Mycoplasma and Ureaplasma

DESCRIPTION OF MOLLICUTES Back to top

Mollicutes are smaller than conventional bacteria in cellular dimensions as well as genome

size, making them the smallest free-living organisms known. Mycoplasmas associated with

humans range from coccoid cells of about 0.2 to 0.3 μm in diameter, as in Ureaplasma spp.

and Mycoplasma hominis (112), to tapered rods 1 to 2 μm in length and 0.1 to 0.2 μm in

width, as in the case of Mycoplasma pneumoniae (152). Mollicutes are contained by a

trilayered cell membrane and do not possess a cell wall. The permanent lack of a cell wall

barrier makes the mollicutes unique among prokaryotes and differentiates them from

bacterial L forms for which the lack of the cell wall is but a temporary reflection of

environmental conditions. Lack of a cell wall also renders the mollicutes insensitive to the

activity of beta-lactam antimicrobials, prevents them from staining by the Gram reaction,

and is largely responsible for their pleomorphic form. The extremely small genome (<600 kb

in the case of Mycoplasma genitalium) and limited biosynthetic capabilities explain the

parasitic or saprophytic existence of these organisms, their sensitivity to environmental

conditions, and their fastidious growth requirements which can complicate cultural detection.

Mollicutes require enriched growth medium supplemented with nucleic acid precursors.

Except for acholeplasmas, asteroleplasmas, and mesoplasmas, mollicutes require sterols in

growth media, supplied by the addition of serum. Growth rates in culture medium vary

among individual species, with generation times of approximately 1 hour

for Ureaplasma spp., 6 hours forM. pneumoniae, and 16 hours for M. genitalium (68).

Typical mycoplasmal colonies vary from 15 to 300 μm in diameter. Colonies of some species,

such as M. hominis, often exhibit a “fried egg” appearance owing to the contrast between

deeper growth in the center of the colony, with more shallow growth at the periphery (Fig.

1). Other species, such as M. pneumoniae, produce spherical colonies (Fig. 2). Whereas

colonies of mycoplasmal species may be observed with the naked eye, those produced by

ureaplasmas are typically 15 to 60 μm in diameter and require low-power microscopic

magnification for visualization (Fig. 3).

Mycoplasmas of human origin can be classified according to whether they ferment glucose,

utilize arginine, or hydrolyze urea (Table 2). Except for hydrolysis of urea, which is unique

for ureaplasmas, these biochemical features are not sufficient for species distinction.

Anaeroplasmas and asteroleplasmas, which occur in ruminants, are strictly anaerobic, while

most other mollicutes are facultative anaerobes.

Attachment of M. pneumoniae to host cells in the respiratory tract of humans is a

prerequisite for colonization and infection. Cytadherence, mediated by the P1 adhesin and

other accessory proteins, described in detail in recent reviews (139, 147), is followed by

induction of chronic inflammation and cytotoxicity mediated by hydrogen peroxide, which

also acts as a hemolysin. M. pneumoniae stimulates B and T lymphocytes and induces

formation of autoantibodies which react with a variety of host tissues and the I antigen on

erythrocytes, which is responsible for production of cold agglutinins (147). Recently, an ADPribosylating

toxin with significant sequence homology to the pertussis toxin S1 subunit, now

known as the community-acquired respiratory distress syndrome toxin that causes

vacuolation and ciliostasis in cultured host cells, was described in M. pneumoniae (74). M.

genitalium also possesses a terminal structure, the MgPa adhesin, which facilitates its

attachment to epithelial cells (60). Factors involved with Ureaplasma and M.

hominis attachment have not been characterized to the same extent as for M.

pneumoniae, and these mycoplasmas do not have prominent attachment organelles. Henrich

et al. (58) demonstrated the presence of the variable adherence-associated antigen that is

believed to be a major adhesin of M. hominis and could also assist in evasion of host immune

responses through antigenic variation. Ureaplasmas also attach to a variety of cell types,

mediated by adhesin proteins expressed on the surface of the bacterial cell. The multiplebanded

(MB) antigen contains serotype-specific and cross-reactive epitopes and is a

prominent antigen recognized during human ureaplasmal infections (146). Ureaplasmas also

produce immunoglobulin A (IgA) protease and release ammonia through urealytic activity

(146).

EPIDEMIOLOGY AND TRANSMISSION Back to top

Mollicutes are common in practically all mammalian species as well as many other

vertebrates in which they have been sought. Although most mollicutes have species-specific

host-organism associations, some mycoplasmas and acholeplasmas of animal origin occur in

a wide variety of different hosts. Mollicutes in the genera Spiroplasma, Mesoplasma,

Entomoplasma, and Acholeplasma can be isolated from insects and plants.

In humans, mycoplasmas and ureaplasmas are associated with the mucosa, residing

predominantly in the respiratory or urogenital tract, rarely penetrating the submucosa,

except in cases of immunosuppression or instrumentation, when they may invade the

bloodstream and disseminate to various organs and tissues. Many Mycoplasma spp. exist as

commensals in the oropharynx (Table 2) and are associated with invasive disease only in

very rare circumstances. Oral commensal mycoplasmas may occasionally spread to the lower

respiratory tract but should not cause diagnostic confusion with M. pneumoniae if

appropriate means of organism identification are employed. Mycoplasma fermentans has

been detected in various body sites, including the urogenital tract, throat, lower respiratory

tract, and other body locations, including joints (146), but its primary site of colonization and

true disease potential are incompletely understood. The frequent occurrence of pathogenic

species such as M. hominis and ureaplasmas in the lower urogenital tract in healthy men and

women has complicated understanding of their disease-producing capabilities. Mycoplasma

primatumand Mycoplasma spermatophilum have been detected in the urogenital tract but

have not been associated with disease. PCR assays have demonstrated the frequent

occurrence of M. genitalium in the urogenital tract in men with urethritis and in the lower and

upper genital tract sites in women and of Mycoplasma penetrans in the urine of homosexual

males with human immunodeficiency virus (64, 84). Although mycoplasmas are generally

considered extracellular organisms, intracellular localization is now appreciated for M.

fermentans, M. penetrans, M. genitalium, and M. pneumoniae (11, 33, 139, 147).

Intracellular localization may be responsible for protecting the organisms from antibodies

and antibiotics as well as contributing to disease chronicity and difficulty in cultivation in

some cases. Variation in surface antigens of M. hominis and Ureaplasma spp. may be related

to persistence of these organisms at invasive sites. In humans, mycoplasmas and

ureaplasmas may be transmitted by direct contact between hosts, i.e., venereally through

genital-genital or oral-genital contact, vertically from mother to offspring either at birth or in

utero, by respiratory aerosols or fomites in the case ofM. pneumoniae, or even by

nosocomial acquisition through transplanted tissues.

CLINICAL SIGNIFICANCE Back to top

Respiratory Infections

M. pneumoniae causes approximately 20% of all community-acquired pneumonias in the

general population and up to 50% of pneumonias in certain confined groups (139, 147).

Although M. pneumoniae has long been associated with pneumonias in school-aged children,

adolescents, and young adults, in recent years this organism was also shown to occur

endemically and occasionally epidemically in older persons, as well as in children under 5

years of age (147). The most typical clinical syndrome is tracheobronchitis, often

accompanied by upper respiratory tract manifestations, such as acute pharyngitis.

Pneumonia develops in about one-third of persons who are infected. The incubation period is

generally 2 to 3 weeks, and spread throughout households is common. The organism can

persist in the respiratory tract for several months after initial infection and sometimes for

years in hypogammaglobulinemic patients, possibly because it attaches strongly to and

invades epithelial cells. Disease tends not to be seasonal, subclinical infections are common,

and the disease is ordinarily mild. However, severe infections requiring hospitalization and

even death are known to occur (147).

Extrapulmonary complications of M. pneumoniae infections may include meningoencephalitis,

ascending paralysis, transverse myelitis, Bell’s palsy, possibly some cases of involuntary

movements, pericarditis, hemolytic anemia, arthritis, nephritis, and mucocutaneous lesions

(123, 139, 147). An autoimmune response is thought to play a role in some extrapulmonary

complications. However, M. pneumoniae has been isolated directly from cerebrospinal,

pericardial, and synovial fluids as well as other extrapulmonary sites, and additional evidence

of direct invasion by this organism has been documented by the use of the PCR assay (123).

Clinical manifestations are not sufficiently unique to allow differentiation from infections

caused by other common bacteria, particularly Chlamydophila pneumoniae. Data from

animal models as well as clinical studies suggest a role for M. pneumoniae and C.

pneumoniae as etiologic or exacerbating factors in bronchial asthma (94, 138), and

additional clinical studies linked these microorganisms to stable as well as exacerbating

disease (56, 72).

M. fermentans has been recovered from the throats of children with pneumonia, some of

whom had no other etiologic agent identified, but the frequency of its occurrence in healthy

children is not known (128). It has been detected in adults with an acute influenza-like

illness (86) and in bronchoalveolar lavage specimens, peripheral blood lymphocytes, and

bone marrow from patients with AIDS and respiratory disease (3, 4). Respiratory infection

with M. fermentans is not necessarily linked with immunodeficiency, but it may also behave

as an opportunistic pathogen.

Very little is known about Mycoplasma amphoriforme beyond what has been described in the

initial reports of its detection in the lower respiratory tract by culture and/or PCR in a series

of patients with antibody deficiency and chronic bronchitis or bronchiectasis (151). Its

biochemical reactivity, colonial appearance, growth characteristics, and gliding motility (57)

are similar to these features of M. pneumoniae, but it is distinct genetically. Repeated

isolations over time and clinical improvement after antimicrobial therapy which resulted in

the elimination of the mycoplasma suggest a possible pathogenic role, but more work must

be done to determine the extent of disease that may be due to this organism.

Genitourinary Infections

Following puberty, Ureaplasma spp. and M. hominis can be isolated from the lower genital

tract in many healthy sexually active adults, but there is evidence that these organisms play

etiologic roles in some genital tract diseases. Results of human and animal inoculation

studies and observations of immunocompromised persons are supportive of ureaplasmas

being a cause of nonchlamydial, nongonococcal urethritis (NGU) in men, with further

evidence supplied by therapeutic and serologic studies (128). Since identification of two

distinct biovars of Ureaplasma urealyticum, now considered separate species, biovar 2 (U.

urealyticum) has been implicated in NGU, whereas biovar 1 (Ureaplasma parvum) has not

been implicated in this manner according to some investigators (38), although doubt about

this species specificity in association with NGU has been raised by others (19). Evidence

that M. hominis causes NGU is lacking. M. genitalium has been detected by PCR technology

significantly more often in urethral specimens from men with acute NGU than from those

without urethritis and is now considered to be one of the causes of the disease

(63, 64, 127). M. genitalium-positive men have been found to have symptomatic urethritis

significantly more often than those infected with Chlamydia trachomatis (53). Antibody

responses have been detected in some men with acute disease, and this mycoplasma has

also produced urethritis in nonhuman primates (127). M. genitalium also could be a rare

cause of conjunctivitis associated with urethritis (16). M. fermentans, M.

penetrans, andMycoplasma pirum were not detected in the urethras of men with urethritis by

PCR assays, suggesting that these organisms are unlikely to have a pathogenic role in this

condition (36). In women, there is no evidence that M. hominis is a cause of the urethral

syndrome, but ureaplasmas may be involved (121).

M. hominis and Ureaplasma spp. have not been detected by culture of prostatic biopsy

samples from patients with chronic abacterial prostatitis (39), and M. genitalium has been

found rarely by using a PCR assay (80). In contrast, ureaplasmas have been recovered in an

epididymal aspirate from a patient suffering with nonchlamydial, nongonococcal acute

epididymo-orchitis accompanied by a specific antibody response (62) and may be an

infrequent cause of this disease. Ureaplasma spp. produce urease and induce crystallization

of struvite and calcium phosphates in urine in vitro and calculi in animal models

(55, 137).They have been found in urinary calculi of patients with infection-type stones more

frequently than those with metabolic-type stones (55). M. hominis has been isolated from

the upper urinary tract only in patients with symptoms of acute pyelonephritis, often with an

antibody response, and may cause about 5% of cases of this disease (133). Obstruction or

instrumentation of the urinary tract may be predisposing factors. Ureaplasmas have not been

associated in the same way.

Mollicutes do not cause vaginitis but are among various microorganisms that proliferate in

patients with bacterial vaginosis (BV). Some studies suggest that M. hominis may contribute

independently to BV, but evidence is lacking for an independent association of ureaplasmas

with BV (146). BV may lead to pelvic inflammatory disease, and M. hominis has been

isolated from the endometrium and fallopian tubes of about 10% of women with salpingitis

accompanied by a specific antibody response (137). Serological evidence suggests that M.

hominis could be an independent factor in tubal factor infertility (7). Ureaplasma spp. have

been isolated directly from affected fallopian tubes but not alone. This latter observation,

together with the negative results of serologic tests, animal models, and fallopian tube organ

cultures (128), does not support a causal relationship for ureaplasmas in pelvic inflammatory

disease. M. genitalium, however, may play a role, as indicated by its significant association

with cervicitis (52) and endometritis (30). In addition, there is serologic evidence that M.

genitalium causes some cases of tubal infertility (28). That ureaplasmas might cause

infertility still remains speculative (126).

Ureaplasmas have been isolated from the internal organs of spontaneously aborted fetuses

and from stillborn and premature infants more often than from induced abortions or normal

full-term infants (146). The results from some serologic and therapeutic studies have also

supported a role for these organisms in fetal morbidity (25). BV is a possible confounding

factor which must be considered in the association between ureaplasmas in the chorioamnion

and low birth weight. Ureaplasmas at this site are directly associated with inflammation (51)

and may invade the amniotic sac early in pregnancy in the presence of intact fetal

membranes, causing persistent infection and adverse pregnancy outcome (23, 129).

The notion that M. hominis causes fever in some women after abortion, or after normal

delivery, is based on its isolation from the blood of about 10% of such women but not from

afebrile women who had abortions or from healthy pregnant women (127). In addition,

antibody responses have been detected in about half of febrile aborting women but in few of

those who remain afebrile (127). Similar observations have been made for the isolation

of Ureaplasma spp. which may be responsible for some cases of postpartum endometritis

(26). The ability of U. parvum and M. hominis to upregulate amniotic fluid leukocytes,

proinflammatory cytokines, prostaglandins, metalloproteinases and uterine activity to induce

chorioamnionitis, a systemic fetal inflammatory response, and contribute to preterm labor

and fetal lung injury is supported by experimental studies in rhesus monkeys (100). There

are conflicting opinions about the importance of U. urealyticum as opposed to U. parvum in

premature delivery, a situation that needs to be resolved (146). There is no evidence that M.

genitalium is a cause of preterm labor or abortion (87, 101).

Neonatal Infections

Colonization of infants by genital mycoplasmas may occur by ascension from the lower

genital tract of the mother at the time of delivery or in utero earlier in gestation and may be

transient and without sequelae. The rate of vertical transmission may be 18 to 55% among

infants born to colonized mothers (146). Ureaplasmaspp. and M. hominis may be isolated

from neonates born to mothers with intact membranes and delivered by cesarean section

(146). Congenital pneumonia, bacteremia, progression to chronic lung disease of prematurity

with the development of inflammatory cyto kines in tracheal aspirates, and even death have

occurred in very-low-birth-weight infants due to ureaplasmal infection of the lower

respiratory tract (118, 146). A meta-analysis of the literature accumulated since the 1980s

supports the association of ureaplasmal infection with development of chronic lung disease,

but so far there has been no evidence of a reduction in the incidence of chronic lung disease

or death when preterm infants have been treated with erythromycin (118). Both M.

hominis and Ureaplasma spp. have been isolated from maternal and umbilical cord blood as

well as the blood of neonates. Both species can also invade the cerebrospinal fluid of

neonates (146). Either mild, subclinical meningitis without sequelae or neurological damage

with permanent handicaps may ensue. Colonization of healthy full-term infants declines after

3 months of age, and fewer than 10% of older children and sexually inexperienced adults are

colonized with genital mycoplasmas (146).Vertical transmission of M. genitalium from

mother to neonate has been reported (88), but its significance in neonates is unknown.

Routine screening of neonates for genital mycoplasmas is not clinically justified based on the

available evidence that many healthy neonates may be colonized without consequence.

However, if there is clinical, radiological, or laboratory evidence of pneumonia, meningitis, or

overall instability, particularly in preterm neonates in whom there are no obvious alternative

etiologies, infection with M. hominis or Ureaplasma spp. should be considered.

Systemic Infections and Immunosuppressed Hosts

Extrapulmonary and extragenital mycoplasmal infections probably occur more often than

currently recognized.M. hominis is alone among pathogenic mycoplasmas of human origin

which may occasionally be detected in routine bacteriologic cultures, so there have been

many instances of accidental discovery when mycoplasmas were not specifically sought. The

number of published case reports implicating Mycoplasma and Ureaplasmaspecies in a

variety of systemic infections involving persons with and without impaired host defenses has

increased in recent years as a result of a more widespread utilization of universal PCR

primers when infection is suspected and no conventional microbes are detected by culture.

Mollicutes can cause invasive disease of the joints as a result of dissemination from the

genital or respiratory tracts in immunosuppressed persons, especially individuals with

hypogammaglobulinemia (137). M. hominis bacteremia has been demonstrated after renal

transplantation (114), trauma, and genitourinary manipulations and also in brain abscesses,

osteomyelitis lesions (95), and wound infections (128). Numerous mycoplasmal species,

including M. fermentans, U. urealyticum, and Mycoplasma salivarium, have been detected by

culture and/or PCR in synovial fluid of persons with various arthritides, although the precise

contribution of these organisms to these disease conditions is still uncertain (71, 116, 117).

The significance of M. fermentans and other mycoplasmas in persons with AIDS and as

possible agents of Gulf War syndrome has received a great deal of attention. However, there

is no credible evidence supporting an association or causal role in either condition (136).

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

Specimen Type and Collection

Body fluids appropriate for mycoplasmal culture or detection by noncultural methods include

blood, synovial fluid, amniotic fluid, cerebrospinal fluid, urine, prostatic secretions, semen,

wound aspirates, sputum, pleural fluid, bronchoalveolar lavage fluid, or other

tracheobronchial secretions, depending on the clinical condition and organisms of interest.

Swabs from the nasopharynx, throat, cervix/vagina, wounds, and urethra are also

acceptable. Tissue from biopsy or autopsy, including placenta, endometrium, bone chips, and

urinary calculi can also be used. When swabs are used, care must be taken to sample the

desired site vigorously to obtain as many cells as possible, since mycoplasmas are cell

associated. Urine specimens may sometimes prove more sensitive than urethral swabs for

detection of fastidious mycoplasmas such as M. genitalium by PCR (65). If determination of

the localization of mycoplasmas in the genitourinary tract is desired, urine specimens can be

obtained at various stages during urination or after prostatic massage. Care should be taken

to avoid collection of specimens that are contaminated by lubricants or antiseptics commonly

used in gynecologic practice. Dacron or polyester swabs with aluminum or plastic shafts are

preferred. Wooden shaft cotton swabs should be avoided because of potential inhibitory

effects. Swabs should always be removed from specimens before transportation to the

laboratory.

Successful isolation of mycoplasmas from blood can be achieved by inoculating blood, free of

anticoagulant, into liquid mycoplasmal growth media at the bedside in a 1:5 to 1:10 ratio,

using as much blood as possible (at least 10 ml is desirable for adults). Mycoplasmas are

inhibited by sodium polyanethol sulfonate, the anticoagulant used in most commercial blood

culture media, but the inhibitory effect can be overcome by addition of gelatin (1% [wt/vol])

(108). Use of commercial blood culture media with or without automated blood culture

instruments is not recommended for detection of mycoplasmas. None of the newer

continuously monitored automated blood culture systems will flag bottles containing M.

hominis, even when additional metabolic substrate and gelatin are added. The organism may

survive in these media for several days, however (141).

Transport and Storage

Mycoplasmas are extremely sensitive to adverse environmental conditions, particularly

drying and heat. Specimens should be inoculated at bedside whenever possible, using

appropriate transport and/or culture media. Specific mycoplasma media such as SP4 or

Shepard’s 10B broth or 2 SP (10% heat-inactivated fetal calf serum with 0.2 M sucrose in

0.02 M phosphate buffer, pH 7.2) are acceptable transport media. Other media available

commercially for transport and storage of specimens are Stuart’s medium, Trypticase soy

broth with 0.5% bovine serum albumin, and Mycotrans (Irvine Scientific, Irvine, CA). A3B

broth (Remel, Inc.) is available as a transport medium, whereas Remel arginine broth, 10B,

and SP4 transport broths also serve as growth media. Liquid specimens do not require

special transport media if cultures can be inoculated within 1 hour, provided the specimens

are protected from evaporation. Tissues can be placed in a sterile container which can be

tightly closed and delivered to the laboratory immediately. Otherwise, tissue specimens

should be placed in transport media if delay in culture inoculation is anticipated. Specimens

should be refrigerated if immediate transportation to the laboratory is not possible. If

specimens must be shipped and/or if the storage time is likely to exceed 24 h prior to

processing, the specimen in transport medium should be frozen at −80°C to prevent loss of

viability and to minimize bacterial overgrowth. Mollicutes can be stored for long periods in

appropriate growth or transport media at −80°C or in liquid nitrogen. Frozen specimens can

be shipped with dry ice to a reference laboratory if necessary. Storage at −20°C is

deleterious to detection, even by nonculture methods. When frozen specimens are to be

examined, they should be thawed rapidly in a water bath at 37°C.

DIRECT EXAMINATION Back to top

Microscopy

Lack of a cell wall precludes visualization of mycoplasmas by Gram staining, but this

procedure may prove useful to exclude contaminating bacteria. A DNA fluorochrome stain

such as Hoechst 33258 (ICN Biomedicals, Costa Mesa, CA) or acridine orange stain may be

useful to assist in organism visualization when applied to body fluids such as amniotic fluid

after cytocentrifugation, but it is not specific for mycoplasmas.

Antigen Detection

Rapid methods for antigenic detection of M. pneumoniae were developed in the 1980s, but

these techniques were hampered by low sensitivity and cross-reactivity with other

commensal mycoplasmas. This approach for rapid diagnosis has now been abandoned in

favor of nucleic acid amplification methods.

Nucleic Acid Detection Techniques

PCR systems have been developed for all of the clinically

important Mycoplasma and Ureaplasma species that infect humans. The advantages they

have over culture and serology include the ability to complete the procedure in 1 day,

utilizing a single specimen containing organisms that do not have to be viable, as well as the

ability to detect nucleic acid in preserved tissues.

Some examples of gene targets used in PCR assays include 16S rRNA

(65, 73, 90, 104, 117, 157); other repetitive sequences such as the insertion-like elements

of M. fermentans and repMp1 of M. pneumoniae (47,150); the P1 adhesin (34, 69, 110),

ATPase operon (15), and tuf genes of M. pneumoniae (89); gap in M. hominis (8); and the

MgPa adhesin (34, 70) and gyrA genes (18) of M. genitalium. Urease genes (17, 157) and

the MB antigen gene (78, 107) have been used as targets in Ureaplasma spp. Real-time PCR

assays have been described for M. pneumoniae (93), M. genitalium (66, 73, 120), M.

hominis (8), and Ureaplasma spp. (92). Advantages of real-time PCR assays include more

rapid turnaround time, less handling of PCR product, and improved diagnostic sensitivity. For

slowly growing organisms, such as M. pneumoniae, and especially for extremely fastidious

species for which optimum cultivation techniques are not established, such as M.

genitalium and M. fermentans, the use of PCR assays may be the only practical means of

detecting their presence in clinical specimens. The sensitivity of PCR is very high,

theoretically corresponding to a single organism when purified DNA is used.

Comparison of the PCR technique with culture and/or serology, in the case of M.

pneumoniae, has yielded varied results that are not always in agreement. Positive PCR

results in culture-negative persons without evidence of respiratory disease suggest

inadequate assay specificity, persistence of the organism after infection, or its existence in

asymptomatic carriers. Positive PCR results in serologically negative persons may be due to

an inadequate immune response or from the collection of specimens before specific antibody

synthesis could occur. Negative PCR results in culture or serologically proven infections

raises the possibility of inhibitors or other technical problems with the assay. Use of a second

PCR assay with a different gene target may help interpret results and resolve such

discrepancies. This is particularly important in the setting of a positive PCR assay when

culture and/or serology is negative. If mycoplasmacidal antibiotics have been administered,

PCR results may be negative even though serology is positive. There is some evidence

suggesting that PCR inhibition may occur more commonly with nasopharyngeal specimens

than throat swabs being assayed for M. pneumoniae (42, 111). Commercial reagents

available for purification of nucleic acid can be helpful in overcoming PCR inhibition.

Multiplex PCR assays have been developed to detect M. pneumoniae and other respiratory

pathogens (110). Assays to detect M. genitalium and other urogenital pathogens (91) have

also been described. A PCR-microtiter plate hybridization assay (158) and a microwell-platebased

PCR assay have been developed for large-scale screening for M. genitalium (48).

Strong associations between serology and PCR for M. genitaliumhave been described (149),

but the analytical sensitivity of a single PCR assay for M. genitalium was questioned by

Baseman et al. (10), who reported that 61% of culture-positive women tested negative by

PCR, despite apparently good quality control parameters for their assay. Assuming culture

positivity was not due to cross-contamination, this highlights the fact that reduced sensitivity

of a single PCR assay may be related to quality of the specimen or presence of inhibitors.

PCR technology is less valuable for routine diagnostic purposes in the case of the more

rapidly growing and relatively easily cultivable organisms, such as M.

hominis and Ureaplasma spp., but this method can be valuable in clinical studies of

ureaplasmal infections. The newest PCR-based techniques permit identification and

differentiation of U. urealyticum versus U. parvum (75, 78, 79, 92, 107). PCR is also a very

good tool for identification of an unknown mycoplasma previously obtained by culture.

PCR is now the diagnostic method of choice for detection of M. pneumoniae infection in

laboratories that can develop their own assays, but no complete PCR diagnostic kits are sold

commercially for diagnostic use in the United States. However, several complete PCR assays

are sold commercially in various European countries. Recent comparisons to in-house

techniques were generally favorable (46, 134).

ISOLATION PROCEDURES Back to top

Biosafety Considerations

M. pneumoniae, M. hominis, and ureaplasmas are considered category 2 pathogens. Work

with these microorganisms and other mycoplasmas of human origin can be safely

undertaken on the laboratory bench and/or in a class 2 safety cabinet.

Growth Media and Inoculation

Growth of mycoplasmas pathogenic for humans requires the presence of serum, growth

factors such as yeast extract, and a metabolic substrate. No single formulation is ideal for all

species due to different properties, optimum pH, and substrate requirements. SP4 broth and

agar (pH 7.5) are the best media overall and can be used for both M. pneumoniae and M.

hominis, provided arginine is added for the latter. Shepard’s 10B broth (pH 6.0) can be used

for M. hominis and Ureaplasma spp., with A8 as the corresponding solid medium. Penicillin G

should be added to minimize bacterial overgrowth. The addition of a pH indicator, such as

phenol red, is important for detection because mycoplasmas usually do not produce turbidity

in broth culture owing to their small cell size. Media formulations are provided elsewhere

(140).

For self-prepared media, quality control is crucial for each of the main components. These

controls must consist of the quantitative growth of a mycoplasma strain(s) in two media that

differ only in the component to be tested. New lots or batches of broth are satisfactory if the

numbers of organisms that grow are within one 10-fold dilution of the reference batch. Agar

plates should ideally support growth of at least 90% of the colonies that are supported by

the reference media. The sterility of commercially purchased media components, such as

horse serum, must be confirmed prior to their use. Quality control test organisms should

include type strains and low-passage clinical isolates of the species of interest. When testing

ureaplasmas, it is recommended to include at least one serovar representative from each of

the two species. Testing inhibitory properties of media against growth of various other

organisms likely present in specimens from nonsterile sites may also be worthwhile to

prevent loss of mycoplasmas due to overgrowth of contaminating organisms.

Specimens should always be mixed well before inoculating media, fluids should be

centrifuged (600×g for 15 min), and the pellet should be inoculated. Urine can be filtered

through a 0.45-μm-pore-size filter if bacterial contamination is suspected. Furthermore, it is

wise to mince, not grind, tissues in broth prior to dilution. Serial dilution of specimens in

broth to at least 10-3, with subculture of each dilution onto agar, is an extremely important

step in the cultivation process, since it will help overcome possible interference by

antibiotics, antibodies, and other inhibitors, including bacteria that may be present in clinical

specimens. Omission of this critical dilution step can be one reason why some laboratories

have difficulty in recovering organisms. Dilution also helps to overcome the problem of rapid

decline in culture viability, which is particularly common with ureaplasmas, and it also

provides information about the number of organisms present.

Incubation Conditions and Subcultures

Broths should be incubated at 37°C under atmospheric conditions. Agar plates yield the best

growth if they are incubated in an atmosphere of room air supplemented with 5 to 10%

CO2 or in an anaerobic environment of 95% N2 plus 5% CO2. The relatively rapid growth

rates of M. hominis and Ureaplasma spp. make identification of most positive cultures

possible within 2 to 4 days, whereas M. pneumoniae may require up to 3 weeks or longer. All

broths that change color should be subcultured into a fresh tube of the corresponding broth

(0.1 ml into 0.9 ml) and onto agar (0.02 ml). Subcultures of Ureaplasma spp. must be

performed soon after the color change occurs because the culture can lose viability within a

few hours. Subculture also increases the diagnostic yield, since some strains may not grow

sufficiently from the original specimen inoculated initially onto solid media. Blind subculture

periodically during incubation may improve the yield of M. pneumoniae, since a color change

may not always be evident, even if growth occurs, but culture is still relatively insensitive for

detection of this mycoplasma. Cultures should be incubated for at least 7 days before being

designated negative for genital mycoplasmas and 4 weeks for M. pneumoniae. The growth

rate of M. fermentans is similar to that of M. pneumoniae. However, for M. fermentans, M.

genitalium, and mycoplasmas of human origin other than M. pneumoniae, M.

hominis, or Ureaplasma spp., cultivation conditions are not well established.

Development of Colonies

Broth cultures for Ureaplasma spp. should be examined for color change resulting from

hydrolysis of urea twice daily for up to 7 days because of the steep death phase of this

organism in culture. This is less critical forMycoplasma spp., for which once daily inspection

of broth cultures is sufficient. Agar plates should be examined, using a stereomicroscope at a

magnification of ×20 to 60, daily for Ureaplasma spp., at 1- to 3-day intervals for M.

hominis, and every 3 to 5 days for M. pneumoniae and other slower-growing

species.Ureaplasma colonies (Fig. 3) can be identified on A8 agar by urease production in the

presence of CaCl2indicator contained in the medium. The larger M. hominis colonies are

urease negative and often have the typical fried-egg appearance (Fig. 1). Other species,

such as M. pneumoniae and M. genitalium, will produce much smaller spherical colonies

which may or may not demonstrate the fried-egg appearance (Fig. 2). Methylene blue stain

applied directly to the agar plate to turn the colonies blue is sometimes useful if there is

uncertainty about whether or not mycoplasmal colonies are present. M. hominis is the only

pathogenic mycoplasma of humans cultivable on bacteriological media such as chocolate

agar or blood agar. However, the pinpoint translucent colonies are easily overlooked, and

routine bacterial cultures may be discarded sooner than the time needed for M.

hominis colonies to develop, which may be 4 days or more in some cases. Occurrence of

suspicious colonies warrants subculture to appropriate mycoplasma media.

Commercial Media and Culture Kits

A variety of kits for detection, quantitation, identification, and antimicrobial susceptibility

testing of Ureaplasmaspp. and M. hominis from urogenital specimens are available in Europe.

Some of these products, such as Mycoscreen Plus and Mycofast US (Wescor Inc., Logan, UT),

are now being sold commercially in the United States along with older products such as

Mycoscreen GU, Mycotrim GU agar, and the Mycotrim GU triphasic flask system (Irvine

Scientific). A comparable system, Mycotrim RS, has been adapted for detection of M.

pneumoniae in respiratory specimens. Remel, Inc., has developed several formulations of

transport and growth media, including 10B broth, A7 agar, A8 agar, SP4 broth, and SP4

agar.

Some kits and other commercial products and media have been evaluated by independent

investigators (1,20, 27, 29, 106, 119, 154). Commercial products and kits may be of

particular value if the need to detect mycoplasmas arises infrequently in laboratories which

do not specialize in mycoplasma detection, but users should be aware of the potential

limitations of existing products. If commercially prepared media are to be utilized, it is

advisable that laboratories perform internal quality control tests.

IDENTIFICATION Back to top

Even though the numerous large-colony mycoplasmal species which may be isolated from

humans cannot be identified based on colonial morphology or a particular biochemical profile,

the body site of origin and rate of growth, in conjunction with biochemical features and

colonial appearance, give some clues. Utilization of glucose by a mycoplasma in SP4 broth

will produce an acidic shift (red to yellow), whereas utilization of arginine will produce a red

to deeper red color change in this broth in the presence of the phenol red pH indicator. Urea

or arginine hydrolysis in 10B broth causes an alkaline shift of orange to deep red. Thus, a

slow-growing glycolytic organism from the respiratory tract that produces spherical colonies

on SP4 agar after approximately 5 to 20 days of incubation is likely to be M. pneumoniae. An

alkaline color change which occurs after overnight incubation without turbidity in 10B broth

containing urea is almost certainly due to Ureaplasmaspp., whereas a urogenital specimen

that produces an alkaline reaction within 24 to 72 hours in broth supplemented with arginine

is likely to contain M. hominis. Examination of colonial morphology is sufficient to

identify Ureaplasma spp., and it is important to keep in mind that these organisms often

coexist with M. hominis in urogenital specimens.

To identify a large-colony mycoplasma completely to species level, a number of different

techniques are available, but PCR is now considered the best overall choice for species

identification, since it is much simpler to perform than other methods and it does not require

immunological reagents that are not readily available. The PCR assay is also less subjective

to interpret than some of the older methods such as epi-immunofluorescence.

TYPING SYSTEMS Back to top

Several methods for typing mollicutes are described and used to study the epidemiology

of M. pneumoniaeand the differential pathogenicity for the two genomic clusters and 14

serotypes of Ureaplasma spp. Techniques used initially to serotype ureaplasmas from clinical

specimens include monoclonal and polyclonal antibodies (49, 159), immunofluorescence

(98), immunoperoxidase (109), and agar growth inhibition (130). Results of earlier studies

have been varied and inconsistent due to the inefficient and imprecise methods available,

occurrence of multiple cross-reactions, and the fact that many persons may harbor more

than one serotype in their urogenital tract in the presence or absence of disease.

Development of monoclonal antibodies enabled identification of MB antigens responsible for

ureaplasma serotype specificity on the cell surface (159). PCR-based assays have enabled

more accurate characterization of the two genomic clusters ofUreaplasma spp. that led to

their designation as two separate species. Pulsed-field gel electrophoresis has been applied

to Ureaplasma spp. to determine the size of the genome (99, 113), and this technique can

distinguish among the majority of the 14 Ureaplasma serotypes and detect differences within

serotypes (97).

Restriction fragment length polymorphism, multiple-locus variable-number tandem-repeat

analysis, Western blotting, two-dimensional gel electrophoresis, nucleic acid sequence-based

amplification, and other types of PCR assays have been used to characterize M.

pneumoniae clinical isolates (31, 35, 41, 45, 61, 102, 115). Most evaluations have

determined that there are two major genomic groups or subtypes distinguishable by analysis

of the P1 adhesin gene, ORF6 gene, P65 gene, and typical DNA restriction fragment pattern.

Typing of human mycoplasmas or ureaplasmas for diagnostic or epidemiological purposes is

not recommended at the present time, and the methods are unavailable except in specialized

research or reference laboratories.

SEROLOGIC TESTS Back to top

M. pneumoniae Respiratory Disease

Historically, serology has been the most common laboratory means for diagnosis of M.

pneumoniae respiratory tract infections. Although culture and PCR are also used to detect

the presence of M. pneumoniae in respiratory specimens, persistence of the organism for

variable lengths of time following acute infection makes it difficult in some cases to assess

the significance of a positive culture or PCR assay without additional confirmatory tests such

as seroconversion.

M. pneumoniae has both lipid and protein antigens which elicit antibody responses that can

be detected after about 1 week of illness, peaking at 3 to 6 weeks, followed by a gradual

decline, allowing several different types of serologic assays based on different antigens and

technologies. Serology is a very useful epidemiologic tool in circumstances where the

likelihood of mycoplasmal disease is high, but it is less suited for assessment of individual

patients in a timely manner. Its main disadvantage is the need for both acute- and

convalescent-phase paired sera collected 2 to 3 weeks apart that are tested simultaneously

for IgM and IgG to confirm seroconversion. This is especially important in adults over 40

years of age who may not mount an IgM response, presumably because of reinfection

(132, 147). Moreover, IgM antibodies can sometimes persist for several weeks to months,

making it risky to base diagnosis of acute infection on a single assay for IgM even in children

(132, 147). Antibody production may also be delayed in some infections, or even absent if

the patient is immunosuppressed. False-negative tests for IgM can also occur if serum is

collected too soon after the onset of illness. Since M. pneumoniae is a mucosal pathogen, IgA

is typically produced early in the course of infection. Measurement of serum IgA has been

suggested as an alternative approach for diagnosis of acute infection because of its rapid rise

and decline, but very few commercial assays include reagents for its detection (139).

Complement fixation (CF) was the primary method for serological testing for M.

pneumoniae in the past. Although CF measures mainly the early IgM response, the test does

not differentiate among antibody classes. Cross-reactions with other organisms, most

notably M. genitalium (82), are well recognized, and false-positive results due to crossreactive

autoantibodies induced by acute inflammation from other unrelated causes may

occur. In most clinical laboratories, CF has been replaced by alternative techniques. Table

3 provides examples of various assays used to detect M. pneumoniae infection. More detailed

descriptions of individual assay kits are available in other reference texts (148).

Immunofluorescent antibody (IFA) assays, direct and indirect hemagglutination using IgM

capture, and other particle agglutination antibody assays (PAs) have been developed to

detect antibody to M. pneumoniae (5, 50,81). IFA assays consist of M. pneumoniae antigen

affixed to microscope slides and measures IgM and IgG separately. This assay is technically

simple to perform but subjective in interpretation, it requires a fluorescent microscope, and

the presence of M. pneumoniae-specific IgG may interfere with IgM results (5). Qualitative

and semiquantitative PAs using either latex beads or gelatin that detect IgM and IgG

simultaneously can be technically easy to perform. However, PAs do not offer any significant

advantages over enzyme immunoassays (EIAs) and are not available commercially in the

United States.

EIAs have become the most widely used methods for detection of M. pneumoniae antibody in

the United States. All are classified as having either moderate or high complexity according

to the Clinical Laboratory Improvement Amendment. They may be qualitative or quantitative

and may or may not require specialized equipment. EIAs are more sensitive than CF and can

be performed with very small volumes of serum. A membrane-based EIA specific for IgM, the

ImmunoCard (Meridian Diagnostics, Cincinnati, OH), was developed for rapid detection of

acute M. pneumoniae infection using a single serum specimen. However, a recent study

found this EIA had a sensitivity of only 31.8% when a single serum was analyzed from

seropositive children with pneumonia, increasing to 88% when paired sera were analyzed

(103). The Remel EIA (Remel, Inc.) is another rapid point-of-care qualitative assay that

detects both IgM and IgG simultaneously in an easy-to-read format without the need for

instrumentation. This test has shown good sensitivity and specificity when compared to other

EIAs, IFA assays, and CF. Several comparison studies have been performed, evaluating each

of the EIA kits listed in Table 3 and various others (5, 6, 9, 13, 50, 81, 122, 131). A

comprehensive evaluation of 12 commercial EIAs and PAs using PCR as a reference standard

found that most assays had problems with sensitivity and specificity. This evaluation

indicated limitations for their use in the diagnosis of acute infections, reaffirmed the

necessity of testing both IgM and IgG in paired sera from adults, and suggested the PCR

assay may be a better diagnostic approach (13). Another study (32) reported sera from a

substantial proportion of healthy blood donors have measurable antibody against M.

pneumoniae, suggesting cross-reactivity of the antigens used in some of the commercial

EIAs and the likelihood their use results in overdiagnosis of mycoplasmal infections. A

combination of IgM or IgA serology and PCR can be a logical diagnostic approach if only a

single specimen is available, but it may be less useful in adults who do not mount an IgM

response and would add considerable cost to laboratory testing (139). Cold agglutinins,

detected by agglutination of type O Rh-negative erythrocytes at 4oC, occur in association

with M. pneumoniae infection in about 50% of cases (24). Titers of 64 to 128 or a fourfold or

greater rise in titer suggest a recent M. pneumoniae infection, but the test is nonspecific and

is not recommended for diagnostic use.

Infections Due to Genital Mycoplasmas

Serological tests for M. hominis and Ureaplasma spp. using metabolism inhibition,

microimmunofluorescence, and EIA have been described previously (21, 22, 85, 125, 130). A

microimmunofluorescence assay for M. genitalium has also been developed (54) and was

shown to detect antibody responses in men with NGU (124) and women with sal pingitis

(96). A sensitive and specific serological assay for M. genitalium using lipid-associated

membrane proteins as antigens has been used in combination with Western immunoblots to

assess the immunoreactivity of women who were regarded as culture positive for M.

genitalium (10). No serological tests for genital mycoplasmas have been standardized and

made commercially available for diagnostic use in the United States. Therefore, they cannot

be recommended for routine diagnostic purposes.

ANTIBIOTIC SUSCEPTIBILITIES Back to top

Methods Used for Testing

Several methods of susceptibility testing used for conventional bacteria have been employed

for testing mycoplasmas. Agar dilution has been used as a reference method (77). It has the

advantages of a relatively stable endpoint over time, the inoculum size does not have a great

effect, and it allows detection of mixed cultures readily. However, this technique is not

practical for testing small numbers of strains or occasional isolates which may be

encountered in diagnostic laboratories. Agar disk diffusion is not useful for testing

mycoplasmas, since there has been no correlation between inhibitory zones and MICs, and

the relatively slow growth of some of these organisms further limits this technology.

Microbroth dilution to determine MICs is the most practical and widely used method. It is

economical and allows several antimicrobials to be tested in the same microtiter plate, but it

has numerous disadvantages in that preparation of antimicrobial dilutions is labor-intensive

and the endpoint tends to shift over time (140). Limited comparisons of agar dilution versus

microbroth dilution indicated that the two methods provided similar results for various

antimicrobials tested against Ureaplasma spp. and M. hominis (59, 76, 145).

Studies using the Etest (bioMerieux, Durham, NC) agar gradient diffusion technique for

detection of tetracycline resistance in M. hominis yielded results comparable to microbroth

dilution (143). Additional comparative studies have also validated this method for

determination of susceptibilities of M. hominis to fluoroquinolones (142) and susceptibilities

of ureaplasmas to various antimicrobials (43).The Etest has the advantages of simplicity of

agar-based testing, has an endpoint which does not shift over time, does not have a large

inoculum effect, and can easily be adapted for testing single isolates.

There have been no universally accepted standards for pH, media, incubation conditions, or

duration of incubation for performing mycoplasmal or ureaplasmal susceptibility tests. No

MIC breakpoints specific for these organisms are endorsed by any regulatory agency. Lack of

specific guidelines for susceptibility testing methods, quality control reference strains, MIC

ranges, and interpretation of results has led to diverse and often inconsistent susceptibility

profiles. The Human Mycoplasma Susceptibility Testing Subcommittee of the Clinical and

Laboratory Standards Institute (CLSI) has submitted recommendations for standard methods

for agar- and broth-based susceptibility testing of human mycoplasmas and ureaplasmas. A

forthcoming CLSI guideline will also designate quality control reference strains, expected MIC

ranges, and proposed MIC interpretive breakpoints for selected drugs.

MIC assays must include control strains for validation purposes. M. pneumoniae strain M129

ATCC 29342, M. hominis strain PG21ATCC 23114, and U. urealyticum (serovar 9) ATCC

33175 have been shown to provide reproducible MICs for several antimicrobials by multiple

laboratories participating in studies to collect data for the proposed CLSI guideline. An

inoculum of 104 to 105 CFU/ml has been recommended as the optimum inoculum for brothbased

testing (140, 144). Nonstandardized conditions at low pH (6.0) can affect MICs,

especially for macrolides, but such conditions are required for growth of Ureaplasma spp.

Step-by-step procedures for performance of in vitro susceptibility tests for mycoplasmas and

ureaplasmas of human origin have been published previously (144). These procedures have

been modified somewhat in the forthcoming CLSI guideline.

Commercial 10B and SP4 broths (Remel, Inc.) perform in an acceptable manner for

determining broth dilution antimicrobial susceptibilities of Ureaplasma spp. and M.

pneumoniae, respectively. A modified nonproprietary Hayflick’s broth has been

recommended for testing M. hominis by the Mycoplasma Susceptibility Testing Subcommittee

of the CLSI. Agar-based tests can utilize A8, SP4, and modified Hayflick’s media for these

same organisms, respectively.

Tetracycline-resistant M. hominis and Ureaplasma spp. can easily be distinguished by brothor

agar-based methods, since the resistant strains generally have MICs of ≥2 μg/ml.

Commercial MIC test kits are available in some countries. Details on these products are

provided in reference texts (148).

Susceptibility Profiles and Treatment

A comparison of MICs for several antimicrobial agents is shown in Table 4. Mollicutes are

innately resistant to all beta-lactams, sulfonamides, trimethoprim, and rifampin. Resistance

to macrolides and lincosamides is variable according to species, with M. hominis being

resistant to erythromycin and other 14- and 15-membered macrolides but susceptible to

clindamycin. For Ureaplasma spp., the reverse is true. Newer macrolides and ketolides have

shown in vitro activity comparable to that of erythromycin for M. pneumoniae.

M. pneumoniae has historically been predictably susceptible to fluoroquinolones,

tetracyclines, and macrolides, so susceptibility testing has not been recommended except for

the in vitro evaluation of new and previously untested agents. However, recent studies from

Japan, China, France, and the United States found that high-level macrolide resistance in M.

pneumoniae due to mutations in domain V on the 23S rRNA gene is increasing in patients

with acute respiratory infections (83, 105, 153, 155, 156). This resistance is greatest in

China, where the percentage of resistant organisms has exceeded 80% (83, 156). Molecularbased

methods to detect mutations in rRNA directly in clinical specimens by PCR enables

monitoring resistance trends without having to isolate M. pneumoniae in culture and can

provide rapid diagnostic information (105, 153, 155). Tetracycline resistance has been well

documented in both M. hominis and Ureaplasma spp. since the mid-1980s, mediated by

the tet(M) determinant which codes for a protein that binds to the ribosomes, protecting

them from the actions of these drugs. The extent to which tetracycline resistance occurs

in M. hominis andUreaplasma spp. varies geographically and according to prior antimicrobial

exposure in different populations but may approach 40 to 50% (146). High-level macrolideresistant

U. parvum in which there was a deletion of 2 amino acids in the L4 ribosomal

protein was recently reported from the United Kingdom, but such resistance is believed to be

rare (14). We have recently encountered occasional Ureaplasma isolates in the United States

with high-level macrolide resistance that have mutations in the 23S rRNA gene. Men with M.

genitalium NGU and women with cervicitis respond better to azithromycin than tetracycline,

possibly because of the lower MICs, but there has also been documentation of clinically

significant macrolide-resistant M. genitalium due to rRNA gene mutations (67).

Fluoroquinolones such as levofloxacin and moxifloxacin are usually active against all human

mycoplasmal and ureaplasmal species. Occasional fluoroquinolone-resistant strains of M.

hominis, Ureaplasma spp., and M. genitalium with mutations in the DNA gyrase and/or

topoisomerase IV genes have been reported (12, 37, 44). Other agents such as

streptogramins, aminoglycosides, and chloramphenicol may show in vitro inhibitory activity,

but these agents are rarely used to treat infections caused by these organisms.

Oxazolidinones are inactive in vitro against mycoplasmas.

Extragenital infections, often in immunocompromised hosts, may be caused by multidrugresistant

mycoplasmas and ureaplasmas, making guidance of chemotherapy by in vitro

susceptibility tests important in this clinical setting. Eradication of infection under these

circumstances can be extremely difficult, requiring prolonged therapy, even when the

organisms are susceptible to the expected agents. This difficulty highlights the facts that

mollicutes are inhibited but not killed by most commonly used bacteriostatic antimicrobial

agents in concentrations achievable in vivo and that a functioning immune system plays an

integral part in their eradication. Treatment of mycoplasmal and ureaplasmal infections has

been described in detail elsewhere (138).

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

Tests offered through diagnostic microbiology laboratories should focus on the species known

to cause human disease and for which cultivation techniques are best defined. Unusual

organisms, or those for which cultivation conditions are not established, may be detectable

by PCR technology offered through specialized research or reference laboratories. Such

organisms should be sought only after consultation with clinicians and personnel from the

reference laboratory. Except for Ureaplasma spp., which can be identified by urease

production and distinct colonial morphology, and until species identification can be

confirmed, a preliminary report of “large-colony Mycoplasma species” is appropriate. In

many instances, as in culturing specimens from the lower genital tract, this may be

sufficient. Isolates from normally sterile sites and/or from immunosuppressed persons should

be identified to species level by PCR if possible.

M. pneumoniae

Detection of M. pneumoniae in culture is time-consuming, not overly sensitive, and rarely

performed. However, isolation of the organism from respiratory tract specimens is clinically

significant in most instances and should be correlated with the presence of clinical

respiratory disease, since a small proportion of asymptomatic carriers can exist. Detection by

PCR is becoming more widely available, but a positive result must still be correlated with

clinical events. A fourfold rise in antibody titer between acute- and convalescent-phase sera

is considered diagnostic of acute infection. In children, adolescents, and young adults, a

single positive IgM result using appropriate immunoglobulin class-specific reagents can be

considered diagnostic of acute infection in most, but not necessarily all, cases because of the

possibility of prolonged IgM elevation that sometimes occurs. Mild respiratory infections due

to M. pneumoniae may not merit a costly and time-consuming microbiological work-up, since

empiric treatment will be effective in most instances. However, the emergence of clinically

significant macrolide resistance may influence choices of empiric antimicrobial agents.

M. hominis

M. hominis can be detected in culture within a few days. It may occasionally be discovered in

routine bacteriologic media from appropriate clinical material, but this should not be relied

upon. Its isolation in any quantity from normally sterile body fluids or tissues is significantly

associated with disease, but quantitation of organisms may be of value in other

circumstances. When mycoplasmas are detected in nonsterile sites, such as the female lower

genital tract in numbers exceeding 105 organisms, they are likely to be associated with BV.

Ureaplasma Species

Isolation of Ureaplasma spp. in any quantity from normally sterile body fluids or tissues is

significantly associated with disease. Fewer than 104 organisms in the male urethra are

unlikely to be significant. Distinguishing between the 2 Ureaplasma spp. by PCR may become

more important in view of possible differences in pathogenicity in some circumstances such

as NGU. The presence of Ureaplasma spp. in the lower respiratory tract of neonates with

respiratory distress may be clinically significant, but there are no definitive guidelines for

antibiotic treatment (2, 38, 40).

M. genitalium

Growing evidence for the role of M. genitalium as a urogenital pathogen has generated

interest in the development of diagnostic methods for its detection, though no molecular

biology-based assays for direct detection or serology test kits are sold commercially thus far.

Even though cultivation techniques for M. genitalium have been described previously

(10, 68, 135), relatively few clinical isolates have actually been attained since the initial

description of this mycoplasma in the early 1980s. The slow growth, requiring 6 weeks or

longer, makes culture impractical. The potential importance of this organism in sexually

transmitted urogenital infections underscores the need for improved and standardized

methods for its detection. At present, noncommercial, nonstandardized PCR-based assays

are all that are available. When M. genitalium is detected in clinical specimens from the

urogenital tract, such as the male urethra or female cervix, in persons with clinical evidence

of urethritis or cervicitis, it should be considered medically significant.