Cumitech 19A: Laboratory Diagnosis of Chlamydia Trachomatis Infections

JANUARY

1999

Laboratory Diagnosis of Chlam ydia trachomatis Infections ANN WARFORD, MAX ELLENA M. PETERSON COORDINATING

CURT

CHERNESKY,

EDITOR

A. GLEAVES

Cumitech CUMULATIVE

TECHNIQUES

AND

AND PROCEDURES

IN CLINICAL

MICROBIOLOGY

Cumitech

1B

Blood Cultures III

Cumitech Cumitech Cumitech Cumitech

2B 3A 4A 5A

Laboratory Diagnosis of Urinary Tract Infections l November 1998 Quality Control and Quahty Assurance Practices in Climcal Microbiology l May 1990 Laboratory Diagnosis of Gonorrhea l Aprtll99.3 Practical Anaerobic Bacteriology l December 2 992 New Developments in Antimicrobial Agent Susceptibihty Testing: a Practical Guide l February 1993

Cumitech GA Cumitech 7A Cumitech 8 Cumitech 9 Cumitech Cumitech

10 11

Cumitech Cumitech

12A

Cumitech Cumitech Cumitech Cumitech

14A 15A 16A 17A

l

Aprrl 2 997

Laboratory Diagnosis of Lower Respiratory Tract Infections l September 2987 Detection of Microbial Antigens by Counterimmunoelectrophoresis l December Collection and Processmg of Bacteriological Specimens l August 1979

1978

Laboratory Diagnosis Practical Methods for August 1980 J.aboratory Diagnosis Laboratory Diagnosis

of Upper Respiratory Tract Infections l December 1979 Culture and Identification of Fungi in the Clinical Microbiology

Laboratory Laboratory Laboratory

Diagnosis Diagnosis Diagnosis

of Central Nervous System Infections l March 1993 of Viral Infections l August 2 994 of the Mycobacterioses l October 1994

Cumitech 18A Cumitech 19A Cumitech 20

J.aboratory Laboratory Laboratory Therapeutic

Diagnosis of Female Gemtal Tract Infections l june 3 99.3 Diagnosis of Hepatitis Viruses l November 1998 Diagnosis of Chlamydla trachomatzs Infections *January 1999 Drug Monitoring: Antimicrobial Agents l October 2984

Cumitech 21 Cumitech 22 Cumitech 23

Laboratory Diagnosis of Viral Respiratory Disease l March 2 986 Immunoserology of Staphylococcal Disease l August 2 987 Infections of the Skin and Subcutaneous Tissues l ]une 2 988

Cumitech Cumitech Cumitech Cumitech

Rapid Detection of Viruses by Immunofluorescence l August 2 988 Current Concepts and Approaches to Antimicrobial Agent Susceptibihty Testing l December 2988 Laboratory Diagnosis of Viral Infections Producing Enteritis l September 2989 Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Compamon and Laboratory Ammals l February 1996 Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Compamon and Laboratory Animals l February 1996 Laboratory Safety m Chrncal Microbiology l jury 2 996

13A

24 25 26 27

Cumitech 28 Cumitech 29 Cumitech 30 Cumitech 3 1

l

l

l

Aprzl 1992 September 2994

Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract l September 1996 Verification and Validation of Procedures m the Clinical Microbiology Laboratory l February 2 997

Cumitechs should be cited as follows, of Chlamydia trachomatis infections. Editorial Jamison.

of Bacterial Diarrhea of Ocular Infections

Laboratory

e.g.: Warford, A., M. Chernesky. Coordinating ed., C. A. Gleaves.

and E. M. Peterson. 1999. Cumitech American Society for Microbiology,

Board for ASM Cumitechs: Frederick S. Nolte, Cha;rman;Vickie Karen Krisher, Brenda McCurdy, Allan Truant, Alice S. Weissfeld,

The purpose of the Cumitech series is to provide consensus recommendations procedures for clinical microbiology laboratories which may lack the facilities given are not proposed as “standard” methods. Copyright 0 1999 American Society 1325 Massachusetts Avenue NW Washington, DC 20005-4171

for Microbiology

Baselski, Stephen

Lorraine Clarke, A. Young.

by the authors for fully evaluating

19A, Laboratory diagnosis Washington, D.C.

Curt A. Gleaves,

Janet Hindler,

Richard

as to appropriate state-of-the-art operating routine or new methods. The procedures

Laboratory Chlamydia

Diagnosis of trachomatis Infections Ann Warford

Diagnostic

Virology,

Stanford

University

Hospital,

Stanford,

California

94305

Max Chernesky McMaster

University Regional Virology and Chlamydiology Laboratory, St. Joseph’s Hospital, Hamilton, Ontario, Canada LSN 4A6

Ellena M. Peterson Division

of Medical Microbiology, California Irvine

Department of Pathology, University of Medical Center, Orange, California 92668

COORDINATING EDITOR: Curt A. Gleaves Infectious

Disease

Reference

Laboratory,

Providence

Portland Portland,

Medical Oregon

Center, 972 13

. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 2 Introduction Biology of C. trachomatis . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 2 Chlamydial Infections Infections in Men . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*....................................... Infections in Women . . . . ..*........................................................................................... Infections in Infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...*.... Trachoma . . . . . . . . . . . . . . . . ..*...................................................*........................................... LGV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*..........................................*...........................*......*...........

Sampling

for C. trachomatis

2 3 4 4 4

. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 4

Lower Genital Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.......... Collection of Lower Genital Tract Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*............... Other Collection Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.......................

5 6 6

Cell Culture . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 6 ............................................................................................. 8 Serology Antigen Detection in Clinical Specimens ................................................. 9 DFA (Slide) Assay .................................................................................................... EIA .........................................................................................................................

10 10

Nucleic

10

Acid Detection

DNA Hybridization Amplified Nucleic

Quality

in Clinical

Specimens

........................................

.................................................................................................... Acid Assays ...................................................................................

Assurance

for Chlamydia

Assays

..............................................

Cost-Benefit Considerations for Diagnostic Techniques or Screening for ..................................................................... C. trachomatis Infections Appendix: Chlamydia Transport Medium (CTM) and Chlamydia Isolation Medium ............................................................................................. References

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.......................................................

10 10

11 12 12 13

2

Warford

CUMITECH

et al.

C

umitech 19, published in 1984, was designed to assist microbiology laboratories in the diagnosis of infection due to chlamydiae and mycoplasmas. Over the last 14 years, several significant changes which have further enhanced our ability to diagnose infections caused by these agents have occurred. This Cumitech serves to update the discussion of laboratory diagnosis of Ch llamydia trachomatis presented in Cumitech 19. The diagnosis of Mycoplasma and the other two species of Chlamydia (C. psittaci and C. pneumoniae) will be discussed in future Cumitechs. BIOLOGY OF C. TRACHOMATIS Members of the genus Chlamydia are obligate intracellular bacteria responsible for a wide range of human and animal infections. The three species responsible for human infections are C. trachomatis, C. psittaci, and C. pneumoniae. These three species share many si.milarities but differ in host range, disease spectrum, and genetic composition (69). Human C. psittaci infections are mainly respiratory in nature and occur as a result of exposure to an infected avian species. C. pneumoniae, thought to be spread by the respiratory route, is responsible for infections that range from self-limiting pharyngitis to fatal pneumonia. C. trachomatis, the topic of this Cumitech, is a sexually transmitted pathogen which in the United States is responsible for approximately 4 million to 6 million new cases annually (19, 93). Members of the genus Chlamydia share common epitopes within the lipopolysaccharide (LPS), which are the basis for the genus-specific serological assays. The LPS is heatstable with a ketodeoxyoctanoic acid as the reactive moiety. The major outer membrane protein (MOMP), the predominant protein in the outer membrane of this organism, while similar in basic structure varies among and within the species (15, 106). It is primarily these antigenic differences in the MOMP that are exploited in species- and subspecies-specific microimmunofluorescent (MIF) assays. All rapid antigen detection tests are based on the detection of MOMP or LPS. DNA amplification techniques used in the diagnostic laboratory for C. trachomatis are also based on the detection of gene sequences of MOMP, the 7.5kDa plasmid that is present (with few exceptions) in all isolates of C. trachomatis, or rRNA VW C: trachomatis is divided into 15 main serovars, A through K, Ba, Ll, L2, and L3. The serovars are grouped based on serological relatedness into the B complex (B, Ba, D, E, Ll, L2, L3) and B-related complex (F, G) and the C complex (A, C, G, H, I, J)

19A

and C-related complex (K) (70, 113). These 15 serovars are also grouped into 3 biovars on the basis of biological differences: the lymphogranuloma venereum (LGV) biovar (Ll, L2, and L3), the trachoma biovar (A to K and Ba), and the mouse pneumonitis biovar. The LGV strains cause systemic illness and are more virulent in mice and more invasive in vitro, whereas the trachoma strains are associated with localized cervicitis, salpingitis, infertility, urethritis, pelvic inflammatory disease (PID), conjunctivitis, and trachoma (2, 77). The growth cycle of Chlamydia (Fig. l), as shown by in vitro studies, takes from 36 to 72 h (89,93). The infectious elementary body (EB) enters the host cell where, during the initial 8 to 12 h inside an inclusion, it reorganizes into the noninfectious reticulate body (RB). After th .is latent phase there is rapid growth of the inclusion, during which the RBs divide by binary fission. The RBs begin to reorganize back into EBs from 24 to 72 h, with formation of an inclusion containing approximately 100 to 1,000 EBs. The mature inclusion can occupy the majority of the host cell cytoplasm with eventual cell lysis and release of the EBs, which are capable of initiating another round of replication.

C. trachomatis is the most common bacterial sexually transmitted disease (STD) in the world ( 11,89). In the United States it is estimated that about 4 million to 6 million new chlamydial infections occur each year, and the worldwide estimate is more than 50 million infections per year (20). While the major impact of the disease caused by C. trachomatis is associated with the female reproductive tract, C. trachomatis does cause infections in men (36) and children. In both sexes, conjunctivitis, pharyngitis, and rectal infections have also been shown to be due to C. trachomatis. The majority of C. trachomatis infections are acquired during the teens and early twenties. Therefore, it is this high-risk population, as well as low-risk populations such as pregnant women, that is the target of many public health screening programs (l&20, 34). Infections in Men C. trachomatis is the most common cause of nongonococcal urethritis in men and may occur twice as frequently as gonorrhea (gonococcal urethritis) in some populations (89). Because up to 30% of men with gonorrhea may also have chlamydial infection, the Centers for Disease Control and Prevention (CDC) guidelines call for routine antichlamydial therapy for all men with gonorrhea (19). Postgonococcal urethritis is a persistent or recurrent urethritis that is the result of a dual infection in a person who is treated only for gonorrhea. Asymptomatic urethral infection

CUMITECH ‘l9A

Chhmydia

trachomatr’s Infections

3

EB enters host cell

EB reorganizes into RB 8-12 h

EBs

RBs divide by binary fission 12-36 h Inclusion grows containing RBs and EBs 36-72 h FIGURE 1.

Developmental

cycle of Chlamydia spp. Reprinted with permission

in young, sexually active males is relatively common. Someof these men will have an asymptomatic pyuria, while others show no signs of inflammation (103). Epididymitis is the most important complication of chlamydia urethritis in men. While acute epididymitis is most often caused by Escherichia coli in men over the age of 3.5, C. trachomatis causesone of every two casesin younger men in the United States (9). Chlamydiae can infect the epididymis singularly or in conjunction with such other pathogens as Neisseria gonorrhoeae. The diseasegenerally develops through the spread of the infection from the urethra through the spermatic cord. Reiter’s syndrome is a painful systemic illness in men. In addition to urethritis, clinical manifestations classically include conjunctivitis and arthritis. The causal relationship between C. trdchomatis and Reiter’s syndrome is not fully elucidated. However, it is known that one of two patients with untreated venereal Reiter’s syndrome has a chlamydial urethral infection and that chlamydial antibody responsesoccur in some of these patients (9,46, 5 1, 88).

from reference

78.

Infections in Women C. trachomatis causes more than 20% of cases of mucopurulent cervicitis, and asymptomatic casesoccur more frequently than symptomatic ones. The cervix may appear normal or reddened and friable, and there are few differences between chlamydial and gonococcal cervicitis. About one-half of women with gonococcal cervicitis have a concomitant chlamydial infection. C. trachomatis is one cause of the urethral syndrome in young women. Dysuria, urgency, increased urinary frequency, and suprapubic pain are the usual symptoms, and a concomitant chlamydial cervicitis may be present (10.5). Chlamydial organisms may ascend from the cervix to the lower abdomen, where they can cause PID (89). C. trachomatis has been isolated from the endometrium. The precise incidence of endometritis complicating cervicitis is not known, but it is estimated that about one-half of all women with cervicitis have endometritis. Ascending infection occurs quite early in the course and is often asymptomatic. Intermenstrual

4

Warford

et al.

uterine bleeding and pelvic pain may accompany endometritis (93). Upper abdominal pain is the predominant symptom of perihepatitis. Other symptoms include fever, tenderness, and spasm of the abdominal wall. Both C. trachomatis and N, gonorrhoeae can cause perihepatitis. This condition occurs almost exclusively in women in whom the infecting organisms have spread to the surface of the liver from inflamed fallopian tubes. Salpingitis involves inflammation of the fallopian tubes and is often associated with infection of the cervix and endometrium. C. trachomatis is detected in the fallopian tubes of approximately 1 of 10 women with chlamydia cervicitis. Salpingitis is a major cause of ectopic pregnancies and infertility. Ectopic pregnancies, which cause 10% of all maternal deaths, are 8 to 10 times more likely in women who have had salpingitis (19). Infertility occurs in as many as 20% of women who have had salpingitis. The risk of sterility resulting from salpingitis increased to 75% in women who have had three or more episodes. The severity of the disease, either by clinical presentation or by direct visualization of the inflammatory changes in the fallopian tubes, may also be associated with a poor prognosis for fertility. It has also been shown that over 50% of women who are infertile due to scarred fallopian tubes have serological evidence of prior chlamydial infection (118). Infections in Infants During vaginal delivery, up to two-thirds of neonates born to mothers with chlamydial genital infections will become infected. Inclusion conjunctivitis and pneumonia are the two principal manifestations of neonatal chlamydia infections. About 30% of infants born to women with genital tract chlamydial infections develop inclusion conjunctivitis. Onset generally occurs between 5 and 19 days after delivery, while gonococcal ophthalmia neonatorum usually appears within 5 days. Conjunctival scarring and pannus formation are unusual complications even without treatment. Topical treatment clears up the conjunctivitis but does not eradicate the organisms or prevent subsequent development of pneumonia (93). Chlamydial pneumonia occurs in 10 to 20% of infants born to infected mothers. C. trachomatis is responsible for 20 to 60% of all pneumonias during the first 6 months of life, making it a major cause of pneumonia in this age group (8). Most cases appear during the second or third month of life. Approximately half of the infants with chlamydial pneumonia also have conjunctivitis. Respiratory failure due to chlamydial pneumonia occurs rarely; the illness is generally of mild to moderate severity. A staccato cough and absence of fever are usual. Because wheezing and bronchospasm may be prominent, th .e infec-

CUMITECH

19A

tion may be misdiagnosed as bronchiolitis. Chlamydial pneumonia is characterized by interstitial, peribronchiolar, and perivascular infiltration by lymphocytes, plasma cells, eosinophils, and neutrophils. Mucus and inflammatory cells plug the airways, predisposing to atelectasis. When evaluated at seven to eight years of age, most children who experienced untreated chlamydial pneumonia as infants show signs of injured airways. Approximately one-third have asthma while another one-third have abnormal respiratory function tests. Trachoma Trachoma is the leading cause of preventable blindness in some underdeveloped countries. Transmission of the organism has been linked to flies, which act as passive vectors, dirty faces, and poor economic conditions (89). In devel .oped countries it is usually sexually tra nsmitted and responsible for a variety of conditions (l&89). Ocular infection can occur at an early age, and through repeated exposure, permanent conjunctival and cornea1 scarring can occur. The mechanism(s) that leads to scar formation is not clearly understood but is believed to be immunologically mediated. It is often difficult to recover C. trachomatis by cell culture from active cases of trachoma, and antigen detection and DNA hybridization methods have been more successful (42). This finding may be a reflection of the stage of the disease in which C. trach oma tis may be present in a persistent noninfectious state. LGV LGV is a sexually transmitted infection that occurs in low frequency in North America and Europe but is common in Africa, Asia, and South America (77,89). This infection is characterized by the appearance of a small painless ulcer or vesicle that heals spontaneously. The lesion usually occurs on the external genitalia, vaginal mucosa, or cervix. Two to six weeks after the lesion is healed, suppurative lymphadenopathy deve lops and may be accompanied by systemic symptoms of chills and fevers. The nodes involved depend on the location of the primary lesion. About 10 to 20% of untreated patients develop severe lymphatic obstruction and lymphedema. Chronic infection can result in draining sinuses and genital ulcerations. SAMPLING

FOR C. TRACH0/W2H‘W

Specimen collection and transport are very important for the accuracy of C. trachomatis diagnostic testing. For all diagnostic C. trachomatis tests, culture or nonculture, the sensitivity and specificity are directly related to the adequacy of the specimen collected (46,

CUMITECH Table 1.

19A

Chlamydia

Specimen

sites and test methods

Disease syndrome

Specimen

for laboratory

Bubo aspirate Endocervix swab or brush Female FVU Conjunctiva NP swabs, lower respiratory Endocervix Female FVU Fallopian tube biopsy Rectal lesion swab Endourethral swab Male FVU

Buboes (inguinal) Cervicitis Conjunctivitis Pneumonia (infant) PID

Proctitis Urethritis

diagnosis

siteW

of Ch/amy&

trachomatis

Infections

trachomatis

Assayi@

tract aspirates

5

Comment

Cell culture All methods NAA Cell culture, DNA probe, DFA, EIA Cell culture All methods NAA Cell culture, NAA Cell culture All methods NAA, EIA

IgM MIF serology

Obtained with proctoscope

a Specimens should be columnar epithelial cells. NP, nasopharynx. b NAA, nucleic acid amplification (PCR, LCR, etc.).

67,96). The choice of which specimen type to collect may well be governed by the test technology used in the laboratory to which the specimens will be sent. Collection and transport procedures have been well documented and described for C. trachomatis (78). Additionally, specimen test sites and test methods and collection of specimens for C. trachomatis are summarized in Tables 1 and 2, and they are briefly explained below. Lower Genital Tract To diagnose lower genital tract chlamydial infection, endocervical or endourethral swabs or urine should Table 2.

Collection

Specimen

be collected. (It should be noted that Chlamydia infects tissue, and epithelial cells must be collected, not pus or mucus.) If the laboratory is using a cell culture system to diagnose chlamydial infection, the specimen of choice will be an endocervical swab and, in some cases, endourethral swabs from women and men. Urine is not a suitable sample for cell culture. A number of different swab materials have been used for the collection of specimens, but problems have been reported with certain swab types (58, 63). Wooden stick swabs are unacceptable for culture because chemicals such as turpenes leach out of the wood into the transport medium. Calcium alginate swabs show

of specimens

type

Collection

method

Comments

Conjunctival

swab

Remove purulent exudate if present. tip over conjunctival surface.

Endocervical

swab

Remove and discard exocervical mucus and pus. Insert clean swab l-2 cm into endocervical swab and rotate for 10 s.

Collection of both cervical and urethral swabs may increase detection of positives.

Endourethral

swab

Express and discard exudate. Insert fine-shafted swab 2-4 cm into urethra, rotate swab gently 2-3 times and withdraw.

Patient should not urinate for 1 h or more prior to sample collection.

First-void urine

Collect into urine collection cup first IO-20 ml at any time (preferably 2 h following the last void).

Tested by EIA or NAA for men but only NAA for women

Fluids and aspirates from epididymis, fallopian tube(s), respiratory tract, and seminal fluid

Fluids and aspirates should be mixed with an equal volume of medium such as transport or cell culture medium.

Usually toxic to cell culture unless diluted

Nasopharyngeal

Insert flexible fine-shafted swab through nostril into nasopharynx and rotate.

Collection of both right and left nasopharyngeal swabs may increase yield.

Rectal swab

Insert swab into rectum and roll against mucosal surface to collect cellular material.

Ideally, cells are collected while visualizing rectal lesions. Avoid feces.

Tissue samples from endometrium, fallopian tube, lung

Place tissue sample into sterile screw-cap container and add transport medium to prevent drying.

Vaginal swab

Rotate swab against mucosal surface for 10 s.

swab

Rotate swab

In prepubertal

females,

only for culture

6

Warford

considerable lot-to-lot variation in toxicity to chlamydiae or cell culture. Ideally, swabs should be of cotton, dacron, or rayon material on a plastic or metal shaft and, if possible, pretested in the chlamydia detection system used to ensure the absence of toxicity or interference (78). If the laboratory is using an antigen detection test, enzyme immunoassay (EIA), or direct fluorescent antibodies (DFA), the choice of specimens for diagnosis is wider. The specimens of choice for women are still endocervical and endourethral swabs. The specimen choice for men is a urethral swab; first-void urine (FVU) may also be submitted if symptoms of urethritis are present (22,26, 84, 102). If chlamydia diagnosis is being performed with nucleic-acid-based technology, swabs may be submitted for testing using DNA probe hybridization methods. Swabs can also be used with amplified nucleic acid assays (that is, PCR, ligase chain reaction [LCR], and transcription-mediated amplification [TMA]). All of the amplification techniques allow the processing of FVU from both men and women to diagnose chlamydia infection of the lower genital and urinary tracts (23,27, 30,61). Collection

CUMITECH

et al.

of Lower Genital Tract Specimens

Collection of urethral or endocervical samples is diagramed in Fig. 2. For endocervical specimens, prior to the insertion of the actual collection swab the cervix is first cleaned of mucus and pus with a separate sometimes-larger cotton swab. The swab should be inserted in the cervix 1 to 2 cm deep just past the squamocolumnar junction, rotated for 5 to 10 s to strip off columnar epithelial cells, and removed. Cytologic brushes can be used as well; brushes collect more cells then swabs and are thought to improve isolation rates. However, brushes are more invasive and can induce bleeding, which may inhibit some nonculture methods (11). If the specimen is to be cultured, it should be placed into a transport medium conducive to survival of the organism such as 2SP or M4 (see the appendix and reference 87). For culture, the specimen needs to be transported at 4OC and inoculated within 24 to 72 h in the laboratory to permit a diagnosis (1, 58, 87). For urethral swabs from males, a dry small swab is placed 2 to 4 cm into the urethra and rotated before removal (Fig. 2). The patient should have urinated within the previous hour because urination will wash out infected columnar cells. For culture, the same requirements as stated above apply. Antigen and nucleic acid detection technology has less stringent transportation needs. Smears for DFA are prepared in the clinic, or the swabs are placed into specific transport containers, which are usually provided with the particular commercial kit. The optimal

19A

performances of these commercial kits (as is the case for cell culture) are dependent on optimal sample collection and laboratory processing and testing. Collection of FVU means the first 10 to 20 ml of a micturition. The urine is collected in a sterile screwcap jar for transport to the laboratory. The package insert for commercial products will have instructions for. processing the samples for testing. Other Collection

Sites

Samples from the lower genitourinary tract have low sensitivity and predictive value for diagnosis of upper tract infections. Biopsy of the upper tract (endometrial, fimbrial) may become useful for this purpose. Samples from the eye (Fig. 3) should be swabs or scrapings from the conjunctival surfaces. As was mentioned with endocervical samples, remove any pus or discharge before collecting the epithelial cells (11). For nasal swabs, rectal swabs, and tissue specimens, refer to Table 2. CELL CULTURE While cell culture has served for years as the “gold standard” for the detection of C. trachomatis, it is a procedure that is dependent on many variables (102). At present there is not an established standard or uniform procedure (i.e., a National Committee for Clinical Laboratory Standards [NCCLS] standard); therefore, the performance of the cell culture can vary from lab to lab. However, there are several very good protocols for the isolation of C. trachomatis in the literature (78,102). Many cell lines have been used for the isolation of C. trachomatis, but the most widely used are McCoy cells, BGMK cells, and HeLa 299 cells. Monolayers are made either on U-mm round coverslips in l-dram shell vials or in 24- or 96-well microtiter plates. Advantages of the individual shell vials are the lower risk of cross-contamination between specimens, ease of reading the stained monolayer, and reports that it is the most sensitive of the culture methods (91). The microtiter format saves time because there is less manipulation required of preparing the cell monolayer, inoculating the specimens, and staining the microtiter plate. In situations where there is a high culture volume this latter method has been frequently used (120). As stated for specimen sampling, the type of swab used to collect the specimen and the transport media can also influence the culture result. Specimens should be transported to the laboratory at 4OC after collection and cultured within 24 h for maximum recovery (1,58). If specimens cannot be cultured within 72 h of collection, they should be frozen at -7OOC. However, the detection of specimens with low numbers of viable C. trachomatis may be compromised when specimens

CUMITECH

Chlamydia

19A

trachomatis

Infections

7

PRELIMINARY l

l

l

Obtain cotton of Dacron swab for cleaning exocervix. Use large or small Dacron swab for specimen collection.

Remove excess mucus from exocervix.

l

l

Patient should not urinate 1 hour before sampling. Use small swab with narrow shaft.

Patient should not urinate 1 hour before sampling.

l

CLEAN AREA

I

I

l

INSERT DEVICE

Insert small swab 2-4 cm into

GATHER

l

Rotate 5-10 seconds.

l l

l l

Do not touch vaginal walls. Place swab in transport tube immediately.

SAMPLE

Gently rotate swab to dislodge cells Rest 2 seconds. l

Place swab in transport tube immediately.

l

For best results, transport and store specimens at 2-8°C.

SPECIMEN TRANSPORT

Collect 2 or more swabs. Place swabs in appropriate transport medium. Tighten caps securely.

SUBMIT SPECIMENS PROMPTLY TO MICROBIOLOGY. FIGURE 2. Specimen collection instructions for genitourinary samples for detection of chlamydiae. Specimen collection is critical in any diagnostic procedure. Several references on specimen collection are available. Abbreviated procedures are presented in the figure. The goal for chlamydial specimen collection is to obtain an adequate number of columnar or cuboidal epithelial cells. These are the cells that C. trachomafis infects.

are frozen or culture is delayed (54). Swabs generally are placed in 3 ml of holding medium for transport, but prior to culture the specimen should be vortexed vigorously for 2 min before being removed from the transport medium. Alternatively, sonication can also be used to remove the specimen from the swab and break up cellular material (116). Biopsy and tissue specimens should be homogenized in transport medium using a tissue grinder or stomacher (63). Before being inoculated, the cell monolayers should be checked for confluency and sterility. In some labo-

ratories, monolayers are pretreated with DEAE-dextran, which has been shown to increase the infectivity of certain serovars; however, the ability to increase the detection rate of positive clinical specimens using this method has not been evaluated (52, 102). The medium bathing the monolayers is removed and two shell vials are each inoculated with 0.1 to 0.2 ml of specimen, or approximately 10% of the specimen. When microtiter plates are used, two or three wells are inoculated with 0.1 ml each. With specimensthat can be toxic to the monolayers, especially tissues,

8

Warford et al.

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. Gently remove pus or discharge. . Swab inside of lower, then upper lid as pictured.

Collect infected epithelial cells (not pus) on swabs for viral and chlamydial detection.

. Use dacron swabs supplied with viral transport Do NOT inhibitory

medium. use calcium alginate or wood swabs as these are to viruses and chlamydiae.

. Collect multiple

swabs from both eyes (if affected) and place in transport medium. Collection of throat or nasopharyngeal swabs may increase detection.

. Submit specimens promptly FIGURE 3.

SDecimen collection

instructions

for conjunctival

biopsy, and semen samples, dilutions of the samples should also be inoculated. Shell vials are centrifuged for 1 h at 1,000 to 3,000 X g at temperatures which can range from room temperature to 37°C. The speed at which microtiter plates can be centrifuged is usually limited by the manufacturer of the plates or the centrifuge carriers. It is important that the centrifugation not be performed at refrigerated temperatures because at colder temperatures the EBs can attach to the host cell but are not internalized and thus will not form an inclusion (110). After centrifugation, the inoculum may be removed if the specimenstend to be toxic to the monolayers, or in the case of microtiter plates due to the limiting volume. Cell monolayers are then fed with media containing cycloheximide at a concentration that ranges from 0.5 to 1 pg/ml. Addition of eucaryotic cell inhibitors, such as cycloheximide, to the medium has been shown to result in larger and more numerous chlamydia inclusions (82, 83, 117). In addition to cycloheximide, several other methods have been described for inhibiting the host cell metabolism; however, cycloheximide is most commonly employed due to its widespread availability and ease of use. Inoculated monolayers are then incubated for 48 to 72 h at 37°C. If microtiter plates are used, 5% CO, is required for incubation; this is not necessarywith tightly capped shell vials. Blind passageof chlamydial cultures remains controversial (95). While reports of the yield of positive cultures detected with one serial passagevary, it appears that one passagecan, on the average, identify from 0 to 10% of additional cultures, with not much benefit from more than one passage.Cross-contamination, especially when microtiter plates are used, can be a problem with blind passage.When a culture is passed in shell vials or a well of a microtiter plate, it can be scraped directly into the culture fluid, and 0.1 to 0.2 ml can be transferred to a fresh monolayer and processed as described above. Alternatively, media can be removed and the monolayer can be scraped into fresh media and transferred to a new monolayer. Should a culture be contaminated, it can be treated with a mixture of vancomycin and streptomycin for

samples for detection

to Microbiology

Laboratory.

of chlamydiae.

30 min before being transferred to a new monolayer. The original sample can also be treated with antibiotics before a new culture is performed. After incubation, the medium is removed and the monolayers are fixed with either methanol or ethanol, depending on the staining reagents used. Three main stains have been used to detect chlamydial inclusions: iodine, Giemsa stain, and monoclonal antibodies tagged with a fluorescent dye or an enzyme such as horseradish peroxidase. Iodine and Giemsa stains, while inexpensive in comparison to monoclonal antibody stains, are less sensitive (104). The majority of experienced laboratories use fluorescent-tagged monocional antibodies for cell culture confirmation. The monoclonal antibodies that are commonly used are directed either to the genus-specific LPS component of the chlamydia organism or to a C. trachomatis-specific epitope on the major outer membrane protein. Monoclonal antibodies to either structure are similar in their ability to detect chlamydial inclusions. The entire surface of the stained coverslips or wells of the microtiter plates are inspected under a magnification of ~200 or x400. As seen in Fig. 1, chlamydial inclusions can take up most of the cytoplasm of the host cell, often appearing to displace the nucleus. However, inclusions can vary in size depending on the host cell, length of incubation, the presence of cycloheximide, and strain of chlamydia. In casesof sexual abuse, especially in children, cell culture is the assayrecommended by the CDC for the detection of C. trachomatis. This is mainly due to the problems associated with a false-positive result with the nonculture methods (18, 20, 26, 31, 39). However, new molecular tests with restriction fragment length polymorphism techniques may prove to be useful for identifying Chlamydiu serovar similarities or differences in these cases(14,119). SEROLOGY

In general, serological assaysare not recommended to aid in the diagnosis of lower chlamydial genital tract infections. This is becauseof the background titers in

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Ch/amyd!‘a

Table 3. Generally accepted standard for comparison8

sensitivities

for chlamydia

detection

methods

Avg sensitivity Specimen

using an expanded

Antigen detection

80-85(47-95jb

DNA probe 75-80(61-96)

75-80(38-94)

Infections

9 gold

(%)

Culture DFA

Cervix Urine (female) Urethra (male) Urine (male)

based on reports

trachomatr’s

EIA 75-80(60-96) 35-40(19-62) 70-75(43-87) 75-80(44-96)

Nucleic acid amplification (LCR, PCR, and TMA)

75-80(60-93) 70-75(70-89)

90-95(81-100) 85-95(77-100) 96-98 (95-l 00) 90-95(84-100)

B References: 3, 4, 5, IO, 23, 28, 30, 33, 38, 44, 47, 48, 50, 55, 64, 65, 68, 70, 75, 97, 101, 112. b Numbers in parentheses are the ranges of sensitivities reported in the literature.

the population due to infection or exposure to Chlamy&a. In addition, in some infected individuals the antibody response to a genital infection is modest or nonexistent ( 102). However, these assays are valuable epidemiology tools and can help in the diagnosis of systemic infections such as pneumonia, lymphogranuloma venereum, tubal factor infertility, or ectopic pregnancy, where titers are often elevated. The presence of immunoglobulin M (IgM) or a fourfold rise in IgG titer between acute- and convalescent-phase sera is evidence of an acute infection and may be useful for the diagnosis of neonatal chlamydia pneumonia (90, 94) There are serological assays that detect antibodies common to all members of the genus. The most common are complement fixation (CF), whole inclusion immunofluorescence (WIF), and recombinant enzyme-linked immunosorbent assay (rELISA), which are mainly based on the detection of LPS. The CF assay is a standardized test using LPS as the antigen. In the WIF test, infected cells, usually serovar L2 infected McCoy cells, are grown on multiwell slides, fixed, and used to titer patient sera (102). A positive reaction is one in which chlamydial inclusions within infected cells are clearly visible. The rELISAs (IgG, IgM, and IgA) have recently become commercially available (Medac, Hamburg, Germany) and have few published evaluations at this time. The MIF assay, which is species or subspecies specific, is based on the visualization of EBs or RBs instead of intact inclusions. Individual preparations of enriched formalin-fixed EBs are mixed with egg yolk sac fluid, spotted on slides, and then probed with patient sera followed by fluorescein conjugate against human immunoglobulin. The MIF assay can utilize all main serovars, and in some cases it has been modified and performed with a few representative serovars (113-l 15). In the original description of the MIF assay, a positive reaction was defined as the visualization of sharply delineated individual EBs. It is now believed that the ability of this test to distinguish among species and even within a species is based on the antigenic differences of the MOMP. The LPS

antigen is also apparent in this test, and inexperienced readers might mistake fluorescence due to LPS (fluorescent haze in the area of the EB inoculum) as a positive signal. This assay, which can measure responses to subclasses IgM, IgA, and IgG, is technically demanding, requiring a well-trained and experienced reader, and thus should be performed only in highly specialized laboratories (9 1). Immunoblotting and EIA for the determination of antibodies to the heat shock protein of C. trachomatis (hsp60) have been described and shown some predictive promise for TFI (111) and ectopic pregnancy (13).

ANTIGEN DETECTION SPECIMENS

IN CLINICAL

The antigen detection assays (nonculture) commercially available for the detection of C. trachomatis in clinical specimens utilize either monoclonal or polyclonal antibodies directed against chlamydia genusspecific LPS or specific C. trachomatis MOMPs as capture or detector reagents. One of several types of chemical labels is conjugated to the detector antibody or antibodies in the assay. Fluorescein isothiocyanate (FITC) is conjugated to the detector in the DFA method, whereas various enzymes may be used in the EIA or microparticle immunoassays (MEIA) in which 96-well plates, beads, or microparticles are used to trap the C. trachomatis antigens. Depending on the substrate used for the enzyme to act on, the signal measured in the EIAs is a color change or chemiluminescence. All of these nonculture antigen methods have similar sensitivities and specificities when compared to the optimal cell culture method as the gold standard. The sensitivity and specificity reported in evaluations of the DFA assay ranged from 5.5 to 96% overall and 82 to 99% in moderate prevalence populations (< 10% prevalence) (2, 26, 32, 46, 102). EIA methods have reported sensitivities and specificities of 44 to 92% overall and 92 to 98% in similar populations (Table 3) .

10

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et al.

DFA (Slide) Assay The DFA assay was the first commercially available nonculture chlamydia detection method to be cleared by the Food and Drug Administration (FDA). The DFA is a simple assay to perform with commercial test kits containing a monoclonal antibody-FITC conjugate. Exfoliated columnar epithelial cells on providerprepared slides are fixed, the monoclonal conjugate is applied to the smear, it is incubated for 30 min and the unbound conjugate is washed off. A thorough examination of the stained preparation at x250 to x400 by a trained microscopist with a good fluorescent microscope permits sensitive and specific identification of chlamydia EBs. The major advantages of this method are the ability to (i) microscopically evaluate the cytologic specimen submitted for adequate number of appropriate types of columnar epithelial cells, (ii) visualize the size and shape of the fluorescing organisms to ensure that the morphology is consistent with EBs of chlamydiae, and (iii) complete the test within 30 to 45 min. However, training and expertise are mandatory for optimal results; cross-reactions and nonspecific binding of immunoglobulin with other microorganisms, such as Staphylococcus aureus, do occur and false-positive results have been reported particularly with rectal specimens (84, 85). Also, the DFA assay is impractical for high volume testing because of the subjective nature of the assay and microscopist fatigue. EIA Commercially available EIAs possess multistep procedures requiring from 30 min to 4 h and are available in a variety of formats. The end products of enzyme activity can be viewed visually or with an automated spectrophotometer or chemiluminometer. If accurately performed, the reactions are proportional to the antigen in the specimen. The major advantage of these methods is automation, which permits high-volume batch testing, low direct kit costs, and detection of nonviable organisms. However, evaluation of specimen quality is not possible and poorly collected acellular samples may lead to erroneous results. Also, assay specificity can be low, which results in an unacceptable number of false positives in low prevalence populations. False positives may be due to poor technical performance of the assay, nonspecific binding of immunoglo bulins, or cross-reactions with other gram-negative organisms including Acinetobacter, members of the family Enterobacteriaceae, Gardnerella, neisseriae, and salmonellae (26, 37, 73, 8486) Numerous studies have shown improvement in the specificity of EIAs with the use of various confirmatory assays (7, 20, 21, 24, 26, 67). Two of the most common methods of confirming initial EIA positives

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are (i) use of a neutralizing antibody with repeat EIA testing to determine amount of specific neutralization in a blocking test and (ii) performance of a monoclonal DFA test on a centrifuged EIA sample and microscopic search for morphologically typical chlamydia EBs. The CDC has recommended the use of a confirmatory test, which detects a different antigenic determinant for all nonculture assays, performed in low prevalence populations or whenever false-positive results will adversely affect the patient tested (19). NUCLEIC ACID DETECTION SPECIMENS

IN CLINICAL

DNA Hybridization The first DNA-based assay was cleared by the FDA for commercial use in 1989. The assay used DNA probes to the rRNA of C. trachomatis (Gen-Probe). These probes, along with those to Neisseria golzorrhoeae, can be used to assay the same sample, an attractive feature for laboratories with a large volume of specimens and testing populations where both organisms are in high prevalence. This one specimen feature is also very attractive with physicians, and this test is being performed in both large and small laboratories with varied prevalence rates for both organisms. The assay is fairly labor-intensive, taking approximately 2 to 3 h to complete. The hybridization is measured by a luminometer that measures the intensity of chemiluminescence resulting from the hybridization of the specimen rRNA and the assay DNA probe that is tagged with an acridinium ester. Published evaluations of this assay vary as to the sensitivity (60 to 80%) and specificity (95 to 99%) depending on the reference method used (Table 3) (12, 49, 64, 79). A borderline zone may be established at individual laboratories in an attempt to increase the sensitivity of the assay; specimens falling into this zone are retested. While the specificity of the DNA probe test is high, a confirmatory assay is also available that can be used to determine the specificity of the assay. Briefly, the assay is a probe competition assay (PCA), and similar to an EIA blocking assay, PCA uses the same target for confirmation that the DNA probe test uses for detection. It is reported to confirm a high rate of DNA probe positive results (11, 50). Amplified

Nucleic Acid Assays

The FDA has cleared three commercial assays for the detection of C. trachomatis by three novel methodologies using enzymatic in vitro nucleic acid amplification: PCR, LCR, and TMA. The bacterial enzymes DNA Taq polymerase and DNA ligase are used in PCR and LCR, respectively, to amplify a short targeted segment of chlamydial plasmid DNA into millions of identical copies. These copies are generated by

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Chlamydia trachomatis

19A

repeated thermal cycling at temperatures that promote optimal annealing of nucleotide primers, and ligase or polymerase activity to create complementary nucleic acid segments followed by specific probe hybridization (29, 56). Chemical labeling of detector or capture probes permits calorimetric assay of specifically hybridized nucleic acid product in a manner similar to enzyme immunoassay (l&29,56, 74). The major advantages of the new PCR and LCR assays are sensitivity of detection, which exceeds optimal cell culture by 15 to 20%, and semiautomated detection methods for batch processing. Both assays have incorporated the use of chemicals to prevent nucleic acid product contamination of incoming specimens. However, technologist training and expertise are mandatory for accurate results because of the small volumes of reactants and exponential amplification of initial target DNA in the sample. Published sensitivities for the first-generation commercial PCR kit are 81 to lOO%, as shown in Table 3 (3,5, 6? 10, 17, 25, 28, 30, 38, 44, 47, 48, 55, 64, 65, 75, 101, 107-109). Commercially available amplification assays for the detection of C. trachomatis in clinical specimens include LCR (Abbott Diagnostics) and PCR (Roche Molecular Systems). In 1997, a third amplification assay, TMA, was cleared by the FDA. TMA (GenProbe) is based on isothermal amplification of the 23s ribosomal RNA target via DNA intermediates (27, 33, 76). All 3 assays have been cleared for the detection of C. trachomatis in endocervical and urine specimens from women and urethral and urine specimens from men. At this time, the FDA has cleared no other specimens (such as nasal, rectal, ocular, or tissue specimens) for use in these assays. Other amplification approaches such as Q beta replicase, nucleic acid sequence-based amplification, and strand displacement amplification are imminent. Plasmid DNA amplification has become the most popular approach because of its inherent theoretically higher sensitivity, which is due to the presence of 7 to 10 plasmids per C. trachomatis EB (60). Cryptic plasmid PCR has received extensive evaluation in female cervical swabs and male urine specimens. In both of these specimens, the sensitivity of the commercial assay has been reported to be approximately 20% higher than culture, antigen, or DNA hybridization (nonamplified) detection methods. Because this technology is more sensitive than the previous reference methods, several approaches for confirming the extra positives have been developed. This usually has been done by performing a second PCR whose primers are directed against a totally different gene or to a different fragment of the same gene (59,6 1,62). PCR rarely demonstrates a sensitivity of 100% because of inhibitors of amplification found in clinical specimens. The

Infections

11

specificities of all three assays (PCR, LCR, and TMA) are usually above 99% (Table 3). The rate of appearance of these yet-to-be-identified substances probably varies accord ing to specimen type and may also be different according to gender. Inhibitors of Taq polymerase have been found that disappear during storage and can be concentrated by centrifugation. A study recently published showed an association with urinary substances in female urine specimens that caused inhibition with all three amplification assays (57). LCR testing of female cervical specimens has ranged in sensitivity from 8 7 to 97%, and female FVU specimens have identified as many infected patients as cervical cultures (or more) in the limited number of studies reported thus far (4,23,53,66,97,112). Both PCR and LCR have proved to be effective when performed on male FVU. The tests have identified more infected men than culture or EIA, and the presence or absence of symptoms of urethritis has not affected the positivity rate. TMA has recently been shown to be just as sensitive and specific as PCR and LCR for the above specimens, with reported sensitivities of 8 8.5 to 93.5% in FVU samples and 100% in both female and male swab samples (27, 33, 76). The exquisite sensitivity of amplified nucleic acid assays provides a 20 to 30% increase in the numbers of infected patients identified (4, 23, 76, 97, 112). Cell culture was once considered the gold standard for detecting C. trachomatis. However, with the introduction of nucleic acid amplification techniques it is clear that cell culture sensitivity varies among laboratories and on the average is 80 to 85% (Table 3). For this reason, an expanded gold standard which should include both culture and a nucleic acid amplification method is evolving (16, 23, 68). It is very possible that, with effective protocols and amplification methods, using small amounts of male and female FVU (centrifuged and washed) may lead to screening programs in highrisk populations. QUALITY ASSAYS

ASSURANCE

FOR CHLAAMO/ll

The CDC has recommended three measures to ensure the quality of assays for laboratory diagnosis of C. trachomatis (20). Use of nonculture assays is recommended only for certain sites and populations, as shown in Table 1. Verification of all positive results by confirmatory testing is recommended for low prevalence populations, asymptomatic patients, and those patients for whom a false-positive result would have adverse effects. Also, periodic cytologic evaluation of specimen quality is recommended for non-DFA tests to ensure proper specimen collection. Cell culture is recommended for genital samples from children and all nasopharyngeal and rectal samples. The U.S. Federal Clinical Laboratory Improvement

12

Warford

et al.

Act of 1988 (CLIA-88) has specific requirements for licensed clinical laboratories performing moderateand high-complexity testing that includes diagnostic tests for C. trachomatis by culture, antigen detection, DNA hybridization, and amplified DNA testing (40, 41,45). These CLIA-88 requirements encompass the preanalytical or specimen collection and transport, analytical or laboratory specimen processing and testing, and the postanalytical processes of reporting and interpretation of laboratory test results. Standards have been established for patient specimen collection and transport; personnel qualification, training, and competency assessment; written procedures; facilities, equipment, and reagent maintenance; and test quality control and test records and reports. Additionally, even FDA-approved methods must be verified in each laboratory as meeting manufacturer-established performance specifications for accuracy, precision, and reportable range of patient test results, usually assessed by parallel testing between old and new methodologies with resolution of discordant results. All manufacturers’ instructions for test performance, reagent storage, and equipment preventive maintenance must be followed. Finally, all laboratories performing diagnostic testing must be enrolled in external proficiency test programs such as the College of American Pathologists surveys (32). In addition to the recommendations and requirements from CDC and CLIA-88 and test kit manufacturers, the NCCLS has issued an approved guideline for molecular diagnostic methods that lists specific guidelines for equipment utilized in molecular testing such as types and numbers of calibrations, functional checks, temperature verifications, and preventive maintenance schedules (32, 71). COST-BENEFIT CONSIDERATIONS FOR DIAGNOSTIC TESTING OR SCREENING FOR C. TRACHOMATIS INFECTIONS With such a high degree of asymptomatic infection, C. trachomatis is easily transmitted to sexual partners. Infants are infected at the time of vaginal delivery. This frequently results in extensive and expensive complications including hospitalization of women with PID or its sequelae, infertility, ectopic pregnancy, or chronic pelvic pain. As an alternative to testing patients with symptoms or contacts, several studies have reported that it is medically cost-effective to screen high prevalence populations, particularly sexually active teenagers and pregnant women (16, 20, 34, 35, 43, 72, 81, 92, 93, 98, 99). Use of an assay with a low direct cost of reagents to the laboratory may not be medically cost-effective to the patient, hospital, or third-party payer if it is not acceptably sensitive and specific. Some issues for consideration before selecting an assay for the local laboratory set-

CUMITECH

19A

ting include assay performance characteristics, prevalence of infection, types of samples and patients, and cost and benefit. Although somewhat higher in direct supply costs, amplified DNA assays offer increased sensitivity and specificity and a wider spectrum of acceptable patient and sample types, which may lower overall medical costs and improve patient outcomes (16) . APPENDIX: CHLAMYDlA (CTM) AND CHLAMYDIA This information

TRANSPORT MEDlUM lSOLATlON MEDIUM

is adapted from reference 78.

1. CTM: 2-W (0.2 M sucrose, 0.02 M phosphate) a. Dissolve the following ingredients separately in approximately 300 ml of deionized water. K,HPO 4*..*...*.....*...*...*....................** KH,PO 4.....................................*...... Sucrose . . . . . . . . . . . . . . . . . . . . . ...*.........**..*..***.

2.01 g 1.01 g 68.46 g

b. Combine the solutions and add deionized water up to a total of 1,000 ml. Adjust the pH to 7.2 to 7.4. 1;: Sterilize by Millipore filtration through a 0.2~pmpore-size filter. e. Aseptically add sterile antimicrobial solutions. The choice and concentration of antimicrobial agents may vary depending on the individual laboratory experience with contamination. Antimicrobial agents (and final concentration) frequently used include gentamitin (50 kg/ml), vancomycin (100 pg/ml), and amphotericin B (2.5 kg/ml). Nystatin (25 U/ml) may be used in place of amphotericin B; nystatin is not soluble in aqueous form, and care must be taken to keep this material in suspension while dispensing. Do not inelude penicillin in CTM. f. Dispense l- to 2-ml volumes into sterile screw-cap vials, Three or four sterile S-mm glass beads added to each vial will facilitate disruption of cells prior to inoculation. Glass beads may be sterilized by autoclaving or by dry heat (160°C for 60 min). Store at -20°C for up to 6 months or at 4°C for 1 week. h. Note: Some laboratories include 3 to 10% heat-inactivated fetal bovine serum in CTM. This may help retain viability of the cells during freezing. However, each lot of serum must be shown to be free of chlamydial inhibitors and antibodies. 2. Chlamydial

Isolation Medium

a. Aseptically tions:

combine the following

sterile stock solu-

Eagle’s minimal essential medium (EMEM), 10X, in Earle’s salts without L-glutamine.. . . . . . . . . . . . . . . . . . . . . . .50 ml L-Glutamine (200 mM) . . . . . . . . . . . . . . . . . . . . . . .5 ml Fetal bovine serum (heat inactivated [56”C, 30 min]) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50 ml Cycloheximide ( 100 X stock). . . . . . . . . . . . . . . .5 ml Gentamicin, sufficient volume to yield.. ..50 pg/ml HEPES (N-Zhydroxyethylpiperazine-N’-2-ethane-

CUMITECH

Chlamydia trachomatis Infections

19A

sulfonic acid) may be included as an additional buffer to yield a final concentration of 20 mM. Add sterile deionized water to a final volume of 500 ml. The medium should be reddish orange to cherry red, indicating a pH of 7.2 to 7.4. Dispense lOO- to SOO-ml volumes into sterile screwcap bottles. Store at 4°C for up to 2 weeks once glutamine and cycloheximide have been added. Without these components, the medium may be stored for up to 3 months at 4°C with the addition of glutamine and cycloheximide when the medium is used. 3. Cycloheximide

( 100 X stock)

a. Ingredients Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg 100 ml Deionized water ..*....................*.......... b. Dissolve cycloheximide in water, and sterilize by filtration through a O.+m-pore-size filter. c. Aliquot in small volumes, and store frozen in the dark at -20°C for up to 1 year. d. The final concentration of cycloheximide in the chlamydial isolation medium is usually 1 @ml; however, this may vary with each new lot or batch of cycloheximide. Using a C. trachomatis stock culture, assay each new batch at several concentrations of cycloheximide ranging from 0.5 to 2.5 &ml. The optimal concentration is that which yields an inclusion number equal to or greater than the inclusion number obtained with the cycloheximide currently in use. e. Cycloheximide is toxic. Avoid inhalation or exposure to skin. 4. Streptomycin-Vancomycin

Stock (20 x )

A stock solution of 2 mg/ml can be prepared, aliquoted, and frozen at -2OOC. To use, add 0.1 ml of solution to each 2.0 ml of specimen to be decontaminated.

5 Commercial Sources for C. trachomatis Transport Media

N. S. Silverman, and J. M. Bondi. 1993. Clinical evaluation of a new polymerase chain reaction assay for detection of Chlamydia trachomatis in endocervical specimens. J. Clin. Microbial. 31:2648-2653.

4. Bassiri, M., H. Y. Hu, M. A. Domeika, J. Burczak, L. 0. Svensson, H. H. Lee, and I?. A. Mardh. 1995. Detection of Chlamydia trachomatis in urine specimens from women by ligase chain reaction. J. Clin. Microbial. 33:898 -900.

5. Bauwens, J. E., A. M. Clark, M. J. Loeffelholz, S. A. Herman, and W. E. Stamm. 1993. Diagnosis of Chlamydia trachomatis urethritis in men by polymerase chain reaction assay of first-catch urine. J. Clin. Microbial. 31:3013-3016.

6. Bauwens, J. E., A. M. Clark,

and W. E. Stamm. 1993. Diagnosis of Chlamydia trachomatis endocervital infections by a commercial polymerase chain reaction assay. J. Clin. Microbial. 31:3023-3027.

7. Beebe, J. L., M. I? Rau, and K. D. Albrecht.

1993. Confirmatory testing of Chlamydia trachomatis Syva enzyme immunoassay gray zone specimens by Syva direct fluorescent antibody test. Sex. Transm. Dis.

20:140-142. 8. Beem, M. O., and E. M. Saxon. 1977. Respiratory tract colonization and a distinctive pneumonia syndrome in infants infected with Cblamydia tracbomatis. AT. Engl.]. Med. 296:306-310.

9. Berger, R. E., E. R. Alexander,

G. D. Monda, J. Ansell, G. McCormick, and K. K. Holmes. 1978. Cblamydia tracbomatis as a cause of “iodiopathic” epididymitis. N. Engl. J. Med. 298:301-304.

10. Bianchi, A., C. Scieux, N. Brunat, D. Vexiau,

M. Kermanach, I? Pezin, M. Janier, P. Morel, and P. H. Lagrange. 1994. An evaluation of the PCR Amplicor Cblamydia tracbomatis in male urine and female urogenital specimens. Sex. Transm. Dis. 21:196-200.

diagnosis of Cblamydia tracbomatis Microbial. Rev. 10:160-184.

infections. Clin.

12. Blanding, J., L. Hirsch, N. Stranton, T. Wright,

Media

1. Aarnaes, S. A., E. M. Peterson, and L. M. de la Maza. 1984. The effect of media and temperature on the storage of Chlamydia trachomatis. Am. J. Clin. Pathol. 81:237-239. 2. Barnes, R. C. 1989. Laboratory diagnosis of human chlamydial infections. Clin. Microbial. Rev. 3: 119 -

136.

3. Bass, C. A., D. L. Jungkind,

11. Black, C. M. 1997. Current methods of laboratory

Multi-Microbe Media (M4) Transport Medium Micro Test, Inc. 4325 Business Park Court Lilburn, GA 30047 Phone: (800) 646-6678 Web site: http://micro.home.mindspring.com “Flex Trans” Viral and Chlamydia Transport Intracel (Bartels) 2005 NW Sammamish Road, Suite 107 Issaquah, WA 98027 Phone: (800) 227-8357

13

S. Aarnaes, L. de la Maza, and E. M. Peterson. 1993. Comparison of the Clearview Chlamydia, the PACE 2 and culture for detection of Cblamydia tracbomatis in a low prevalence population. J. Clin. Microbiol. 3 1: 1622-1625.

13. Brunham, R. C., R. Peeling, I. MacLean, M. L. Kosseion, and M. Paraskevas. 1992. Cblamydia tracbomatis associated ectopic pregnancy: serologic and histologic correlates. J. Infect. Dis. 165:1076-1081.

14. Brunham,

R., C. Yang, I. Maclean, J. Kimani, G. Maitha, and F. Plummer. 1994. Cblamydia tracbomatis from individuals in a sexually transmitted disease core group exhibit frequent sequence variation in the MOMP (ompl) gene. J. Clin. Invest. 94:458 -

463. 15. Caldwell,

H. D., and J. Schachter. 1992. Antigenic

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