Chlamydiaceae

TAXONOMY AND NOMENCLATURE Back to top

Chlamydia spp. are nonmotile, obligate intracellular prokaryotic pathogens characterized by

a unique biphasic developmental cycle bearing two chlamydial forms that differ essentially in

terms of morphology and function. According to the Approved List of Bacterial

Names, published in 1980, the Chlamydiaceae contained one genus with just two

species, Chlamydia trachomatis and Chlamydia psittaci, which were separated by their

capability to accumulate glycogen in inclusions (Fig. 1A) and their susceptibility to

sulfadiazine. In the 1990s, the application of DNA-based classification methods contributed

to the recognition of the emerging human pathogen Chlamydia pneumoniae (31) and

of Chlamydia pecorum (21), a pathogen of ruminants, as new species of the Chlamydiaceae.

Phylogenetic analyses of the 16S and 23S rRNA genes were the rationale for the proposal of

an emended description of the order Chlamydiales and a revised taxonomy of the

familyChlamydiaceae in 1999 (19). According to this proposal, members of the

order Chlamydiales are obligately intracellular bacteria that have the unique chlamydia-like

developmental cycle and more than 80% sequence identity with chlamydial 16S rRNA genes

and/or 23S rRNA genes. The emended order now includes four families: Chlamydiaceae,

Parachlamydiaceae, Simkaniaceae (46), and Waddliaceae (83).

DESCRIPTION OF THE CHLAMYDIACEAE Back to top

The Chlamydiaceae contain the known human pathogens C. trachomatis, C.

pneumoniae, and C. psittaci as well as organisms such as C. abortus and C. felis that have

been associated only rarely with human infections. Members of the Chlamydiaceae show less

than 10% overall 16S rRNA gene diversity and less than 10% overall 23S rRNA gene

diversity. The genome sizes of the Chlamydiaceae range from 1.0 to 1.24 Mbp, with a G+C

content of about 40%.

The cell wall harbors a common lipopolysaccharide (LPS) that differs from the LPS of other

bacteria in its relatively low endotoxic activity. Accounting for about 60% of the protein

mass, the 40-kDa chlamydial major outer membrane protein (MOMP) encoded by

the ompA gene, is an important structural component of the organisms’ outer membrane.

The variable domains (VD1 through VD4) lead to multiple C. trachomatis serovars associated

with different clinical manifestations of oculogenital infections. In contrast, C.

pneumoniae isolates possess a strikingly high MOMP homology, and serovars of C.

pneumoniae have not been described.

Members of the Chlamydiaceae share a unique biphasic developmental cycle leading to

important consequences in laboratory diagnosis, clinical course, and antibiotic therapy. The

elementary body (EB) of chlamydiae infects eukaryotic host cells and can survive for only a

limited period of time outside the host cell. Once inside the host cell, EBs differentiate to

metabolically active reticulate bodies (RB) that multiply by binary fission (Fig. 1D) within

vacuoles that are continuously growing and that develop into large intracytoplasmic

inclusions (Fig. 1C). Reticulate bodies reorganize back to EBs at the end of the chlamydial

developmental cycle (Fig. 1D). After 48 to 72 h, hundreds of EBs are released from the host

cell using two mutually exclusive pathways to perpetuate the infectious cycle (41). Genomic

transcriptional analysis of the chlamydial developmental cycle revealed a small subset of

genes that control the differentiation stages of the cycle and have evolutionary origins in

eukaryotic lineages (2).

Evidence is accumulating that factors including gamma interferon, antibiotics, and nutrient

deprivation may drive chlamydiae into a state of persistence. Persistent chlamydial forms are

morphologically characterized by aberrant enlarged RBs located within small intracellular

inclusions that are arrested in a viable but noninfectious state. It was proposed that

persistence is an alternative life cycle used by chlamydiae to avoid the host immune

response. As a consequence, chronic infections have been attributed to chlamydial

persistence (14). However, the clinical significance of chlamydial persistence is still a matter

of debate because diagnostic tools to detect persistence in the human host are lacking.

The chlamydiae can elicit the induction of apoptosis under some circumstances and actively

inhibit apoptosis under others (9). This points to an important strategy that chlamydiae have

evolved to promote their survival through the modulation of programmed cell death

pathways in infected host cells.

Sequence information is now available for C. trachomatis, including lymphogranuloma

venereum (LGV) isolates (95, 101), C. pneumoniae (47), C. caviae (78), C. abortus (102),

and a Chlamydia-like endosymbiont ofAcanthamoeba (39), and is in progress for C.

muridarum. Genome analysis of this environmental chlamydial strain showed that about 700

million years ago the last common ancestor of pathogenic and symbiotic chlamydiae was

already adapted to intracellular survival in early eukaryotes and contained many virulence

factors found in modern pathogenic chlamydiae, including a type III secretion system (39).

Comparison of theC. pneumoniae genome with the C. trachomatis genome has provided an

understanding of the common biological processes required for infection and survival in

mammalian cells. Prominent comparative findings include expansion of a novel family of 21

sequence-variant outer membrane proteins, conservation of a type III secretion virulence

system, three serine/threonine protein kinases and a pair of paralogous phospholipase D-like

proteins, additional purine and biotin biosynthetic capability, a homologue for aromatic

amino acid (tryptophan) hydroxylase, and the loss of tryptophan biosynthesis genes (47).

CLINICAL SIGNIFICANCE, EPIDEMIOLOGY, AND

TRANSMISSION Back to top

C. trachomatis

Based on the antigenic reactivity of the MOMP, C. trachomatis is currently divided into 18

serovars. Serovars A, B, Ba, and C can be isolated from patients with clinical trachoma in

areas of endemicity in poor countries in Africa, the Middle East, Asia, and South America.

Acute manifestations of trachoma include primarily a follicular kerato-conjunctivitis, while

late-stage manifestations include tarsoconjunctival scarring with trichiasis, entropium, and

subsequent loss of vision (93). According to estimates of the World Health Organization

(WHO), approximately 1.3 million people in the world suffer from preventable blindness due

to trachoma. Trachoma is transmitted under poor hygienic conditions between members of

the same family or between families with shared facilities via discharges from the eyes of

infected patients. Flies feeding from the mucopurulent eye discharges of infected and

weakened humans may carry the organisms on their legs from one person to another across

relatively long distances. A WHO global initiative aims to eliminate blinding trachoma by

2020.

The C. trachomatis serovars D through K, including the serovars Da and Ia and the

genovariant Ja, are associated with genital tract disease and are among the most common

sexually transmitted bacterial organisms in industrialized countries. According to surveillance

data of the Centers for Disease Control and Prevention (CDC), these organisms are

responsible for an estimated 2 to 3 million new cases every year in the United States, with

1,108,374 cases reported in 2007 in the United States. They typically cause nongonococcal

urethritis in men and cervicitis in women. Infection of the urethra and the lower genital tract

may cause dysuria, whitish or clear urethral or mucopurulent vaginal discharge, and

postcoital bleeding. Urethritis and the rarer manifestations proctitis and conjunctivitis are

observed with both men and women. The majority of infections are asymptomatic and

therefore remain undetected (73). This can result in ascending infections, such as

epididymitis in men and endometritis, salpingitis, pelvic inflammatory disease, and

perihepatitis (Fitz-Hugh-Curtis syndrome) in women. Manifestations of upper genital infection

in women are irregular uterine bleeding, pelvic discomfort, or chronic abdominal pain.

Salpingitis may lead to tubal scarring and severe reproductive complications, such as tubalfactor

infertility and ectopic pregnancy. Tubal-factor infertility attributable to C.

trachomatis is the most frequent form of infection-induced infertility. C. trachomatis-infected

pregnant women may transmit the organisms during delivery to the infants, who are

therefore at risk to develop neonatal conjunctivitis and/or pneumonia (81, 82).

Sequelae of C. trachomatis infection in both men and women can involve HLA-B27-

associated reactive arthritis, presenting most frequently as an acute asymmetric

oligoarthritis with or without enthesopathic and extramusculoskeletal symptoms (117). The

young age of sexually active people is strongly associated with infection, with the highest

prevalence in those 25 years or less (11). Additionally, sex workers, persons with a new sex

partner, or persons who have had several sex partners are at increased risk of infection. In

addition, high prevalence rates were found among incarcerated females entering juvenile and

adult correctional facilities (43). Screening women who are at risk for C.

trachomatis infection can prevent serious complications such as pelvic inflammatory disease

(73). Consequently, female screening programs have been established in some European

countries and the United States to identify and treat infections of asymptomatic individuals

and their partners. Screening asymptomatic men has been discussed, and guidance for

screening men was issued by the CDC in 2007

(http://www.cdc.gov/std/chlamydia/ChlamydiaScreening-males.pdf). However, the USPSTF

concluded that the current evidence is insufficient to assess the balance of benefits and

harms of screening for chlamydial infection for men

(http://www.ahrq.gov/clinic/uspstf07/chlamydia/chlamydiars.htm#clinical).

The C. trachomatis serovars L1, L2, L2a, and L3 cause LGV, a systemic sexually transmitted

disease that is endemic in parts of Africa, Asia, South America, and the Caribbean but rare in

industrialized countries. However, ongoing reports about outbreaks with the newly identified

variant L2b in Europe, Australia, and the United States show that health care providers

should be vigilant for LGV especially among men who have sex with men (30, 70, 94, 114).

The primary lesion, a small, painless papule that tends to ulcerate at the site of inoculation,

often escapes attention. Proctitis is more common in people who practice receptive anal

intercourse, and elevated white blood cell counts in anorectal smear specimens may predict

LGV in these patients (105). Ulcer formation favors transmission of human immunodeficiency

virus and other sexually transmitted and blood-borne diseases. The cardinal feature of LGV is

the presence of painful inguinal and/or femoral lymphadenopathy. Complications of LGV

include development of coalescing fluctuant lymph nodes (buboes) that result in discharging

sinuses and fistula formation. If untreated, fibrosis can lead to lymphatic obstruction causing

elephantiasis of the genitalia.

C. pneumoniae

C. pneumoniae causes infections of the upper and lower respiratory tract, such as sinusitis,

pharyngitis, bronchitis, and pneumonia (51). C. pneumoniae was identified as the causative

agent in 10 to 15% of cases of community-acquired pneumonia in adults (54) as well as in

children (77) . However, data from studies yielding prevalence rates under 1% for C.

pneumoniae pose the question of whether its role in community-acquired pneumonia is

overestimated (97, 113). Severe and life-threatening C. pneumoniae infections have been

described for patients with acute leukemia and treatment-induced neutropenia (35). Chronic

infection with C. pneumoniae was reported among patients with chronic obstructive

pulmonary disease and could also play a role in the natural history of asthma, including

exacerbations. The clinical symptoms of C. pneumoniae infection are nonspecific and do not

differ significantly from those caused by respiratory viruses and Mycoplasma pneumoniae.

Persistent cough seems not to be strongly associated with C. pneumoniae (108). Primary

infection occurs mainly in school-age children, while reinfection has been observed with

adults. Seroprevalence rates from 40 to 70% show that C. pneumoniae is a widely spread

organism in industrialized as well as developing countries.

The role of C. pneumoniae in the etiology of atherosclerosis, a chronic inflammatory disease

of the artery vessel wall (80), has been discussed since 1988 when Saikku and coworkers

presented serological evidence of an association of C. pneumoniae with coronary heart

disease and acute myocardial infarction (85). In subsequent studies, the organisms were

identified in atherosclerotic lesions of patients by culture, PCR, immunohistochemistry, and

transmission electron microscopy; however, the discrepancies of study results (112),

including those of animal studies and the failure of large-scale treatment studies (32), have

raised skepticism about the organism’s causative role in atherosclerosis (42). In addition, a

heterogeneous spectrum of extrapulmonary diseases have been linked to C.

pneumoniae, including multiple sclerosis, Alzheimer’s disease, and chronic fatigue syndrome;

however, a causal relationship between these diseases and C. pneumoniae infection has not

been substantiated.

C. psittaci

Psittacine birds and a wide range of other avian species can act as natural reservoirs for C.

psittaci. In the C. psittaci taxon (19), only the avian chlamydial strains previously

designated Chlamydia psittaci are retained. Others have been placed into several animalassociated

species, such as C. abortus, C. muridarum, C. suis, C. felis, and C. caviae. All

birds are susceptible; however, pet birds (parrots, parakeets, macaws, and cockatiels) and

poultry (turkeys and ducks) are the most frequently involved in C. psittaci transmission to

humans. Exposure is greatest for poultry breeders and the processing workers, as well as in

households with pet birds. Infectious forms of the organisms are shed from symptomatic and

from apparently healthy birds and may remain viable for several months. C. psittaci can be

readily transmitted to humans either by direct contact with infected birds or following

inhalation of aerosols from nasal discharges and from infectious fecal or feather dust.

Transmission from person to person has been suggested but has never been proven.

Symptomatic C. psittaci infection in humans may present as a severe chronic pneumonia

(18), although mild illness and asymptomatic infections in persons exposed to infected birds

have also been observed (66). Typical symptoms include fever, chills, muscular aches and

pains, severe headache, hepato- and/or splenomegaly, and gastrointestinal symptoms.

Cardiac complications may involve endocarditis and myocarditis. Fatal cases were common in

the preantibiotic era. Due to quarantine of imported birds and improved veterinary-hygienic

measures, outbreaks and sporadic cases of psittacosis are rarely observed nowadays. Since

1996, fewer than 50 confirmed cases have been reported in the United States each year.

C. abortus

Chlamydiae associated with ruminant abortion and formerly contained within the Chlamydia

psittaci taxon were transferred to a new species: C. abortus (107). C. abortus has been

acknowledged as a cause of abortion and fetal loss in sheep and has also been detected

broadly in calves. There are a number of reports of pregnant women who have had

spontaneous abortions following exposure to animals infected with C. abortus (76, 109). The

incidence of this animal-acquired infection is not known, but sheep and goats during the

birthing season represent a potential risk to pregnant women. Obstetricians should consider

this diagnosis along with early antibiotic treatment and cesarean section delivery in the

context of the patient’s case history.

Environmental Chlamydiae

The host range of chlamydiae was further broadened with the discovery of Chlamydia-related

endosymbionts in free-living amoebae. The so-called environmental chlamydiae that have

been placed in the familyParachlamydiaceae share the chlamydial developmental cycle and

represent an evolutionary early-diverging sister of the pathogenic chlamydiae (39).

Environmental chlamydiae were discussed as potential emerging pathogens (33); however,

clinical evidence for their importance in human infection is still pending. Simkania

negevensis, currently the only member of the Simkaniaceae, is a recently

discovered Chlamydia-like intracellular agent which has been associated with respiratory

infections in infants (46). The natural host of Simkania is not known; however, the

organisms were successfully grown in various cell lines as well as in free-living amoebae and

were identified in drinking water and in reclaimed wastewater.

COLLECTION, TRANSPORT, AND STORAGE Back to top

General Comments

Since chlamydiae are obligate intracellular pathogens, the objective of specimen collection

should usually be to include the host cells that harbor the organisms. Outside their host,

chlamydiae survive only briefly, and efforts must be undertaken to maintain the organisms’

viability for successful culture. Commercial diagnostic nonculture assays do not require the

presence of viable chlamydiae in the specimen; nevertheless, the instructions of the

manufacturer given in the package insert should be followed for appropriate collection,

transport, and storage of specimens. This includes the use of swabs and transport media

specified by the manufacturer.

For successful culture of chlamydiae, the time between collection and processing of the

specimens in the laboratory should be minimized while keeping specimens cold (4 to 8°C).

Specimens should be forwarded to the laboratory within 24 h in a special chlamydial

transport medium, such as 2-sucrose phosphate or sucrose phosphate glutamate

supplemented with fetal bovine serum (5 to 10%), gentamicin (10 μg/ml), vancomycin (25

to 100 μg/ml), and amphotericin B (2 μg/ml) or nystatin (25 U/ml). Tetracyclines,

macrolides, and penicillins cannot be used in the transport media since they have activity

against chlamydiae. If specimens cannot be processed within 24 h, storage at −70°C in

transport media is acceptable. Specimens for culture should not be stored at -20°C or in

frost-free freezers. Swab specimens should be collected on swabs with a Dacron tip and an

aluminum or plastic shaft. Swab tips made of calcium alginate and swabs with wooden shafts

may inhibit the growth of chlamydiae. It is recommended to check new lots of swabs that are

used to collect specimens for culture of chlamydiae for possible inhibition of chlamydial

growth (16).

C. trachomatis

The type and anatomical site of specimen collection for laboratory diagnosis of C.

trachomatis infection depend on both the clinical picture and the laboratory test selection, as

comprehensively reviewed elsewhere (4, 23,44, 56, 93). Table 1 gives an overview about

ranges of sensitivity and specificity for common diagnostic tests for C. trachomatis in

urogenital specimens. Noninvasively collected specimens such as first-void urine (FVU; first

10 to 30 ml of urine) and vaginal swab specimens are excellent for diagnosis of C.

trachomatis genital tract infection by nucleic acid amplification techniques (NAATs).

Sensitivity and specificity of NAATs for C. trachomatison noninvasively collected specimens

are similar to those obtained on samples collected directly from the cervix or urethra

(25, 88). Patients and clinicians may prefer self-sampling to the standard collection methods

(40). FVU specimens should be obtained at least 2 h after the last micturition. Ambienttemperature

storage of fresh unprocessed urine should not exceed 24 h to avoid

denaturation of chlamydial DNA. Subsequent processing of the urine specimens for NAAT

varies depending on the manufacturer’s instructions. Neither urine nor vaginal specimens are

recommended for testing by culture and nonamplification assays, such as enzyme

immunoassay (EIA), direct fluorescence assay (DFA), and nucleic acid hybridization (NAH),

because of their relatively very low sensitivity with these assays (44).

Traditional sites for specimen collection in C. tracho matis genital tract infection involve the

endocervix in females and the urethra in males. Newly recommended specimen additions

include vaginal samples for women and urine for men, but only when highly sensitive nucleic

acid amplification tests are used (23, 86). Proficient specimen collection including speculum

examination in females is required to obtain appropriate samples that contain sufficient

columnar or squamocolumnar cells. Purulent discharges have to be cleaned before a swab is

inserted 1 to 2 cm into the cervical os past the squamocolumnar junction, rotated more than

two times, and removed without touching the vaginal mucosa (4). Urethral specimens from

males are collected by placing a dry swab 3 or 4 cm into the urethra and rotating prior to

removal. Urination prior to specimen collection may reduce test sensitivity by washing out

infected columnar cells. Since culture and older less-sensitive tests are falling into disuse,

the preferred samples recommended for screening asymptomatic women are vaginal swabs,

which can be self-collected for some assays. Self-obtained vaginal swabs are gaining in

practical use and have been recommended as highly accurate and acceptable samples by the

NIH and CDC (38). It was shown that for women, self-collected vaginal swabs had a clearly

higher mean chlamydial load than did first-void urine specimens (115).

In women with salpingitis, samples may be collected by needle aspiration of the involved

fallopian tube. Endometrial specimens have also yielded chlamydiae. Further appropriate

sites include the conjunctiva in chlamydial eye infection (trachoma, inclusion conjunctivitis,

and newborn conjunctivitis) and the nasopharynx and deeper respiratory tract of infants in

newborn pneumonia. For men who have sex with men, screening of rectal and pharyngeal

specimens is recommended since some reports support the utility of commercial NAATs as a

screening test for this population (49, 89). In cases of suspected LGV, ulcer swabs, aspirates

of bubo fluid, and rectal or urethral swabs should be collected in transport medium. Buboes

of LGV may contain only small amounts of thin milky fluid, and it may be necessary to inject

2 to 5 ml of sterile saline to obtain any fluid by aspiration (56).

C. pneumoniae

The optimal sites for specimen collection in C. pneumoniae infection are poorly defined.

Respiratory specimens from which the organisms were cultured include sputum,

bronchoalveolar lavage fluid, nasopharyngeal aspirates, throat washings, and throat swabs

(tonsil area). Swab specimens should be collected using a Dacron tip and a plastic shaft (16)

and placed immediately in transport medium. Specimens need to be kept at 4 to 8°C in

chlamydial transport medium, since the organisms are inactivated rapidly at room

temperature. Rapid freezing or freezing and thawing of specimens should be avoided (51).

Liquid specimens are collected in transport medium at a specimen-to-medium ratio of 1:2

(16). Testing of vascular tissue specimens and blood samples, except for research studies, is

of questionable value.

C. psittaci

C. psittaci strains seem to be the most stable organisms among the pathogenic chlamydiae.

Nevertheless, specimens should be collected in chlamydial transport medium. Appropriate

specimens include sputum, bronchoalveolar lavage fluid, pleural fluid, blood, and tissue

biopsy specimens from various anatomical sites. Culture is no longer recommended because

of the potential for laboratory acquired infections. There are only single commercial

nonculture tests for C. psittaci (7) available; however, a panel of research nucleic acid

amplification assays has been published (28, 57, 64).

DIRECT EXAMINATION Back to top

Nucleic Acid Amplification Techniques

C. trachomatis

Due to their high sensitivity and specificity, NAATs are the tests of choice for diagnosis of

genital C. trachomatis infections in routine clinical laboratories. NAATs can be used to

detect C. trachomatis without a pelvic examination or intraurethral swab specimen by testing

self- or clinician-collected vaginal swabs or urine, respectively (13, 86). This facilitates the

establishment of screening programs in asymptomatic individuals and may enhance the

compliance for testing asymptomatic contact persons of infected individuals. NAATs on urine,

with confirmation, were shown to be adequate for use as a new forensic standard for

diagnosis of C. trachomatis and Neisseria gonorrhoeae in children suspected of being sexual

abused (5). Increasing experience is available for the use of NAATs in conjunctival,

oropharyngeal, and rectal samples (49, 89) and in LGV (12,98). Thus far, no commercial

company has an FDA-cleared test for these alternative sample types, but it is possible for

laboratories to use these samples for testing by NAATs if they perform a verification study to

indicate their performance. If such verification is performed, CLIA compliance can be

demonstrated

(http://www.aphl.org/aphlprograms/infectious/std/Pages/stdtestingguidelines.aspx).

Commercial NAATs seem to work in newborn conjunctivitis (82), but no one has an FDA

claim. For research studies of trachoma patients, NAATs have been recommended as the

“gold standard” and are now being used by many research laboratories. However, the

commercial assays presently available may be too expensive and too complex for use in

some national trachoma programs (93). In many evaluations, NAATs detected 20 to 30%

more positive specimens than could be detected by earlier technologies.

Licensed NAATs for detection of C. trachomatis include (in the order of their introduction) the

PCR-based Roche Amplicor (Roche Diagnostics, Basel, Switzerland), the Aptima

transcription-mediated amplification assay (Gen-Probe, Inc., San Diego, CA) and the BD

ProbeTec strand displacement amplification (SDA) assay (Becton Dickinson and Company,

Diagnostic Systems, Franklin Lakes, NJ). The former, frequently used Abbott LCx ligase chain

reaction was withdrawn from the commercial market by the manufacturer in 2003. Licensed

assays working on fully automated platforms for use in high-volume laboratories include the

Cobas TaqMan, Abbott m2000, BD ProbeTec (Viper), and Aptima (Tigris) (53, 61).

Both the PCR and SDA assay amplify nucleotide sequences of the 7.5-kbp cryptic plasmid

of C. trachomatis,which is present in an average copy number of about four plasmids per

chromosome in EBs and up to seven plasmids per chromosome in replicating RBs (74). C.

trachomatis strains that do not harbor the cryptic plasmid have been isolated sporadically

from urethral specimens. A new variant of C. trachomatis with clinical and epidemiological

relevance was recently discovered in Sweden. Due to a 377-bp deletion in the target

sequence for nucleic acid amplification, this strain has initially escaped detection by some of

the licensed NAATs (37, 65), but manufacturers affected by this discovery moved quickly to

modify their primers to enable this variant’s detection. The transcription-mediated

amplification-based assays target specific sequences of the 23S rRNA, which is also present

in multiple copies. Each of the three commercially available NAAT systems offers the option

for combination testing of C. trachomatis and Neisseria gonorrhoeae in the same specimen.

The transcription-mediated amplification platform is also offered as individual assays for

chlamydia infection or gonorrhea.

Considering the multiplicity of target sites for the amplification procedures being used,

NAATs should be able to produce a positive signal from less than one EB; however, the

actual sensitivity with clinical specimens is lower because of sampling variability and

inefficient nucleic acid isolation. Since inhibitor problems of NAATs can be reduced by dilution

of specimens, heating, freeze-thaw cycles, or overnight storage at 4°C, the use of internal

inhibitor controls of the amplification assays (as supplied by the manufacturers of PCR and

SDA) is helpful for identification of clinical specimens containing inhibitory factors (59).

Extraction of nucleic acids by target capture and magnetic bead procedures by secondgeneration

NAATs has almost completely eliminated the presence of inhibitors in processed

clinical samples. All these NAAT assays are highly specific for chlamydiae if problems with

cross-contamination, labeling errors, and mistakes in specimen collection can be avoided

(Table 1). Confirmatory testing of positive specimens was recommended by the CDC if a low

positive predictive value was expected (<90%) or if a false-positive result would have

serious psychosocial or legal consequences (44). However, supplemental testing is no longer

recommended for chlamydia

(http://www.aphl.org/aphlprograms/infectious/std/Pages/stdtestingguidelines.aspx) or for

diagnosis of C. trachomatis and Neisseria gonorrhoeae in children suspected of having been

sexually abused (5, 87).

In settings where resources are limited, including developing countries, the concept of

pooling to detect C. trachomatis by NAATs has proved to be a simple, accurate, and costeffective

procedure compared to individual testing (45, 55). Specimen pools may consist of

aliquots from 4 to 10 processed specimens (FVU or genital swab) combined into one

amplification tube. Subsequent testing of individual samples is required only if the pooled

sample gives a positive result. Following this strategy, considerable savings of reagent costs

can be obtained, especially in low-prevalence populations.

C. pneumoniae

A vast number of PCR-based protocols using different formats and target genes have been

developed in research laboratories for detection of C. pneumoniae in both respiratory and

nonrespiratory samples (50). However, the lack of a reliable gold standard for C.

pneumoniae infection has made it difficult to evaluate the published protocols thoroughly.

Broad application of NAATs for diagnosis of C. pneumoniae infection has been hampered

because many PCR protocols are not reliable or robust enough to provide reproducible

results in routine clinical laboratories. Even in specialized laboratories, there seems to be a

substantial interlaboratory variation in the performance of C. pneumoniae NAATs, and the

need for quality control and standardization of these assays has been recognized (16, 50).

Subsequently, specific recommendations for standardizing C. pneumoniae PCR assays were

made, and it was suggested that the performance of newly developed PCR protocols be

compared with that of at least one of four recommended assays that target

the PSTI fragment (10), the ompA gene (104), or the 16S rRNA gene (24, 57). However, all

of these assays must be considered research tools (16), because commercial FDA-cleared

assays are currently not available. Real-time PCR technology provides promising results that

warrant further evaluation of this approach for detection of C. pneumoniae infection

(1, 79, 103). A recent review again stated that standardization and validation, particularly of

PCR assays, are urgently needed because the true role of the organism in respiratory

infections as well as in extrapulmonary diseases cannot be ascertained at the moment (50).

C. psittaci

NAATs could be helpful for detection of avian C. psittaci strains from clinical samples since

culture of these organisms is dangerous and requires biosafety level 3 (BSL-3) facilities and

is not recommended. Some PCR-based assays have been developed for diagnosis of human

ornithosis (7, 18, 57, 63, 104), and a commercially available DNA microarray assay for

detection and species identification of human and zoonotic chlamydiae has been introduced

(7). Due to the rarity of the disease, the performance characteristics of these assays have

been poorly evaluated in clinical specimens.

Nucleic Acid Hybridization

Two NAH tests are commercially available for detection of C. trachomatis. The Gen-Probe

PACE 2 test (Gen-Probe, Inc.) hybridizes to a species-specific sequence of chlamydial 16S

rRNA that is present in a high copy number in replicating chlamydiae. Available data suggest

that it is about as sensitive as the better antigen detection and cell culture methods and is

relatively specific. However, it was shown that commercial NAATs improved the detection of

infections in women by 17 to 38% compared to PACE 2 (6). The second NAH test, the Digene

Hybrid Capture II, is a nucleic acid probe-signal amplification assay (Digene Corp.,

Gaithersburg, MD) that uses RNA hybridization probes for DNA sequences encoding both

genomic and cryptic plasmid sequences of C. trachomatis. This assay was shown to reach

the sensitivity of a commercial NAAT when cervical specimens were investigated (106). NAHs

are considered highly robust test methods for detection ofC. trachomatis. NAH tests have

been recommended for endocervical swabs or urethral swabs from men when a NAAT is not

available or not economical. As is the case with other non-NAATs, NAH tests have not been

recommended for use in noninvasive-collection specimens, such as urine and vaginal swabs

(44). Both NAH systems also offer a test format that enables detection of C.

trachomatis and N. gonorrhoeae in a single specimen. However, their use is rapidly being

replaced by NAAT assays, which is now the test platform of choice for chlamydia tests.

Antigen Detection Assays

DFA

The presence of typical intracytoplasmic inclusions in columnar epithelial cells of the

conjunctiva, urethra, or cervix of infected patients can be demonstrated when air-dried

smears are fixed on a slide with absolute methanol and stained with Giemsa stain.

Cytological testing was particularly useful in diagnosing acute inclusion conjunctivitis of the

newborn, but the more sensitive immunofluorescence procedures have largely replaced this

method. DFAs use fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies

directed at a C. trachomatis-specific epitope of the MOMP (Chlamydia Cel [Cellabs,

Brookvale, Australia] or Pathfinder [Bio-Rad Laboratories, Redmond, WA]). DFAs are based

on detecting EBs in smears, although staining of inclusions can also succeed if intact infected

host cells are collected. Checking for the presence of columnar cells allows assessment of the

adequacy of the sample. The procedure offers rapid diagnosis, taking only 30 min to

perform, making DFAs useful, especially for laboratories that test only a limited number of

specimens. However, this method requires an experienced microscopist who can distinguish

between fluorescing chlamydial particles and nonspecific fluorescence. The DFA has

approximately 75 to 85% sensitivity and 98 to 99% specificity compared with culture and a

lower sensitivity than NAATs (8, 69). DFAs can be another alternative for testing

endocervical swabs from females or urethral swabs from males when a NAAT is not available

or not economical. In addition, DFAs have been recommended for use with conjunctival

specimens and for testing of individuals with possible rectal and pharyngeal exposure to C.

trachomatis, if a C. trachomatis MOMP-specific antibody is used (44). Nontrachomatis

chlamydial conjunctivitis should be considered if the DFA reveals the presence of chlamydial

LPS but not C. trachomatis-specific MOMP.

EIA

EIAs for the detection of C. trachomatis use either monoclonal or polyclonal antibodies to

detect chlamydial LPS, which is more soluble than MOMP. Although they can theoretically

detect all chlamydiae, EIAs have not been well evaluated for the diagnosis of infections

with C. pneumoniae or C. psittaci. The performance characteristics of EIAs for laboratory

diagnosis of C. trachomatis have been reviewed comprehensively elsewhere (4). Using

cultures as reference standards, the sensitivities of EIAs applied to endocervical swabs were

in a range from 62 to 72% (69). EIAs are never recommended for testing of noninvasively

collected specimens, such as urine and vaginal swabs. EIAs are now considered substandard

and are not recommended for use as a diagnostic platform by the CDC.

Rapid or point-of-care (POC) tests designed for office- or clinic-based settings have been

developed and provide test results in less than 30 min for C. trachomatis infection in women.

Similar to EIAs, they also use antibodies against chlamydial LPS, with the potential to yield

false-positive results due to cross-reaction with other gram-negative bacteria. Current POC

tests are not recommended in laboratory settings because sensitivity and specificity are

lower, quality controls are less rigorous, and costs are higher than for tests designed for

laboratory use (44). Although some POC assays are FDA cleared, they were compared to

culture as the gold standard and now that the new gold standard is NAATs, the package

inserts often overstate sensitivities. When compared to PCR, the Clearview POC

demonstrated a sensitivity of 32.8% for vaginal swabs and 49.7% for cervical swabs (116).

New POC assays are being developed and appear promising but are not yet FDA cleared

(58).

ISOLATION PROCEDURES Back to top

Biosafety Considerations

C. pneumoniae and C. trachomatis are BSL-2 organisms, whereas C. psittaci is a BSL-3

organism. Transmission of the organisms from patient specimens or infected cell cultures can

occur through aerosols, splashes onto the mucous membranes of the eyes, and hand-to-face

actions. In recent years, fewer laboratory-acquired infections have been reported, probably

due to the common usage of class II biosafety cabinets in laboratories that work

with Chlamydia-infected cell cultures. Use of a class II biosafety cabinet protects laboratory

staff from exposure to aerosols as well as specimens and cell cultures from contamination.

Additional means of preventing laboratory-acquired infection include the use of gloves,

alcohol-based hand disinfectants, safety centrifuge caps, and face protection, if appropriate.

Laboratory infections with C. trachomatis usually manifest as follicular conjunctivitis. The

LGV strains are more invasive, and severe cases of laboratory-associated pneumonia and

lymphadenitis are reported. C. psittaci must be considered a potentially dangerous organism,

requiring appropriate BSL-3 facilities. Laboratory-acquired C. pneumoniae infections might

be underestimated since the mild clinical course may not prompt infected laboratory workers

to seek medical attention.

Specimen Processing

Ocular and Genital Tract Specimens

For culture of chlamydiae from ocular and genital tract sites, only swabs that are rapidly

forwarded to the laboratory in a special chlamydial transport medium are acceptable (see

above). Specimens to be assayed by commercial EIA, DFA, NAH, or NAAT should be

processed as directed by the manufacturer.

Bubo Pus

To prepare bubo pus, the aspirate fluid of fluctuant lymph nodes is ground and then

suspended in nutrient broth or cell culture medium to at least 20% by weight. Even when the

pus is not viscous, dilution is advisable. The material should be tested for bacterial

contaminants and inoculated onto monolayer cultures of McCoy or HeLa 229 cells.

Blood

Blood samples from clotted blood tubes have been used in the past for diagnosis of C.

psittaci endocarditis. The blood clot was ground, and cell culture medium was added to make

a 10% solution. However culture is no longer recommended for C. psittaci due to the

possibility of laboratory acquired infections, so this method is reserved for specialized

research laboratories

Sputum, Throat Washings, and Other Secretions from the Respiratory

Tract

Sputum and other respiratory samples are suspended in antibiotic-containing transport

medium or cell culture medium at a ratio of specimen to medium of 1:2 to 1:10, depending

on specimen consistency. Specimens are homogenized by adding sterile glass beads to the

sample and vigorously vortexing for 1 to 2 min in a tightly stoppered container. Extracts

should be centrifuged for 20 to 30 min at 100× g to remove coarse material before the

supernatant fluid is inoculated onto cell monolayers. Serial dilutions may be required if the

inoculum is toxic to cells.

Fecal Samples

Human rectal swabs for C. trachomatis and avian material for C. psittaci are suspended in

chlamydial transport medium or antibiotic-containing cell culture medium. The suspension is

shaken thoroughly and centrifuged at 300× g for 10 min, and the supernatant is removed. It

may be further diluted (1:2 and 1:20) with medium before being inoculated into cell culture.

Rectal swabs for a commercial NAAT are processed in accordance with the corresponding

protocol of the manufacturer.

Tissue Samples

Frozen tissue is thawed in a refrigerator at 4°C. The specimen is weighed, minced with

sterile scissors or a scalpel, and ground with a mortar and pestle or homogenizer. A volume

of cell culture medium required to make a 10 to 20% suspension is added, and the

suspension is thoroughly mixed. For tissue specimens, serial dilutions (1:10 to 1:100) are

often required for inoculation to prevent toxicity.

Isolation

Cell culture was considered the gold standard for diagnosis of genital C. trachomatis infection

because its sensitivity and specificity were thought to be close to 100%. Problems associated

with cell culture isolation of chlamydiae, including technical complexity and long turnaround

time, and stringent requirements related to collection, transport, and storage of specimens

have driven the development of commercially available nonculture methods that have found

widespread application in many routine laboratories. With the advent of antigen detection

methods, it became clear that the sensitivity of culture was substantially lower than

previously thought, probably due mostly to the presence of nonviable chlamydiae that died

during transport and processing. Culture for detection of chlamydiae in clinical specimens is

now performed generally only in specialized laboratories (4). Culture is recommended in

treatment failures (when a viable isolate is needed for susceptibility testing) and in cases

related to possible sexual assault for medicolegal reasons (44), although NAATs on urine

have been shown to be adequate for children suspected of being sexually abused (5).

Historically, chlamydiae were cultivated in the yolk sac of embryonated eggs. The yolk sac

method (for details, see reference 91) is still used for preparing antigens for the

microimmunofluorescence (MIF) test. The ability to propagate chlamydiae in the laboratory

has greatly increased the understanding of diagnosis and pathogenesis of chlamydial

infections (92). For isolation of chlamydiae from clinical specimens, appropriately collected

and transported samples are inoculated onto preformed cell monolayers. A number of

susceptible permanent cell lines, including McCoy, HeLa 229, HEp-2, HL, BGMK, Vero, and L

cells, have been used. Clinical samples are centrifuged onto monolayers to enhance

infection. Strains of C. psittaci and LGV biovars are capable of serial growth in cell culture

without centrifugation. Cultures are incubated for 48 to 72 h in the presence of the host cell

protein synthesis inhibitor cycloheximide. McCoy and HeLa 229 cells are most commonly

used for C. trachomatis. HL and HEp-2 cells seem to be more sensitive for recovery of the

fastidiousC. pneumoniae from clinical specimens. Visualization of cell culture-grown

chlamydiae is achieved by the immunostaining of inoculated cell monolayers for

intracytoplasmic inclusions. A positive culture shows one or more typical intracellular

inclusions (Fig. 1B).

Cell culture methods can vary among laboratories. Host cells are plated either onto 12-mm

glass coverslips contained in 15-mm-diameter (1 dram [1 dram = 3.697 ml]) disposable

glass vials (shell vial method) or in 6-, 12-, or 24-well tissue culture plates. The cells are

seeded in concentrations of 1 × 105 to 2 × 105 cells/ml to give a healthy and confluent

monolayer after 24 to 48 h of incubation. For optimal results, cell monolayers should be

inoculated with patient specimens within 24 h after reaching confluency. Clinical specimens

are thoroughly vortexed with glass beads in tightly closed screw-cap vials to facilitate release

of chlamydiae before inoculation. The cell culture medium of the cell monolayers to be

inoculated is discarded and replaced by a volume of 0.2 to 2 ml of the vortexed specimen.

The inoculated specimen is centrifuged onto the cell monolayers at 900 to 3,000× g for 1 h

at 22 to 35°C. Cells are incubated at 35°C for 1 to 2 h to allow uptake of chlamydiae before

the medium is replaced with chlamydial isolation medium consisting of the cell culture

medium supplemented with fetal calf serum (10%), L-glutamine (2 mM), cycloheximide (1 to

2 μg/ml), gentamicin (10 μg/ml), vancomycin (25 μg/ml), and amphotericin B (2 μg/ml).

Cultures are incubated at 35°C in 5% CO2 for 48 to 72 h. Then, one coverslip per specimen

is removed for immunostaining of inoculated monolayers. Both cell detritus and toxic effects

of the inoculum may make it difficult to read slides. Dilution of cell-rich material (bubo pus,

sputum, tissue samples, and rectal swabs) and blind performance of subpassages can be

helpful for microscopic interpretation of slides.

If a blind subpassage or passage of positive material is to be performed, the corresponding

cell monolayers of duplicate wells are scraped and disrupted by vortexing with glass beads.

Cell debris of harvested material is removed by low-speed centrifugation (300× g) for 10

min, and the supernatant is passed onto preformed cell monolayers as described above.

For C. pneumoniae, most laboratories agree that at least two passages are needed to

maximize the recovery of the organisms from respiratory specimens. Modifications of the

standard procedure, including use of serum-free culture medium, pretreatment of cell

monolayers with polyethylene glycol or diethylaminoethyl-dextran, and extension of culture

times, have not been sufficiently tested to warrant their routine recommendation (16).

Laboratories processing large numbers of specimens may use flat-bottom 48- or 96-well

microtiter plates onto which cells are plated directly. Processing and incubation are as

described above, but microscopy is modified because cells are stained directly in the well,

requiring use of inverted microscopes and long working objectives.

Continuous quality control is important for maintaining a sensitive and specific culture

system. Because of its technical complexity, there are multiple opportunities to modify

factors in the culture system that may impact the isolation efficiency (92, 93). Therefore,

positive controls with a known number of inclusion-forming units should be run routinely to

check the sensitivity of the culture system. Negative controls with uninfected human cells

may help to evaluate episodes of cross-contamination as a result of handling positive patient

specimens or positive controls. Routine testing of cell culture systems

for Mycoplasma contamination has been recommended because Mycoplasma contamination

may impair the growth of chlamydiae and may decrease the sensitivity of the culture system

(16).

IDENTIFICATION Back to top

The basic procedure for detection of isolated chlamydiae involves demonstration of

intracytoplasmic inclusions by fluorescent-antibody staining that provides both morphological

and immunological identification of chlamydiae. Screening of cultures can be performed with

a commercially available FITC-conjugated monoclonal anti-LPS antibody (Pathfinder; Bio-

Rad), which recognizes all chlamydiae known to cause infections in humans. Confirmation of

positive genital cultures can be done by the use of a C. trachomatis MOMP-specific

monoclonal antibody (Fig. 1B). For respiratory cultures, a C. pneumoniae-specific monoclonal

antibody may additionally be appropriate. Monoclonal antibodies specific for C. psittaci are

not commercially available. Using DFA procedures, inclusions of C. trachomatis-infected cells

are visible at 24 h postinfection. Less expensive but also less sensitive methods that were

commonly used before the advent of monoclonal antibodies include Giemsa staining (which

needs an experienced and well-trained microscopist for interpretation) and iodine staining for

identification of glycogen- containing inclusions that are produced by C. trachomatis but not

by C. psittaci or C. pneumoniae (Fig. 1A).

Identification of replicating chlamydiae can also be done by fluorescence in situ hybridization

using fluorescently labeled oligonucleotide probes complementary to order-, genus-, and

species-specific target sites on the chlamydial 16S rRNA molecules (75). The risk of falsepositive

signals caused by nonspecific binding of the fluorescent dyes to nontarget organisms

or structures of the host cells can be minimized by the simultaneous application of multiple

probes with hierarchical specificity labeled with different dyes, leading to a characteristic

hybridization pattern (Fig. 1E)

TYPING SYSTEMS Back to top

Serotyping and genotyping procedures are important tools for epidemiological studies. They

are of clinical use if medicolegal issues are involved or if LGV is suspected. The most

convenient method for serotyping C. trachomatis isolates appears to be the microwell typing

system (100), in which inclusions in microtiter plates are stained with pools of monoclonal

antibodies (available at Washington Research Foundation, Seattle, WA) that recognize

serovar- and subspecies-specific epitopes of the MOMP. Genotyping of C.

trachomatis isolates usually involves either restriction fragment length polymorphism

analysis of the MOMP-encoding ompA gene or sequence analysis of the VDs in

the ompA gene. These variable regions include the peptides responsible for species, serovar,

and serogroup specificities. PCR amplification and sequencing of ompA using extracted DNA

from patient specimens, such as urine or genital samples, allows direct genotyping from C.

trachomatis-positive individuals without isolation of the organisms. In addition, new highresolution

genotyping methods applying a multilocus variable number tandem repeat assay

or multilocus sequence typing have been introduced (71). ompA-based procedures (27),

including real-time PCR with high-resolution melt analysis (64) and DNA microarray

technology (84) are used to identify all known and additional new genotypes of avian C.

psittaci strains. Different serotypes or genotypes of C. pneumoniae have not been described.

SEROLOGIC TESTS Back to top

Serologic testing may be helpful in the diagnosis of human ornithosis, LGV, neonatal

pneumonia caused by C. trachomatis, and respiratory C. pneumoniae infections. Serological

testing for diagnosis of uncomplicated genital infections of the urethra and the lower genital

tract as well as for C. trachomatis screening in asymptomatic individuals is not recommended

(44). C. trachomatis antibody testing has been proposed as the first screening test for tubal

factor subfertility (15). Antibodies to C. trachomatis were independently associated with

reduced rates of pregnancy and elevated rates of recurrent pelvic inflammatory disease (68).

Since a reference standard has not been defined, the diagnostic value of some serological

assays for detection of chronic or persistent chlamydial infections is difficult to estimate.

General problems of chlamydial serodiagnosis arise from the difficulty in obtaining paired

serum samples, the high seroprevalence of C. pneumoniae in adult populations, and the lack

of standardized species-specific test methods. The most commonly used serological assay

formats include the complement fixation (CF) test, the MIF test, and the EIA to detect

immunoglobulin M (IgM), IgA, IgG, or total classes of antibodies, with either family, species,

or serotype specificity. Some of these assays have been commercialized and are being used

by clinical laboratories, although their performance characteristics have been evaluated only

in a limited number of studies.

CF Test

The CF test is based on antibody reactivity to the chlamydial LPS antigen common to all

members of theChlamydiaceae. The CF test may be useful in diagnosing LGV in patients who

present compatible clinical symptoms. A titer of ≥256 strongly supports the clinical

diagnosis, while a titer of <32 rules it out except in the very early stages of the disease (56).

In addition, the CF test is useful for diagnosis of psittacosis; however, in the absence of a

typical patient history (exposure to birds), C. pneumoniae infection might be considered for

patients with positive test results. However, due to its potential for cross-reactivity and its

low sensitivity for reinfection, CF is not recommended for serodiagnosis of C.

pneumoniae infections (16). The CF test also lacks sensitivity for the diagnosis of trachoma,

inclusion conjunctivitis, and uncomplicated genital infections caused by C. trachomatis. The

CF test is widely becoming unavailable in many laboratories, which may limit its usefulness

in the near future.

MIF Test

The MIF test developed by Wang and Grayston in the early 1970s is still considered the

method of choice for serodiagnosis of chlamydial infections. With this procedure, speciesand

serovar-specific antibody responses in human chlamydial infection can be detected. The

MIF test allows quantitative detection of IgM and IgG antibodies that may be helpful in

distinguishing recent from past infections.

The MIF test is the diagnostic test of choice for C. trachomatis pneumonitis in infants

because elevated levels of IgM antibodies are regularly associated with disease (4). A single

IgM titer of ≥32 may support the diagnosis of neonatal pneumonia caused by C. trachomatis.

IgG antibodies are less useful because infants may present with typical symptoms when they

still have a high level of maternal IgG. In LGV-infected individuals, a MIF IgG titer of ≥128

strongly supports the clinical diagnosis, although invasive genital infection with C.

trachomatis serovars D through K, such as pelvic inflammatory disease, salpingitis, or

epididymitis, can also give rise to high serum titers of antichlamydial antibody (56). The MIF

test may be useful in the diagnosis of psittacosis and is the serological testing method of

choice for diagnosis of acute C. pneumoniae infection. Criteria for acute infection of C.

pneumoniae generally include paired sera demonstrating at least a fourfold rise in titer and

single serum samples with IgM titers of ≥16 and/or IgG titers of ≥512. However, single IgG

titers of ≥512 should be interpreted with caution because elevated IgG titers may persist for

several years in the absence of clinically apparent disease (16). IgG titers in the range of 16

to 256 are suggestive of past infection. The usefulness of IgA as a diagnostic marker in acute

or chronic C. pneumoniae infections has not been substantiated (50).

The MIF assay is performed using purified formalinized EBs of representative strains or

serovars of C. trachomatis, C. psittaci, and C. pneumoniae that are dotted in a specific

pattern onto glass slides. MIF antigens are commercially available from the Washington

Research Foundation. Serial dilutions of patient sera are placed over the fixed antigen dots

and incubated, and bound antibody is detected with fluorescein-conjugated anti-IgG or anti-

IgM antibody (Fig. 1F). A more detailed description of the MIF procedure has been

summarized elsewhere (110). In addition, recommendations for standardizing the MIF assay

in terms of antigen preparation, testing, interpretation of results, and quality assurance

should be followed (16).

The MIF assay format is technically demanding, time-consuming, and less useful for higher

volume testing. In addition, subjective reading of titers may contribute to intra- and

interlaboratory variation in MIF assay results (72). For these reasons, well-trained and

experienced laboratory staff are required. A few standardized kits based on the MIF format

have been developed and marketed (Focus Diagnostics, Cypress, CA; Labsystems Oy,

Helsinki, Finland; and Savyon Diagnostics Ltd., Ashdod, Israel). Initial studies suggest that

their performance characteristics are similar and seem to correspond well to those of the

classical MIF method (3). However, at the time of this writing, none of these assays are

cleared by the FDA for use in the United States for the diagnosis of C. pneumoniae or C.

trachomatis infection.

Enzyme Immunoassay

To overcome the problems associated with MIF testing, EIAs have been developed that offer

a more automated workflow and objective end points for serodiagnosis of chlamydial

infections. EIAs based on synthetic peptides from the VD4 of the C. trachomatis MOMP have

been marketed for detection of C. trachomatis-specific IgG and IgA antibodies (CT-EIA

[Labsystems], SeroCT [Savyon Diagnostics], and CT pELISA [Medac, Wedel, Germany]).

These assays performed as well as the MIF assay in a few studies (67); however, little is

known regarding how long specific antibodies may persist in individuals with resolved

infections. For this reason they cannot reliably differentiate current and past infections. They

are not useful inC. trachomatis infections of the lower genital tract for which adequate

specimens for direct detection of the organisms can be noninvasively obtained. Further

studies are needed to clarify if C. trachomatis species-specific antibody tests based on

recombinant antigens are convenient tools for the diagnosis of upper-genital tract infections

(20).

The major antigenic determinants of C. pneumoniae that are broadly immunodominant

among infected individuals are elusive. Commercial assays designed for specific diagnosis

of C. pneumoniae infection are based on either whole elementary bodies (Savyon

Diagnostics) or (to obtain more specificity) on LPS-extracted EB preparations (Labsystems

and Medac). Most kits have been compared only to MIF (36), but none has been evaluated

adequately with sera from culture- or PCR-positive patients. Thus, their diagnostic value for

acute C. pneumoniae infections remains to be determined (16).

ANTIMICROBIAL SUSCEPTIBILITIES AND TREATMENT Back

to top

Evaluation of antimicrobial resistance and potential clinical treatment failure in chlamydial

infection is hampered by the lack of standardized antimicrobial susceptibility tests and the

fact that in vitro resistance does not correlate with the patient’s clinical outcome (99). For

these reasons, antimicrobial susceptibility testing of Chlamydia organisms has little clinical

utility and is currently performed only in some research laboratories. Antimicrobial

susceptibility testing of chlamydiae requires growing the organisms in epithelial cells cultured

in medium containing increasing concentrations of antibiotics. Cells are stained with an FITClabeled

anti-chlamydial antibody, and the lowest concentration of antibiotic that inhibits

inclusion formation after 48 h of incubation is reported as the MIC (99, 111). The minimum

chlamydicidal concentration has been defined as the lowest concentration of antibiotic

producing no viable bacterial progeny as determined after passage from antimicrobialcontaining

medium to antimicrobial-free medium. However, variation of antimicrobial

susceptibility results is common because they depend on many factors, including the cell

type used, the inoculum size, and the time between infection and the addition of an

antimicrobial.

Tetracyclines, macrolides, fluoroquinolones, and rifampin are commonly used for antibiotic

treatment of chlamydial infections. A single 1-gram dose of azithromycin has been shown to

be as effective for the treatment of uncomplicated genital C. trachomatis infections in adults

as a standard 7-day course of doxycycline (29, 52). Alternative regimens include a 7-day

course of erythromycin, ofloxacin, or levofloxacin. More data and clinical experience are

available to support the efficacy and safety of azithromycin in pregnant women (29).

Cotreatment or testing for chlamydiae should be considered among gonorrhea-infected

patients because of the frequency of coinfection. Systemic treatment with erythromycin has

been recommended for ophthalmia neonatorum as well as for infant pneumonia caused by C.

trachomatis. In the treatment of adult inclusion conjunctivitis, a single azithromycin dose

was as effective as a standard 10-day treatment with doxycycline (48). Doxycycline for 21

days is the antibiotic treatment of choice for both bubonic and anogenital LGV (62).

Doxycycline, azithromycin, erythromycin, levofloxacin, and newer macrolides, such as

clarithromycin and roxithromycin, have been recommended for treatment of C.

pneumoniae infection; however, evidence from clinical trials supporting their use is limited.

Chlamydial resistance to recommended antimicrobial agents appears to be rare and confined

to only a few clinical isolates of C. trachomatis and has not yet been reported for C.

pneumoniae or C. psittaci infections. Nevertheless, concern has been raised about resistance

because recurrent or persistent chlamydial infections were observed with women adequately

treated for C. trachomatis infection and in a few cases of C. pneumoniae infections (34).

In vitro, chlamydial resistance to fluoroquinolones, macrolides, tetracyclines, and rifampin

can be induced with large numbers of organisms cultured in the presence of antimicrobials.

In an animal model, persistence of C. pneumoniae after antimicrobial therapy has been

demonstrated (60). The emergence of Chlamydia suis strains isolated from livestock and

displaying a chromosomally stable tet(C) resistance gene raises concern about the issue of

antibiotic use in animal feeds (17).

INTERPRETATION AND REPORTING OF RESULTS Back to top

Licensed commercially available NAATs enable the reliable detection of uncomplicated

genital C. trachomatisinfection, even from noninvasively obtained specimens such as first

void urine and (self-collected) vaginal swabs. These specimens are also recommended for

screening asymptomatic individuals. Reporting of test results for chlamydiae should include

the type of test used and a clinical interpretation if possible. Sexual partners of infected

patients should be notified, examined, and treated for C. trachomatis. Patients and their

partners should be instructed to abstain from sexual intercourse until therapy is completed.

Due to the presence of nonviable bacteria, nonculture tests for C. trachomatis, especially

NAATs, may remain positive when performed ≤3 weeks after completion of therapy (22).

The use of EIAs, DFAs, and NAH-based assays are increasingly discouraged due to their

relatively low sensitivity compared to those of NAATs. In cases of repeated treatment failure,

isolation should be attempted and specimens should be forwarded to a specialized reference

laboratory.

NAATs could also be helpful for diagnosis of C. pneumoniae and C. psittaci infections.

However, commercial FDA-cleared assays are currently not available. Therefore, respiratory

specimens of patients with clinical suspicion of ornithosis or C. pneumoniae infection should

be directed to a specialized laboratory.

Interpretation of serological results is particularly challenging with chlamydial infections.

Serological testing may be helpful for diagnosis of human ornithosis, LGV, neonatal

pneumonia, and respiratory C. pneumoniaeinfections. A reliable serologic marker for chronic

or persistent chlamydial infection is not available. Especially inC. pneumoniae, there is poor

agreement between the presence of chlamydial antibody and direct markers of current

infection, such as culture or PCR (26). Single-point serology for diagnosis of C.

pneumoniae infection is discouraged, except when specific IgM antibodies are positive. Paired

sera should be tested in the same assay on the same day, and seroconversion or a fourfold

rise or fall in titer is diagnostic for a recent infection. Obviously, there is a general lack of

reliable and standardized assays for laboratory diagnosis of C. pneumoniae, and this

basically hampers the current understanding of the organism’s true prevalence and role in

respiratory infections as well as in extrapulmonary diseases.