Epididymitis Pneumonia of newborns Perihepatitis (Fitz-Hugh-Curtis syndrome)

Humans

L1, L2, L2a, L2b, L3 Lymphogranuloma venereum

Pharyngitis Influenza-like febrile illness

Humans

Endocarditis Abortion

Birds

*Predominant serovars associated with disease.

estimates that 1.3 million people are blind because of trachoma Prevention includes either or both antimicrobial treatment and a simple surgical procedure on the eyelid

Lymphogranuloma Venereum C trachomatis serovars

L1, L2, L2a, L2b, and L3 cause LGV, a sexually transmitted disease (STD); these serovars are more invasive than the others In LGV, patients have inguinal and anorectal symptoms (Figure 24-4) The serovars causing LGV are able to survive inside mononu-clear cells The bacteria enter the lymph nodes near the genital tract and produce a strong inflammatory response that often

results in bubo formation and subsequent rupture of the lymph

node LGV is uncommon in the United States and is usually seen

in immigrants from and returning travelers to countries in which the disease is endemic, typically the tropics and subtropics The LGV serovars have also been linked to Parinaud oculoglandular conjunctivitis

Pneumocystis The incubation period is variable, but symptoms

generally appear 2 to 3 weeks after birth

Other Urogenital Diseases C trachomatis infections in

adult men include nongonococcal urethritis (NGU), epididymitis,

and prostatitis Serovars D through K are associated with these

clinical infections, which can be persistent and subclinical, as

well as acute and demonstrable 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 chlamydial infection are also

infected The same serovars can produce a conjunctivitis in males

and females

Infections in adult women include urethritis, follicular

cervi-citis (leukorrhea hypertrophic cervical erosion), endometritis,

proctitis, salpingitis, pelvic inflammatory disease (PID), and

perihepatitis Reiter syndrome (urethritis, conjunctivitis,

poly-arthritis, and mucocutaneous lesions) in adults is believed to be

caused by C trachomatis Salpingitis can lead to scarring and

dysfunction of the oviductal transport system, resulting in

infer-tility or ectopic pregnancy In the United States, this is a major

cause of sterility Most infections in women and men can remain

asymptomatic, which facilitates the spread of the bacteria by

unprotected sexual contact

C trachomatis is the most common sexually transmitted

bac-terial pathogen in the United States In 2011, a total of 1,412,791

cases of genital infections were reported, but many infections are

undiagnosed, and the Centers for Disease Control and Prevention

(CDC) estimates that 2 to 3 million new cases occur annually in

the United States The number of reported cases has been

increas-ing by over 5% annually since 1997 Only genital warts, caused

by the human papillomavirus, is a more common sexually

trans-mitted disease in the United States Neisseria gonorrhoeae is a

distant third, with 321,849 confirmed cases in 2011 and an

esti-mated 600,000 new cases annually The reported rate of

chla-mydial infections in women increased from 496.5 cases/100,000

women in 2005 to 610.6 cases/100,000 women in 2010, an

increase of about 23% The rate in males was 233.7 The CDC

attributes the higher rate in women and the continued increase in

the reported national C trachomatis infection rate in women to

improved screening, increased use of nucleic acid amplification

tests, better reporting, and ongoing high burden of disease

Chlamydial Infection in the Newborn Traveling

through an infected birth canal, infants can be infected with

Chlamydia spp Chlamydial infection in an infant delivered by

cesarean section is rare, and infection from seronegative mothers

has not been reported Infants suffering from chlamydial

infec-tion can experience conjunctivitis, nasopharyngeal infecinfec-tions,

and pneumonia Table 24-4 shows selected features associated

with neonatal inclusion conjunctivitis The portal of entry is

ocular or aspiration, with colonization of the oropharynx being

a necessary event before infection Between 20% and 25% of

neonates born to Chlamydia culture–positive mothers develop

conjunctivitis, 15% to 20% develop nasopharyngeal infection,

and 3% to 18% develop pneumonia Otitis media is a less

fre-quent infection Infants born in the United States receive

prophy-lactic eyedrops, generally erythromycin, to prevent eye infections

by C trachomatis and N gonorrhoeae.

Clinically, it is believed that pneumonia in infants younger

than 6 months is associated with C trachomatis, unless proven

otherwise This pneumonia also can occur as a mixed

infec-TABLE 24-4 Inclusion Conjunctivitis in the Neonate

Caused by Chlamydia trachomatisCharacteristic Comments

Incubation period 4-5 days Signs Edematous eyelids Discharge Copious, yellow Course Untreated, weeks to months Complications Corneal panus formation, conjunctival scarring

✓ 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 neo- nate’s history are suggestive of C trachomatis infection.

• Knowledge of the population at risk

• Capability and facilities available for testing

• Cost of assays

• Ability to batch specimen types

• Experience of laboratory scientistPrevalence in the population to be tested is an important cri-terion in determining which method or combination of methods should be used For any assay, the positive predictive value increases (assuming optimum 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 epithelial cells and not exudate First-void urine and vaginal swab specimens are excellent for detecting infection 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 24-7

lists the optimum specimens for detection of Chlamydia spp in

patients with a variety of clinical manifestations

Direct Microscopic Examination Direct specimen ination by cytologic methods primarily involves trachoma and inclusion conjunctivitis (Figure 24-5) Investigators have esti-mated this method as almost 95% sensitive, but it is technically demanding and influenced by the quality of the specimen and

exam-TABLE 24-5 Appropriate Chlamydia trachomatis Assays for Selected Patient Population

Assay

Patient Population

Legal Applicability (Rape or Child Abuse?) Test of Cure Low Risk High Risk Eye Throat Low Risk High Risk

Culture A/B A B A A/B B Yes Yes

A, Most useful, stands alone; B, probable, but needs verification or complementary assay recognizing different Chlamydia trachomatis macromolecules,

i.e., LPS (EIA) vs MOMP (DFA) or competition assay for DNA probes; CFI, complement fixation; DFA, direct fluorescent antibody; EIA, enzyme immunoassay; IUO, investigational use only; LGV, lymphogranuloma venereum; MIF, microimmunofluorescence; MOMP, major outer membrane protein; NA, not available; OIA, optical immunoassay; PCR, polymerase chain reaction; SDA, strand displacement amplification; TMA, transcription-mediated amplification.

*A low-risk population is defined as one with a <5% incidence, such as in an obstetrics-gynecology or family practice patient group (e.g., birth control, annual gynecologic examination) A high-risk population is defined as one with a >10% incidence, such as those in sexually transmitted disease clinics, university or college student health centers, and emergency department patients.

TABLE 24-6 Detection Capabilities of Various Methods for Chlamydia trachomatis

Nonculture, nonamplified

DFA 70-95 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, GC,

Acinetobacter

Verify with complementary assay

DFA, Direct fluorescent antibody; EIA, enzyme immunoassay; FA fluorescent antibody; LA limited availability; NPV, negative predictive value; PCR, polymerase

chain reaction; PPV, positive predictive value; SDA, strand displacement amplification; SENS, sensitivity; SPEC, specificity; TMA, transcription-mediated

amplification.

*Range—low to high prevalence as described in the text.

difficult to use with large numbers of specimens, it does offer

rapidity in selected cases, particularly in detecting ocular

infec-tion in newborns When direct fluorescent antibody (DFA) testing

is used for endocervical or urethral specimens, the sensitivity is

80% to 85% Characteristic fluorescence of EBs is suggestive,

but verification by alternative methods using a different epitope

is needed Direct specimen examination offers one additional

important advantage—it allows for immediate quality control of

the specimen, revealing whether columnar epithelial cells are present Figure 24-6 shows inclusion bodies demonstrated by direct examination of cytologic stains of endocervical smears

Cell Culture Until the development of PCR assays, mydial cell culture was considered the gold standard for detect-

chla-ing C trachomatis infection; however, cell culture usefulness has

been limited because of the inherent technical complexity, time and specimen handling requirements, expense, and labile nature

Fluorescein-labeled monoclonal antibodies can be used to detect the chlamydial inclusions Alternatively, iodine or Giemsa stain can be used, but these methods are less sensitive and spe-cific (Figure 24-7) There are a number of commercially available fluorescent antibodies Some researchers use species-specific monoclonal antibodies that bind to the MOMP, whereas others prefer the family-specific antibody, which binds to an LPS com-ponent Monoclonal antibodies against the MOMP are reported

to offer the brightest fluorescence, with consistent bacterial phology and less nonspecific staining than monoclonal antibod-ies against the LPS

mor-Immunoassays The most commonly used rapid antigen

assay for the detection of C trachomatis is the enzyme

immu-noassay (EIA) Depending on the manufacturer, the EIA detects the outer membrane LPS chlamydial antigen or the MOMP Many commercial kits are available, all having similar advan-tages These include the ability to do the following: screen large volumes of specimens, obtain objective results, have test results available in 3 to 5 hours, and use various specimen types EIAs, however, are not recommended for testing urine or vaginal swab specimens A summary of the published sensitivity, specificity, and negative and positive predictive values, as well as test speci-mens, is listed in Table 24-6 However, none of them equals the sensitivity of culture, and most are significantly less sensitive Discrepancies in sensitivity could be based on differences in sample size, disease prevalence, population characteristics, col-lection sampling techniques, and laboratory standards One addi-tional caution must be observed when EIA is used for chlamydial antigen detection A positive result must be considered prelimi-nary and should be verified, because antigen detection methods may give a false-positive result when used in low-prevalence (<5%) populations Because of these limitations, the CDC con-

siders EIAs substandard for the detections of C trachomatis, and

of the organism Even under the most stringent and optimal

conditions, isolation of chlamydiae is only approximately 80%

sensitive Cell lines commonly used for the detection of

chla-mydiae include McCoy, HEp-2, HeLa, and buffalo green monkey

kidney The cell lines are grown on coverslips in 1-dram shell

vials or on the surface of multiwell cell culture dishes containing

cell culture media with cycloheximide Because multiple blind

passes are not necessary to maximize the isolation rate in a

1-dram vial, the shell vial technique (see Chapter 29) has been

found to be more sensitive than the microwell method The

speci-men is centrifuged onto the cell monolayer and incubated for

TABLE 24-7 Appropriate Specimens for Detection of Chlamydial Infections

Inclusion conjunctivitis and trachoma Conjunctival swab, scraping with

spatula or tears

Specimen collection in neonates is difficult Urethritis Urethral swab In males, >4 cm; do not use discharge Epididymitis Epididymis aspirate

Cervicitis Endocervical swab Remove exudate first.

Salpingitis Fallopian tube (lumen) or biopsy

Lymphogranuloma venereum Bubo or cervical lymph node aspirate

Infant pneumonia Throat swab, nasopharyngeal aspirate,

or lung tissue Sexually transmitted disease, male sex

partner

Urine Noninvasive diagnostic procedure;

EIA antigen detection is 80% accurate, PCR, 98%

Psittacosis Sputum, lung tissue

Chlamydophila pneumoniae pneumonia

or pharyngitis

Sputum, throat swab, or lung tissue Tissue culture isolation and direct

immunofluorescence are relatively new and need further evaluation.

Sexually transmitted disease, result

clarification

Rectal, vaginal swabs May be used for supplemental

information and in clarifying previous isolates or diagnostic dilemmas

EIA, Enzyme immunoassay; PCR, polymerase chain reaction.

(BD Diagnostic Systems, Sparks, MD), are commercially able Although commercial tests differ in their amplification methods and target nucleic acid sequences, the increased sensi-tivity of NAATs is ascribed to their ability to produce positive signals from as little as a single copy of the target DNA or RNA All three commercial systems offer the ability to simultaneously

avail-detect N gonorrhoeae infection Because of improvements

in the NAATs, confirmation of positive results is no longer recommended

Nucleic Acid Hybridization and Amplification Assays

The newest advances in Chlamydia spp identification have dealt

with the detection of nucleic acids Initially, only one probe was

commercially available, a nonisotopically labeled DNA probe

that detected C trachomatis rRNA (PACE 2; Gen-Probe, San

Diego, CA) in urogenital specimens The sensitivity, specificity,

and positive and negative predictive values are higher than those

reported for EIA and cultures DNA probe assays can have the

added advantage of detecting two STDs in one sample—

gonorrhea and C trachomatis infection.

Nucleic acid amplification tests (NAATs) have become the

preferred diagnostic method for C trachomatis genital

infec-tions They 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 Results can be obtained quickly and

testing is less technically demanding than culture However, no

NAAT has been approved for use on conjunctival,

oropharyn-geal, or rectal specimens

NAATs amplify and detect organism-specific DNA or RNA

sequences In-house PCR tests and FDA-approved systems for

the detection of C trachomatis in clinical specimens, such as the

PCR-based Roche Amplicor (Roche Molecular Systems,

India-napolis), APTIMA transcription-mediated amplification assay

(Gen-Probe), and ProbeTec strand displacement amplification

FIGURE 24-6 A, B, Cytologic examination of endocervical specimens demonstrating inclusion

In the Case in Point, the diagnosis of C trachomatis infection was

con-firmed by a nucleic acid amplification test These assays are generally rapid and highly sensitive and specific.

Antibody Detection Serologic assays can be used in the

detection of C trachomatis infections Historically, these were

thought to be limited and problematic Many individuals have chlamydial antibodies from previous infections, and because chlamydial infections tend to be localized, they do not cause the traditional fourfold rise in antibody titer between acute and con-valescent specimens Serologic testing of uncomplicated genital

Reporting Results

With such great latitude in current testing choices, it is important for each laboratory to clearly report and define results Some key points in the development of an approach to ordering and report-

ing results of tests for C trachomatis and related organisms in a

patient specimen are as follows:

• Agreeing in advance with the obstetrics-gynecology and emergency departments on which organisms are associated with which clinical syndrome and then testing accordingly, using profiles

• Reporting which tests were and were not performed for each patient profile

• Reporting unusual observations Pure isolates of nas, Haemophilus, Neisseria meningitidis, and yeast are not

Pseudomo-normal, and the physician needs to be aware of their presence

Chlamydophila pneumoniae

Chlamydophila pneumoniae was formerly known as Chlamydia

sp., strain TWAR; it was originally identified in 1965 from a conjunctival culture of a child (TW) enrolled in a Taiwan tra-choma vaccine study In 1983, at the University of Washington,

a similar organism was isolated in HeLa cells from a pharyngeal

specimen of a college student (AR) Today, C pneumoniae is

recognized as an important respiratory pathogen It is known to

be a cause of acute respiratory disease, pneumonia, and gitis It also has been isolated from patients with otitis media with effusion, pneumonia with pleural effusion, and aseptic pharyngi-

pharyn-tis Infection with C pneumoniae has been established as a risk

factor for Guillain-Barré syndrome, an immunologically ated neurologic disease There also appears to be a relationship

medi-between sarcoidosis and C pneumoniae, but considerable work

needs to be done to establish the existence and degree of this

relationship To date, only a single C pneumoniae serovar has

been found

C pneumoniae has been implicated as a possible factor in

asthma and cardiovascular disease The organism has been lated from atherosclerotic tissue, but its possible pathogenic role remains under investigation Association of this organism with other vascular diseases, such as abdominal aortic aneurysm, has also been considered Because of the evidence implicating

iso-C pneumoniae with the development or outcome of

cardiovas-cular disease, antimicrobial therapy was recommended for ing vascular disease by up to 4% of physicians in the United States, according to a 1999 survey Results from clinical studies, however, have not shown benefits of antimicrobial therapy in individuals with coronary heart disease Furthermore, results suggest that conventional antimicrobial therapy may not eradi-cate the organism or reduce mortality in these patients, although

treat-C pneumoniae remains a potential risk factor in cardiovascular

disease

Clinical Infections

Although probably 90% of infections are asymptomatic or mildly

symptomatic, infection with C pneumoniae is thought to be

fairly common, with an estimated 200,000 to 300,000 cases/year

in the United States In some populations, antibodies have been

infections and screening of asymptomatic individuals is not

rec-ommended Currently, the interpretation and significance of

sero-logic assays are being reevaluated, and serosero-logic testing is

growing as a complementary diagnostic tool in certain situations,

such as the following:

• With microimmunofluorescence (MIF), when a specific IgM

response to a different serovar of C trachomatis is observed,

new infections can be diagnosed in patients who have had

previous infections with other serovars

• Ascending infections by C trachomatis involving the

fallo-pian tubes and other organs of the upper female genital tract

are almost never detected by endocervical cultures Hence,

patients at risk for chronic infections would be missed with

the standard screening methods using a cervical swab

Sero-logic testing of women with subfertility has been proposed as

a screening test

• Complement fixation (CF) detects family-reactive antibody,

including elevated levels of antibody in systemic infections,

such as LGV Diagnosis of LGV is supported by CF titers of

1 : 64 or more (Table 24-8) It must be noted, however, that

CF generally is not useful in nonsystemic chlamydial

con-junctivitis or routine urogenital tract infections

The MIF is considered the method of choice for detecting

antibodies to C trachomatis MIF detects antibodies to

chla-mydial EBs; these antibodies are serovar-specific antibodies

Hence, high levels of chlamydial IgM by MIF are diagnostic of

systemic C trachomatis infection in infants Same-day diagnosis

is possible; therefore, IgM MIF is the method of choice for

diag-nosis of C trachomatis pneumonia in infants, preferable even to

culture Furthermore, infants with inclusion conjunctivitis

nor-mally do not have detectable IgM antibodies unless they have a

systemic infection Chlamydial IgG is generally not useful in

infants, because rising titers are seldom observed, and when high

titers are detected they probably reflect maternal antibody EIA

and complement fixation methods have also been described to

detect antibodies to C trachomatis.

TABLE 24-8 Detection of Chlamydia Species by

Various Serologic Methods

A/C, Acute/convalescent sera; CFI, complement fixation (using LPS common

to all members of the Chlamydiaceae); MIF, microimmunofluorescence.

and bronchitis but is rarely accompanied by sinusitis Fever is relatively uncommon, and radiographs show isolated pneumoni-

tis C pneumoniae is recognized as the third most common cause

of infectious respiratory disease It accounts for approximately 10% to 15% of community-acquired cases of pneumonia The mode of transmission, incubation period, and infectiousness of

C pneumoniae infections are still largely unknown No animal

reservoir or vector is known Table 24-10 summarizes situations and/or populations at risk that would benefit from the detection

of C pneumoniae, usually by serologic methods.

Laboratory Diagnosis

Specimens collected for the detection of C pneumoniae include

sputum, bronchial lavage fluid, nasopharyngeal aspirates, throat washings, and throat swabs (Table 24-7) C pneumoniae may be

cultured on selected cell lines and visualized with conjugated monoclonal antibodies Human lines and HEp-2 from the human respiratory tract are the most sensitive Monoclonal

fluorescein-antibodies specific for C pneumoniae are used to identify

inclu-sions in cell culture It should be noted that a family-reactive

monoclonal antibody can identify C pneumoniae inclusions but

cannot differentiate this organism from the other chlamydiae

Attempts to culture C pneumoniae, if undertaken, should take into account the organism’s lability C pneumoniae seems to be considerably more labile than C trachomatis, although its viabil-

ity is relatively stable at 4° C An indirect fluorescent antibody

method has been reported for detecting C pneumoniae in

respira-tory secretions; the antibody reacts with the MOMP (Figure24-8) This same antibody can be used to identify infected cell culture monolayers

Given the difficulty of and lack of standardization for isolation

of C pneumoniae, serologic tests have been the method of choice

for diagnosis A CF test had been the traditional assay most often

used for C pneumoniae detection, but it is rarely used today The

present method of choice is the MIF assay, which is more tive and specific than CF Furthermore, it does not cross-react

sensi-with C trachomatis and C psittaci MIF also can distinguish an

IgM from an IgG response Single-titer evaluations, although not diagnostic, may be suggestive An IgM titer greater than 1 : 32 or

an IgG single titer greater than 1 : 512 may suggest C moniae as a recent causative agent, warranting further evaluation

pneu-An IgG titer of 1 : 16 or higher but less than 1 : 512 is evidence

of past infection or exposure

antibody detectable in children younger than 5 years It is thought

that the attack rate is highest between the ages of 6 and 20 years,

with a particular emphasis in college-age students Unlike viral

respiratory diseases, there seems to be no seasonal incidence,

although some Scandinavian data have indicated the possibility

of epidemics every 4 to 6 years 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 24-9

The clinical picture in college-age students, although it may

be varied, is a biphasic clinical course C pneumoniae infection

results in prolonged sore throat (5 to 7 days) and hoarseness,

followed by flulike lower respiratory tract symptoms (8 to 15

days) Because of its striking clinical similarity to bacterial

phar-yngitis, the result of a streptococcal antigen test often is thought

to be falsely negative The second phase of the biphasic illness

often results in pneumonia (approximately one in nine infections)

TABLE 24-9 Summary of Key Epidemiologic and

Clinical Features of Chlamydophila

pneumoniae Infections

Almost no antibody detectable

before 5 years of age

Antibodies present in >50% of

adults

Attack rate highest between the

ages of 6 and ≈25 yr, often

Biphasic illness—prolonged sore throat, crouplike hoarseness, followed by lower respiratory (flulike) 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 24-10 Evaluating for Chlamydophila pneumoniae

Pneumonias requiring hospitalization (age

6-20 yr)

C pneumoniae–specific IgM and IgG: acute

and convalescent, use MIF IgM, single visit

12% antibody prevalence Pharyngitis in college students 9% antibody prevalence

Retrospective, undiagnosed outbreaks in

young adults, college, or military

CF or MIF, IgG-specific Serious pneumonia, undiagnosed; clinically

presents like Mycoplasma pneumoniae

respiratory pathogens (i.e., Mycoplasma pneumoniae), establish cause and impact

on diagnosis-related group reimbursement

CF, Complement fixation; IgG, immunoglobulin G; IgM, immunoglobulin M; MIF, microimmunofluorescence.

physicians need to know the tests that are most appropriate for differentiating these microorganisms.

Isolation of C psittaci in culture, although diagnostic, is

difficult, dangerous, and not routinely used or recommended

Therefore, almost all diagnoses of C psittaci are based on

sero-logic evaluation A single antibody titer greater than 1 : 32 is suggestive of acute illness in a symptomatic patient during an outbreak of psittacosis The rise in antibodies is usually not demonstrable until the acute illness is over, however, and it

is often weak or absent if appropriate antimicrobial therapy is given This is most often a so-called rule-out disease If

C pneumoniae– and C trachomatis–specific IgG and IgM are

not detected by MIF and a fourfold rise in chlamydiae antibodies

is detected by CF, then C psittaci should be strongly suspected

A good history is paramount in evaluating bird exposure, bation time, and disease process The results of PCR-based assays have been published, but lack of a gold standard for comparison has made evaluation difficult No commercially prepared NAATs are available

incu-Rickettsiaceae and Similar Organisms

The genera Rickettsia and Orientia belong to the family

Rickett-siaceae Most members of the rickettsial group are borne, obligately intracellular pathogens that can grow only in the cytoplasm of host cells These bacteria have become 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 arthro-pods by transovarial transmission Because of this mechanism, arthropods are not only vectors for rickettsioses but also reser-voirs Second, arthropods directly inoculate new hosts with rick-ettsiae during feeding An exception to this pattern occurs with

arthropod-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

mono-layer cell lines Rickettsia spp are divided into three groups

according to the types of clinical infections they produce The

typhus group contains only two species, R prowazekii and

R typhi The spotted fever group includes a number of species generally recognized as human pathogens, such as R rickettsii,

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 bioterror agents

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 first described in the western

United States during the latter part of the nineteenth century It

Two antibody response patterns have been identified for

C pneumoniae infections In the primary response, most often

seen in adolescents, university students, and military trainees,

CF antibodies usually appear first By MIF, C pneumoniae–

specific IgM does not appear until 3 weeks after onset of

symp-toms, and often C pneumoniae–specific IgG does not reach

diagnostic levels for 6 to 8 weeks Therefore, the traditional

convalescent serum obtained approximately 14 to 21 days after

onset does not contain MIF-detectable C pneumoniae antibody

In contrast, during reinfection, a CF antibody change is not

detected, but by MIF, an IgG titer of 1 : 512 or more can appear

within 2 weeks IgM levels may be detectable but are low

Currently, no commercial kits are FDA-approved Laboratories

wishing to use MIF for C pneumoniae must develop their own

in-house protocols Recently, some partially automated

enzyme-linked immunosorbent assays (ELISAs) have become

commer-cially available, but they too are not yet FDA-approved Studies

have shown a concurrence between the ELISAs and MIF test

results The ELISAs have major advantages, namely being less

time-consuming, and the method does not rely on the quality

of the fluorescent microscope used or the experience of the

laboratory scientist

Chlamydophila psittaci

Chlamydophila psittaci is the cause of psittacosis among

psitta-cine birds, also known as ornithosis or parrot fever The former

mammalian C psittaci strains that cause feline conjunctivitis,

rhinitis, and respiratory infections among cats, guinea pig

con-junctivitis, and abortion among ruminants have been replaced in

three new species—Chlamydophila felis, Chlamydophila caviae,

and Chlamydophila abortus, respectively Diagnosis of

psittaco-sis is usually based on a history of exposure to psittacines and a

fourfold rise in antibody to the chlamydial group LPS antigen

In the United States, fewer than 50 cases of C psittaci are

reported annually Retrospective serologic testing of sera from

patients with acute respiratory disease have shown that many

people previously thought to have C psittaci infections because

of transient bird exposure were actually infected with C

pneu-FIGURE 24-8 Chlamydophila pneumoniae detection from

direct sputum smear using fluorescent-labeled monoclonal

antibody, highlighting cytoplasmic inclusion (×400) (Courtesy

DAKO Reagents, Carpinteria, CA.)

disseminated intravascular coagulation The mortality rates for untreated or incorrectly treated patients can be as high as 20%, although correct antimicrobial therapy with tetracycline or chlor-amphenicol lowers the rates to 3% to 6%.

Boutonneuse Fever Boutonneuse fever, also known as

Mediterranean spotted fever, caused by R conorii, 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 tick-borne, and its reservoirs include ticks and dogs

Boutonneuse fever is also clinically similar to 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 Also in contrast with RMSF, this disease is characterized by the presence of taches noires (black spots) at the primary site of infection Taches noires are lesions caused by the

introduction of R conorii into the skin of a nonimmune person

As the organism spreads to the blood vessels in the dermis, damage occurs to the endothelium Edema secondary to increased vascular permeability reduces blood flow to the area and results

in local necrosis

Typhus Group

The typhus group of rickettsiae includes the species R typhi (endemic typhus, also referred to as murine typhus) and R

prowazekii (epidemic louse-borne typhus and Brill-Zinsser

disease) Generally, the typhus rickettsiae differ from the other

rickettsial groups in that they replicate in the cytoplasm of the host cell and cause cell lysis, thereby releasing the rickettsiae Other rickettsiae pass directly through an uninjured cell

Murine Typhus The arthropod vector for R typhi is the oriental rat flea Xenopsylla cheopis, and the rat (Rattus exulans)

is the primary reservoir Apparently, the cat flea, Ctenocephalides felis, can also harbor the organism Because this flea infests a

large number of domestic animals, it may be an important factor

in the persistence of infection in urban areas

The rickettsiae also survive in nature, to a lesser extent, by transovarial transmission When a flea feeds on an infected host, the rickettsiae enter the flea’s midgut, where they replicate in the epithelial cells They are eventually released into the gut lumen Humans become infected when fleas defecate on the surface of the skin while feeding The human host reacts to the bite by scratching the site, allowing direct inoculation of the infected

feces into abrasions R typhi can also be transmitted to humans

directly from the flea 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 in this country but continues to be a problem in areas of the world in which rats and their fleas are present in urban settings As is the case 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 usually occurs

on the trunk and extremities Rash on the palms of the hands occurs rarely Complications are rare, and recovery usually occurs without incident

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 had to discount

a viral cause, because the agent was not filterable The organism

was first seen using light microscopy in 1916

RMSF is a zoonosis, and humans typically acquire the

infec-tion by tick bites Ticks are the principal vector and reservoir for

RMSF The most common tick vectors are Dermacentor

varia-bilis (Figure 24-9) in the southeastern United States and

Derma-centor andersoni in the western part of the country Other species

of ticks, however, can be vectors Ticks transmit the organism

into humans via saliva, which is passed into the host during the

tick’s feeding Once in the host tissue, the rickettsiae are

phago-cytosed into endothelial cells (cells that line blood vessels),

where they replicate in the cytoplasm of the host cell Replication

in the nucleus also occurs The rickettsiae pass directly through

the plasma membranes of infected cells into adjacent cells

without causing damage to the host cells The rickettsiae are

spread throughout the host hematogenously and induce vasculitis

in internal organs, including the brain, heart, lungs, and kidneys

Clinically, the patient experiences flulike symptoms for

approximately 1 week, which follows an incubation period of

approximately 7 days The symptoms include fever, headache,

myalgia, nausea, vomiting, and rash The rash, which may be

hard to distinguish in individuals of color, begins as

erythema-tous patches on the ankles and wrists during the first 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

macu-lopapular patches eventually consolidate into larger areas of

ecchymoses

Once disseminated, the organisms cause vasculitis in

the blood vessels of the lungs, brain, and heart, leading to

pneumonitis, central nervous system manifestations, and

myo-carditis The patient experiences symptoms secondary to

vascu-litis, including decreased blood volume, hypotension, and

FIGURE 24-9 Dorsal view of Dermacentor

variabilis, the Amer-ican dog tick, a vector for Rocky Mountain spotted fever

(×20,000) (Courtesy Janice Carr, Centers for Disease Control

and Prevention, Atlanta, GA.)

mite (chigger) bite The incubation period is about 10 days, after which a papule forms at the site of inoculation The papule pro-gresses to a pustule and then to an indurated eschar The patient becomes febrile as the rickettsiae are disseminated throughout the body via the bloodstream The patient also experiences head-ache, nausea, and chills 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 symp-toms resolve without medical attention.

Orientia

Scrub typhus is a disease that occurs in India, Pakistan, Burma,

eastern Russia, Asia, and Australia The causative agent is entia (formerly Rickettsia) tsutsugamushi The vector is the chigger, Leptotrombidium deliensis, and the main reservoir is the

Ori-rat The bacteria are transmitted transovarially in chiggers

The transmission of O tsutsugamushi to the human host is

followed by an incubation period of approximately 2 weeks A tache noire, similar to that of boutonneuse fever, forms at the site

of inoculation The normal rickettsial symptoms of fever, ache, and rash are also present The rash starts on the trunk and spreads to the extremities 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 Without treatment, mortality approaches 30%

head-Laboratory Diagnosis of Rickettsial Diseases

Because of their infectious nature, isolation of the rickettsiae is not recommended and should only be attempted by biosafety level 3 laboratories If culture is attempted, blood should be col-lected as early in the disease as possible The immunohistochemi-cal detection of rickettsiae is an established method for diagnosis

of these infections Monoclonal antibodies directed against the spotted fever or typhus group have been used, but no antibody is commercially available PCR assays have also been described, but they too are not readily available

Typically, serologic assays are the only laboratory tests formed for the diagnosis of rickettsial diseases Unfortunately, these methods can only confirm a diagnosis in convalescent speci-mens and offer little help in diagnosing acute infections that could guide antimicrobial therapy The immunofluorescent 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-specific antibody is available Antibodies to certain rickettsial species are known to cross-react with bacteria in the genus

per-Proteus This gave rise to the Weil-Felix agglutination test

Because the assay does not use rickettsial antigen, it is nonspecific and rarely used in the United States However, because of its low cost, it is used in some other countries An agglutination test using latex beads coated with rickettsial antigens is commercially avail-able for the diagnosis of RMSF (Panbio, Baltimore)

Anaplasmataceae

Ehrlichia

Ehrlichiosis was first noted in France in the 1930s when dogs infected with brown dog ticks became ill and died Postmortem examination revealed rickettsial-like inclusions in the monocytes

Louse-Borne Typhus Louse-borne (epidemic) typhus is

caused by R prowazekii The vectors include the human louse

(Pediculus humanus; Figure 24-10), squirrel flea (Orchopeas

howardii), and squirrel louse (Neohaematopinus sciuriopteri)

The reservoirs are primarily humans and flying squirrels located

in the eastern United States The louse often dies of its

rickettse-mia, unlike vectors of other rickettsiae

Louse-borne 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 louse-borne 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

origi-nating in Africa Louse-borne typhus is similar to 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 lumen of the gut When the louse

feeds on another human, it defecates, and the infected feces are

scratched into the skin, just as in murine typhus The disease

progression is similar to 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 a rash 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 louse-borne 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

than louse-borne typhus, and death is rare Patients with latent

infections constitute an important reservoir for the organism

Transitional Group

Rickettsialpox Caused by R akari, the reservoir is the

common house mouse, and the vector is the mouse mite

Liponys-soides sanguineus Rickettsialpox occurs in Korea and the

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

infec-FIGURE 24-10 The female head louse, Pediculus humanus,

which is a vector for Rickettsia

prowazekii, the agent of epi-demic typhus (×40) (Courtesy Dr Dennis D Juranek, Centers

for Disease Control and Prevention, Atlanta, GA.)

sensitive (29%) The bacteria are primarily found in monocytes 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 the most frequently used method for direct detection

of E chaffeensis The bacteria have also been isolated from

peripheral blood in cell monolayers Most cases of HME are diagnosed retrospectively by serologic testing; IFA is the most widely used method

Anaplasma Anaplasma phagocytophilum, formerly known as Ehrlichia

phagocytophilum, causes a disease referred to as human

granu-locytic anaplasmosis (HGA) The disease is identical to that

which Ehrlichia equi causes in horses and Ehrlichia philum causes in ruminants All three of these organisms are now classified as A phagocytophilum The incubation period for HGA

phagocyto-is 5 to 11 days The symptoms closely resemble those of HME; less than 11% of infected individuals have a rash

HGA is not a reportable disease in all states, so the number

of cases is probably underreported In Wisconsin and necticut, the average annual incidence ranges from 24 to 58 cases/100,000 people Cases have increased steadily from 348 cases in 2000 to 1761 cases in 2010 As of 2010, over 7000 cases were reported nationwide Most cases are identified in the upper Midwest and Northeast United States Natural hosts include deer,

Con-rodents, horses, cattle, and humans Tick vectors include Ixodes scapularus and I pacificus.

As with HME, staining of peripheral blood and buffy coats can be used to diagnose HGA The morulae are found in granu-locytes (Figure 24-11), and the sensitivity is about 60% because

of a large number of infected white blood cells Diagnosis can also be made by 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

Rick-ettsiaceae and Anaplasmataceae For example, although C netii is an obligate intracellular parasite, it develops within the

bur-phagolysosomes of infected cells The acidic environment

acti-vates its metabolic enzymes Spore formation by C burnetii

allows it to survive harsh environmental conditions In addition,

C burnetii is generally not transmitted by arthropods, although

it is known to infect more than 12 genera of ticks and other arthropods The bacteria can infect fish, birds, rodents, livestock, and other mammals

C burnetii is the causative agent of Q (query) fever, a disease

found worldwide Q fever is highly contagious and, as such, is considered a potential bioterror agent (see Chapter 30) Most infections are spread by the inhalation of dried birthing fluids The ingestion of unpasteurized milk is also a recognized risk factor Acute Q fever generally has an abrupt onset of an undifferentiated febrile disease consisting of high fever that can be accompanied by headaches, myalgia, arthralgia, cough and, rarely, a rash Patients may present with elevated liver enzyme levels, increased erythrocytic sedimentation rate, and thrombocytopenia Because of the rapid dissemination of the

named Rickettsia canis They were obligately intracellular,

arthropod-borne, gram-negative coccobacilli They differ from

the other members of the rickettsiae in that they multiply in the

phagosomes of host leukocytes and not in the cytoplasm of

endo-thelial cells

Because these organisms grew within host cell vacuoles, they

were reclassified into a new genus, Ehrlichia, in 1945 The

ehrlichiae have a developmental cycle similar to that of the

chla-mydiae The infective form of the organism is the EB, which

replicates in the phagosome and prevents phagolysosome

forma-tion These bodies give rise to inclusions with initial bodies

inside As the inclusions mature, they develop morulae

(mulberry-like bodies; Figure 24-11) Morulae are round to oval

clusters of bacteria 1 to 3 µm in diameter As the host cell

rup-tures, the morulae break into many individual EBs, which

con-tinue the infective cycle

Ehrlichia chaffeensis causes human monocytic ehrlichiosis

(HME), which occurs in the United States, Europe, Africa, and

South and Central America In the United States, most cases are

found in the southeastern and south central states, as well as in

the Mid-Atlantic states Oklahoma, Missouri, and Arkansas

account for about 35% of the cases Ehrlichia ewingii produces

a disease indistinguishable from E chaffeensis, and no currently

available serologic test can distinguish these agents Ehrlichiosis

cases have increased from about 200 in the year 2000 to 961 in

2008, although cases may be underreported Reported cases

decreased to 740 in 2010 A total of 6100 cases were reported

through 2010 Natural hosts of the organism include dogs and

deer, as well as humans, with the lone star tick (Amblyomma

americanum) being the primary vector.

Many patients with HME may experience asymptomatic

infection The organism has an incubation period of 5 to 10 days

Patients often experience high fever, headache, malaise, and

myalgia Nausea, vomiting, diarrhea, cough, joint pain, and

con-fusion are rarely present As many as 67% of the pediatric

patients infected with E chaffeensis have a rash; however, adults

rarely experience a rash Patients may also have evidence of

leukopenia and neutropenia, thrombocytopenia, and elevated

liver enzyme levels Patients can experience severe

complica-tions, including toxic shock–like syndrome, central nervous

system involvement, and adult respiratory distress 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

FIGURE 24-11 Anaplasma morula (arrow) in an infected white

blood cell (×1000)

Beninati T, et al: First detection of spotted fever group rickettsiae in

ricinus from Italy, Emerg Infect Dis 8:983, 2002.

Bengis RG, et al: The role of wildlife in emerging and re-emerging

zoonoses, Rev Sci Tech 23:497, 2004.

Centers for Disease Control and Prevention: Ehrlichiosis: statistics and

index.html Accessed August 14, 2012.

Centers for Disease Control and Prevention: Anaplasmosis: statistics and

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Centers for Disease Control and Prevention: Summary of notifiable

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bacteria, a number of tissues can be infected, resulting in a

chronic disease The cardiovascular system is most susceptible

The laboratory diagnosis of Q fever can be made by direct

immunofluorescence assays of infected tissue,

immunohisto-chemistry However, with the exception of heart tissue in cases

of endocarditis, infected tissue contains low numbers of bacteria

NAATs, such as the PCR assay, have also been successful in

diagnosing infections; whole blood and buffy coats are often

successful in detecting the organism C burnetii is highly

conta-gious; isolation in cell cultures should be attempted only in

bio-safety level 3 facilities Several serologic assays have been

described for detecting antibodies in acute and chronic cases IFA

is the method of choice EIA kits are commercially available and

have sensitivities and specificities comparable to those of IFA

Learning Assessment Questions

1 What organisms should be considered as possible causes of

neonatal conjunctivitis?

2 What stains should be performed on the discharge or

conjunc-tival scraping for microscopic examination?

3 For the infant described in the Case in Point, what other clinical

conditions could be due to the causative organisms?

4 What STD is caused by Chlamydia trachomatis serotypes L1, L 2 ,

7 What is psittacosis or ornithosis?

8 What is the most common laboratory method used to diagnose

rickettsial diseases? Explain.

9 What cells do the Ehrlichia and Anaplasma species typically infect

in humans?

10 How does Coxiella burnetii differ from the Rickettsia spp.?

Points to Remember

■ Chlamydiae and rickettsiae are obligate intracellular organisms.

bacterial pathogen, and certain serovars are associated with

tra-choma, which can result in blindness.

■ NAATs are better assays for the diagnosis of C trachomatis

infec-tions than cultures.

pathogen considered responsible for many cases of

community-acquired pneumonia It has also been linked to chronic illnesses

such as atherosclerosis, coronary heart disease, and stroke.

parrot fever or ornithosis This bacterium produces lower

respira-tory tract infections in humans.

■ The Rickettsia spp are important human pathogens responsible for

a number of diseases including Rocky Mountain spotted fever,

rickettsialpox, and typhus.

■ The Rickettsia, Orientia, Ehrlichia, and Anaplasma are typically

transmitted to humans by the bites of arthropods.

cells: mononuclear cells and granulocytes, respectively.

often transmitted by inhalation of dried birthing fluids The

inges-tion of unpasteurized milk is also a risk factor.

CHAPTER

Donald C Lehman and Connie R Mahon *

■ GENERAL CHARACTERISTICS

■ CLINICAL INFECTIONS

Mycoplasma pneumoniae

Mycoplasma hominis and Ureaplasma Species

Other Mycoplasma Species

■ LABORATORY DIAGNOSIS

Specimen Collection and Transport

Direct Examination

CultureSerologic Diagnosis

■ ANTIMICROBIAL SUSCEPTIBILITY

■ INTERPRETATION OF LABORATORY RESULTS

CHAPTER OUTLINE

OBJECTIVES

After reading and studying this chapter, you should be able to:

1 Describe the general characteristics of the mycoplasma 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, and Ureaplasma urealyticum

4 Compare the pneumonia caused by Mycoplasma pneumoniae with

that caused by Streptococcus pneumoniae

5 Identify the preferred stain for demonstration of the mycoplasmas

6 Discuss the possible roles of M hominis and U urealyticum in

infections of low-birth-weight and high-risk neonates

7 Discuss the clinical manifestations of other Mycoplasma spp in immunocompromised patients

8 Analyze the diagnostic methods appropriate for the detection and identification of mycoplasmal and ureaplasmal infections

9 Discuss the use of serologic assays for diagnosing M pneumoniae infections

10 Name two selective media for the detection of the mycoplasmas

11 Explain the effects of antimicrobial therapy on mycoplasmal infections

12 Provide recommendations for the proper interpretation and reporting for Mycoplasma and Ureaplasma

*My comments are my own and do not represent the view of the Health Resources

and Services Administration of the Department of Health and Human Services.”

Case in Point

A premature male infant 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 Results of a

white blood cell count of the cerebrospinal fluid were negative,

the Gram stain was reported as “no organisms seen,” and

routine culture at 3 days was “no growth.” The infant was still

symptomatic at this time, and the pediatric infectious disease

physician, after consultation with the microbiology laboratory,

performed another spinal tap 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

Pleuropneumonia-like organism (PPLO)

Nongonococcal urethritis (NGU)

Primary atypical pneumoniaT-strain mycoplasma

This chapter discusses a group of organisms once thought

to be viruses because of their size Mycoplasmas are the smallest self-replicating organisms in nature This group

of bacteria belongs within the class Mollicutes Mycoplasma and Ureaplasma are the two genera in the family Mycoplasmataceae

At least 16 species of mollicutes have been isolated from humans

Although there are numerous species of Mycoplasma and

aerobic M pneumoniae and the more rapidly growing plasma hominis The mollicutes produce small colonies ranging

in size from about 15 µm to over 300 µm in diameter plasma spp often grow embedded beneath the surface of solid

Myco-media; therefore, transferring colonies with a loop is ineffective

On solid media, some species (e.g., M hominis) form colonies

with slightly raised centers giving the classic fried egg ance (Figure 25-1) In the laboratory, mycoplasmas are common and hard to detect contaminants of cell cultures

appear-FIGURE 25-1 Typical large Mycoplasma colony showing fried

egg appearance (Courtesy Bionique Testing Laboratories, Saranac Lake, NY.)

Ureaplasma identified in plants and animals, the following

species are the most significant human pathogens (Table 25-1):

• Mycoplasma pneumoniae, which causes respiratory disease

• Mycoplasma hominis, associated with urogenital tract disease

• Ureaplasma urealyticum, associated with urogenital tract

disease

General Characteristics

Mycoplasmas are pleomorphic organisms that do not possess a

cell wall, a characteristic that makes them resistant to cell wall–

active antibiotics such as the penicillins and cephalosporins

Because of the permanent absence of a cell wall, they were

originally grouped under the general term cell wall–deficient

bacteria They are not, however, classified as L-forms, which

are bacteria that have temporarily lost their cell wall as a result

of environmental conditions The mollicutes, a common name

used to describe members of the class Mollicutes, are

character-ized by permanently lacking a cell wall They range in size for

coccoid forms from approximately 0.2 to 0.3 µm in diameter to

tapered rods of approximately 1 to 2 µm in length and 0.2 to

0.3 µm in diameter Eight genera and over 200 species of

mol-licutes have been described Table 25-2 compares features of

three genera known to be pathogenic for humans

Mollicutes are generally slow-growing, highly fastidious,

fac-ultative anaerobes requiring complex media containing

choles-terol and fatty acids for growth; important exceptions include

TABLE 25-1 Divergent Ecosystems Inhabited by Genera of the Class Mollicutes

+, Present in ecosystem; −, rarely associated with ecosystem.

TABLE 25-2 Pathogens in the Class Mollicutes

Lack of cell wall induced in hypertonic

solution and penicillin, lysozyme, or salts − − − Exists in nature as free-living organism − − +

Other shared characteristics Smaller than other bacteria; close in size to myxoviruses

Smaller genome than other bacteria Lower guanidine-to-cytosine (G/C) ratio than most bacteria Limited metabolic activity (i.e., fastidious)

Many mollicutes contain DNase.

military personnel Epidemics are known to occur in these lations Infection is not considered seasonal, but many cases occur in autumn and early winter Outbreaks also have been noted when adolescents return to school in the fall Transmission

popu-is probably through aerosol droplet spray produced while coughing

Many infections are completely asymptomatic or very mild The most common presentation is tracheobronchitis; however, about one third of infected patients demonstrate clinically appar-ent pneumonia (Figure 25-4) The incubation period is usually 2

to 3 weeks, and early symptoms are nonspecific, consisting of headache, low-grade fever, malaise, and anorexia Sore throat, dry cough, and earache are accompanying symptoms Extrapul-monary complications, including cardiovascular, central nervous system, dermatologic, and gastrointestinal problems, are rare

occurrences M pneumoniae is not associated with infections

of the urogenital tract It has, however, been implicated as a

FIGURE 25-2 Electron micrographs showing effect of Mycoplasma

pneumoniae on ciliated tra-cheal cells A, Infected animal model B, Uninfected animal model (×20,000)

FIGURE 25-3 Electron micrograph of Mycoplasma

trachea (×100,000) c, Cilia; mv, microvilli; m, mycoplasma

mv

m

c

The first Mycoplasma was isolated in the late 1800s from a

cow with pleuropneumonia Later, a mycoplasma was isolated

from humans and was referred to as a pleuropneumonia-like

organism (PPLO) and the Eaton agent, after the researcher who

first isolated it from humans This human isolate became known

as Mycoplasma pneumoniae The mycoplasmas adhere to the

epithelium of mucosal surfaces in the respiratory and urogenital

tracts and are not eliminated by mucous secretions or urine flow

epithelial cells before and after M pneumoniae adherence Figure

25-3 is an electron micrograph demonstrating the shape of

M pneumoniae and its orientation of attachment Mycoplasma

spp indigenous to humans are listed in Table 25-3 The human

mycoplasmas are susceptible to adverse environmental

condi-tions, such as heat and drying Transmission of mycoplasmas and

ureaplasmas in humans can occur via direct sexual contact, 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

Mycoplasma pneumoniae may cause bronchitis, pharyngitis, or

a relatively common respiratory infection known as primary

atypical pneumonia, or walking pneumonia The clinical

mani-festations resemble those caused by Chlamydophila pneumoniae

The disease differs from the typical pneumonia caused by

Strep-tococcus pneumoniae in that it is milder, has a higher incidence

in young adults, and is not seasonal M pneumoniae does not

occur as a normal commensal; therefore, its isolation is always

significant and pathognomonic M pneumoniae causes

approxi-mately 20% of reported pneumonias in the general population

and up to 50% in confined populations, such as those in military

settings School-age children and young adults are especially

susceptible to infection Clinical disease is uncommon in very

young children and older adults Other groups at risk include

closed-in populations such as prisoners, college students, and

disease In addition, it has been reported that among sexually active individuals, the rate of colonization is directly related to the number of sexual partners Higher rates of colonization have been noted in adults of lower socioeconomic status The organ-isms do not persist in infants colonized at birth Table 25-4 summarizes the known association of genital mollicutes with urogenital and newborn diseases.

Mycoplasma hominis is found in the lower genitourinary

tracts of approximately 50% of healthy adults and has not been

reported as a cause of nongonococcal urethritis (NGU)

Ure-thritis in males not caused by Neisseria gonorrhoeae The

organ-ism may, however, invade the upper genitourinary tract and cause salpingitis, pyelonephritis, pelvic inflammatory disease (PID), or

postpartum fevers Ureaplasma parvum (U urealyticum biovar 1) and U urealyticum (U urealyticum biovar 2) do not cause

disease in the female lower genital tract but have been associated with approximately 10% of cases of NGU in men, as well as with

upper female genitourinary tract disorders The role of U parvum

in NGU has been questioned U urealyticum has been recovered

from more than 60% of normal sexually active females and has been associated with reproduction disorders, chorioamnionitis, congenital pneumonia, and the 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-birthweight infants with respiratory disease; 14% of infections were in newborns deliv-ered by cesarean section, thus indicating that infection occurred

in utero and not during passage through the birth canal

M hominis and Ureaplasma spp can be transmitted to the

fetus at delivery and have been recovered from the cerebrospinal fluid (CSF) of certain high-risk newborns, including preterm and low-birthweight babies (see Table 25-4) In particular, U parvum

has been linked to respiratory distress in premature infants It has been recommended that culture for these organisms be attempted when the CSF specimen from a newborn with evidence of men-ingitis is negative for bacteria on Gram stain and routine bacte-

co-infection or cofactor in epidemic group A meningococcal

meningitis (Neisseria meningitidis) and infant pneumonitis.

Mycoplasma hominis and

Ureaplasma Species

Although the mollicutes do not cause vaginitis, both M hominis

and U urealyticum are associated with infections of the

urogeni-tal tract and might play a role in bacterial vaginosis They are,

however, frequently isolated from asymptomatic sexually active

individuals, making interpretation of a positive culture difficult

Because they are opportunistic pathogens, the immune status of

FIGURE 25-4 Typical chest radiograph of a patient with a

3-week course of atypical pneumonia Note nonspecific inter-stitial pneumonia and a patchy infiltrate delineated by a

feathery outline

TABLE 25-3 Mycoplasma Species Indigenous to Humans

Urogenital tract Uncommon

peripheral zone Urogenital tract Common (9% to 50% women)

30% to 50% men)

Tiny, spherical, fried egg, granular

*Relative sizes: large, >100 nm; small, 50-100 nm; tiny, <50 nm.

TABLE 25-4 Summary of Association of Genital Mollicutes with Urogenital and Newborn Diseases

Disease, Target Population Mycoplasma hominis Ureaplasma urealyticum Comments

Nongonococcal urethritis None Strong Ureaplasmas cause some cases, but the proportion

is unknown.

Prostatitis Weak None An association with a few cases of chronic disease

has been reported; a causal relation is unproven Epididymitis None None Mycoplasmas are not an important cause.

Reiter disease None None The role of ureaplasmas should be studied.

Bartholin gland abscess Weak None M hominis may cause some disease but is not an

important cause.

Vaginitis and cervicitis None None M hominis is often associated with disease, but a

causal relation is unproven.

Pelvic inflammatory disease Strong Weak M hominis causes some cases, but the proportion

Urinary calculi None Weak Ureaplasmas cause calculi in male rats, but no

convincing evidence exists that they cause natural human disease.

Pyelonephritis Strong None M hominis causes some cases.

Involuntary infertility None Weak Ureaplasmas are associated with altered motility

of sperm.

Repeated spontaneous abortion

and stillbirth

None Weak Maternal and fetal infections have been associated

with spontaneous abortion, but a causal relation is unproven.

Chorioamnionitis None Strong An association exists, but a causal relation is unproven Low birth weight None Strong An association exists, but a causal relation is unproven Neonatal infections, including

sepsis, pneumonia, meningitis

Strong Strong Further clarification is needed, but importance is

growing in a selected prenatal population.

Neonatal period, particularly

preterm delivery, very low

birth weight; clinical signs

compatible with meningitis

(CSF), pneumonia (trachea),

sepsis (blood)

Strong Strong These findings need further clarification because most

neonatal infections resolve without therapy, but in low socioeconomic groups the diagnostic workup of newborns should include CSF and 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 fluid.

✓ Case Check 25-1

In the Case in Point, the cerebrospinal fluid was culture negative for the

more common cause of neonatal meningitis: group B Streptococcus,

Escherichia coli, and Listeria monocytogenes Both M hominis and U

urealyticum have been isolated from the cerebrospinal fluid of

low-birth-weight infants Because of the absence of a cell wall, these bacteria do

not Gram stain, so the lack of bacteria seen in a direct Gram stain

sup-ports the diagnosis.

Other Mycoplasma SpeciesMycoplasma genitalium, first isolated in 1980, has been associ-

ated with NGU, cervicitis, endometriosis, and PID There is

evidence linking M genitalium to some cases of tubal sterility

Its prevalence is not known, but it may be primarily a resident

of the gastrointestinal tract that occurs secondarily in the urinary or respiratory tracts Using polymerase chain reaction

genito-(PCR) assays, M genitalium has been found more frequently in

urethral samples taken from men with acute NGU than in those from men without urethritis

Mycoplasma fermentans has been noted as a likely

oppor-tunistic respiratory pathogen It is not known how often

M fermentans occurs in the respiratory tracts of healthy children,

but it has been detected in throats of patients with lower tory tract infection, in some of whom a specific causative agent

respira-was not identified Other groups of patients from whom M mentans has been recovered include adult patients with respira-

fer-tory illness and those with acquired immunodeficiency syndrome

(AIDS) M fermentans has been isolated from tissue in patients

In immunocompromised individuals, bacteremia and invasive

disease of the joints and respiratory tract caused by mycoplasmal

species have occurred U urealyticum has been reported to cause

chronic inflammatory diseases such as arthritis and cystitis in

hypogammaglobulinemic patients Mycoplasma isolates have

been intermittently associated with patients with endocarditis,

sternal wound infections, and arthritis

and wooden shafts should be avoided because of possible tory effects Most references recommend that swabs be made of Dacron polyester or calcium alginate with aluminum or plastic shafts and that swabs be removed when the sample is placed in

inhibi-a trinhibi-ansport medium On inhibi-arrivinhibi-al in the linhibi-aborinhibi-atory, the specimens should be frozen at −70° C if plating within 24 hours is not possible

Direct ExaminationBecause they lack a cell wall, the mollicutes will not be visible

by Gram staining A DNA fluorescent stain (e.g., acridine orange) can be used, but this is not specific for the mollicutes Antigen detection assays have been used but are generally low in sensitiv-ity and are not recommended The PCR assay has been described for the detection of many mollicutes with varied results Patients

with M pneumoniae infections can persistently harbor the

organ-ism for varied lengths of time after the acute infection Therefore,

it is difficult to interpret a positive PCR result No commercial kits are currently available

Culture

Media

Several media have been developed for the recovery of licutes, and no single medium is suitable for all species isolated from humans Penicillin can be added to minimize bacterial con-

mol-tamination 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’s 10B arginine broth) It

is difficult to maintain 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 Because mycoplasmas do not produce turbidity in broth media, a pH indicator such as phenol red should be added to detect growth

A8 agar can be used as a solid medium to recover M hominis and Ureaplasma spp.

Recovery of mycoplasma from blood can be performed by placing uncoagulated blood into mycoplasmal broth media A

with and without AIDS who died of systemic infection M

fer-mentans has also been isolated from synovial fluid of patients

with rheumatoid arthritis M penetrans has been demonstrated

in urine of homosexual males with human immunodeficiency

virus-associated disease M salivarium has been recovered from

culture or detected by PCR assay in synovial fluid from patients

with rheumatoid arthritis; however, the significance of this

organ-ism in this disease condition is unclear

Laboratory Diagnosis

Because recovery from culture is difficult (sensitivity ≅ 40%),

isolation of M pneumoniae from respiratory sites is infrequently

attempted Growth may take several weeks, and technical

exper-tise is necessary M hominis and Ureaplasma spp are less

strin-gent in their growth requirements 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,

tra-ditionally with acute and convalescent sera collected 2 to 3 weeks

apart to demonstrate a fourfold rise 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 nonspecific, and a thorough understanding

of the disease process is necessary for interpretation of serum

and culture results

Specimen Collection and Transport

Specimens for mycoplasmal culture include body fluids such as

blood, sputum, synovial fluid, CSF, amniotic fluid, and urine, as

well as wound aspirates and nasopharyngeal, cervical, and

vaginal swabs Tissue samples may also be submitted for culture

Because of the lack of a cell wall, all mycoplasmas are extremely

sensitive to drying and heat Ideally, specimens should be

inocu-lated at bedside If this is not possible, specimens should be

delivered immediately to the laboratory in a transport medium,

such as SP4 (sucrose phosphate buffer, Mycoplasma base, horse

serum [20%], and neutral red) or Shepard’s 10B broth or 2SP,

which are designed for the mycoplasma Cotton-tipped swabs

TABLE 25-5 Major Clinical and Corresponding Diagnostic Manifestation of Mycoplasma pneumoniae

With antimicrobial treatment 104° F 104° F 102° F 100° F Absent

Without antimicrobial treatment 104° F 100° F Absent Absent Absent

Chest radiograph +2 +3 +2 +2 +1

Mycoplasma culture with or without antibiotic treatment + + + + + +

Complement fixation (titer) ≤8 8 32 64 256 256 128 Mycoplasma-specific Ig

Commercial culture media and kits for the detection and recovery of mycoplasmal organisms have been developed and are available in Europe, and a few are now sold in the United States Such products may detect, quantify, identify, and deter-mine the antimicrobial susceptibility of genital mycoplasmas

from urogenital specimens and M pneumoniae from respiratory

secretions

Isolation and Identification

Once inoculated, broth media should be placed at 37° C under atmospheric conditions, whereas solid agar media may be incu-bated in an environment of room air enhanced with 5% to 10%

CO2, or in an anaerobic atmosphere of 95% N2 with 5% CO2 Incubation in a candle jar is adequate

M hominis and Ureaplasma spp colonies may appear within

2 to 4 days, whereas M pneumoniae may take 21 days or longer

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% (wt/vol) of gelatin may help

overcome the inhibitory effect of SPS; nevertheless, the use of

commercial blood culture media, whether or not used in

auto-mated instruments, is not recommended Figure 25-5 presents

a schematic representation of media and methods used in the

traditional procedures for isolation and identification of

Mycoplasma spp.

Fluids should be centrifuged and the pellet resuspended in a

small volume of liquid for media inoculation It is important that

specimens be diluted in broth up to 10−3 before plating each

dilu-tion This helps minimize the inhibitory effects of antimicrobial

agents, antibodies, and other inhibitors that may be present in the

GP–RBC–HAD §

(not M pneumoniae)

*SP4, Sucrose phosphate buffer, Mycoplasma base, fetal bovine serum (20%), phenol red Medium stabilizes and

decontaminates specimen Storage at –70° C for repeat 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.

§ GP–RBC–HAD = Guinea pig red blood cell hemadsorption b-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 can be used for identification.

Inoculate transport medium onto SP4 agar and incubate at 35° to 37° C

in CO 2 ; perform weekly microscopic observation for small (10 to 100 µm), grainy colony with thin “apron,” †

hold 4 weeks before reporting as negative Incubate SP4 broth

at 35 ° to 37° C (no CO 2 )

✓ 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.

Mycoplasma-like colonies are stained with the Dienes or

methy-lene 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 the periphery staining a light blue and the center dark blue

colony presentation on primary isolation when examined with a

stereomicroscope (Figure 25-7)

Although not conclusive, growth rate, body site recovered

from, and colony appearance can aid in the identification of

mycoplasma Glucose utilization in SP4 broth will cause an acid

shift producing a yellow color, whereas arginine metabolism will

produce a rise in pH, changing the indicator to a deeper red color

In 10B broth, urea or arginine utilization 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 specimen is suggestive of U urealyticum, whereas

an alkaline shift in media with arginine within 24 to 72 hours is

mono-M pneumoniae antibody is flooded on colonies on the plate;

the plate is then washed and examined for immunofluorescence The Chen assay is a fluorochrome method used to identify

Mycoplasma-infected cell cultures It uses a DNA fluorochrome stain (Hoechst 33258), which highlights Mycoplasma spp as

small ovoid bodies distributed throughout the glacial acetic acid–fixed cell culture Figure 25-8 shows Vero cells (a monkey

kidney cell line) artificially infected with Mycoplasma orale

(see Figure 25-8, A ) and M salivarium (see Figure 25-8, C) Note the differences in morphotypes and distribution Vero cell nuclei, which are rich in DNA, fluoresce with Hoechst 33258 stain in the negative control (see Figure 25-8, B) and in the infected cell cultures This method offers a unique way for diag-nostic and clinical virology laboratories to perform quality control on their continuous cell cultures The characteristic of guinea pig red blood cells (0.4% in saline) adhering to colonies

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, which are common inhabitants of the upper respiratory tract PCR-based assays have also been described

Ureaplasma spp., once called T-strain mycoplasma (T for

“tiny”), form extremely small colonies that are difficult to see with the naked eye; hence, mycoplasmal cultures on solid media should always be examined with a stereomicroscope Figure 25-9

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

Both 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 Although uncommon, extragenital M hominis infections

are emerging; this organism should be considered whenever many polymorphonuclear cells are seen on Gram stain but there

is no growth on routine bacterial culture M hominis grows well

anaerobically and will appear as pinpoint (0.05 mm), clear, tening, raised colonies on Columbia colistin–nalidixic acid agar

glis-or anaerobic blood agar (Centers fglis-or Disease Control and tion formula) in 48 hours Under these anaerobic conditions, the colonies do not display the fried egg morphology feature The anaerobic plate should be examined using oblique light Those colonies that do not 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

Preven-out the infection or suggest additional evaluations The cold agglutinin antibody titer had been used for many years as an indicator of primary atypical pneumonia but is insensitive and

nonspecific 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 commonly used technique for

demon-stration of M pneumoniae–specific antibodies was the

micro-method complement fixation assay, which was time-consuming and had inherent technical problems Several commercially available enzyme immunoassays and microimmunofluorescence 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 Detection methods were added for comparative analysis and completeness It is important to remember that demonstration of

a significant rise in antibody titer in conjunction with culture isolation is preferable for definitive diagnosis Serologic methods

are available for M hominis and U urealyticum but are generally

performed only by reference laboratories and are not mended for routine diagnosis

recom-Antimicrobial SusceptibilityBecause they lack a cell wall, the mollicutes are inherently resis-tant to the β-lactams—penicillins and cephalosporins—as well

Serologic Diagnosis

Because of the inherent difficulties of cultures and interpretations

of a positive PCR assay, M pneumoniae has historically 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 and convalescent measurements;

however, this often is not practical With newer methods, single

serum samples collected during the course of the disease can rule

FIGURE 25-8 Identification of Mycoplasma-infected cell culture using DNA-fluorochrome stain

FIGURE 25-9 Mixed isolation of Mycoplasma hominis and

Ureaplasma urealyticum showing why U urealyticum was

originally called “T” for tiny strain (arrow) (×40)

Interpretation of Laboratory Results

M pneumoniae detected by any method from pulmonary or

non-pulmonary specimens should be considered significant and a pathogen The high sensitivity of PCR means that a positive result must be correlated with the clinical picture Interpretation

of M hominis isolation is not as obvious; differentiation from

colonization and infection requires detailed clinical analysis and potentially repeat cultures Isolation from a normally sterile site

is significant

U urealyticum is the most difficult to assess In urogenital

specimens, it has been reported to colonize up to 70% of men and 45% of women with no apparent infection Its isolation is not indicative of pathogenicity, and it is incumbent on the labora-tory to educate the physician, usually including a statement with culture results suggesting its potential for colonization versus pathogenicity In these specimens, quantification is important

In sterile specimens, particularly CSF isolates, it is reasonable to assume that isolation is significant

Respiratory specimens received in the laboratory often provide limited clinical information Specimens are processed and inoculated onto the appropriate media given the most likely candidate for the disease, clinical presentation, age of patient, and seasonability, recognizing that there is a certain predictability with selected pathogens Table 25-7 presents laboratory methods used to diagnose infections caused by several pathogens—

Mycoplasma, Chlamydia, Legionella, mycobacteria, fungi, and

viruses—in various age groups All respiratory specimens should

be stored at −70° C Acute sera should also be stored frozen for

as sulfonamides, trimethoprim, and rifampin M pneumoniae has

remained susceptible to the tetracyclines, newer

fluoroquino-lones, and the macrolides (e.g., erythromycin) However, there

have been scattered reports of high-level macrolide resistance

Because of side effects, tetracycline is used only for the treatment

of adults M hominis, which is more resistant than M

pneu-moniae, is usually resistant to erythromycin but susceptible to

clindamycin and lincomycin, whereas U urealyticum is

gener-ally resistant to clindamycin and lincomycin and sensitive to

erythromycin Both organisms are often sensitive to tetracycline,

but high-level resistance is emerging and is common in some

geographic areas

Standard methods have not been developed for susceptibility

testing of mycoplasma, and protocols have varied considerably

among laboratories The agar dilution has been regarded as the

reference method; however, because of the high degree of

techni-cal expertise required and the few mycoplasmal isolates, this

assay is not offered by most hospital laboratories The broth

microdilution is the most commonly used method to determine

minimal inhibitory concentrations With antimicrobial resistance

reportedly increasing, the availability of newer, broad-spectrum

antimicrobials, and the emergence of more infections caused by

the mycoplasma, antimicrobial susceptibility testing methods are

being reevaluated Because of the variable susceptibility pattern

of M hominis, antimicrobial susceptibility testing is usually

rec-ommended for clinically significant isolates; these isolates should

be forwarded to a reference laboratory M pneumoniae has a

more predictable sensitivity pattern, so antimicrobial

susceptibil-TABLE 25-6 Comparative Features of Various Laboratory Methods Used to Detect Mycoplasma pneumoniae,

Mycoplasma hominis, and Ureaplasma urealyticum

Nonserologic

Culture Traditionally difficult Method of choice, but must

differentiate infection from colonization

Method of choice using urease detection, but must differentiate infection from colonization Indirect

Complement fixation Traditional assay but <50%

seroconvert; need fourfold rise between acute and convalescent sera >32 single titer may be suggestive Immunofluorescent

antibody

Measures IgG and IgM separately Measures IgG and IgM separately;

not recommended Latex agglutination IgM/IgG IgG only

Enzyme immunoassay Method of choice

M pneumoniae reactive IgM, IgG,

and IgA, but IgM may remain elevated for 1 yr

IgG, Immunoglobulin G; IgM, immunoglobulin M.

Cuccuru MA, et al: PCR analysis of Mycoplasma fermentans and M

penetrans in classic Kaposi’s sarcoma, Acta Derm Venereol 85:459,

2005.

Cultrera R, et al: Molecular evidence of Ureaplasma urealyticum and

Ureaplasma parvum colonization in preterm infants during

respira-tory distress syndrome, BMC Infect Dis 6:166, 2006.

Dhawan B, et al: Evaluation of the diagnostic efficacy of PCR for

Urea-plasma urealyticum infection in Indian adults with symptoms of

genital discharge, Jpn J Infect Dis 59:57, 2006.

Shyh-Ching L, et al: Mycoplasma penetrans infections and

seroconver-sion in patients with AIDS: identification of major mycoplasmal

antigens targeted by host antibody response, Immunol Med Microbiol

■ The most significant species of Mycoplasmataceae include M

pneumoniae, M hominis, and Ureaplasma spp., although others

are beginning to be recognized as opportunistic pathogens.

pneumonia.

com-monly diagnosed by culture, although polymerase chain reaction

technology is also available.

■ Because of the lack of a cell wall, the mycoplasmas are inherently

resistant to the β-lactam antibiotics.

Learning Assessment Questions

1 From what source did the infant described in the Case in Point

likely acquire the infection?

2 Would routine prenatal culture of the mother have yielded this

organism?

3 Why was the Gram stain negative?

4 How does primary atypical pneumonia caused by Mycoplasma

pneumoniae differ from pneumonia caused by Streptococcus

pneumoniae?

5 Name the four common species of mollicutes associated with the

genitourinary tracts of humans.

TABLE 25-7 Laboratory Detection of Frequent Respiratory Pathogens

Age

Organism

Frequently

Newborn 1, 3, 4 Pneumonia, aseptic

workup

Tracheal suction Gram Traditional, plus

mycoplasmal Grade school 2, 4 Atypical pneumonia Fall Sputum Gram, acid-fast

bacillus

Traditional, plus mycoplasmal

EIA, mycoplasmal, IgM

College student 1, 2 Biphasic disease with

pharyngitis and later, bronchitis

Spring? Sputum DFA Cell culture,

mycoplasmal

EIA, mycoplasmal IgM

Gomori methenamine silver, toluidine blue, and/or calcofluor white

Traditional plus fungal, acid-fast bacillus

1, Chlamydophila pneumoniae; 2, Mycoplasma pneumoniae (outbreak); 3, Mycoplasma hominis; 4, viral (outbreak): adenovirus, respiratory virus, influenza

(seasonal); 5, other—acid-fast bacilli, fungus, Legionella, or Pneumocystis pneumonia; 6, Streptococcus pneumoniae; EIA, enzyme immunoassay; DFA, direct

fluorescent antibody; IgG, immunoglobulin G; IgM, immunoglobulin M.

6 What special stain is used on suspected colonies of Mycoplasma?

7 What culture media are used to isolate Mycoplasma pneumoniae,

M hominis, and Ureaplasma urealyticum?

8 Why is cold agglutinin titer not a useful diagnostic tool for M pneumoniae?

9 What current serologic assays are available to demonstrate M pneumoniae antibodies?

10 Why are the mollicutes universally resistant to penicillin?

Slowly Growing Species

Rapidly Growing Species

■ SUSCEPTIBILITY TESTING OF MYCOBACTERIUM TUBERCULOSIS

■ IMMUNODIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS INFECTION

Skin TestingSerology

CHAPTER OUTLINE

OBJECTIVES

After reading and studying this chapter, you should be able to:

1 Compare the general characteristics of mycobacteria to those of

other groups of bacteria

2 Discuss the clinical significance of nontuberculous mycobacteria

3 Discuss the safety precautions to be followed while working in a

mycobacteriology laboratory

4 Describe the appropriate specimen collection and processing

procedures to recover mycobacteria from clinical samples

5 Justify the digestion and decontamination of certain clinical

specimens for the isolation of mycobacteria

6 Describe the principles and procedures for the stains used to

demonstrate mycobacteria in clinical samples and isolates

7 Compare the different culture media used for the isolation of mycobacteria

8 Discuss the different tests used to identify mycobacteria

9 Compare continuous monitoring systems to those of conventional media for detecting mycobacterial species in clinical samples

10 Develop a protocol for the isolation and identification of Mycobacterium tuberculosis from a sputum specimen

11 Discuss the clinical disease caused by Mycobacterium tuberculosis

12 Describe the use of the tuberculin skin test

Case in Point

A 56-year-old white man came to the emergency department

complaining of fatigue and weight loss (10 lb) over the past 12

months The patient also complained of a cough for 3 months

that produced red-tinged sputum He indicated a history of

night fever and chills but denied dyspnea or chest pain The

patient had a family history of pulmonary tuberculosis from his

original home in Mexico He reported that his last purified

protein derivative (PPD) skin test, approximately 5 years ago, was

negative Vital signs included temperature of 36.5° C (97.7° F),

pulse of 63 beats/m, respirations 15/m, and blood pressure

infiltrate in the right upper lobe A computed tomography scan

of the chest showed a nodular patchy opacity in the right upper lobe The patient was admitted for further evaluation A PPD skin test showed a 10- × 7-mm induration Three sputum samples were obtained over a 3-day period for acid-fast bacilli (AFB) smears and culture Direct smears on all three samples were reported as no organisms seen Processed samples were inoculated onto Löwenstein-Jensen medium and into BACTEC 12B bottles After 12 to 14 days of incubation, the BACTEC bottles from all three specimens were positive Stained smears

of the bottles revealed AFB by Kinyoun stain Polymerase chain reaction DNA amplification for Mycobacterium tuberculosis

General CharacteristicsMycobacteria are slender, slightly curved or straight, rod-shaped organisms 0.2 to 0.6 µm × 1 to 10 µm in size They are nonmotile and do not form spores The cell wall has extremely high lipid content; thus, mycobacterial cells resist staining with commonly used basic aniline dyes, such as those used in the Gram stain,

at room temperature Mycobacteria take up dye with increased staining time or application of heat but resist decolorization with

acid-ethanol This characteristic is referred to as acid fastness—

hence, the term AFB—and is a basic characteristic in ing mycobacteria from most other genera

distinguish-Mycobacteria are strictly aerobic, but increased carbon dioxide (CO2) will enhance the growth of some species The pathogenic mycobacteria grow more slowly than most other bac-teria pathogenic for humans The rapidly growing species gener-ally grow on simple media in 2 to 3 days at temperatures of 20° C

to 40° C Most mycobacteria associated with disease require 2 to

6 weeks of incubation on complex media at specific optimal temperatures One of the mycobacteria pathogenic for humans,

M leprae, fails to grow in vitro.

Clinical Significance of the Mycobacterium tuberculosis Complex

The MTB complex consists of M tuberculosis, M bovis

(including the vaccination strain bacillus Calmette-Guérin), M

BOX 26-1 Usual Clinical Significance of

Mycobacterium Species Isolates

Pathogen

Mycobacterium tuberculosis Mycobacterium bovis Mycobacterium ulcerans

Often Pathogen, Potential Pathogen

Mycobacterium avium complex Mycobacterium kansasii Mycobacterium marinum Mycobacterium haemophilum Mycobacterium xenopi Mycobacterium genavense Mycobacterium abscessus subsp abscessus Mycobacterium chelonae

Usual Saprophyte, Rare Pathogen

Mycobacterium gordonae Mycobacterium flavescens Mycobacterium gastri Mycobacterium nonchromogenicum Mycobacterium terrae

Mycobacterium phlei Mycobacterium smegmatis Mycobacterium vaccae Mycobacterium thermoresistibile

The genus Mycobacterium is composed of approximately

100 recognized and proposed species The most familiar

of the species are MTB and Mycobacterium leprae, the

causative agents of tuberculosis (TB) and Hansen disease

(leprosy), respectively Both diseases have long been associated

with chronic illness and social stigma TB remains a major

cause of morbidity and mortality in the world today Also, the

growing number of immunocompromised patients worldwide

has led to a resurgence of TB and diseases caused by

nontuber-culosis mycobacteria (NTM), or mycobacteria other than

M tuberculosis.

In addition to TB and Hansen disease, Mycobacterium spp

produce a spectrum of infections in humans and animals A large

group of mycobacteria, excluding the M tuberculosis complex

and M leprae, normally inhabit the environment and can cause

disease that often resembles TB in humans These organisms are

sometimes referred to as atypical mycobacteria or mycobacteria

other than the tubercle bacillus (MOTT) The term

nontubercu-lous mycobacterium is used here Box 26-1 shows the usual

clinical significance of Mycobacterium spp isolates.

Epidemiologic changes have led to challenges in the

myco-bacteriology laboratory, including rapid identification of all

clini-cally significant mycobacteria and antimicrobial susceptibility

testing of Mycobacterium spp Fortunately, new developments in

the field of clinical mycobacteriology are helping meet these

challenges Rapid methods may eliminate the need for lengthy

culturing for isolation and protracted biochemical methods of

identification Future developments in the application of

molecu-lar biology to mycobacteriology may further diminish the time

required for identification, increase accuracy and reproducibility,

ease performance, and reduce cost

PhotochromogenicPott diseasePurified protein derivative (PPD)

ScotochromogenicZiehl-Neelsen stain

antituberculosis regimen of isoniazid, rifampin, pyrazinamide,

and ethambutol was recommended

Issues to Consider

After reading the patient’s case history, consider:

■ What might be significant about this patient’s family

history

■ What are the characteristic symptoms of tuberculosis

■ What is the typical length of time for a culture to yield

pathogenic Mycobacterium

response is less organized, and no granuloma is formed Without granuloma or necrosis, lesions may heal without obvious pathol-ogy With necrosis, a caseous material may be present at the site

of the primary lesion as a result of solid or semisolid amorphous material laid down at the site of necrosis After healing of first-degree infection, the bacilli are not totally eradicated but can remain viable in granulomas for months or years In infected individuals, there is a potential for reactivation of TB

Clinical diagnosis of primary TB is usually limited to signs

and symptoms and a positive PPD skin test Children may

dem-onstrate a nonproductive cough and fever, with or without ness of breath; these symptoms are unusual in adults Chest radiographs are usually normal although, rarely, there may be infiltrates without cavitation in the anterior segment of the upper, middle, or lower lobe, with hilar or paratracheal lymphadenopa-thy Along with these limited clinical findings, patients with primary TB can have a paucity of bacteriologic findings If sputum or bronchial washings are cultured during the primary infection, the yield is only 25% to 30% positive

short-A small percentage of individuals who are infected with TB develop progressive (active) pulmonary disease, usually from a failed cellular immune response and hence a failure to stop mul-tiplication of the bacilli In young children or older adults who are primarily infected, and in people with an underlying immu-nodeficiency, massive lymphohematogenous dissemination may occur and lead to meningeal or miliary (disseminated) TB In addition, 10% of young adults may progress to active disease from their primary infection This will resemble reactivation TB

in older adults; the only way to differentiate it is by finding a positive PPD in a previously negative individual

africanum, M canettii, and M microti M africanum has been

associated with human cases of TB in tropical Africa, and M

microti has been linked to TB in immunocompetent and

immu-nocompromised individuals The latter three species are rarely

encountered in the United States

Mycobacterium tuberculosis

MTB was first described by Robert Koch in 1882; however, TB

is one of the oldest documented communicable diseases In 2011,

the World Health Organization (WHO) estimated that 12 million

people worldwide suffered from TB; this is a decrease of 36%

since 1990 However, as many as one third of the world’s

popula-tion might be harboring the bacteria There were an estimated 8.7

million new cases and 1.4 million deaths in 2011 In the United

States, before 1985, the number of TB cases continually declined

at a rate of about 5%/year In 1985, this decline ended, and the

number of documented TB cases annually in the United States

increased until the early 1990s The increase in the early 1980s

could be attributed to several factors, including the acquired

immunodeficiency syndrome (AIDS) epidemic, which increases

a person’s risk for TB, and greater spread among inhabitants of

closed environments, such as nursing homes, correctional

facili-ties, and shelters for the homeless In the 1990s, the U.S Centers

for Disease Control and Prevention (CDC) allocated additional

funds for public laboratories to improve identification and

sus-ceptibility testing of mycobacteria As a result, the U.S incidence

of TB again steadily declined, from a high of 26,673 in 1992 to

10,528 (a rate of 3.4/100,000) in 2011 Asians, for the first time,

exceeded all other racial or ethnic groups with the largest

per-centage of total cases (30%) Currently, in the United States,

more cases are associated with foreign-born individuals from

endemic areas than with U.S.-born individuals

Primary Tuberculosis

After exposure to M tuberculosis, whether a person develops TB

is determined by his or her cellular immune response, amount of

exposure, and virulence of the strain TB is usually a disease of

the respiratory tract Tubercle bacilli are acquired from persons

with active disease who are excreting viable bacilli by sneezing

or talking Airborne droplets containing bacteria, 1 to 5 µm in

size, enter the respiratory tract of an exposed individual and reach

the lung alveoli M tuberculosis cells are phagocytized by

alveo-lar macrophages and are capable of intracellualveo-lar multiplication

In a person with adequate cellular immunity, T cells arrive within

4 to 6 weeks owing to macrophage-activating polypeptides

termed lymphokines This enables the macrophage in the area of

infection to destroy the intracellular mycobacteria There is then

a regression and healing of the primary lesion and any

dissemi-nated foci of infection by the M tuberculosis organisms.

In many exposed individuals, the immune system does not

eliminate the bacteria The pathologic features of TB are the

result of a hypersensitivity reaction to mycobacterial antigen If

there is little antigen and a strong hypersensitivity reaction, a

hard tubercle or granuloma may be formed The granuloma is an

organization of lymphocytes, macrophages, fibroblasts, and

cap-illaries With granuloma formation, healing occurs, as well as

fibrosis, encapsulation, and calcification, with scar formation as

a reminder of the past infection If the antigen load and

hyper-✓ Case Check 26-1

A positive PPD skin test only indicates past exposure to M tuberculosis;

it does not imply a recent infection In the Case in Point, the patient reported a negative skin test about 5 years ago, thus establishing a negative baseline During his current evaluation, he exhibited a positive skin test, indicating exposure and subsequent immune response some time in the last 5 years.

no symptoms, but most patients eventually have cough, chest pain, and productive sputum Hemoptysis, indicating cavitation

pleural fluid, but cultures may be positive in 20% to 50% of cases; pleural biopsies offer a higher yield of microbiologic diagnosis.

Lymphadenitis is usually a disease of children, appearing as painless head or neck swellings Lymph node involvement, par-ticularly mediastinal, has been a common extrapulmonary mani-festation in patients with AIDS Genitourinary TB can involve the kidneys and genital organs Renal TB accounts for 2% of all cases of TB and manifests as typical urinary tract symptoms and sterile pyuria Cultures may be positive in up to 80% of cases Male genital TB usually appears as a scrotal mass and frequently occurs along with renal TB In men and women, hematogenous spread is generally the source of genitourinary TB Skeletal TB

of the spine is referred to as Pott disease Back pain is the most

common characteristic Cultures of bone and tissue are needed

to confirm the diagnosis Peripheral skeletal bones and joints also may be involved, with the hip and knee being the most common sites

Meningitis caused by M tuberculosis is usually the result of

a rupture of a tubercle into the subarachnoid space and not usually via hematogenous spread In childhood, it occurs rarely after primary pulmonary infection Most infections occur at the base of the brain; patients may develop very thick, gelatinous, masslike lesions there With more chronic disease, a fibrous mass may surround cranial nerves Involvement of arteries can cause infarctions Cerebrospinal fluid (CSF) examination usually reveals an elevated protein level, decreased glucose level, and a predominance of lymphocytes

Identification of Mycobacterium tuberculosis

Colonies of this slowly growing species are typically raised, with

a dry, rough appearance The colonies are nonpigmented and classically described as being buff-colored (Figure 26-1) Elaboration of cord factor can result in characteristic cord forma-tion Optimal growth occurs at 35° C to 37° C

Biochemically, M tuberculosis is characteristically positive

for niacin accumulation, reduction of nitrate to nitrite, and duction of catalase, which is destroyed after heating (heat-stable, catalase-negative) Isoniazid-resistant strains may not produce

pro-catalase at all M tuberculosis is inhibited by nitroimidazopyran

or p-nitroacetylamino-β-propiophenone (NAP) This species can

be distinguished from M bovis by the inhibition of M bovis by

thiophene-2-carboxylic acid hydrazide (T2H) and dase activity

pyrazinami-with increased linear densities extending to the hilum;

thick-walled cavities without air-fluid levels usually are found in apical

or posterior segments of the upper lobe or superior segment of

the middle lobe of the lung If there is bronchogenic spread of

the bacilli, multiple alveolar densities will be seen; rarely is there

enlargement of the lymph nodes

In chronic disease, fibrosis, loss of lung volume, and

calcifica-tions will be demonstrated The PPD skin test may be negative

in up to 25% of these cases; diagnosis is confirmed by stained

smear and culture of sputum, gastric aspirates, or bronchoscopy

specimens Fiberoptic bronchoscopy has been found to yield a

95% recovery of the bacteria; postbronchoscopic sputa are also

usually positive In any case of pulmonary TB disease, there can

be complications if diagnosis and treatment are delayed These

include empyema, pleural fibrosis, massive hemoptysis, adrenal

insufficiency (rare), and hypercalcemia (up to 25% of cases) In

patients with AIDS and TB with drug-resistant bacilli, the risk of

progression to disease from infection is quite high, although the

clinical findings may vary from those in the non-AIDS patient

with reactivation TB The diagnosis is usually made by stained

smears and culture, with a rate of sensitivity similar to that in the

non-AIDS patient

Extrapulmonary Tuberculosis

Extrapulmonary TB occurred much less commonly than

pulmo-nary TB (<15%) before the AIDS epidemic; however, as cases

of pulmonary TB in the United States declined, the number of

cases of extrapulmonary TB remained constant Cases of

extra-pulmonary disease are a common presentation in individuals with

human immunodeficiency virus (HIV) infection, although it is

most often in association with pulmonary disease

Miliary TB refers to the seeding of many organs outside the

pulmonary tree with AFB through hematogenous spread This

usually occurs shortly after primary pulmonary disease but can

take place at any point in the course of acute or chronic TB The

most common sites of spread of M tuberculosis are the spleen,

liver, lungs, bone marrow, kidney, adrenal gland, and eyes,

usually in that order of occurrence Other forms of

extrapulmo-nary TB include pleural, lymphadenitis, gastrointestinal (GI),

skeletal, meningeal, peritoneal, and genitourinary infections

Almost any organ of the body can be infected by M tuberculosis;

additional uncommon manifestations include peritonitis,

cutane-ous TB, laryngitis, otitis, involvement of the adrenal glands and

eyes, and breast infections

Overall, children account for most cases of miliary TB, but it

is also a common form of TB in HIV-infected individuals The

mortality is 20% or higher in most studies; the finding of

men-ingitis is an extremely poor prognostic indicator Up to 70% of

HIV-infected patients may have extrapulmonary TB alone or,

usually, in combination with pulmonary disease The most

common extrapulmonary sites in this population are lymph nodes

(especially mediastinal), genitourinary tract, and the abdominal

cavity Bacteremia is not uncommon

Pleurisy, an unexplained pleural effusion with mononuclear

pleurocytosis, manifests as cough, fever, and chest pain,

resem-bling the presentation of bacterial pneumonia; it occurs in about

5% of all cases of TB In endemic areas, pleurisy presents in

young persons; in the United States, middle-aged to older persons

are most affected Resolution is common AFB are rarely seen in FIGURE 26-1stein-Jensen medium Mycobacterium

tuberculosis growing on Löwen-MDR-TB requires an extended treatment period compared with drug-susceptible isolates For cases of resistance to isonia-zid or rifampin, second-line antituberculosis drugs may include aminoglycosides and fluoroquinolones In communities in which

at least 4% of the isolates are drug-resistant, a regimen of four drugs is usually recommended With the numbers of cases of

MDR M tuberculosis increasing, newer agents are being tested

in vitro to determine their efficacy

Mycobacterium bovis

Mycobacterium bovis produces TB primarily in cattle but also in

other ruminants, as well as in dogs, cats, swine, parrots, and humans The disease in humans closely resembles that caused by

M tuberculosis and is treated similarly In some areas of the

world, a significant percentage of cases of TB are caused by

M bovis, but in the United States the number of isolates of this

organism is very low

M bovis is closely related taxonomically to M tuberculosis and belongs to the M tuberculosis complex It grows very

slowly on egg-based media, producing small, granular, rounded, nonpigmented colonies with irregular margins after 21 days

of incubation at 37° C On Middlebrook 7H10 medium,

colo-nies are similar to those of M tuberculosis but slower to mature Most strains of M bovis are niacin-negative, do not

reduce nitrate, and do not grow in the presence of T2H,

char-acteristics that distinguish the species from most strains of M tuberculosis.

Clinical Significance of Nontuberculous Mycobacteria

Most NTM are found in soil and water They have been monly implicated as opportunistic pathogens in patients with underlying lung disease, immunosuppression, or percutaneous trauma AIDS has contributed greatly to the incidence and aware-ness of NTM disease Chronic pulmonary disease resembling TB

com-is the usual clinical presentation associated with these organcom-isms, although a few species are more often associated with cutaneous infections Infections caused by the NTM are not considered transmissible from person to person

Slowly Growing Species

Mycobacterium avium Complex

Epidemiology Mycobacterium avium and M

intracellu-lare are part of the Mycobacterium avium complex (MAC)

These organisms are common environmental saprophytes and have been recovered from soil, water, house dust, and other environmental sources Certain areas, such as coastal marshes,

have higher concentrations of the organism M avium is a cause

of disease in poultry and swine, but animal to human sion has not been shown to be an important factor in human disease Environmental sources, especially natural waters, seem

transmis-to be the reservoir for most human infections Moreover, there appears to be a close association between many of the organisms that cause disease in humans and those commonly found in animals, suggesting that some infections may qualify as a zoo-nosis A large increase in MAC infections occurred in the past

Treatment

The treatment of TB involves the use of more than one

antimy-cobacterial agent For pulmonary TB, treatment typically involves

a 9-month course of therapy with isoniazid and rifampin, usually

once per day the first month and twice a week thereafter Many

regimens also include a 2- to 8-week initial course of

streptomy-cin or ethambutol Most individuals clear their sputum of AFB

within the first 2 months Pyrazinamide (PZA) may be added to

the regimen if there is a suspicion of lowered cellular immunity

and a need to obtain bactericidal levels of antimycobacterial

activity intracellularly in macrophages PZA is usually

recom-mended for a shorter course, initially along with isoniazid and

rifampin

Multidrug-Resistant Mycobacterium tuberculosis

The incidence of multidrug-resistant (MDR) TB in the United

States decreased from 2.5% of isolates in 1993 to 1.3% in 2011

Since 1997, the percentage of U.S.-born patients with MDR-TB

has remained low, less than 1.0% However, of the total number

of reported MDR-TB cases, the proportion occurring in

foreign-born persons increased from 25.5% in 1993 to 82% in 2011

Within any population of M tuberculosis, resistance to a

single agent can develop at a fairly well-defined rate For example,

with isoniazid and streptomycin, the chance that a resistant

isolate will develop is approximately 1 in 106 The rate of

spon-taneous mutation of resistance to both drugs in one cell is the

product of the rates of resistance to the individual drugs or 1 in

1012 In a patient with pulmonary TB, the pulmonary cavity may

contain 107 to 109 bacterial cells Random drug resistance has a

good likelihood of developing when only one antimycobacterial

agent is used or if the patient is on multidrug therapy and fails

to complete the course of medication Therefore, the use of

com-bination therapy (i.e., two or more drugs) to treat mycobacterial

infections is common

Risk factors for drug resistance may include previous

treat-ment for TB, residence in an area endemic for drug resistance,

or close contact with an individual who is infected with MDR-TB

Drug resistance is usually acquired by spontaneous mutations as

a result of the inappropriate use of antimicrobial agents to treat

M tuberculosis and the lack of patient compliance If compliance

is an issue, directly observed therapy is recommended to ensure

proper treatment Otherwise, resistance may be assumed and

tested for in vitro

MDR-TB is defined as resistance to at least isoniazid and

rifampin, drugs recognized as the primary treatments for drug

susceptible M tuberculosis Extensively drug-resistant TB

(XDR-TB) is defined as resistance to isoniazid and rifampin

plus resistance to any fluoroquinolone and at least one of three

injectable second-line anti-TB drugs—the aminoglycosides

ami-kacin, kanamycin, or capreomycin In the United States, 6

cases of XDR-TB were reported in 2011, bringing the total

to 12 since 2008 Of the 12 XDR-TB cases, 11 were in

foreign-born persons Clusters of XDR-TB have been reported in other

areas of the world Because of the threat of MDR-TB and

XDR-TB, it is important for laboratories to identify

Mycobac-terium spp rapidly and perform antimicrobial susceptibility

testing so that appropriate therapy can be administered as

The significance of in vitro tests for predicting clinical response and recommendations for testing individual isolates have yet to

M avium subsp paratuberculosis or M avium subsp silvaticum

M avium subsp paratuberculosis is difficult to cultivate because

of its very slow growth rate (3 to 4 months) and its need for a mycobactin-supplemented medium for primary isolation Myco-bactin is an iron-binding hydroxamate compound produced by other mycobacterial species

Mycobacterium kansasii

Epidemiology Mycobacterium kansasii is second to MAC

as the cause of NTM lung disease In the United States, most

cases of M kansasii infections have been reported from the

southern states of Texas, Louisiana, and Florida; from Illinois

and Missouri in the Midwest; and from California M kansasii

strains have been isolated from water, but the natural source of human infection is not clear As with other NTM, infections are not normally considered contagious from person to person

Clinical Infections The most common manifestation is chronic pulmonary disease involving the upper lobes, usually with evidence of cavitation and scarring Extrapulmonary infec-tions, including lymphadenitis, skin and soft tissue infections, and joint infection, have been reported occasionally Dissemi-

nated M kansasii infection rarely occurs in immunocompetent

individuals but has been reported in severely mised patients, particularly those with AIDS

immunocompro-When tested in vitro using the current drug concentrations

recommended for M tuberculosis, most strains of M kansasii

are susceptible to rifampin and ethambutol, partially resistant to isoniazid and streptomycin, and resistant to pyrazinamide For

treatment of pulmonary disease caused by M kansasii, a

multi-drug regimen of isoniazid, rifampin, and ethambutol is currently recommended

Laboratory Diagnosis M kansasii is a slow-growing

organism that appears as long rods with distinct crossbanding

M kansasii has an optimal growth temperature of 37° C, and

colonies appear smooth to rough, with characteristic wavy edges and dark centers when grown on Middlebrook 7H10 agar Some cording can usually be seen with low-power magnification Colo-

nies are photochromogenic (Figure 26-2), meaning that they form a pigment when exposed to light but are nonpigmented in the dark With prolonged exposure to light, most strains form dark red crystals of β-carotene on the surface of and inside the

colony Scotochromogenic (produce pigment in light and dark) and nonchromogenic strains are rarely isolated Most strains are

strongly catalase-positive (>45 mm in semiquantitative test); strains that are low catalase producers (<45 mm) are less com-monly isolated

Characteristics that distinguish this species are a growth

rate similar to that of M tuberculosis at 37° C, strong

patients with AIDS MAC is now the most common NTM

causing TB in the United States

Clinical Infections Pulmonary disease resulting from

MAC infection presents a clinical picture similar to that of TB—

cough, fatigue, weight loss, low-grade fever, and night sweats

Radiologic examination demonstrates cavitary disease in most

patients, whereas solitary nodules or diffuse infiltrates may be

observed in others Disseminated disease is common, usually

occurring in immunocompromised patients or patients with

hematologic abnormalities MAC infections are the most common

systemic bacterial infection in patients with AIDS The loss of

CD4+ T cells reduces the activation of the macrophage to kill

MAC organisms

The clinical outcome of MAC lung disease is unpredictable,

so the management of affected patients can be difficult

Obser-vation, therapy for underlying pulmonary disease (e.g.,

bron-chodilators, broad-spectrum antimicrobials, smoking cessation),

and periodic sputum cultures may be all that is required for

most patients For patients with significant symptoms and

advanced or progressive radiographic disease, multidrug therapy

is indicated For children with cervical lymphadenitis from

MAC, excisional surgery without chemotherapy is usually

suc-cessful A combination of surgical excision and chemotherapy

is the usual treatment for adults with localized nonpulmonary

disease Most cases of disseminated disease in

immunosup-pressed patients without AIDS respond to multidrug regimens

Multidrug therapy consisting of ethambutol, rifampin (or

rifabu-tin), clofazimine, and an aminoglycoside has resulted in

symp-tomatic and clinical improvement in most (but not all) patients

with AIDS

Laboratory Diagnosis Because the two species in the

MAC are so similar, most laboratories do not distinguish between

them but report isolates of both species as MAC On primary

isolation media, these organisms grow slowly, producing thin,

transparent or opaque, homogeneous smooth colonies A small

proportion of strains may exhibit rough colonies Usually the

colonies are nonpigmented, but they may become yellow with

age Rarely are the colonies pigmented from the onset of

detect-able growth Optimal growth temperature is 37° C

On microscopic examination, the cells are short,

coccobacil-lary, and uniformly stained, without beading or banding Long,

thin, beaded bacilli resembling Nocardia spp may be seen in

stains of very young cultures or under certain other conditions

MAC species are inactive in most physiologic tests used to

iden-tify the mycobacteria Exceptions are the production of a

heat-stable catalase and the ability to grow on media containing 2 µg/

mL of T2H Nucleic acid probes are available for the

identifica-tion of MAC and the two individual species

Susceptibility Testing In laboratory tests, members of the

MAC are generally resistant to the relatively low concentrations

of anti-TB drugs currently used for testing M tuberculosis

Treat-ment recommendations have been based largely on empiric data

rather than in vitro susceptibility testing For this reason, routine

agar dilution susceptibility testing with the anti-TB agents, as

currently performed for testing M tuberculosis, is not

recom-mended for MAC isolates Currently, the usefulness of testing at

higher drug concentrations than those used for M tuberculosis

or determination of minimum inhibitory concentration (MIC) is

being evaluated In vitro susceptibility studies using

combina-tions of drugs have shown significant synergism between drugs