44

Nucleic Acid
Amplification Tests
for Detection
of Chlamydia
trachomatis and
Neisseria
gonorrhoeae
AMY L. LEBER, GERRI S. HALL, AND
WILLIAM D. LEBAR

COORDINATING EDITOR
SUSAN E. SHARP

Cumitech
CUMULATIVE TECHNIQUES AND PROCEDURES IN CLINICAL MICROBIOLOGY
Cumitech 1C Blood Cultures IV (2005) Cumitech 30A Selection and Use of Laboratory Procedures
Cumitech 2B Laboratory Diagnosis of Urinary Tract for Diagnosis of Parasitic Infections of the
Infections (1998) Gastrointestinal Tract (2003)
Cumitech 3B Quality Systems in the Clinical Cumitech 31 Verification and Validation of Procedures
Microbiology Laboratory (2005) in the Clinical Microbiology Laboratory
(1997)
Cumitech 7B Lower Respiratory Tract Infections (2004)
Cumitech 10A Laboratory Diagnosis of Upper Respiratory Cumitech 32 Laboratory Diagnosis of Zoonotic
Tract Infections (2006) Infections: Viral, Rickettsial, and Parasitic
Agents Obtained from Food Animals and
Cumitech 12A Laboratory Diagnosis of Bacterial Diarrhea
Wildlife (1999)
(1992)
Cumitech 13A Laboratory Diagnosis of Ocular Infections Cumitech 33 Laboratory Safety, Management, and
(1994) Diagnosis of Biological Agents Associated
with Bioterrorism (2000)
Cumitech 16A Laboratory Diagnosis of the
Mycobacterioses (1994) Cumitech 34 Laboratory Diagnosis of Mycoplasmal
Infections (2001)
Cumitech 18A Laboratory Diagnosis of Hepatitis Viruses
(1998) Cumitech 35 Postmortem Microbiology (2001)
Cumitech 19A Laboratory Diagnosis of Chlamydia Cumitech 36 Biosafety Considerations for Large-Scale
trachomatis Infections (1999) Production of Microorganisms (2002)
Cumitech 21 Laboratory Diagnosis of Viral Respiratory
Cumitech 37 Laboratory Diagnosis of Bacterial and
Disease (1986)
Fungal Infections Common to Humans,
Cumitech 23 Infections of the Skin and Subcutaneous Livestock, and Wildlife (2003)
Tissues (1988)
Cumitech 38 Human Cytomegalovirus (2003)
Cumitech 24 Rapid Detection of Viruses by
Immunofluorescence (1988) Cumitech 39 Competency Assessment in the Clinical
Microbiology Laboratory (2003)
Cumitech 26 Laboratory Diagnosis of Viral Infections
Producing Enteritis (1989) Cumitech 40 Packing and Shipping of Diagnostic
Cumitech 27 Laboratory Diagnosis of Zoonotic Specimens and Infectious Substances (2004)
Infections: Bacterial Infections Cumitech 41 Detection and Prevention of Clinical
Obtained from Companion and Laboratory Microbiology Laboratory-Associated Errors
Animals (1996) (2004)
Cumitech 28 Laboratory Diagnosis of Zoonotic Cumitech 42 Infections in Hemopoietic Stem Cell
Infections: Chlamydial, Fungal, Viral, and Transplant Recipients (2005)
Parasitic Infections Obtained
from Companion and Laboratory Animals Cumitech 43 Cystic Fibrosis Microbiology (2006)
(1996) Cumitech 44 Nucleic Acid Amplification Tests for
Cumitech 29 Laboratory Safety in Clinical Microbiology Detection of Chlamydia trachomatis and
(1996) Neisseria gonorrhoeae (2006)

Cumitechs should be cited as follows, e.g.: Leber, A. L., G. S. Hall, and W. D. LeBar. 2006. Cumitech 44, Nucleic Acid Amplification Tests for Detection of
Chlamydia trachomatis and Neisseria gonorrhoeae. Coordinating ed., S. E. Sharp. ASM Press, Washington, D.C.
Editorial Board for ASM Cumitechs: Alice S. Weissfeld, Chair; Maria D. Appleman, Vickie Baselski, B. Kay Buchanan, Mitchell l. Burken, Roberta Carey,
Linda Cook, Lynne Garcia, Susan L. Mottice, Michael Saubolle, David L. Sewell, Daniel Shapiro, Susan E. Sharp, James W. Snyder, Allan Truant.
Effective as of January 2000, the purpose of the Cumitech series is to provide consensus recommendations regarding the judicious use of clinical micro-
biology and immunology laboratories and their role in patient care. Each Cumitech is written by a team of clinicians, laboratorians, and other interested
stakeholders to provide a broad overview of various aspects of infectious disease testing. These aspects include a discussion of relevant clinical consid-
erations; collection, transport, processing, and interpretive guidelines; the clinical utility of culture-based and non-culture-based methods and emerging
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 Nucleic Acid Amplification
Tests for Detection of
Chlamydia trachomatis
and Neisseria gonorrhoeae

Amy L. Leber
Quest Diagnostics, Nichols Institute, 33608 Ortega Highway,
San Juan Capistrano, CA 92690
Gerri S. Hall
Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195
William D. LeBar
Hospital Consolidated Laboratories-Providence Hospital,
23775 Northwestern Highway, Southfield, MI 48075

COORDINATING EDITOR: Susan E. Sharp

Kaiser Permanente, 13705 Airport Way, Portland, OR 97230

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Biology of the Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Chlamydia trachomatis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Neisseria gonorrhoeae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Epidemiology and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Chlamydia and Gonorrhea Infections in Women . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Chlamydia and Gonorrhea Infections in Men . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Other Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Review of Testing Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Enzyme Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
NAATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Specimen Types and Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Endocervical Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Inhibition with Endocervical Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Urethral Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Noninvasive Specimen Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Urine Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Liquid-Based Cytology Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Vaginal Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Nonurogenital Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Specimen Pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Review of Current NAATs for C. trachomatis and
N. gonorrhoeae Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Ligase Chain Reaction: LCx Probe System (Abbott Laboratories, Abbott Park, Ill.) . . . . . . . 10
PCR: COBAS AMPLICOR CT/NG Test (Roche Diagnostics Corporation, Indianapolis, Ind.) . 11
Transcription-Mediated Amplification: APTIMA (Gen-Probe Inc., San Diego, Calif.) . . . . . . 16
Strand Displacement Amplification: BD ProbeTecET (Becton Dickinson and Company,
Sparks, Md.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Hybrid Capture: Hybrid Capture 2 CT/GC DNA Tests (Digene Corporation,
Gaithersburg, Md.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
New Technologies and Laboratory-Developed Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Interpretation and Reporting of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Test of Cure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Children and Sexual Abuse or Assault Cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

1
2 Leber et al. CUMITECH 44

Reporting Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Kit Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
External Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Positivity Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Proficiency Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Contamination Concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Considerations for Laboratories Performing NAATs . . . . . . . . . . . . . . . . . . . 33
Verification of a Test Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Guidelines for Screening and Testing for Infection . . . . . . . . . . . . . . . . . . . . 37
Symptomatic Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Asymptomatic Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Laboratory Guidelines for Screening Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Effectiveness of Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Coding and Reimbursement Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

INTRODUCTION BIOLOGY OF THE ORGANISMS

All the world loves loving, but it entails the bother-
some inconveniences of pregnancy and disease.
Richard Gordon, The Alarming History of Medicine
(St. Martin’s Press, New York, N.Y., p. 143, 1993)
Chlamydia trachomatis
Organisms in the genus Chlamydia are gram-negative,
nonmotile, obligate intracellular parasites of eukary-
otic cells. The chlamydiae do not carry out energy
metabolism and are entirely dependent on the host
cell to supply them with ATP. C. trachomatis is clas-

S
exually transmitted diseases impact millions of
people worldwide, and Chlamydia trachoma-
tis and Neisseria gonorrhoeae represent two of
the most common of these infections. Laboratory
diagnosis has evolved from culture, to direct detection
by staining and enzyme immunoassay, and then to
use of nucleic acid probes to detect both organisms.
With the advent of nucleic acid amplification tests
(NAATs) for the detection of C. trachomatis and N.
gonorrhoeae, the quality of testing for both organisms
has improved greatly, and these tests have arguably
become the standard of care for diagnosis. Amplified
techniques may present new challenges to laboratori-
ans. The exquisite sensitivity of these methods is both
a blessing and a curse. Changes to the work practices
and physical facility may be necessary, along with a
vigilant quality control program.
This Cumitech is intended as a guide for those
interested in either bringing C. trachomatis and N.
gonorrhoeae amplified testing into their laboratories
for the first time or improving the testing processes
already in place. All aspects of the testing process are
covered in this document, including a review of those
assays that are commercially available. The commer-
cial tests tend to change rapidly, but it is hoped that
much of the information provided will be applicable
for laboratories regardless of the particular NAAT
used.
sified in the order Chlamydiales and family Chlamy-
diaceae, which has recently been divided into two
genera, Chlamydia and Chlamydophila (71). Four
species are recognized, C. trachomatis, Chlamydia
muridarum, Chlamydia suis, and Chlamydophila
pneumoniae.
The chlamydiae have a distinctive dimorphic life
cycle that takes place within the cytoplasm of the
host cell. Stages in this cycle consist of a nonreplicat-
ing infectious particle called the elementary body and
a replicative form called the reticulate body found
within the cytoplasm of infected cells. The chlamy-
dial developmental cycle is initiated by the ingestion
of the infective elementary body by the host cell. The
elementary body is a metabolically inert, electron-
dense particle 0.2 to 0.6
m in diameter that contains
the genome and a cryptic plasmid (164). RNA poly-
merase, ribosomes, and ribosomal subunits are also
present in the elementary body (10). Following
attachment and endocytosis, the elementary body
differentiates into a noninfectious reticulate body
within a cytoplasmic vacuole in the infected host cell.
Transcription, protein synthesis, and DNA replica-
tion all take place within this structure. The reticu-
late body divides by binary fission, forming particles
that develop into the infectious elementary bodies.
After multiple division cycles, the newly developed
infectious elementary bodies are released by rupture
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
of the host cell. The chlamydial development cycle is
completed within 48 to 72 h (170).
All members of the Chlamydiaceae express a family-
specific lipolysaccharide epitope (25), and structural
integrity is maintained by three proteins, including a
40-kDa major outer membrane protein, a cysteine-
rich 60-kDa protein, and a low-molecular-weight
cysteine-rich lipoprotein (97, 174). It is the major
outer membrane protein complex that is responsible
for antigenic differences between species and sub-
species. Based on antigenic differences, C. trachomatis
is divided into 15 main serovars comprising two
human biovars, the lymphogranuloma venereum
(LGV) biovar (L1, L2, L3) and the trachoma biovar
(A–K, B, Ba) (220).
The Chlamydiae, in general, have a small genome.
In addition to chromosomal DNA, most strains of C.
trachomatis contain a 7,500-base pair plasmid com-
mon to all serovars (164). This plasmid is the target
used in many of the DNA-based amplified molecular
assays. Ribosomal RNA, specifically 23S rRNA, is
also used as a target for amplified nucleic acid hybri-
dization procedures. All 15 of the C. trachomatis
serovars may be detected by commercially available
DNA or RNA amplification methods (64, 125).
Neisseria gonorrhoeae
N. gonorrhoeae, the agent of gonorrhea, was first
described by Albert Neisser in 1879 (158). Members
of the genus Neisseria are gram-negative coccal
organisms that may occur in pairs or short chains.
The gonococcus has a tendency to occur in pairs, with
adjacent sides flattened. All members of the genus are
aerobic, produce cytochrome oxidase (except Neisse-
ria elongata), grow best at 35 to 37°C and do not
form spores (108). Capneic incubation and increased
humidity may stimulate growth, whereas some gono-
coccal strains have an obligate requirement for CO2.
Gonococci infect nonciliated columnar epithelial
cells. Once attached, they enter the cell by endocyto-
sis and multiply on the basement membrane. Gono-
coccal adherence is mediated by the antigenic char-
acteristics of surface proteins and pili. Pili are
hairlike appendages that extend from the surface of
the cell and are composed of protein subunits called
pilin (98). These structures are important in the ini-
tial attachment of the organism to the host cell and
also impart increased resistance to phagocytosis. Pili
can undergo both phase and antigenic variation, which
assists in the evasion of the immune system. Once
attached, other surface proteins including Opa (Pro-
tein II, P.II) assist in the adhesion of gonococci to
mucosal surfaces (193).
Protein I (P.I, Por) is a porin protein located in the
outer membrane of the gonococcus and is involved in
the penetration of the host cell. Organisms express
3
one of two forms of P.I—IA or IB—which differ anti-
genically from strain to strain. Antibodies to P.I ac-
quired during infection are bactercidal and prevent
repeated attacks of salpingitis with strains of the
same P.I type (17, 18, 175, 193).
All strains of N. gonorrhoeae contain an outer
membrane P.III (Rmp) found in complex with P.I and
bacterial lipooligosaccharide (LOS) (18). This LOS
antigen is released by autolysis of cells and mediates
the inflammatory response associated with the symp-
toms of gonorrhea (89). Gonococcal LOS is respon-
sible for mucosal damage in fallopian tube organ cul-
tures through local release of tumor necrosis factor
alpha. In strains that cause systemic infection, LOS
binds serum sialic acid, preventing complement acti-
vation and blocking antibodies from binding to sur-
face proteins (17, 175). Antibodies also may be pro-
duced against P.III that may react on the gonococcal
surface by blocking bactericidal antibodies directed
against LOS and P.I.
Iron acquisition is necessary to support bacterial
growth and invasion. Gonococci obtain host iron
complexes through the expression of transferrin
(Tbp1, Tbp2) and lactoferrin (Lbp) receptors in their
outer membrane. These receptors are induced under
low iron conditions and are able to extract iron from
transferrin, lactoferrin, heme, and hemoglobin. The
transferrin receptor has been shown to be required
for experimental urethral infection in male volun-
teers (187).
N. gonorrhoeae may also produce immunoglobu-
lin A (IgA) proteases (110). These proteases cleave
the heavy chain of the IgA1 at specific points within
the hinge region. Once cleaved, the IgA loses its
activity. Although products of IgA1 cleavage have
been found in genital secretions of females with gon-
orrhea, it has been shown that N. gonorrhoeae does
not require IgA1 protease activity to produce experi-
mental urethritis in males.
EPIDEMIOLOGY AND DISEASE
Chlamydia and gonorrhea are the two most com-
monly reported notifiable diseases in the United
States, with an estimated 3 million new C. tracho-
matis and 1 million new N. gonorrhoeae infections
annually (34, 70). In 2003, 877,478 chlamydial in-
fections and 335,104 cases of gonorrhea were re-
ported to the Centers for Disease Control and Pre-
vention (CDC) from all 50 states and the District of
Columbia. The case count corresponds to a rate of
304.3 cases of C. trachomatis infection per 100,000
population, a 5.1% increase over 2002. Increases
have been seen for greater than 10 years in reported
C. trachomatis infections in women and are likely
attributable to expanded screening programs and the
4 Leber et al.
use of more sensitive testing such as NAATs. The
case rate for N. gonorrhoeae in 2003 was 116.2 cases
per 100,000 population, the lowest ever reported and
a 10% decrease since 1999. The rate of coinfection is
less clear, but patients infected with N. gonorrhoeae
often are coinfected with C. trachomatis. Miller et al.
(149) found a prevalence of 0.3% coinfection in a
representative population of young adults in the
United States. Among persons with N. gonorrhoeae
infection, the prevalence of C. trachomatis infection
was very high at 70%, and those infected with C. tra-
chomatis had a prevalence of N. gonorrhoeae of
7.3%. These data support the recommendation that
patients treated for gonococcal infection should also
be treated routinely with a regimen effective against
uncomplicated genital C. trachomatis infection (35).
Infections with C. trachomatis and N. gonorrhoeae
are probably significantly underreported because
both infections are often asymptomatic (201). Also,
most data are collected from select populations that
may not reflect all susceptible individuals (34). In a
recent study of a nationally representative sample of
14,322 young adults aged 18 to 26 years in the United
States, the overall prevalence of C. trachomatis infec-
tion was 4.19%, and for N. gonorrhoeae infections,
prevalence was 0.43% (149). The authors state that
these rates are higher than those proposed by the
CDC.
Risk factors for infection with these organisms
include lower socioeconomic status, early sexual
activity, multiple sexual partners, non-white race,
illicit drug use, prostitution, and younger age (15 to
25 years) (149). Within a given population there may
be reservoirs of infection called “core group trans-
mitters” who are more likely to become infected and
transmit infection (173). Treatment focused on such
individuals would be a useful strategy for effective
disease control.
Transmission of C. trachomatis and N. gonor-
rhoeae is both horizontal and vertical. Sexual con-
tact, including vaginal, anal, and oral contact, is the
most common mode of transmission; infection
passed from mother to infant during birth also
occurs. C. trachomatis infections of the genital tract
are caused by biovars D–K. The organism targets the
squamocolumnar epithelial cells of the endocervix
and upper genital tract in women. The bacteria also
infect the conjunctiva, urethra, and rectum in men
and women. Infants are commonly infected in the
epithelial cells of the respiratory track, leading to
pneumonia. The squamocolumnar epithelial cells in
mucous membranes are the site of gonoccocal infec-
tions in adults. Mothers can infect infants during
delivery; the most common manifestation is gono-
coccal conjunctivitis (ophthalmia neonatorum), but
infection also can lead to arthritis and septicemia.
CUMITECH 44
Chlamydia and Gonorrhea Infections in Women
As stated previously, urogenital infection is often
asymptomatic or minimally symptomatic in women
infected with C. trachomatis or N. gonorrhoeae.
Symptomatic infection may result in abnormal vagi-
nal discharge, bleeding, abdominal pain, and dsyuria.
Symptoms usually appear within 1 to 3 weeks after
exposure for C. trachomatis and 10 days for N. gon-
orrhoeae. Infections can extend to the endometrium
and fallopian tubes, resulting in abdominal pain, low
back pain, nausea, fever, abnormal bleeding, and pain
during intercourse. Women generally suffer more seri-
ous complications than men, including pelvic inflam-
matory disease, infertility, and ectopic pregnancy. Up
to 40% of women with untreated urogenital chlamy-
dia infection develop pelvic inflammatory disease
(PID). Among women with PID, 18% will suffer from
chronic pain, 20% will be left infertile, and 9% will
have tubal pregnancies (223). Perihepatitis (Fitz-
Hugh–Curtis syndrome) can occur with both organ-
isms. It is more commonly associated with C. tra-
chomatis than N. gonorrhoeae, and evidence of
concomitant salpingitis may or may not be present.
In one study, a prior cervical C. trachomatis infection
was associated with an increased risk for develop-
ment of invasive cervical cancer (219). More data are
needed to make a conclusive link between these two
conditions.
Chlamydia and Gonorrhea Infections in Men
Urethral infection with C. trachomatis and N. gon-
orrhoeae can result in asymptomatic or symptomatic
infection in males. Chlamydial urethritis is more
likely to be asymptomatic than gonococcal urethritis:
nongonococcal urethritis and postgonococcal ure-
thritis. N. gonorrhoeae infections in men are often
symptomatic, with a shorter incubation period (aver-
age 4 days) compared to that of C. trachomatis (7 to
14 days). Men with symptomatic infection with
either organism may present with dysuria and ure-
thral discharge. Pain and swelling in the testicles are
uncommon. Complications of untreated infection
include epidimitis, prostatitis and proctitis, and sys-
temic disease. An additional concern is the finding
that human immunodeficiency virus transmission is
facilitated by infection with C. trachomatis and N.
gonorrhoeae (51, 75).
Other Diseases
Sexual exposure to C. trachomatis or N. gonor-
rhoeae can result in rectal infections in both men and
women. Women may have anal infections by exten-
sion from the cervix. Rectal infection in both men
and women may be symptomatic with discharge,
anal itching, soreness, bleeding, or tenesmus. Rectal
infection may also cause no symptoms. Oropharyn-
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
geal infections occur after N. gonorrhoeae exposure
and may result in sore throat but are most often
asymptomatic and resolve without intervention, and
transmission to another individual is uncommon
(187). Pharyngitis caused by C. trachomatis has been
reported and correlates with recent sexual contact
(113). Studies using serology testing to link C. tra-
chomatis infection to nonstreptococcal community-
acquired pharyngitis were most likely detecting cross-
reacting C. pneumoniae antibodies (189).
Conjunctivitis in adults occurs and is linked with
autoinoculation of the eye with genital secretions.
For C. trachomatis infection, the disease is very sim-
ilar to milder trachoma. It is seen in approximately
1% of cases with proven genital infections (171).
Gonococcal conjunctivitis tends to be more severe
and necessitates prompt therapy (187). Rarely, geni-
tal chlamydial infection can cause disseminated dis-
ease. In men and women, acute aseptic arthritis can
result from untreated urogenital infection. In men,
this reactive arthritis in combination with skin
lesions, conjunctivitis, and urethritis is called Reiter’s
syndrome. Approximately 1% of men presenting with
chlamydial urethritis have the arthritis syndrome, and
one-third of these have Reiter’s syndrome (117). Dis-
seminated gonococcal infections occur in 0.5 to 3% of
infected individuals; the most common manifestations
are septic arthritis and dermatitis (101). Among sexu-
ally active young adults, disseminated gonococcal
infection is a common cause of infective arthritis.
Birth of an infant to a mother who is infected with
C. trachomatis or N. gonorrhoeae can result in infec-
tion in the baby, with organisms being detectable
from the conjunctiva, anus, oropharynx, urethra, and
vagina. As noted above, C. trachomatis infection
most commonly manifests as pneumonia, but it is also
a common cause of neonatal conjunctivitis. Neonatal
infection with N. gonorrhoeae can result in conjunc-
tivitis and other less common but serious diseases.
Trachoma and Lymphogranuloma Venereum
Two additional diseases caused by C. trachomatis are
trachoma and LGV. These diseases have been re-
viewed extensively elsewhere and are covered briefly
in this document (137, 138, 186, 222). Trachoma is
the leading cause of preventable blindness in the world
and is caused by C. trachomatis biovars A, B, Ba, and
C. Infection with serovars L1, L2, and L3 causes LGV,
a sexually transmitted disease that is endemic in
Africa, Asia, and South America and sporadic else-
where. Both LGV and trachoma are uncommon in
the United States; however, a recent outbreak of LGV
occurred in The Netherlands. This finding, along
with cases in Europe, suggests that there may be an
increase in LGV cases in the United States, especially
among men who have sex with men (32).
5
REVIEW OF TESTING METHODS
Methods for the laboratory diagnosis of C. tracho-
matis and N. gonorrhoeae include culture and non-
culture techniques. Below are brief descriptions of
each of the available methods that have been or con-
tinue to be used. Summaries of the various assays and
their advantages and disadvantages for the detection
of C. trachomatis and N. gonorrhoeae infections are
listed in Tables 1 and 2.
Culture
Culture is available for both organisms and is still
considered the recommended method in certain situ-
ations. These situations include testing specimen
types other than urogenital; testing urogenital speci-
mens from infants and children or in cases of possi-
ble sexual abuse; and “test of cure” (in rare instances
when that is indicated). Culture for C. trachomatis
requires that well-collected endocervical or urethral
specimens be centrifuged onto a monolayer of tissue
culture cells, such as McCoy fibroblasts or human
foreskin cells. After cells are incubated for at least 2
days, specific fluorescent antibody stains are used to
detect the inclusions characteristic of cells infected by
C. trachomatis. In many laboratories, if the first stain
is negative, a blind passage of the tissue culture is
made into a new monolayer and the test is repeated.
This is done to enhance recovery of C. trachomatis.
This can be labor-intensive and, compared to the
NAATs that are discussed in this Cumitech, such cul-
tures can be as much as 30% less sensitive than
amplification (15). Culture for the recovery of N.
gonorrhoeae remains a very sensitive method for the
detection of this organism; however, the problems
with transportation inherent in culture methods
often make nonculture methods more convenient,
efficient, and attractive. One significant advantage of
N. gonorrhoeae cultures is the ability to perform
antimicrobial susceptibility testing, which is not pos-
sible with the nonculture techniques. Specificity of
culture for either C. trachomatis or N. gonorrhoeae
is considered to be 100%.
Staining
Staining directly for the presence of C. trachomatis
and N. gonorrhoeae is possible. Cytopathology can
be performed on Papanicolaou’s (Pap) smears or
other tissue specimens and examined for the presence
of inclusions typical of C. trachomatis; however, the
sensitivity of this method is less than that of other
available diagnostic methods. The sensitivity and
specificity of cytology for C. trachomatis in 159
women in a study from Ontario, Canada, were 13.3%
and 90.3%, respectively, although the negative pre-
dictive value was 90.9% (215). Another study
6 Leber et al. CUMITECH 44

Table 1. Advantages and disadvantages of assays for detection of C. trachomatis

Method Advantages Disadvantages

Culture Specificity; any specimen type can be cultured
Cytopathology Performed on Pap smears
Direct fluorescent Specificity; can be used to determine suitability of
antibody stain specimen; can be used in discrepancy testing
when two assays are compared
Enzyme immunoassay Batching of large volumes of specimens, especially
for screening; some success with urine samples
DNA probe Easy to perform and rapid turnaround time; can use
conjunctival specimens as well as endocervical
and urethral specimens
NAAT Sensitive and specific assays; batching of speci-
mens possible; automation possible
Serology Only useful in the diagnosis of LGV infections
Up to 30% less sensitive than NAAT; labor inten-
sive; slow turnaround time; cell lines need to
be purchased or maintained in the laboratory;
experienced technologist required for setup
and reading of cell cultures
Low sensitivity; can be low specificity; labor inten-
sive; need experienced individuals to read
adequately
Reduced sensitivity as compared to NAAT; time-
consuming since each must be read individu-
ally; only genital specimens
Sensitivity can be low; specificity can be less than
needed, unless blocking antibody tests are
done for confirmation; availability of reagents
may be a problem
Less sensitive than NAAT by as much as 30%
Concern about contamination by amplicon; can be
costly; in low-prevalence populations, need to
consider confirmation of positives; manual pro-
cessing of specimens can be time-consuming;
cannot be used in cases of sexual abuse and/or
in children; cannot be used for nongenitourinary
specimens
Positive results could indicate past or present
infection or infection with other species of
Chlamydia

Table 2. Advantages and disadvantages of assays for detection of N. gonorrhoeae

Method Advantages Disadvantages

Culture Ease of performance in most laboratories; can be If transportation of cultures cannot be optimized,
100% sensitive if transport can be controlled; organisms may be nonviable and limit recovery;
specificity great; any specimen can be cultured; requires overnight incubation or longer for
viable organism available for susceptibility detection
testing if needed
Gram stain Easy to perform; can be performed rapidly on Specificity not acceptable for making diagnosis in
most specimens; good sensitivity and specificity female endocervical specimens due to possible
in male urethral specimens presence of other diplococci
Direct fluorescent NAa Not readily available
antibody stain
Enzyme immunoassay NA NA
DNA probe Easy to perform and rapid turnaround time; can May be slightly less sensitive than culture
use endocervical and urethral specimens
NAAT Sensitive and specific assays; batching of speci- Concern about contamination by amplicon; can be
mens possible; automation possible costly; in low-prevalence populations, need to
consider confirmation of positives; manual pro-
cessing of specimens can be time-consuming;
cannot be used in cases of sexual abuse and/or
in children; cannot be used for nongenitourinary
specimens
Serology NA NA
a
NA, not available.
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
demonstrated 100% sensitivity for the Pap smear
versus PCR and an enzyme immunoassay in 125 preg-
nant females; however, the positive predictive value
for the smear in this study was only 75% (9). In the
same study, the sensitivity, specificity, and positive
and negative predictive values were 100% for PCR.
Use of the Gram stain for the detection of N. gonor-
rhoeae is relatively easy, and the finding of intracel-
lular gram-negative diplococci, consistent with N.
gonorrhoeae, can be used to make the diagnosis of
gonorrhea from a male urethral specimen. The sensi-
tivity of the Gram stain has been reported at 89 to
95% versus culture and as high as 99.6% versus the
PACE 2 probe assay in symptomatic male patients;
the specificity is
94% in this same population (114).
Because of problems with specificity in the female
endocervical specimens, Gram stains must be used in
conjunction with culture or other means of confir-
mation for diagnosis.
Direct fluorescent antibody (DFA) staining is avail-
able for C. trachomatis and N. gonorrhoeae, al-
though rarely used for the latter. DFA is the only avail-
able method that allows assessment of the quality of
the specimens that have been submitted, but it can be
a time-consuming, subjective, and labor-intensive
procedure. Sensitivity of the C. trachomatis DFA
assay in most studies is considered less than or equiv-
alent to that of culture (107, 129). Specificity is usu-
ally shown to be
97% (129). The primary use of
the DFA today is for discrepancy testing in studies in
which NAATs or other nonculture methods are being
compared to culture or to each other.
Enzyme Immunoassay
Enzyme immunoassays (EIAs) have been available
for C. trachomatis and N. gonorrhoeae and afford
the capability of batch testing for a less labor-
consuming method of detection. EIA for N. gonor-
rhoeae is no longer commercially available and is not
covered further. The Wampole MicroTrak (Wampole
Laboratories, Princeton, N.J.) for C. trachomatis
detection has been reported most widely in the liter-
ature. The sensitivity ranges from 61 to 98%, with
specificities from 96 to 100% (48). Wampole has
recently discontinued distribution of the MicroTrak
products; Abbott’s Chlamydiazyme (Abbott Labora-
tories, Abbott Park, Ill.) is no longer available in the
United States. The Bio-Rad Pathfinder Chlamydia
EIA (Bio-Rad Laboratories, Hercules, Calif.) for the
detection of C. trachomatis is available in the United
States, but references in the literature are limited.
Specificity of the EIA can be enhanced if one uses a
competitive assay with blocking antibody, for exam-
ple, to confirm a positive result. EIA is not performed
in many laboratories today since the advent of
NAATs. In a study comparing NAATs versus EIA for
7
the detection of C. trachomatis, Forward reported a
46% increase in positive results with a $18 price sav-
ings (Canadian dollars) per test when PCR was uti-
lized as compared to EIA (78).
Probe
The DNA probe (PACE 2; Gen-Probe Inc., San Diego,
Calif.) assay became the procedure used in many lab-
oratories for the detection of C. trachomatis and N.
gonorrhoeae prior to the advent of NAAT. Sensitivity
and specificity for the probes have been shown to be
slightly less than those of culture (78% sensitive ver-
sus culture), but turnaround times are much better
for C. trachomatis in particular (15, 127). For labo-
ratories where transport of specimens for culture is
problematic, the probe assay can be a good alterna-
tive to culture for N. gonorrhoeae. Laboratories
today could combine a NAAT for C. trachomatis, to
enhance recovery, and a probe for N. gonorrhoeae,
to maximize sensitivity, in a cost-effective manner
(40, 45).
NAATs
The commercially available NAATs are displayed in
Table 3. Many studies using NAATs have demon-
strated a sensitivity approaching 100%, with some
variation in specificity, depending on the population
being studied. One of the striking advantages of a
NAAT is the ability to use urine as a specimen.
Although there are limited studies in the literature
that demonstrate use of urine with EIAs, this speci-
men type can only be reliably tested if NAAT is
employed. In analyzing NAATs, or any assays for the
detection of C. trachomatis or N. gonorrhoeae, one
must be careful to note several factors such as the
prevalence of the analyte in the population studied,
the specimen type, whether endocervical or urethral
swabs or urine is used, and the gender of the patients.
All of these variables will affect the overall results
and their analysis. Likewise, to which assay the “new”
or test assay is being compared will strongly influ-
ence comparative results. In most studies today, an
“expanded gold standard” is being used as compara-
tors for NAATs, since no one test can be considered
as the sole “correct” answer. A recent publication by
Katz et al. cautions about the validity of N. gonor-
rhoeae test results when using a NAAT without tak-
ing into consideration disease prevalence and the pre-
dictive positive value of a positive test in the
population being tested (116).
Serology
Serologic assays for C. trachomatis are available in
the form of a complement fixation assay, a microim-
munofluorescence assay, and EIA; however, their util-
ity for the diagnosis of acute infection is very limited.
8 Leber et al.
Table 3. Chlamydia trachomatis and N. gonorrhoeae NAATs available and FDA cleared in the United States (2005)
NAAT Product Principle of assay
Amplicor, COBAS AMPLICOR PCR
CT/NG assays
BD ProbeTec ET CT/GC and Strand displacement amplifi-
CT assays cation (SDA)
APTIMA Combo 2, CT, and Transcription-mediated
GC assays amplification (TMA)
Hybrid Capture 2 CT/NG, CT, Hybrid Capture (HC)
and GC assays
a
FCU, first-catch urine.
b
Roche PCR not cleared for N. gonorrhoeae testing on female urine.
c
PreservCyt liquid Pap medium for collection of cervical cells. Clearance granted to Cytyc Corporation, Boxborough, Mass., not Roche Diagnostics.
Serologic cross-reactivity between Chlamydia species
has been well documented (15, 126). In addition,
antibodies are long-lived, and a positive result cannot
distinguish past or present exposure to the organism.
If one obtains an acute and convalescent titer and a
fourfold rise is present, this may indicate acute,
active infection with C. trachomatis, but it would
require 1 month for the rise to occur. This time
delay renders serology inappropriate for the diagno-
sis of acute infection and administration of appropri-
ate antimicrobials (46). The presence of IgM,
although rarely positive, is not helpful since it could
actually represent exposure to other species of
Chlamydia, specifically C. pneumoniae (15). Serol-
ogy is the method of choice, however, for diagnosis
of LGV, a specific infection with the L biovars of C.
trachomatis. Culture and other nonculture assays are
not available for the specific diagnosis of LGV.
Chlamydia serological assays have also been used in
the evaluation of infertility and other gynecologic
problems, but their accuracy is still under investiga-
tion (111, 151, 199).
SPECIMEN TYPES AND COLLECTION
Proper specimen collection is critical for all methods
used to detect C. trachomatis and N. gonorrhoeae
infections. Specimens for testing by the commercially
available NAATs must be obtained as directed by the
manufacturer in the package insert. Each of these
assays is approved for specific specimen types and has
its own assay-specific collection and transport device
(Table 3). Other specimen types or transport systems
must be validated by the user (50).
The performance characteristics of NAATs have
been shown to be related to specimen adequacy. The
presence of columnar epithelial cells has been associ-
ated with superior assay performance for NAAT and
other methods. Studies have demonstrated that the
sensitivity of commercially available assays is af-
CUMITECH 44
Manufacturer FDA-cleared sample types
Roche Diagnostics, Corp., Female, cervical swab,
Indianapolis, Ind. FCU,a,b PreservCyt: cMale,
urethral swab, FCU
Becton Dickinson, and Company, Female, cervical swab, FCU:
Sparks, Md. Male, urethral swab, FCU
Gen-Probe Inc., San Female, cervical swab,
Diego, Calif. vaginal swab, PreservCyt,
FCU: Male, urethral swab,
FCU
Digene Corporation, Female, cervical brush or
Gaithersburg, Md. swab
fected by the quality of the endocervical specimen;
variations in sensitivities range up to 10% for speci-
mens containing endocervical cells compared to inad-
equate specimens (12, 118, 119, 136, 221). The need
to obtain columnar cells to detect N. gonorrhoeae is
less critical than to detect C. trachomatis (33). The
CDC recommends routine or periodic assessment of
endocervical specimen quality (33).
Endocervical Specimens
The procedure for collecting female endocervical
swabs is well known. Briefly, a cleaning swab should
be used to remove secretions and mucus from the cer-
vical os. An endocervical swab or brush supplied by
the manufacturer should be inserted 1 to 2 cm into
the cervical canal and rotated for 15 to 30 s (33). The
swab should then be placed in the medium supplied
for transport.
Inhibition with Endocervical Specimens
The increased sensitivity of NAATs over culture is
well documented; however, early studies revealed
problems with inhibitors for certain types of amplifi-
cation reactions. Bauwens et al. (11) reported reduced
sensitivity of PCR with endocervical specimens due to
the presence of inhibitors. These inhibitors were con-
sidered labile as many of the repeat tests from falsely
negative PCR reactions became positive after storage.
Verkooyen et al. (211) described factors that were
associated with inhibition of PCR, including a higher
cervical pH (cervical mucosa pH of 7.5) and speci-
men processing on the day of collection. Unlike some
other publications, these authors noted that blood did
not affect PCR amplification.
Many techniques have been used to facilitate
removal of inhibitory substances. Transport of swabs
dry rather than in PCR transport medium resulted in
fewer false-negative specimens (120). Reduction or
removal of inhibitors has been accomplished by dilu-
tion of specimens 1:10, heat treatment (95°C for 10
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
min), a combination of heat treatment and 10-fold
dilution, freeze-thawing, and holding specimens at
4°C (211). These same techniques have been applied
to urine as well (42, 140).
Inhibition of amplification in specimens can be
detected by the use of internal amplification controls.
Currently such controls are available only for PCR
and strand displacement amplification (SDA) assays.
The use of these controls is optional and up to the dis-
cretion of each laboratory. When introducing NAAT
with either of the above methods, it is prudent to
include the amplification control until such time an
inhibitory rate with common specimen types can be
computed. Unpublished data from one of the authors’
own laboratories showed an inhibition rate of 0.37%
in over 52,000 endocervical and urethral specimens
tested by PCR (W. D. LeBar, unpublished data). The
use of an amplification control was found to be of
limited value for use with vaginal swab specimens
(56). Because of difficulties with urine specimen pre-
paration, amplification controls are recommended
when available.
Urethral Specimens
For collection of a male urethral swab, the patient
should not have urinated within the previous 1 h. If
the patient has a discharge, the exudates may be col-
lected and examined by Gram stain for organisms
typical of N. gonorrhoeae. A thin-wired swab is
introduced 2 to 4 cm into the urethra, rotated, with-
drawn, and placed into transport medium for testing.
Noninvasive Specimen Types
NAATs have proved to be more sensitive (with equiv-
alent specificity) than other nonculture technologies
used for the diagnosis of C. trachomatis and N. gon-
orrhoeae infections. This increased sensitivity has
extended the ability to test a variety of noninvasively
collected sample types such as first-catch urine (FCU)
or vaginal swabs from females. Noninvasive samples
are useful in screening at-risk populations and for
epidemiological studies.
Urine Specimens
The use of FCU as a specimen for chlamydial diagno-
sis was first reported by Caul et al. (30), using an EIA
compared to direct fluorescent monoclonal antibody.
Since that time, FCU from both males and females
has been found to be an appropriate specimen for
NAAT. The ease of collection of this sample type has
permitted the expansion of screening programs be-
yond the traditional clinic setting (3, 74, 81, 128,
144, 179, 205, 207). In males, NAATs using FCU
have been found to be sensitive and specific in both
symptomatic and asymptomatic patients and may be
9
considered the test of choice for the diagnosis of ure-
thral chlamydial infection (74, 109, 205, 207).
For females, urine NAATs can be affected by a vari-
ety of factors. The presence of phosphates, nitrates,
crystals, beta-human chorionic gonodotropin, and
hemoglobin is associated with inhibition of amplifi-
cation (41, 42, 140, 161). This inhibition is transient
and can be reduced by removing residual urine from
the pellet after processing and by storing the speci-
men overnight in the freezer or refrigerator (41). The
volume of urine processed has also been shown to
have an effect on NAAT sensitivity. Each NAAT has
specific FCU volume requirements for testing. Mon-
cada et al. (152) recently demonstrated that the sen-
sitivity of NAAT varied drastically when specimen
volumes ranging from 20 to 90 ml of urine were
collected and analyzed. Verkooyen et al. (212) re-
ported the results of a study designed to assess the
abilities of laboratories to detect C. trachomatis in a
panel of urine samples by various NAATs. Although
the specificity for this group of specimens was high,
sensitivity problems occurred frequently, underscor-
ing the need to adhere to all manufacturers’ stated
protocols. A recent review has summarized the Eng-
lish language literature describing chlamydial and
gonococcal testing with self-collected urine or vagi-
nal specimens outside of clinic settings (77).
Liquid-Based Cytology Specimens
The introduction of liquid-based cytology has made
possible the screening of residual materials for
chlamydial and gonococcal infection. Using cervical
scrapings collected for cervical cytology, Lentrichia et
al. (130) demonstrated the feasibility of detecting
chlamydia from a fluid-based sample. Liquid-based
cytology samples collected in Autocyte preservative
fluid from women at high risk for chlamydia infec-
tion and tested by a modified LCx were compared to
the standard Abbott LCx method (Abbott Laborato-
ries, Abbott Park, Ill.). There was agreement for 588
of 590 specimens (6). The collection of endocervical
scrapings into PreservCyt (Cytyc Corporation, Box-
borough, Mass.) has been shown to be an acceptable
process for sample collection for different NAATs
(14, 230). Chlamydial DNA was found to be stable
in PreservCyt for 6 weeks at room temperature (14).
Koumans et al. (124) examined the agreement be-
tween specimens collected in PreservCyt and multiple
NAATs for the detection of C. trachomatis and N.
gonorrhoeae. In this study, test performances were
similar for LCx-PreservCyt, LCx-cervix, and LCx-
urine, with sensitivities from 93 to 99% for C. tra-
chomatis and 81 to 83% for N. gonorrhoeae and
specificities from 95.5 to 99% for C. trachomatis and
99.1 to 99.6% for N. gonorrhoeae using an infected
patient PCR-based standard. At present, collection of
10 Leber et al.
liquid-based cytology samples in PreservCyt fluid is
approved by the U.S. Food and Drug Administration
(FDA) for testing for chlamydia and gonococcal
infection on the COBAS AMPLICOR (Roche Diag-
nostics, Indianapolis, Ind.) and APTIMA Combo 2
(Gen-Probe Inc., San Diego, Calif.).
Vaginal Specimens
A question could be posed as to what is the best spec-
imen to use to detect C. trachomatis and N. gonor-
rhoeae infection in women. After reports of the suc-
cessful application of urine for testing, there were a
number of reports that vaginal or vulvar swabs may be
useful as noninvasively collected samples for clinical
and epidemiological studies (194, 196, 226). Numer-
ous studies have demonstrated that self-collected
vaginal swabs, introital swabs, and tampons are ap-
propriate for diagnosis of genital tract infection, per-
form as well as physician-collected endocervical
swabs, and are suitable for routine screening of asymp-
tomatic women (4, 23, 28, 38, 56, 88, 178, 192, 227).
These noninvasive specimen types have been shown
to be preferable to patients (105, 169, 185, 210). In
January 2004, the Gen-Probe APTIMA vaginal swab
specimen collection kit received FDA clearance. This
collection device allows sample collection by the
physician or by the patient, under medical supervi-
sion, for C. trachomatis and N. gonorrhoeae testing.
Nonurogenital Sites
Collection of samples from ocular, pharyngeal, and
rectal sites follows standard protocols. Currently, no
NAAT is FDA cleared for performance with speci-
mens from any of the above sites. Conjunctival
swabs may be indicated for conjunctivitis in adults
and in newborns or infants with neonatal conjunc-
tivitis or pneumonia consistent with C. trachomatis
infection (33). Pharyngeal swabs may be indicated
from patients exposed during oral sex, neonates with
conjunctivitis or pneumonia consistent with C. tra-
chomatis infection, or in cases of possible sexual
abuse (33). Rectal swabs may be indicated in patients
with a history of receptive anal intercourse or procti-
tis or in cases of possible sexual abuse (33).
PCR has performed comparably to culture for
detecting C. trachomatis in conjunctival and pharyn-
geal specimens from infants with conjunctivitis (69,
96, 197). NAATs have been evaluated for diagnosis
of chlamydial and gonococcal infection in rectal and
pharyngeal specimens. In general, these tests have
performed with comparable sensitivity to culture;
however, there have been few published studies (85,
132, 133, 162, 190, 229). These uses are not FDA
cleared, and consideration needs to be given to poten-
tial cross-reacting species of Neisseria that may pro-
duce false-positive results.
CUMITECH 44
Specimen Pooling
Pooling of specimens has been investigated in public
health laboratories and in facilities processing large
numbers of specimens. Depending on the prevalence
of infection, considerable reagent cost-savings and
technologist time-savings may be achieved by the
pooling when compared to testing individual sam-
ples. This strategy employs screening samples in
groups. If any group is positive, individual specimens
within the group are then retested to find the positive
sample. Most of the chlamydia pooling studies have
been performed with urine specimens; however,
recent studies have been extended to vaginal and
endocervical samples as well (47, 59, 115, 155, 166).
Pooling has been shown to be a sensitive and specific
strategy to reduce the number of tests performed
without sacrificing accuracy. It should be noted that
pooling of specimens has not been FDA cleared for
any nucleic acid amplification method; therefore,
verification must be performed by any laboratory
implementing this strategy (50).
REVIEW OF CURRENT NAATS FOR
C. TRACHOMATIS AND
N. GONORRHOEAE DETECTION
Ligase Chain Reaction: LCx Probe System
(Abbott Laboratories, Abbott Park, Ill.)
The Abbott LCx C. trachomatis and N. gonorrhoeae
assays were removed from the market in 2003. Much
of the literature about other commercial NAATs used
these assays as a comparator; therefore, LCx will be
covered briefly. The LCx assays used ligase chain
reaction (LCR) amplification technology (Abbott
Laboratories, package insert, LCx Probe System
Chlamydia trachomatis Assay, List No. 9B11-91,
2000; Abbott Laboratories, package insert, LCx
Probe System Neisseria gonorrhoeae Assay, List No.
8A48-82; 2001).
The principle of LCR involves the release of single-
stranded DNA from the specimen in transport buffer
by using heat. Separate assays for C. trachomatis and
N. gonorrhoeae meant that individual reactions were
required to detect the two analytes. The prepared
sample is added to the LCR mixture consisting of
oligonucleotide probes, thermostable ligase, poly-
merase enzymes, and individual oligonucleotides.
The four probes are designed in pairs that hybridize
to single-stranded DNA target sequences in the
exposed single-stranded DNA in the sample prepara-
tion. The C. trachomatis assay targets a conserved
DNA sequence on a cryptic plasmid, and the N. gon-
orrhoeae assay targets a DNA sequence of the opa
gene. The polymerase enzyme then fills in the gap
between the hybridized probe pairs. The ligase
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
enzyme covalently joins the pairs of probes to form
an amplification product complementary to the tar-
get sequence. After multiple rounds of replication in
the thermocycler, the amplified product is detected
on the LCx analyzer by means of the immunoreactive
labeling of the probes in the amplicon detected by
microparticle enzyme immunoassay.
LCx C. trachomatis and N. gonorrhoeae assays
were FDA cleared for testing of female endocervical
and male urethral swabs and female and male FCU
specimens from symptomatic and asymptomatic indi-
viduals. The LCx probe system consisted of an LCx
analyzer for product detection, a thermocycler for
product amplification, centrifuges, and a dry bath for
specimen processing. Contamination control was
achieved by unidirectional workflow, surface decon-
tamination with 10% bleach, and chemical inactiva-
tion of amplicons with hydrogen peroxide plus a
metal chelator, resulting in a 107-fold reduction in
product. The LCx probe system contained no inter-
nal inhibition control for individual samples. A neg-
ative result may have been due to lack of target or
target level below the sensitivity of the assay (true
negative) or inhibitors of amplification present in the
specimen (false negative). Studies on urine specimens
for the detection of C. trachomatis demonstrated
that nitrates and phosphate may inhibit LCR (41,
140, 198). Inhibition could be removed by overnight
storage of samples at 4 or 70°C and by dilution
(140, 161, 198). In one study of C. trachomatis
detection in female urines, inhibition of LCR ampli-
fication was seen in approximately 3% of samples.
The authors concluded that most inhibition would
disappear during transport and that additional steps
to remove inhibition for every urine were not war-
ranted (161).
Future Development
Abbott Laboratories currently has no commercial
product available in the United States for the ampli-
fied detection of C. trachomatis and N. gonorrhoeae.
In collaboration with another company, artus GmbH,
they are marketing a commercial real-time PCR test
for detection of C. trachomatis. Known as the
RealArt C. trachomatis PCR, this test is CE-marked
and available throughout the world, except the
United States, Canada, and Japan. Abbott does not
intend to sell this product in the United States.
Abbott also has a fully automated sample prepa-
ration/real-time PCR system in development. It has
agreements with Tecan of Zurich, Switzerland, for
the development and commercialization of two auto-
mated sample preparation systems, the m1000 and
m2000sp. Introduction of C. trachomatis/N. gonor-
rhoeae duplex and C. trachomatis-only NAATs in the
United States is planned using the totally automated
11
m2000 sample preparation system linked to real-time
homogeneous PCR technology. PCR amplification
will be detected homogeneously with random-coiled
oligonucleotide probes using a real-time thermal
cycler/detection instrument codeveloped with Applied
Biosystems. (J. Yu, Abbott Laboratories, personal
communication).
PCR: COBAS AMPLICOR CT/NG Test (Roche
Diagnostics Corporation, Indianapolis, Ind.)
Principle of Procedure
Roche Molecular Systems first marketed a manual
assay (Roche AMPLICOR CT/NG Test) utilizing
PCR for the simultaneous detection of C. trachoma-
tis (CT) and N. gonorrhoeae (NG) in microwell plate
format. In the early 1990s, a semiautomated format,
the Roche COBAS AMPLICOR CT/NG test, was
introduced, and this has become one of the three
most widely used systems for the simultaneous detec-
tion of these two pathogens. Because the majority of
Roche users are performing the COBAS AMPLICOR
method, further discussion of the manual AMP-
LICOR is limited.
The method of amplification for the COBAS is
PCR (Roche Diagnostics, package insert, COBAS
AMPLICOR CT/NG Test for Chlamydia trachoma-
tis, Revision 1.0, 1999; Roche Diagnostics, package
insert, COBAS AMPLICOR CT/NG Test for Neisse-
ria gonorrhoeae, Revision 3, 1999). The COBAS test
detects both C. trachomatis and N. gonorrhoeae
simultaneously and includes an internal control to
detect the presence of any inhibitors in the specimen.
There are four major steps in the procedure: speci-
men processing; PCR of the target DNA; hybridiza-
tion of the amplified DNA to oligonucleotide probes;
and detection of the hybridized probe and amplicon.
For sample processing, specimens are treated with a
detergent solution to release the organism’s DNA. A
second detergent solution is then added to prepare
the lysed specimen for amplification. The processed
specimens are added to the amplification tubes con-
taining a reaction mixture with biotinylated primers,
thermostable Thermus aquaticus DNA polymerase
(Taq Pol), and excess deoxynucleotide triphosphates
(dNTPs). The reaction mixture is heated to denature
the double-stranded DNA, exposing the specific
primer target sequences allowing primers to bind,
and extending the target region by the action of Taq
Pol. The COBAS AMPLICOR analyzer automati-
cally repeats this process (thermocycling), thus dou-
bling the amount of amplicon DNA for each of 35
cycles. The target for C. trachomatis primers is a
region of 270 nucleotides within the cryptic plasmid
DNA and for N. gonorrhoeae, a region of 201 nucleo-
tides within the cryptic plasmid in the M.Ngo P11
gene. Following PCR amplification, the amplicons
12 Leber et al.
are chemically denatured to form single-stranded
DNA. The denatured specimen is transferred to
detection cups along with a suspension of magnetic
particles coated with an oligonucleotide probe spe-
cific for the target region (C. trachomatis, N. gonor-
rhoeae, or internal control). The magnetic particles
are washed to remove unbound material, avidin-
horseradish peroxidase conjugate is added, and the
particles are washed again. The substrate solution
containing hydrogen peroxide and 3,3,5,5-tetra-
methylbenzidine is added to each reaction. In the
presence of hydrogen peroxide, the particle-bound
horseradish peroxidase catalyzes the oxidation of
3,3,5,5-tetramethylbenzidine to form a colored
complex. The absorbance is then measured by the
analyzer at a wavelength of 660 nm.
Instrumentation
Performance of the COBAS assay requires a vortex
mixer, microcentrifuge, heating block, the COBAS
AMPLICOR analyzer, and printer. Specimen process-
ing occurs manually. The COBAS analyzer, a bench-
top system, automates the amplification and detec-
tion steps of the PCR testing process. It combines five
instruments into one (thermal cycler, automatic pipet-
tor, incubator, washer, and reader). The instrument
allows barcode data entry and has a 48-sample capac-
ity per run.
Sample Collection and Transport
The COBAS AMPLICOR CT test is FDA cleared for
testing female endocervical and male urethral swabs
from symptomatic and asymptomatic patients. The
COBAS AMPLICOR NG test is FDA cleared for test-
ing endocervical specimens, male urethral specimens
from symptomatic patients, and male urine speci-
mens. The COBAS AMPLICOR NG test is not
cleared by the FDA for testing on female urine or
urethral swabs from asymptomatic males.
Swabs are collected and transported in Chlamydia
culture transport medium such as 2SP, SPG, Bartels
ChlamTrans (Bartels, Inc.), or M4 Culture Transport
Medium (Microtest, Inc.). Various laboratories have
reported on the use of M4 and M4RT media for
transport (7). M4RT is more convenient for use than
M4 because it does not require refrigeration before
inoculation. Media lots must be qualified for use in
each individual laboratory (Roche Diagnostics, pack-
age insert, COBAS AMPLICOR CT/NG Test for
Chlamydia trachomatis, Revision 1.0, 1999; Roche
Diagnostics, package insert, COBAS AMPLICOR
CT/NG Test for Neisseria gonorrhoeae, Revision 3,
1999). Swabs may be transported at 18 to 25°C pro-
vided that the total time of storage and transport is
less than 1 h. Specimens should be refrigerated if test-
ing is delayed for more than 1 h. If testing is delayed
for longer times, swabs may be stored refrigerated
CUMITECH 44
for 7 days or frozen at 20°C or colder for up to 30
days. For urine specimens, the patient must not have
urinated during the previous 2 h. FCU (first 10 to 50
ml) is collected in a clean polypropylene container.
Urine specimens are stable for up to 24 h at 18 to
25°C. If urine will not be processed within 24 h, it
can be held refrigerated for 7 days or frozen at 20°C
or colder for up to 2 months. The presence of
inhibitor substances in specimens may prevent PCR
amplification. Swabs for the PCR assay are collected
from endocervix or urethra after the external surface
is cleansed to remove excess mucus and debris. If
much of the mucus remains in the specimen that is
being submitted to the laboratory or the specimen is
very bloody, PCR may be inhibited, causing a false-
negative result (200). Urine specimens have also been
shown to contain inhibitory substances that could
result in false-negative results (2, 140, 200).
As with other amplification assays, testing of spec-
imens outside the urogenital tract with COBAS
CT/NG PCR is not FDA cleared. There are some
publications in which specimen types, such as rectal
and pharyngeal, have performed adequately with
COBAS (85, 133). Vaginal samples, including those
that have been self-collected, have been reported to
provide results comparable to results with well-
collected endocervical samples (80, 178).
Liquid cytology transport media, such as ThinPrep
and SurePath, have been used to transport samples
for processing of Pap smears, human papilloma-virus
detection, and C. trachomatis and N. gonorrhoeae
PCR testing, all from the same sample (14, 124). In
2002, Cytyc Corporation announced that FDA clear-
ance was granted for C. trachomatis and N. gonor-
rhoeae testing directly from the ThinPrep Pap Test
collection vial using COBAS AMPLICOR PCR.
Approval was issued to Cytyc, not Roche Diagnos-
tics. Instructions for testing are not provided in the
AMPLICOR product insert but must be obtained
from Cytyc.
Inhibition and Contamination Controls
There is an optional C. trachomatis and N. gonor-
rhoeae internal control (IC), run simultaneously with
each specimen that provides a means of identifying
any substances in the specimens that could inhibit
PCR amplification. The IC is a recombinant plasmid
DNA containing primer regions identical to those of
the C. trachomatis target sequence, a randomized
internal sequence of similar length and base composi-
tion as the N. gonorrhoeae and C. trachomatis target
sequences, and a unique probe-binding region that
differentiates it from the target amplicon. The IC is
introduced into each amplification reaction to be
coamplified with target DNA from the clinical speci-
men. The amount of the IC added is set at 20 copies
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
per reaction; this acts to monitor for adequate ampli-
fication of the lower level of sensitivity of the assay
to prevent the reporting of a false-negative result. If
the IC is negative, the negative results of the clinical
samples should be questioned; repeat testing of the
sample after a freeze-thaw or dilution procedure may
eliminate the inhibition and allow positive IC and
correct results with the clinical samples. As men-
tioned above, use of the IC is optional. The control is
amplified in each reaction, but the user decides
whether the product is detected and reported.
In the presence of large amounts of target DNA,
the PCR reactants in the COBAS CT/NG test may be
consumed, leading to failure of the IC to amplify. A
single set of primer oligonucleotides is used to
amplify both the IC and target C. trachomatis DNA.
All three targets (C. trachomatis, N. gonorrhoeae,
and IC) also draw from a common pool of dNTPs
and polymerase. Thus, IC amplification may be sup-
pressed due to competition in the samples containing
large amounts of target C. trachomatis DNA. One
group of investigators was able to demonstrate this
suppression of the IC signal by testing samples of
known increasing amounts of target C. trachomatis
DNA with IC included. About 5,000 to 50,000 copies
per milliliter of sample were needed to decrease the
IC response; a concentration of 2.5  105 had to be
present to bring the IC down to background levels.
Compared to the IC, C. trachomatis and N. gonor-
rhoeae target DNAs were less sensitive to competi-
tion. Approximately 2.5  105 copies per test sample
of C. trachomatis or N. gonorrhoeae DNA were
required to produce a small decrease in the signal
generated by 25 copies of N. gonorrhoeae or C. tra-
chomatis DNA (172). A second advantage of use of
the IC in the COBAS AMPLICOR system is to mon-
itor for potential competition between the C. tra-
chomatis and N. gonorrhoeae target DNA if both are
present. If only one of the pathogens is determined to
be positive in a sample in which the IC is negative,
results for the second analyte may not be truly nega-
tive. Rather, there may have been a competition
between the two targets. This competition has been
shown to occur only when the target of one is
10-
fold more than that of the second (172).
To avoid contamination of specimens by amplicons,
AmpErase and deoxyuridine triphosphates (dUTPs)
are added to each reaction vessel in the COBAS pro-
cedure. AmpErase is an enzyme, uracil-N-glycosylase,
that recognizes and catalyzes the destruction of DNA
strands containing deoxyuridine, but not DNA con-
taining thymidine. Deoxyuridine is not contained in
naturally occurring DNA, so when it is added as one
of the dNTPs in the Master Mix reagent, amplicons
that contain deoxyuridine triphosphate will be de-
stroyed before amplification of target DNA. Amp-
13
Erase is inactive above 55°C, and hence cannot
destroy target amplicon during the thermal cycling
steps. Following amplification, any residual enzyme
is denatured by the addition of denaturation solution
by the COBAS analyzer. Unidirectional workflow
and environmental monitoring for amplicon contam-
ination are also recommended, as for any NAAT.
Result Interpretation
Results of the COBAS AMPLICOR are read colori-
metrically at 660 nm, and results are displayed as
optical density (OD) readings; a result
3.5 is con-
sidered positive and 2.0 is negative. The assay is
qualitative and no correlation can be made between
the magnitude of a positive absorbance signal and the
number of cells infected with C. trachomatis or the
number of CFU of N. gonorrhoeae. Results between
2.0 and 3.5 are in what is called an equivocal zone,
and the manufacturer recommends that with N. gon-
orrhoeae results in the equivocal zone, the assay
should be repeated two more times and the results of
the two of three or three of three same answers
should be reported. On the repeat samples, a positive

2.0 is accepted as positive. For C. trachomatis, the
manufacturer’s recommendation is to ask for a new
specimen if results are in the equivocal zone. If the
results are found to be inhibitory (i.e., if the IC does
not amplify), and results of the C. trachomatis and/or
N. gonorrhoeae assay are negative, a new specimen
should be requested or repeat testing of specimens
can be attempted. Elimination of inhibition has been
demonstrated after repeat testing subsequent to one
of these three methods: dilution of the samples, freez-
ing of the sample at 70°C, or refrigeration for at
least 24 h (140). If the inhibition is no longer seen,
results can be reported as determined without inhibi-
tion; if inhibition is still present after repeat testing
with one of the suggested methods, then equivocal
results should be reported and repeat specimens
requested.
Performance Characteristics
Sensitivity of the COBAS CT/NG test is excellent as
compared to that of other NAATs. There are vari-
ances between males and females and between geni-
tal swabs and urine samples, but overall a good sen-
sitivity has been seen in most studies. The prevalence
of C. trachomatis and N. gonorrhoeae in the popula-
tion studies accounts for differences between studies,
but again, that is true for all of the amplification
assays. Tables 4 and 5 summarize the performance
characteristics of the COBAS CT/NG tests.
In literature reviews comparing PCR to culture,
EIA, or probe assays, enhanced sensitivity and speci-
ficity for urogenital samples, especially for the detec-
tion of C. trachomatis, have been demonstrated.
Black (15) reviewed over 11 published reports on the
 14

Table 4. Recent studies evaluating the Roche COBAS AMPLICOR assay for detection of C. trachomatis

No. of Specimen Prevalence Reference Sensitivity Specificity Positive Negative

Reference subjects, sex types (%) methoda (%) (%) predictive predictive
value (%) value (%)
Leber et al.

Livengood et al., 654, Fa CX 9.2 Culture and PCR (manual) 93.3 99.7 99.3 96.4
2001 (135)
Van Der Pol et al., 1,098, F (asymptomatic) CX 7.7 Culture or alternate target PCR, DFA 97.6 99.5 94.3 99.8
2000 (207)
Van Der Pol et al., 1,138, F (symptomatic) CX 9.1 Culture or alternate target PCR, DFA 96.1 99.2 92.5 99.6
2000 (207)
Van Der Pol et al., 1,098, F (asymptomatic) FCU 8.1 Culture or alternate target PCR, DFA 93.3 99.6 95.4 99.4
2000 (207) 1,138, F (symptomatic) FCU 9.2 Culture or alternate target PCR, DFA 93.3 98.5 86.0 99.3
Van Der Pol et al., 710, M (asymptomatic) UR 11.7 Culture or alternate target PCR, DFA 98.8 98.7 91.1 99.8
2000 (207)
Van Der Pol et al., 1,230, M (symptomatic) UR 19.0 Culture or alternate target PCR, DFA 97.4 98.7 94.6 99.4
2000 (207)
Van Der Pol et al., 710, M (asymptomatic) FCU 12.5 Culture or alternate target PCR, DFA 92.1 98.4 89.1 98.9
2000 (207)
Van Der Pol et al., 1,230, M (symptomatic) FCU 20.6 Culture or alternate target PCR, DFA 92.5 98.5 94.0 98.1
2000 (207)
Pasternack et al., 442, F UR 11.3 Culture or LCR, alternate target PCR 94 99.2 92 99.2
1997 (165)
Puolakkainen et al., 447, F CX 6 Culture or LCR 78.6 98.8 81.5 98.6
1998 (168)
Puolakkainen et al., 442, F FCU 6.3 96.4 99.8 96.4 99.8
1998 (168)
Puolakkainen et al., 565, M UR 7.8 92.2 99.1 89.9 99.4
1998 (168)
Puolakkainen et al., 565, M FCU 8.8 95.9 99.4 94 99.6
1998 (168)
Vincelette et al., 2,010, F CX 2.4 Patient infected statusb (culture, 96.5 99.4 87.4 99.8
1999 (214) alternate target PCR on swab and urine)
Vincelette et al., 1,248, F FCU 2.4 Patient infected statusb (culture, alternate 95.1 99.8 92.9 99.8
1999 (214) target PCR on swab and urine)
Vincelette et al., 371, M UR 7.2a Patient infected statusb (culture, alternate 100 98.5 89.1 100
1999 (214) target PCR on swab and urine)
Vincelette et al., 254, M FCU 7.2 Patient infected statusb (culture, alternate 94.4 100 100 99.6
1999 (214) target PCR on swab and urine)
a
F, female; M, male, CX, endocervical swab; UR, urethral swab.
b
True positives are defined as two or more positive results with reference tests in any combination of method and sample type.
CUMITECH 44
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae 15

Negative predictive use of the manual AMPLICOR PCR for the detection
value (%) of C. trachomatis. She demonstrated that sensitivity
was 64 to 96% and specificity was
96% for PCR,
100
NA
NA
NA
NA
NA
NA
as compared to culture and other amplification assays
or combinations of the two. There are minimal re-
ported data on the use of the manual AMPLICOR
NG test. In the early 1990s, an automated format,
Positive predictive

the Roche COBAS AMPLICOR C. trachomatis/

N. gonorrhoeae PCR test was introduced, and today
value (%)

99.8
NA
NA
NA
NA
NA
NA

it is more widely used than the AMPLICOR CT/NG

test. Equivalence in performance was found when
COBAS was compared to the manual AMPLICOR
PCR, with a much easier throughput and workflow
than the manual AMPLICOR CT/NG assay (Tables
Specificity

4 and 5). Puolakkainen et al. (168) compared COBAS

99.5
99.8
99.0
99.0
99.9
99.9
(%)

100

to LCx for the detection of C. trachomatis and found

that in 97% of the samples tested, results were iden-
tical between the two “automated” methods.
Sensitivity

Specificity for C. trachomatis in the PCR assays is

96.4
79.6
90.5
99.5
78.6
96.3
(%)

99%; specificity for N. gonorrhoeae is good; how-

96

ever, there have been reported problems with cross-

reactions with species of Neisseria other than N. gon-
orrhoeae (72, 73, 131). The AMPLICOR assay
PCR
PCR
PCR
PCR
PCR
PCR
Table 5. Recent studies evaluating the Roche COBAS AMPLICOR assay for detection of N. gonorrhoeae

targets a sequence in the cytosine DNA methyltrans-

or alternate target
or alternate target
or alternate target
or alternate target
or alternate target
or alternate target
and PCR (manual)

ferase gene of N. gonorrhoeae. Similar sequences are

Comparison

present in some strains of “normal flora” Neisseria

methoda

species, such as N. sicca, N. subflava, N. lactamica,

N. flavescens, and N. cinerea. Although the latter
organisms are usually present in the oropharyngeal
cavity, they may be present in the genital tract and
Culture
Culture
Culture
Culture
Culture
Culture
Culture

may result in false-positive results with the N. gon-

orrhoeae AMPLICOR assay. In a study that corre-
lated specificity of the assay with positive predictive
Prevalence

values (PPV) based on the type of specimen assayed,

4.4
6.3
6.2
2.9

2.0
(%)

29

29

male urine samples achieved as high as 83% PPV as

F, female; M, male; CX, endocervical swab; UR, urethral swab; NA, not available.

compared to a low of 13.3% PPV with female endo-

cervical samples (63). In that same study, a correla-
Specimen

tion was shown between the absorbance values (OD)

types

FCU

FCU
FCU
UR
UR
CX
CX

of the assay (the measurement of positive detection in

the assay) and false-positive results. If the OD reading
was in the positive range, i.e.,
3.5, the likelihood
1,267, M (symptomatic)

1,267, M (symptomatic)
714, M (asymptomatic)

714, M (asymptomatic)

that a positive N. gonorrhoeae result was true was

65% as compared to a 10% likelihood if the OD was
subjects, sex

in the range of 2.5 to 3.5. Results of a positive N.

No. of

gonorrhoeae result in the latter range (2.5 to 3.499),

or what some people would call the “gray zone,”
2,192, F
2,192, F
618, Fa

should be repeated twice, as per the manufacturer’s

package insert instructions. Further confirmation of
any positive N. gonorrhoeae result before reporting
should be considered if NAAT is the method used for
Livengood, 2001 (135)

screening (33). Leslie et al. (131) found a confirma-

Martin, 2000 (145)
Martin, 2000 (145)
Martin, 2000 (145)
Martin, 2000 (145)
Martin, 2000 (145)
Martin, 2000 (145)
Reference

tion rate of 86.2% for urethral swabs in a population

of male patients with a 7% prevalence of N. gon-
orrhoeae. The confirmation rate in cervicovaginal
specimens was only 5.7%, but this represented only
3 of 1,256 patients in a population with 0.24%
a
16 Leber et al.
prevalence. When a real-time PCR assay (Roche
LightCycler), targeting different genes than the
COBAS assay, was used to confirm N. gonorrhoeae
results of 172 endocervical samples from women,
59.8% of the COBAS positive samples confirmed as
positive on the LightCycler assay (195).
Future Directions
Improvements in the sensitivity for detection of C.
trachomatis in the COBAS AMPLICOR system con-
tinue to appear in the literature. Pretreatment of clin-
ical samples with an erythrocyte lysis buffer and
additional centrifugation steps aided in detecting
what were considered to be low-level infections
missed with the routine procedure of sample prepa-
ration (160). For the future, Roche is planning to
automate the sample processing by means of the
MagnaPure for extraction of DNA or use of the more
automated Tecan systems for many of the pipetting
steps of the procedures for setting up the A-rings.
Roche’s TaqMan instrumentation, utilizing real-time
PCR technology, may also become a format for
amplification assays for C. trachomatis and N. gon-
orrhoeae, either in addition to or as a replacement of
the COBAS.
Transcription-Mediated Amplification:
APTIMA (Gen-Probe Inc., San Diego, Calif.)
Principles of Procedure
The APTIMA assays are second-generation target
amplification nucleic acid tests for the qualitative
detection and differentiation of rRNA from C. tra-
chomatis and/or N. gonorrhoeae. The APTIMA
assays combine the technologies of target capture,
transcription-mediated amplification (TMA), and the
dual kinetics assay (DKA) (Gen-Probe Inc., package
insert, APTIMA Combo 2 Assay, IN0097-01 Rev. A,
2003). APTIMA Combo 2 (AC2), which detects C.
trachomatis and N. gonorrhoeae, received FDA
clearance in August 2001. APTIMA CT (ACT) for
detection of C. trachomatis and APTIMA GC (AGC)
for detection of N. gonorrhoeae use the same tech-
nology as AC2. These two tests were FDA cleared in
2005. The first-generation test, Amp CT, utilizes
TMA for the detection of C. trachomatis only; it does
not utilize target capture or DKA. The performance
characteristics of Amp CT served as the basis for
development of the second-generation assay and
have been published elsewhere (57, 74, 84, 156).
In the APTIMA assays, the target rRNA molecules
are released in the specimen transport solution and
then isolated from the samples by the use of capture
oligomers in a method called target capture. This
method differs from other NAATs that use crude lysis
for isolation of nucleic acid. By isolating the rRNA
CUMITECH 44
away from other components in the sample, inhibitor
substances are removed. The capture oligomers con-
tain sequences complementary to specific regions of
the target molecules as well as a string of deoxy-
adenosine residues. A separate capture oligomer is
used for each target. During the hybridization step,
the sequence-specific regions of the capture oligo-
mers bind to specific regions of the target molecules.
The capture oligomer-target complex is then cap-
tured out of solution by decreasing the temperature
of the reaction to room temperature. This tempera-
ture reduction allows hybridization to occur between
the deoxyadenosine region on the capture oligomer
and the polydeoxythymidine molecules that are cova-
lently attached to the magnetic particles. The micro-
particles, including the captured target molecules
bound to them, are pulled to the side of the reaction
vessel by magnets and the supernatant is aspirated.
The particles are washed to remove residual speci-
men matrix that may contain amplification reaction
inhibitors. After the target capture steps are com-
pleted, the specimens are ready for amplification.
The AC2 assay replicates a specific region of the
23S rRNA from C. trachomatis and a specific region
of the 16S rRNA from N. gonorrhoeae via DNA
intermediates. For the ACT and AGC assays, the
rRNA targets are different from those used in AC2;
the C. trachomatis assay replicates a specific region
of the 16S rRNA, and the N. gonorrhoeae target is a
16S rRNA target different from that in AC2. A unique
set of primers is used for each target molecule. The
rRNA amplification product sequences (amplicon)
are detected by nucleic acid hybridization. Single-
stranded chemiluminescent DNA probes, which are
complementary to a region of each target amplicon,
are labeled with different acridinium ester molecules.
The labeled DNA probes combine with amplicon to
form stable RNA:DNA hybrids. The selection reagent
differentiates hybridized from unhybridized probe,
eliminating the generation of signal from unhybri-
dized probe. During the detection step, light emitted
from the labeled RNA:DNA hybrids is measured as
photon signals in a luminometer and is reported as
relative light units (RLU). In DKA, differences in the
kinetic profiles of the C. trachomatis and N. gonor-
rhoeae labeled probes allow the differentiation of sig-
nal; kinetic profiles are derived from measurements
of photon output during the detection read time. The
chemiluminescent detection reaction for C. tracho-
matis signal has very rapid kinetics and has the
“flasher” kinetic type. The chemiluminescent detec-
tion reaction for N. gonorrhoeae signal is relatively
slower and has the “glower” kinetic type. Assay
results are determined by a cutoff based on the total
RLU and the kinetic curve type.
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Instrumentation
The AC2 assay can be performed with two platforms,
the direct tube sampling (DTS) systems and the
TIGRIS DTS system. For the single-analyte tests (ACT
and AGC), the DTS systems are the approved plat-
form. DTS systems include DTS 400, DTS 800, and
DTS 1600. These systems are designed for low-,
medium-, and high-volume laboratories yielding up
to 400, 800, or 1,600 results, respectively, in an 8-h
shift. Equipment for the DTS systems includes three
water baths, a multitube vortexer, a luminometer for
measurement of chemiluminescent emission, and the
target capture system (TCS). More recently a dry
bath-vortexer has been developed to replace the water
baths and vortexer (S. Yamagata, M. Kennedy, F. Li,
K. Dickey, E. Brown, and T. Calderoni, Abstr. 104th
Gen. Meet. Am. Soc. Microbiol., abstr. C-282, p. 175,
2004). The DNA extraction is performed on the TCS
platform: for the DTS 800 and DTS 1600, this in-
volves a modified Tecan workstation.
The TIGRIS DTS system received FDA clearance
in December 2003 and is the first fully automated
molecular testing system for C. trachomatis and N.
gonorrhoeae. Currently only AC2 can be used on this
platform. The TIGRIS system offers direct tube sam-
pling utilizing the specimen collection devices with
piercible caps; both swab and urine collection devices
are directly loaded on the instrument along with
reagents. No other manual manipulation of speci-
mens is required. All steps of specimen processing,
amplification, detection, and result analysis are car-
ried out by the instrumentation. There is positive sam-
ple identification throughout the process, and results
are automatically linked to reagent lot numbers and
can be uploaded to a laboratory information system.
The throughput as reported by the manufacturer is
approximately 500 samples in an 8 h shift and up to
1,000 samples in approximately 13 h. The TIGRIS
DTS analyzer is the size of a common chemistry ana-
lyzer, measuring 69  36  72 inches and weighing
1,300 pounds.
In a study performed by Novak et al., perfor-
mance of the TIGRIS versus the DTS 1600 system
with the AC2 assay was evaluated (S. M. Novak-
Weekly and A. M. Vannier, Abstr. 104th Gen. Meet.
Am. Soc. Microbiol., abstr. C-281, p. 175, 2004).
Using over 10,000 specimens, the authors found ex-
cellent agreement for C. trachomatis and N. gonor-
rhoeae in swabs and urines, with 98% or greater con-
cordance between results on both platforms. Similar
agreement between TIGRIS and DTS results was seen
in a multicenter study with 1,061 urine and swab
specimens (E. W. Hook, P. Fine, M. Cohen, A. Weiss-
feld, D. Willis, Y. Wang, and B. Fugikawa, Abstr.
104th Gen. Meet. Am. Soc. Microbiol., abstr. C-280,
17
p. 174, 2004). These authors also concluded that use
of the TIGRIS reduced operator-related error that
could result in the reporting of incorrect results.
Sample Collection and Transport
The AC2, ACT, and AGC assays are FDA cleared for
testing endocervical, vaginal, and male urethral swab
specimens and female and male urine specimens. The
assays are approved for testing in both symptomatic
and asymptomatic individuals. Rectal, oropharyn-
geal, conjunctival, and female urethral specimens are
not approved for testing.
The APTIMA collection devices contain specimen
buffer and have a penetrable top. Once inoculated,
the tube is not opened for sample processing and is
pierced directly to remove the sample. This feature
minimizes the risk of cross-contamination in the lab-
oratory. Swab specimens are transported at 2 to 30°C
and are stable for 60 days. Urine specimens can be
transported at 2 to 30°C in either the primary collec-
tion container or in the urine specimen transport
tube. Urine specimens must be transferred to the man-
ufacturer’s transport tube within 24 h and before
assay. After transfer, urine specimens are stable for
up to 30 days at 2 to 30°C. Both swab and urine
specimens in transport tubes can be stored frozen at
20 to 70°C for up to 90 days.
The APTIMA vaginal swab specimen collection
kit received FDA clearance in January 2004. It is the
first kit that enables patients to self-collect vaginal
swab specimens, under the direction of medical pro-
fessionals, for C. trachomatis and N. gonorrhoeae
detection with the APTIMA assays. The swab is
inserted into the vagina about 2 inches past the introi-
tus and gently rotated for 10 to 30 s. The swab is then
withdrawn with care not to touch skin and placed in
the transport tube.
The PACE collection device, used for Gen-Probe
nonamplified C. trachomatis and N. gonorrhoeae
probes, can also be used to test male urethral and
endocervical swab samples with the AC2 assay. These
samples require use of the APTIMA adaptor kit for
dilution of the PACE medium. Liquid cytology media
such as PreservCyt (Cytyc Corporation, Marlbor-
ough, Mass.) and SurePath (TriPath Imaging Inc.,
Burlington, N.C.) can be tested; however, only
PreservCyt has been FDA cleared (August 2005). Sev-
eral researchers have demonstrated success testing
both media in AC2 for the detection of C. trachoma-
tis and N. gonorrhoeae. (124; B. Weinbaum, R. Pilla,
M. Alvarez, M. Shih, C. Eng, and R. Johnson, Abstr.
104th Gen. Meet. Am. Soc. Microbiol., abstr. C-285,
p. 175, 2004; R. Johnson, B. Weinbaum, R. Pilla, M.
Alvarez, M. Shih, and C. Eng, Abstr. 104th Gen. Meet.
Am. Soc. Microbiol., abstr. C-283, p. 175, 2004).
18 Leber et al.
Inhibition and Contamination Control
There is no inhibition control for individual samples
in the APTIMA assays. With the use of target capture
for isolation of rRNA, the effect of inhibitors in the
sample is minimized. In spiking studies conducted by
Chong et al. (44), urine from 415 pregnant and non-
pregnant females was spiked with 12 C. trachomatis
elementary bodies and assayed on APTIMA and
LCx. False-negative rates of 0.48% and 13% were
detected with the APTIMA and LCx, respectively.
Repeat testing after overnight storage reduced the
false-negative rate to 0% for APTIMA and 5.4%
with LCx. Additional experiments suggested that the
APTIMA assay has a higher sensitivity and better
nucleic acid extraction or fewer problems with inhi-
bition than LCx (44).
Contamination control is accomplished by unidi-
rectional workflow and amplicon deactivation. The
laboratory should be designed so that work proceeds
from reagent preparation through DKA. The manu-
facturer strongly suggests a separate and dedicated
area for the DKA. After detection has been com-
pleted, the samples are removed from the luminome-
ter and placed in a buffer bleach solution to deacti-
vate amplicons. Work surfaces and equipment are
decontaminated with a 1:1 solution of bleach and
water. Regular monitoring of the environment is rec-
ommended to check for contamination. Swabs of
surfaces and water in water baths are tested in the
APTIMA assays, and any positive results indicate
that additional cleaning is needed.
Result Interpretation
AC2 test results are automatically interpreted by the
manufacturer’s software and are presented as indi-
vidual C. trachomatis and N. gonorrhoeae results.
The final patient results are calculated by using an
algorithm that incorporates several elements, includ-
ing the kinetic output (measured in RLUs) and kinetic
type/curve shape of the light-off reaction. A test result
may be negative, equivocal, positive, or invalid for C.
trachomatis and N. gonorrhoeae. Along with the
qualitative results, the RLU values for each sample
are printed on the sample report. The ACT and AGC
assays have result interpretation and reporting simi-
lar to that of AC2; the result is negative, equivocal,
positive, or invalid and the result is based on RLU
and kinetic type/curve shape.
Individual testing for C. trachomatis or N. gonor-
rhoeae is also possible with the AC2. The software
suppresses the result for the analyte that is not
ordered. With this mode of testing, the RLU for the
reaction, which includes both analytes, is still reported
and may indicate the presence of the unordered ana-
lyte. For example, high RLUs in a C. trachomatis neg-
ative specimen may indicate that N. gonorrhoeae is
CUMITECH 44
present in the sample. However, because the algo-
rithm is based on RLU and curve kinetics, the presence
of N. gonorrhoeae cannot be automatically inferred.
Performance Characteristics
As the newest of the commercially available NAATs
for the detection of C. trachomatis and N. gonor-
rhoeae, there are relatively few peer-reviewed publi-
cations concerning the performance characteristics of
AC2 and the ACT and AGC reagents. Tables 6 and 7
summarize data from publications and abstracts
about the three tests.
Analytical performance has been assessed in stud-
ies, and it appears that the APTIMA technology may
have greater sensitivity than other NAATs. In a study
using urine spiked with C. trachomatis elementary
bodies (EB), AC2 was found to have a limit of detec-
tion of 0.01 EB versus 12 EB for the LCR assay (44).
Another spiking study comparing AC2 to AMP-
LICOR PCR showed an analytical sensitivity of
0.008 EB versus 0.5, respectively (106).
In clinical studies, APTIMA Combo 2 has demon-
strated comparable sensitivity and specificity when
compared to other NAATs. Gaydos et al. (82) com-
pared AC2, BD ProbeTec, and LCR for the detection
of C. trachomatis in 506 FCU specimens from a high-
risk population. They found no statistically signifi-
cant differences in the sensitivity and specificity of
the three NAATs using a rotating gold standard.
Another evaluation by Gaydos et al. (81) found AC2
performance comparable to or higher than that of
other NAATs for the detection of C. trachomatis and
N. gonorrhoeae in female swab and urine specimens.
Unlike reports for other NAATs, they demonstrated
that the sensitivity and specificity were comparable
for the swab and FCU. LCR, PCR, and SDA have
lower sensitivity for urines, probably due to inhibi-
tors present in this sample type.
Using other NAATs as referee methods, re-
searchers have found positive results by AC2 that are
negative by the other methods (152, 177). These
results may be due to a lower specificity or, con-
versely, a high sensitivity that cannot be confirmed by
the less sensitive NAAT (imperfect gold standard).
The use of rRNA in TMA assays increases the num-
ber of target molecules per cell. This increase in tar-
get increases the sensitivity of the test. Also, TKA may
remove inhibitor that can lead to false negatives by
other methods. When the APTIMA reagents for indi-
vidual analytes are used for confirmation of such dis-
crepant samples, many are confirmed as positive. In
a study comparing the performance of ACT and
AGC assays to AC2, LCx, culture, and DFA, the
ACT and AGC reagents were found to be suitable for
confirmation of positive tests in urine and swab spec-
imens (24). There was 100% concordance with the
 CUMITECH 44

Table 6. Recent studies evaluating Gen-Probe APTIMA assay for detection of C. trachomatis

No. of Specimen Prevalence Comparison Sensitivity Specificity Positive predictive Negative predictive
Reference(s)
subjects, sex types (%) methoda (%) (%) value (%) value (%)

Moncado et al., 1,411, Fa CX 13.3 Patient infected statusb (LCR, PCR, and 92.1 97.7 NA NA
2004 (152) TMA on swab and urine)
Gaydos et al., 2004 (82) 506, M and F FCU 14.8 Specimenc (LCR and PCR) 99.4 97.4
LCR and SDA 100 98.8 93.8 100
Gaydos et al., 2003 (81) 1,389, F CX 15.0 Patient infected status (LCR, PCR on swab 94.2 97.6 87.4 99.0
and urine)
Gaydos et al., 2003 (81) 1,391, F FCU 15.0 Patient infected status (LCR, PCR on swab 94.7 98.9 93.8 99.1
and urine)
Martin et al., 2001d 2,457 total NA Patient infected status (LCR, PCR on swab NA NA
and urine)
M UR Patient infected status (LCR, PCR on swab 96.9 97.5
and urine)
M FCU Patient infected status (LCR, PCR on swab 97.9 98.5
and urine)
F CX Patient infected status (LCR, PCR on swab 94.2 97.6
and urine)
F FCU Patient infected status (LCR, PCR on swab 95.3 98.9
and urine)
Ferrero et al., 2001e 1,095, M FCU 25.8 Patient infected status (LCx, PCR on swab 97.9 98.9 95.8 99.3
and urine)
1,391, F FCU 15.0 94.7 98.9 93.8 99.1
Martin et al., 2002f 271, F CX NA Patient infected status (LCx, SDA on swab 91.5 94.6 NA NA
and urine)
FCU NA Patient infected status (LCx, SDA on swab 94.9 99.0 NA NA
and urine)
a
F, female; M, male; CX, endocervical swab; UR, urethral swab; NA, not available.
b
True positives are defined as two or more positive results with reference tests in any combination of method and sample type.
c
True positives are defined as two or more positive results by test methods only for a single sample type.
d
D. H. Martin, E. W. Hook, D. Ferrero, D. Willis, J. Schachter, A. Weissfeld, C. Gaydos, and T. Quinn, Abstr. Int. Soc. Sex. Transm. Dis. Res. p. 97, 2001.
e
D. V. Ferrero, C. A. Gaydos, T. C. Quinn, E. W. Hook, D. H. Martin, J. Schachter, A. Weissfeld, and D. Willis, Abstr. Int. Soc. Sex. Transm. Dis. Res. p. 97, 2001.
f
D. H. Martin, C. L. Cammarata, and B. Smith, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. C-174, p. 131, 2002.
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
19
 20

Table 7. Recent studies evaluating Gen-Probe APTIMA assay for detection of N. gonorrhoeae
Leber et al.

No. of Specimen Prevalence Comparison Sensitivity Specificity Positive predictive Negative predictive
Reference
subjects, sex types (%) method (%) (%) value (%) value (%)

Moncado et al., 2004 1,489, Fa CX 8.7 Patient infected statusb (LCx, PCR on swab 98.5 98.7 NA NA
(152) and urine)
Specimenc (LCR and PCR) 99.2 98.6 NA NA
Gaydos et al., 2003 (81) 1,479, F CX 8.6 Patient infected status (LCR, PCR on swab 99.2 98.7 88.1 99.9
and urine)
Gaydos et al., 2003 (81) 1,484, F FCU 8.6 Patient infected status (LCR, PCR on swab 91.3 99.3 92.1 99.2
and urine)
Martin et al., 2001d 2,546 total NA Patient infected status (culture or LCR and NA NA
PCR on swab and urine)
M UR Patient infected status (culture or LCR and 99.2 97.9
PCR on swab and urine)
M FCU Patient infected status (culture or LCR and 98.1 99.6
PCR on swab and urine)
F CX Patient infected status (culture or LCR and 99.2 98.7
PCR on swab and urine)
F FCU Patient infected status (culture or LCR and 91.1 99.3
PCR on swab and urine)
Ferrero et al., 2001e 1,095, M FCU 29.0 Patient infected status (LCR and culture on 98.5 99.6 99.1 99.4
swab and urine)
1,391, F FCU 8.6 Patient infected status (LCR and culture on 91.3 99.3 92.1 99.2
swab and urine)
Martin et al., 2002f 269, F CX NA Patient infected status (LCR, SDA on swab 100 100 NA NA
and urine)
FCU Patient infected status (LCR, SDA on swab 99.0 100
and urine)
a
F, female; M, male; CX, endocervical swab; UR, urethral swab; NA, not available.
b
True positives are defined as two or more positive results with reference tests in any combination of method and sample type.
c
True positives are defined as two or more positive results by test methods only for single sample type.
d
D. H. Martin, E. W. Hook, D. Ferrero, D. Willis, J. Schachter, A. Weissfeld, C. Gaydos, and T. Quinn, Abstr. Int. Soc. Sex. Transm. Dis. Res. p. 97, 2001.
e
D. V. Ferrero, C. A. Gaydos, T. C. Quinn, E. W. Hook, D. H. Martin, J. Schachter, A. Weissfeld, and D. Willis, Abstr. Int. Soc. Sex. Transm. Dis. Res. p. 97, 2001.
f
D. H. Martin, C. L. Cammarata, and B. Smith, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. C-174, p. 131, 2002.
CUMITECH 44
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Combo 2 assay in both sample types. All of the
APTIMA assays (AC2, ACT, and AGC) detected
more positives than culture, DFA, or LCx. Use of
other NAATs for confirmation of positive samples
with AC2 and AC and AG assays may not be advis-
able due to differences in sensitivity. In a study by
Schachter et al. (177), separate swab and urine sam-
ples were collected from each patient for testing with
AC2, ACT, and BD ProbeTec C. trachomatis assay.
The authors found that the use of BD to confirm AC2
and AC positive C. trachomatis samples resulted in
incorrectly reporting approximately 15% of the AC
and AC2 positive samples as negative. Conversely,
use of the AC2 or AC assays for confirmation of C.
trachomatis positive BD samples resulted in confir-
mation of 96.9% of specimens. Such findings are
important in light of the recommendation to confirm
positive screening tests in low-prevalence popula-
tions. Confirmation must be done with a test that is
at least as sensitive as the initial assay.
Future Directions
With the first fully automated instrument on the mar-
ket, Gen-Probe will hopefully develop other NAATs
for testing on the TIGRIS platform. This may make
it more economically feasible for laboratories to jus-
tify the cost of the instrumentation. Gen-Probe has
stated its intent to standardize its assays on the
TIGRIS platform. Analytes that have been mentioned
include human papillomavirus, Trichomonas, human
immunodeficiency virus, hepatitis C virus, and PCA3
(a prostate cancer marker under investigation) (M.
Bott, personal communication).
Strand Displacement Amplification:
BD ProbeTecET (Becton Dickinson
and Company, Sparks, Md.)
Principles of Procedure
The BD ProbeTecET (BDP) C. trachomatis and N.
gonorrhoeae amplified DNA assays are a second-
generation NAAT system for the detection of chlamy-
dial and gonococcal infection from endocervical, ure-
thral, or urine specimens. The BDP C. trachomatis
and N. gonorrhoeae assays are based on the simulta-
neous amplification and detection of organisms’ tar-
get DNA by isothermal SDA and real-time fluores-
cence detection using a fluorescent detector probe
(134, 218). Targets for amplification are the C. tra-
chomatis cryptic plasmid and the pilin gene inverting
protein sequence of N. gonorrhoeae (134).
SDA is an isothermal process that utilizes a series
of primers, DNA polymerase, and a restriction en-
zyme to exponentially amplify the unique nucleic acid
target sequences. SDA consists of two processes, tar-
get generation and exponential target amplification
21
(134, 218). Throughout the process of target gen-
eration, double-stranded DNA is heat denatured, cre-
ating two single-stranded copies. A series of ampli-
fication primers for copying the base sequence and
bumper primers for displacing the newly created
strands combine with DNA polymerase to form
altered targets capable of exponential amplification.
The exponential amplification process begins with
single-stranded DNA strands with restricted enzyme
recognition sites from the target generation phase.
Amplification primers bind to each strand at its com-
plementary DNA sequence. DNA polymerase uses
the primer to identify a location to extend the primer
from its 3 end, using the altered target as a template
for adding individual nucleotides. The extended
primer forms a double-stranded DNA segment con-
taining a complete restriction enzyme recognition site
at each end. The restriction enzyme binds to the
double-stranded DNA segment at its recognition site.
The restriction enzyme dissociates from the recogni-
tion site after having cleaved only one strand of the
double-sided segment, forming a nick. DNA poly-
merase recognizes the nick and extends the strand
from the site, displacing the previously created strand.
The recognition site is repeatedly nicked and restored
by the restriction enzyme and DNA polymerase with
continuous displacement of DNA strands containing
the target segment. Each displaced strand is then
available to anneal with amplification primers, and
the process continues with repeated nicking, exten-
sion, and displacement of new DNA strands, result-
ing in exponential amplification of the original DNA
target (134, 218).
Single-stranded DNA probes containing fluores-
cein and rhodamine labels are present in the reaction
mixture. A stem-loop structure occupies the space
between the two fluorochromes. Before target ampli-
fication, the fluorochromes are located close enough
to each other so that any excitation of the fluorescein
results in the transfer of the emitted energy to the
rhodamine molecule. This results in very little emis-
sion from the excited fluorescein molecule being
detected. After the SDA reaction, the probe is con-
verted to a double-stranded molecule that is cleaved
by a restriction enzyme. This separates the two fluo-
rochromes to the extent that no energy transfer from
the excited fluorescein to rhodamine can occur. The
fluorescence detected from the excited fluorescein
label indicates amplification of the specific target
sequence (134).
Instrumentation
The BDP system instrumentation components consist
of an expandable, programmable pipettor, lysing
heater and sample rack, priming and warming heater,
and the BD ProbeTecET reader (134). This system
22 Leber et al.
requires manual processing of samples and pipetting
of reagents throughout the testing.
The BD VIPER (VIPER) sample processor has been
developed to minimize pipetting and reduce hands-
on time that is associated with the BDP C. tracho-
matis and N. gonorrhoeae assays. For manual users,
samples are processed, lysed, and then manually
transferred from the sample tubes to the priming and
amplification wells. The VIPER automates the pipet-
ting of samples from sample tubes to the priming
wells and from priming to amplification wells. The
instrument also replaces the current priming and
warming heaters.
In the first comparison of the VIPER to manual
methods, negative cervical and urine pools were
spiked with varying levels of chlamydial elementary
bodies and gonococcal particles. The analytical sen-
sitivities for C. trachomatis and N. gonorrhoeae from
endocervical swabs were not significantly different
from those seen with the manual procedure. For urine
samples, the limits of detection were significantly
lower for both C. trachomatis and N. gonorrhoeae
using the automated procedure (T. Schlitzer, T. Fort,
T. Hansen, and P. Johnson, Abstr. 102nd Gen. Meet.
Am. Soc. Microbiol., abstr. C-161, p. 128, 2002).
The automated assay has been validated by ana-
lyzing specimens previously run with the manual
assay in duplicate for between-run variability and
found to be concordant (D. E. Anamani., L. E.
Burgess, S. Vicki, L. Chaffee, and G. J. Tsongalis,
Abstr. 19th Annu. Meet. Clin. Virol. Symp., abstr.
S48, 2003). In addition, the authors compared
VIPER automation to the Abbott LCx system and
commented on superior throughput, ease of opera-
tion, and maintenance with the BD system. Blackwell
et al. found the sensitivity and specificity of VIPER
automation for C. trachomatis to be comparable to
the manual assay using pools of four endocervical
swabs (G. Blackwell, F. Jamieson, G. Riley, and M.
Gorus, Abstr. 19th Annu. Meet. Clin. Virol. Symp.,
abstr. TM19, 2004). By pooling, this study noted a
cost savings of 30 to 40%, which was attributed to
reduced reagent and consumable usage. Approxi-
mately 15 linear feet are required to accommodate
the VIPER and two ProbeTec instruments. The man-
ufacturer’s estimated throughput on a daily basis
(swab specimens per 8-h shift) is 360 specimens for C.
trachomatis/N. gonorrhoeae with amplification con-
trol and 552 specimens for C. trachomatis/N. gonor-
rhoeae without amplification control.
Sample Collection and Transport
The BDP is approved for the direct detection of C.
trachomatis and N. gonorrhoeae from endocervical
swabs, male urethral swabs, and female and male
CUMITECH 44
urine specimens from asymptomatic and sympto-
matic individuals.
To collect female endocervical samples, a cleaning
swab is used first to remove mucus. Endocervical
specimens are then collected with a polyurethane-
tipped swab (CULTURETTE DIRECT; Becton Dick-
inson). Male urethral specimens are collected using
the MiniTip CULTURETTE DIRECT. Swab speci-
men types can be transported without preservatives
to the testing laboratory at 2 to 27°C and are stable
for 4 to 6 days. At the testing site swabs are
expressed into sample diluent tube and the swab is
discarded. The diluent tubes are recapped and vor-
texed for 5 s. The expressed sample is placed into the
lysing heater (114°C) and cooled for 15 min at room
temperature before the assay is performed.
Urine specimens are collected in sterile, plastic,
preservative-free containers. Collection of the first 15
to 20 ml of voided urine is optimal, with a maximum
volume of 60 ml. The patient should not have voided
for 1 h prior to collection. A urine-processing pouch
is added to the sample cup, and the cup is then
capped and transported to the testing laboratory.
Samples may be held for 2 days at 15 to 27°C or 4 to
6 days at 2 to 8°C. The urine-processing pouch must
be in contact with the specimen for a minimum of 2
h before to processing. Urine specimens are mixed by
swirling, and 4 ml is pipetted into an empty sample
tube. The tubes are capped and centrifuged for 30
min. The supernatant is decanted and 2 ml of sample
diluent is added to each tube. The samples are placed
into the lysing heater (114°C) and cooled for 15 min
at room temperature before the assay is performed.
Inhibition and Contamination Control
An amplification control for inhibition testing is
available in the CT/GC/amplification control reagent
pack for use with the manual method. The control is
included for each specimen and should identify sam-
ples containing amplification inhibitors that could
prevent detection of target DNA. For result interpre-
tation, different MOTA (method other than acceler-
ation) values are used whether or not the amplifica-
tion control is included. The use of the amplification
control is optional and, depending on the type of
specimens tested, may not have a noticeable impact
on the test performance characteristics (56). It is cur-
rently not available for use on the VIPER instrument.
According to the manufacturer, dedicated work
areas are not required because of the assay’s design
(Becton Dickinson, BD ProbeTec ET C. trachomatis
and N. gonorrhoeae Amplified DNA Assays, package
insert L000203, 1999). Contamination is prevented
through standard laboratory precautions such as
changing gloves and decontaminating work surfaces
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
with a 1% solution of bleach in distilled/deionized
water. Plates containing amplification microwells are
sealed before they are moved from the priming and
warming heater to the ProbeTec instrument. The use
of sealed plastic bags is suggested for disposal of
amplification microwells to prevent environmental
contamination with amplicons. After the assay is
completed, all countertops and instrument surfaces
should be disinfected as above. Wipe tests of work
areas and equipment surfaces, including the lysing
heater, rack, priming and warming heater, microwell
carriers, pipettor handles, and ProbeTec instrument,
should be performed at least monthly to monitor for
the presence of DNA contamination. Although sepa-
rate work areas are not mandatory, they are advisable
for sample processing and amplification/detection to
prevent amplicon contamination. Of particular note,
the amplification control generates amplicon that is
indistinguishable from the amplicon generated by N.
gonorrhoeae, thus increasing the risk of false-positive
N. gonorrhoeae results.
Result Interpretation
The presence or absence of C. trachomatis and N.
gonorrhoeae is determined by relating MOTA values
for the sample to predetermined cutoff values (134).
The MOTA is used to determine the magnitude of
the signal generated in the reaction; however, this
does not correlate with the number of organisms in
the specimen. When testing without the amplification
control, MOTA scores of
10,000 are considered
positive for C. trachomatis and/or N. gonorrhoeae
DNA. When testing is performed with the amplifica-
tion control, MOTA scores of
2,000 are considered
positive for C. trachomatis and/or N. gonorrhoeae
DNA. The package insert refers to samples with
MOTA values between 2,000 and 10,000 as “low
positive” samples and states that there is a decreased
likelihood of the results being a true positive as com-
pared to results with MOTA values greater than
10,000. CDC guidelines suggest that consideration
be given to additional testing for persons with posi-
tive screening results for C. trachomatis and N. gon-
orrhoeae in low-prevalence populations, which
results in a lower PPV (33). All nucleic acid amplifi-
cation assays may be prone to problems with repro-
ducibility of positive test results (90, 167). A recent
article described a repeat test algorithm for samples
with MOTA scores greater than or equal to the man-
ufacturer’s cutoff (58). Poor reproducibility (21 of 26
C. trachomatis and 4 of 12 N. gonorrhoeae) was
noted with MOTA scores between 2,000 and 9,999
for both analytes. The reproducibility of both ana-
lytes with initial MOTA scores of
10,000 increased
to 96.7%. It is also noteworthy that Neisseria cinerea
23
and other Neisseria species may cause false positives
in the BDP N. gonorrhoeae assay (163). Confirma-
tory testing in low-prevalence populations or in
patients with history or clinical signs inconsistent
with gonococcal infection is suggested.
Performance Characteristics
A number of published studies have evaluated the per-
formance of the assays. Tables 8 and 9 summarize the
performance characteristics of the BDP C. trachoma-
tis and N. gonorrhoeae assays.
The performance of the BDP has been compared
to other nucleic acid amplification systems and found
to be equivalent. In the first published study using
this technology, the BDP was 100% sensitive and
specific when compared to the LCx (134). The BDP
performs comparably to other amplification tech-
nologies for the detection of C. trachomatis with
male urine and female endocervical specimens (26,
37, 147, 205, 209); however, in a study of a relatively
high-prevalence population the sensitivity of the
assay using female urine specimens was notably
reduced (147). In the largest study comparing the
BDP to culture and LCx, the assays were comparable
for the detection of C. trachomatis and N. gonor-
rhoeae, but indeterminate assays occurred more
often with the BDP assay and urine specimens (205).
Akduman et al. (3) demonstrated greater sensitivity
using the BDP for detecting N. gonorrhoeae in urine
samples when compared to female endocervical and
male urethral specimens.
Future Directions
Becton Dickinson is developing a second-generation
SDA, the C. trachomatis Diplex, which incorporates
an internal amplification control to monitor the pres-
ence of amplification inhibitors and to verify the exis-
tence of proper conditions for amplification (R. A.
McMillian, T. L. Fort, T. L. Hellyer, and R. Moore,
Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr.
C-275, p. 173, 2004). The performance characteris-
tics of the diplex assay were comparable to those of
the currently available assay with both urine and
endocervical swabs.
Addressing automation, an extraction control
based on the binding of DNA and RNA to iron oxide
particles has been developed for use with a prototype
BD VIPER equipped for nucleic acid extraction (J. M.
Harris, T. Brink, and C. Keys, Abstr. 104th Gen.
Meet. Am. Soc. Microbiol., abstr. C-293, p. 177,
2004). The extraction control functions as a process
control and verifies that conditions exist for binding,
washing, and recovering nucleic acids from vaginal
swabs and urine.
 24

Table 8. Recent studies evaluating the BD ProbeTec assay for detection of C. trachomatis

No. of Specimen Prevalence Sensitivity Specificity Positive predictive Negative predictive

Reference Expanded standard methoda
subjects, sex types (%) (%) (%) value (%) value (%)
Leber et al.

Little et al., (134) 122, Fa CX, FCU 8.2 LCR 100 100 100 100
Chan et al., (37) 1,224, M and F FCU 13.8 PCR 95.3 99.3 95.9 99.2
Van Der Pol et al., 2001 (205) 2,109, M and F CX, FCU, 13.0 Patient infected statusb (culture, or 90.0 97.3 NA NA
and UR LCR and DFA on swab or urine)
Van Dyck et al., 2001 (209) 733, F CX 10.2 Culture or two positive tests PCR, 94.7 100 100 99.4
SDA, LCR
Browning et al., 2001 (26) 419, M and F FCU 11.7 Culture (CX, UR) 96 100 100 99.4
McCartney et al., 2001 (147) 715, M FCU 9.2 LCR, PCR 95.5 100 100 99.5
291, F FCU 9.1 LCR, PCR 77.3 100 100 97.3
291, F CX 9.1 LCR, PCR 90.9 100 100 97.3
Ferrero et al., 1999c 657, F CX 5.0 Culture or LCR and SDA positive 100 99.3 89.2 100
588, F FCU 5.0 Culture or LCR and SDA positive 100 98.6 82 100
Leister et al., 1999d 468, F CX 12.4 LCR 96.6 97.3 83.5 99.5
658, M FCU 23.1 LCR 94.1 95.1 85.6 98.1
Lovechik et al., 1999e 294, F CX 10.9 Patient infected statusb (culture, or 96.9 96.6 77.5 99.6
LCR on CX or FCU)
FCU 10.9 Patient infected statusb (culture, or 90.6 98.4 87.8 98.8
LCR on CX or FCU)
170, F CX 12.9 Patient infected statusb (culture, or 100 96.7 81.4 100
LCR on CX or FCU)
FCU 12.3 95.5 96.1 77.7 99.3
Fuller et al., 2000f 377, F CX 12.7 LCR, PCR on CX or FCU 98 100 100 99.6
FCU 12.7 LCR, PCR on CX or FCU 96 100 100 99.4
Chandler et al., 2000g 293, F CX, FCU 10.2 LCR 100 100 100 100
Homick et al., 2000h 272, F CX 11.8 Two or more positives between 90.6 NA NA 98.8
PCR, LCR, SDA, culture
272, F FCU 11.8 Two or more positives between 78.1 NA NA 97.1
PCR, LCR, SDA, culture
a
F, female; M, male; CX, endocervical swab; UR, urethral swab; NA, not applicable.
b
True positives are defined as two or more positive results with reference tests in any combination of method and sample type.
c
D. V. Ferrero, L. Buck-Barrington, H. Meyers, G. S. Hall, M. Tuohy, D. Wilson, J. Schachter, J. Moncada, and F. Pang, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-120, p. 129, 1999.
d
L. S. Leister, K. A. Crotchfelt, B. VanderPol, R. B. Jones, K. Smith, C. Lenderman, E. W. Hook, T. C. Quinn, and C. A. Gaydos, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-115, p. 127, 1999.
e
J. C. Lovchick, L. M. Peralta, C. M. Brown, D. M. Hilligoss, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-119, p. 128, 1999.
f
D. Fuller, T. Davis, P. Lineback, M. Milish, and L. Jasper, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-376, p. 220, 2000.
g
L. Chandler, B. Reisner, J. Fraser, P. Fulcher, D. Fahlen, T. Gateley, and G. Woods, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-375, p. 219, 2000.
h
K. Homick, J. B. Myers, L. A. Garringer, D. Amsterdam, and S. J. Zimmerman, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-377, p. 220, 2000.
CUMITECH 44
 CUMITECH 44

Table 9. Recent studies evaluating BD ProbeTec assays for detection of N. gonorrhoeae

No. of Specimen Prevalence Sensitivity Specificity Positive predictive Negative predictive

Reference method
Reference subjects, sex types (%) (%) (%) value (%) value (%)

Little et al., 1999 (134) 122, Fa CX, FCU 0.8 LCR 100 100 100 100
Chan et al., 2000 (37) 1,224, M and F FCU 2.6 PCR 100 99.7 88.2 100
Van Der Pol et al., 2001 (205) 2,109, M and F CX, FCU, UR Patient infected statusb (culture, or 95.9 98.6 NA NA
LCR on swab or urine)
Van Dyck et al., 2001 (209) 733, F CX 1 Culture or two positive tests PCR, 93.9 100 100 99.4
SDA, LCR
Akduman et al., 2002 (3) 3,544, M and F FCU 3.6 Culture 99.2 99.3 84.9 99.9
Ferrero et al., 1999c 644, F CX 1.5 Culture or LCR and SDA positive 100 99.7 83.3 100
578, F FCU 1.5 Culture or LCR and SDA positive 90 99.6 81.8 99.8
Leister et al., 1999d F, 471 CX 12.1 LCR 96.6 97.3 93.3 99.8
463, F FCU 11.0 84.4 96.9 86.2 98.2
663, M FCU 29.5 94.1 95.1 92.7 98.6
Lovechik et al., 1999e 294, F CX 7.1 Patient infected statusb (culture, or 100 99.6 95.4 100
LCR on CX or FCU)
FCU 7.1 Patient infected statusb (culture, or 84.2 98.8 84.2 98.8
LCR on CX or FCU)
170, F CX 11.7 Patient infected statusb (culture, or 100 98.3 86.9 100
LCR on CX or FCU)
U 10.6 Patient infected statusb (culture, or 88.9 97.5 80 98.7
LCR on CX or FCU)
Fuller et al., 2000f 377, F CX 6.1 Consensus LCR, PCR on CX or FCU 98 100 100 99.7
377, F FCU 6.1 Consensus LCR, PCR on CX or FCU 100 99 95.6 100
Chandler et al., 2000g 293, F CX, FCU 6.5 LCR 100 99.6 95 100
Homick et al., 2000h 272, F CX 8.8 Two or more positives between 100 NA NA 100
PCR, LCR, SDA, culture
a
F, female; M, male; CX, endocervical swab; UR, urethral swab.
b
True positives are defined as two or more positive results with reference tests in any combination of method and sample type.
c
D. V. Ferrero, L. Buck-Barrington, H. Meyers, G. S. Hall, M. Tuohy, D. Wilson, J. Schachter, J. Moncada, and F. Pang, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-120, p. 129, 1999.
d
L. S. Leister, K. A. Crotchfelt, B. VanderPol, R. B. Jones, K. Smith, C. Lenderman, E. W. Hook, T. C. Quinn, and C. A. Gaydos, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-115, p. 127, 1999.
e
J. C. Lovchick, L. M. Peralta, C. M. Brown, and D. M. Hilligoss, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-119, p. 128, 1999.
f
D. Fuller,T. Davis, P. Lineback, M. Milish, and L. Jasper, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-376, p. 220, 2000.
g
L. Chandler, B. Reisner, J. Fraser, P. Fulcher, D. Fahlen, T. Gateley, and G. Woods, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-375, p. 219, 2000.
h
K. Homick, J. B. Myers, L. A. Garringer, D. Amsterdam, and S. J. Zimmerman, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-377, p. 220, 2000.
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
25
26 Leber et al.
Hybrid Capture: Hybrid Capture 2 CT/GC DNA
Tests (Digene Corporation, Gaithersburg, Md.)
Principles of Procedure
The Hybrid Capture 2 (HC2) assays developed by
Digene are nucleic acid hybridization assays with sig-
nal amplification that utilize microplate chemilumi-
nescent detection of C. trachomatis and N. gonor-
rhoeae DNA from endocervical specimens. Three
testing formats are available: CT/GC DNA for the
simultaneous detection of C. trachomatis and N.
gonorrhoeae, CT-ID DNA for detection of C. tracho-
matis, and GC-ID for detection of N. gonorrhoeae
(Digene Corporation, package insert, Hybrid Cap-
ture 2 CT/GC DNA Test Version 2.0, catalog no.
5130-1220, 2001; Digene Corporation, package
insert, Hybrid Capture 2 CT-ID DNA Test Version
2.0, catalog no. 5135-1220, 2001; Digene Corpora-
tion, package insert, Hybrid Capture 2 GC-ID DNA
Test Version 2.0, catalog no. 5140-1220, 2001).
Unlike the other NAATs discussed above, HC involves
a signal amplification rather than target amplifica-
tion. This chemistry, like the other NAAT assays,
achieves improved sensitivity over probe and culture
(150). Specimens containing the target nucleic acid
are denatured to release single-stranded DNA and
then hybridized with RNA probes in solution. The
probe cocktails contain a mixture of complementary
RNA sequences to the genomic and plasmid DNAs.
The CT probes are complementary to approximately
39,300 bp (4%) of the C. trachomatis genomic DNA
and 7,500 bp (100%) of the cryptic plasmid; the NG
probes are complementary to 9,700 bp (0.5%) of the
N. gonorrhoeae genomic DNA and 4,200 bp (100%)
of the cryptic plasmid. There are about 10 copies of
the cryptic plasmid in each C. trachomatis organism,
and 25 per genome in most N. gonorrhoeae isolates,
respectively, hence a natural amplification of signal in
each cell.
The DNA:RNA hybrids are captured onto the sur-
face of a microplate well coated with antibodies spe-
cific for RNA:DNA hybrids. Immobilized hybrids are
then reacted with alkaline phosphatase conjugated
antibodies specific for RNA:DNA hybrids and
detected with a chemiluminescent substrate. Several
alkaline phosphate molecules are conjugated to each
antibody. Multiple conjugated antibodies bind to
each captured hybrid, resulting in substantial signal
amplification. As the substrate is cleaved by the
bound alkaline phosphatase, light is emitted that is
measured as RLUs on a luminometer. The intensity of
the light emitted denotes the presence or absence of
target DNA in the specimen.
Instrumentation
The HC2 CT/GC, CT-ID, and GC-ID assays can be
run in two formats. The manual method involves the
CUMITECH 44
use of a water bath, shaker, plate heater, and lumi-
nometer. For partial automation, the Rapid Capture
System (RCS) is the newest addition to the HC
assays. RCS is a programmable 96-well microplate
processor that integrates liquid handling, shaking,
and washing with software specific to testing HC2
CT/GC. The sample processing is still performed
manually, and after processing on RCS, the micro-
well plates must be manually placed in the lumi-
nometer for reading. Each run of the RCS has the
capacity to test up to four 96-well plates, with 88
wells each for samples plus 8 controls per plate.
However, the flexible format allows any number of
samples to be run from 1 to 352, although maximal
efficiency would be obtained with larger batches.
Sample Collection and Transport
The Digene HC2 CT/GC, CT-ID, and GC-ID DNA
tests are FDA cleared for testing with female cervical
samples collected with the cervical sampler (cervical
brush with specimen transport medium) and the
female swab specimen collection kit (Dacron swab
and specimen transport medium). No other sample
types have received FDA clearance for testing. Speci-
mens are collected after the endocervix is cleaned of
mucus and debris as per other nonculture systems.
Samples may be held up to 2 weeks at room temper-
ature and shipped without refrigeration to the labo-
ratory. At the testing site, the samples may be held at
4°C for an additional 7 days or frozen up to 3 months
before the assay is performed.
Inhibition and Contamination Control
Because HC2 uses signal amplification, no amplicons
are generated as in target amplification techniques
such as PCR, TMA, and SDA. Therefore, the risk of
contamination from amplicons is not present. Care
should be taken, however, to prevent cross-
contamination from sample to sample by utilizing
good laboratory technique and unidirectional work-
flow. Inhibition, as seen with target amplification, is
not a factor in HC2. A specific control for inhibition
of the signal amplification reaction is not included in
the test.
Result Interpretation
The Digene HC2 CT/GC assay is used to screen a
sample for the presence of either or both of the patho-
gens. If positive, the individual HC2 assays, CT-ID
and GC-ID, can be performed to determine whether
C. trachomatis, N. gonorrhoeae, or both are present
in the specimen. The results for each specimen are
generated by the Digene qualitative software based
on the ratio of RLUs for the sample divided by the
cutoff value (RLU/CO). The cutoff value for the test
is the mean number of RLUs of three replicates of a
positive control that is tested in each run. Results are
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae 27

either positive (RLU/CO
1.0) or negative (RLU/CO

Negative predictive
1.0). There are no equivocal or indeterminant

value (%)
results for HC assays.

99.3
99.7

99.7
NA
NA

99
Performance Characteristics
There have been several published studies examining
the performance of HC2 for the detection of C. tra-
chomatis and N. gonorrhoeae. These studies are

Positive predictive
summarized in Tables 10 and 11. Girdner et al. (83)

value (%)

93.3
compared HC2 CT-ID to culture and PCR for the

100
NA
NA

100

98
detection of C. trachomatis from 587 endocervical
samples. A specimen was considered positive if cul-
ture was positive for C. trachomatis or if two of these
tests were positive: HC2 CT-ID, AMPLICOR PCR,

Specificity
or DFA staining. The latter was only performed on

99.2

98.2
99.8

99.8
(%)
discrepant samples that were culture negative but

100

100
positive by HC2 and/or PCR. The Digene CT-ID
assay demonstrated a sensitivity of 95%, a specificity
of 99%, and positive and negative predictive values

Sensitivity

95.6

93.3
97.2

97.7
of 92.5% and 99.4%, respectively. Although much of

(%)

100

97
the processing of the CT-ID in this format was man-
ual, it was well adapted for a high throughput; 90
clinical samples could be processed per microplate in

PACE 2; discrepants with TMA

Culture; discrepants, with DFA

PACE 2; discrepants with PCR

Culture; discrepants with DFA
5 h. In a study comparing the manual HC2 versus the
Recent studies evaluating Digene Hybrid Capture 2 Assays for detection of C. trachomatis

Comparison method
RCS instrument, 352 samples plus 32 controls were
run in a single shift; 3.5 h of that time required no
hands-on involvement from a technologist. The per-

Culture or PCR

Culture or PCR
formance of HC2 RCS was equivalent to that of the
manual HC2. In the same study, the RCS showed
and PCR

and PCR
excellent performance on 330 endocervical samples

F, female; M, male; CX, endocervical swab; UR, urethral swab; PACE 2, Gen-Probe PACE DNA probe.
when compared to PCR and culture for the detection
of C. trachomatis and N. gonorrhoeae. For the detec-
tion of chlamydial infection, HC2-RCS had sensitiv-
Prevalence

4.7; 3.7b
12.6; 7.8b

ity and specificity similar to those of PCR and an

(%)

improved sensitivity compared to that of culture (see

9.6
13.6

13.6

8.4

Tables 10 and 11). For the identification of gonococ-

cal infections, all assays performed similarly (208).
Specimen

In a study by Darwin et al. (60), the HC2 CT/GC

types

CX

UR

combination assay was found to have 94.8% sensi-

CX

CX
CX
CX

Two sites were used in the study with different prevalence rates.

tivity and 99.8% specificity when compared to HC2

CT-ID and GC-ID or culture. Tables 10 and 11 give the
results of using the Digene HC2 CT/GC to detect the
C. trachomatis only
sex, sample site
No. of subjects,

specific pathogens in endocervical specimens. For

N. gonorrhoeae

the detection of C. trachomatis, in relatively high-

All samples

prevalence populations (4 to 14%), sensitivities

1,202, M

93% and specificities of
98% were found in a

1,370, F
1,746, F
669, Fa

330, F

330, F

variety of studies. For N. gonorrhoeae, in populations

with 1% to 15% prevalence, sensitivities
92%
and specificities
98% were found. Excellent predic-
Van Der Pol, 2002c (208)

Van Der Pol, 2002 (208)

Modarress, 1999 (150)

tive values were also found for both pathogens. How-

Schachter, 1999 (176)

Rapid Capture System.

ever, some of these studies compared HC2 to culture

Darwin, 2002 (60)

Darwin, 2002 (61)

Reference

or DNA probe (PACE 2), and not to other NAATs

directly. Although discrepancies were adjudicated
Table 10.

with other NAATs, results for sensitivity may be lower

if direct comparisons are made to available amplifi-
cation assays by testing on all specimens with NAATs.
b
a

c
28 Leber et al. CUMITECH 44

The Digene HC2 CT/GC assays are FDA ap-

Negative predictive
proved for use on female endocervical samples but

value (%)
not for male urethral samples or urine samples from

98.8

99.8
100

100
males or females. However, one study by Darwin et
al. (61) examined their use with 1,202 male urethral
specimens. The authors found that HC2 had excel-
lent performance characteristics comparable to re-
Positive predictive

sults with female genital samples.

The Digene HC2 CT/GC assay might be consid-
value (%)

98.8

99.4
91.3
87.5
ered for use in a laboratory that receives predomi-
nantly female endocervical samples in a relatively
low-prevalence population, in which there would be
few positives that would have to be confirmed by
additional assays. In a population with an overall
Specificity

prevalence of C. trachomatis and N. gonorrhoeae

99.8

99.7
98.5

99.9
99.4
(%)

99

less than 3 to 4%, few GC-ID or CT-ID assays or

other specific tests would have to be performed. On
the other hand, one could process the CT-ID and GC-
Sensitivity

ID assays individually if the volume of samples was

92.2

92.6

98.9
(%)

low, but prevalence higher.

100

100

100

Future Directions
It would be most efficient to be able to perform mul-
PACE 2; discrepants with TMA
Culture; discrepants, with DFA

PACE 2; discrepants with PCR

Culture; discrepants with DFA

tiple tests from a single specimen in addition to C.

Comparison method
Table 11. Recent studies evaluating Digene Hybrid Capture 2 assays for detection of N. gonorrhoeae

trachomatis and N. gonorrhoeae. A study reported in

2000 compared cervical specimens collected in Cytyc
Thin Prep PreservCyt solution to those collected
Culture or PCR

Culture or PCR

using Digene transport medium (K. J. Modarress,

H. E. Richter, J. R. Schwebke, M. Venglarik, B. Cot-
and PCR

or PCR

ton, D. Ball, A. P. Cullen, A. T. Lorincz, and W. J.

F, female; M, male; CX, endocervical swab; UR, urethral swab; PACE 2, Gen-Probe PACE DNA probe.

Payne, Abstr. 100th Gen. Meet. Am. Soc. Microbiol.,

abstr. C-371, p. 218, 2000). Both samples were tested
with the HC2 CT/GC DNA test. Agreement between
Prevalence

3.8; 6.3b

the two specimen types was 98.6% for C. trachoma-

2.8; 0.8
(%)

tis and 98.0% for N. gonorrhoeae. Use of the liquid

6.9
6.4

6.4

14.6

Pap medium would permit testing for human papil-

lomavirus, C. trachomatis, and N. gonorrhoeae from
Specimen

the same sample used for Pap smear preparation.

types

UR
CX

CX
CX
CX

CX

FDA clearance of PreservCyt or other liquid Pap for

Two sites were used in the study with different prevalence rates.

testing with the HC2 assays is still awaited along

330, F; only C. trachomatis/

with FDA clearance for male urethral and male and

female urine specimens.
sex, sample site
No. of subjects,

N. gonorrhoeae

New Technologies and

All samples

Laboratory-Developed Assays
1,202; M
1,746, F

As with other areas of laboratory medicine, nucleic

669, Fa

1357, F
330, F

acid-based techniques have had a major impact on

and will continue to change the way testing is done.
The most significant developments over the past
Van Der Pol, 2002c (208)

Van Der Pol, 2002 (208)

Modarress, 1999 (150)

decade for the detection of chlamydia and gonorrhea

Schachter, 1999 (176)

Rapid Capture System.

infections have involved NAATs. More work is

Darwin, 2002 (60)

Darwin, 2002 (61)

Reference

needed to improve their performance, validate novel

specimen types, and increase automation of testing and
standardization of sample processing. However, there
are numerous publications on new innovations and
technologies for the detection of these organisms.
b
a

c
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Some may be used in development of next-generation
commercial assays and some may be used in labora-
tory-developed tests. Most clinical laboratories in the
United States use one of the FDA-cleared NAATs;
however, if a laboratory chooses to develop its own
assay, the analytical performance characteristics
must be fully verified.
Real-Time PCR
Real-time PCR involves the amplification and detec-
tion of nucleic acid sequences in a closed system (49,
139). Amplicons are generated and detected during
the reaction process, as opposed to postamplification
end-point detection used in conventional techniques.
Real-time PCR assays generally utilize fluorescent re-
porter molecules, and the amount of reporter signal
detected increases proportionally with the number of
amplicons generated. Multiple detection chemistries
exist, including hybridization probes (LightCycler
probes; Roche Molecular Systems, Indianapolis,
Ind.), hydrolysis probes (TaqMan probes; Applied
Biosystems, Foster City, Calif.), molecular beacon
probes, minor groove-binding probes, and double-
stranded DNA-binding dyes such as SYBR green.
There are also several instrumentation platforms pro-
duced by companies such as Roche Diagnostics
(LightCycler and COBAS TaqMan); Cepheid, Sunny-
vale, Calif. (SmartCycler); Applied Biosystems, Fos-
ter City, Calif. (7000, 7500, 7700, and 7900HT);
Corbett Research, Sydney, Australia (Rotor-gene);
Bio-Rad Laboratories, Hercules, Calif. (i-Cycler); and
others.
Several publications have reported the use of real-
time PCR for the detection of C. trachomatis and N.
gonorrhoeae (22, 66, 123, 141, 195, 224). Koenig
et al. (123) compared the BD ProbeTecET C. tracho-
matis and N. gonorrhoeae assay (Becton Dickinson)
to a laboratory-developed real-time PCR assay tar-
geting the portion of the ccp gene present on a cryp-
tic plasmid of N. gonorrhoeae and the 7.5-kb cryptic
plasmid of C. trachomatis. In specimens initially pos-
itive for N. gonorrhoeae by BD ProbeTec, the overall
agreement between the two systems for N. gonor-
rhoeae detection from 155 clinical specimens was
only 77.4%, with agreement particularly low
(24.1%) when the BD ProbeTec MOTA scores
ranged from 2,000 to 19,999. Results for C. tra-
chomatis showed a 91.2% agreement when 114 clin-
ical specimens were tested. The agreement between
the two systems improved to 96% when only MOTA
scores of
30,000 were retested by the laboratory-
developed assays. The authors proposed an algorithm
for selective repeat testing with the real-time PCR
assay for samples giving equivocal BDP C. tracho-
matis results and N. gonorrhoeae results with MOTA
scores of 2,000 to 9,999.
29
Boel et al. (22) compared the use of two laboratory-
developed real-time PCR (LightCycler) assays and
two EIA-based PCR assays for the confirmation of
positive COBAS N. gonorrhoeae results. Of 765 male
and female urogenital and nasopharyngeal specimens
positive for N. gonorrhoeae in the COBAS assay,
only 229 (30%) were confirmed positive. Some of
the samples were determined to contain Neisseria
species other than N. gonorrhoeae. Of the two target
regions examined (16S rRNA and ccp cryptic plas-
mid), only the 16S rRNA target was reliable. With
the cppB assays, the authors found that 5.7% of the
N. gonorrhoeae positive samples lacked the gene,
giving a false-negative results. Both studies described
above demonstrate that real-time PCR assays may be
useful for confirming positive results obtained with
one of the FDA-cleared tests.
NASBA
Nucleic acid sequence-based amplification (NASBA)
(bioMérieux, Inc., Durham, N.C.) is a transcription-
based amplification method that amplifies RNA
from either an RNA or DNA target (36, 121). It is an
isothermal process, similar to TMA, that relies on the
simultaneous activity of three enzymes: avian myo-
blastosis virus-reverse transcriptase, RNase H, and
T7 polymerase. Real-time detection can be performed
with molecular beacon probes, or electrochemilumi-
nescence technology can be used in a postamplifica-
tion detection step.
Several publications have reported the use of
NASBA for the detection of C. trachomatis. Morre
et al. (153), using two different primer sets in NASBA,
found it was more sensitive than conventional PCR
methods. Using both the cryptic plasmid and omp1
targets of C. trachomatis, NASBA gave a limit of
detection (LOD) of 1 inclusion-forming unit (IFU),
whereas the 16S rRNA target gave even better sensi-
tivity with a LOD of 103 IFU. This compared to the
LOD of 102 IFU for the most sensitive PCR with
primers to the plasmid DNA. In another study, Morre
et al. (154) compared the use of 16S rRNA NASBA
and cryptic plasmid-based PCR for their ability to
monitor the effect of antibiotic treatment in women
with genital C. trachomatis infection. The RNA tar-
get was found up to 2 weeks following antimicrobial
therapy in cervical and urine samples compared to
DNA, which was found in some samples for up to 3
weeks. NASBA was more effective in monitoring test
of cure than DNA PCR, which can be present in non-
viable organisms and detectable 3 to 4 weeks after
successful treatment has ended (216).
Mahony et al. (142) compared NASBA for C. tra-
chomatis and N. gonorrhoeae 16S rRNA to C. tracho-
matis PCR and N. gonorrhoeae culture and showed
that the test has excellent analytical sensitivity and
30 Leber et al.
clinical performance characteristics. The authors
found an LOD of 1 IFU for C. trachomatis and 1 CFU
for N. gonorrhoeae with the NASBA assays on bac-
terial stock solutions and 100 copies for 16S rRNA
transcript constructs for both C. trachomatis and
N. gonorrhoeae. When testing clinical specimens,
NASBA resulted in sensitivity and specificity of 100%
and 98.6%, respectively, for C. trachomatis; for
N. gonorrhoeae, the NASBA had sensitivity and
specificity of 97.9% and 98.7%.
Although NASBA looks to be a promising tech-
nology for the detection of C. trachomatis and N.
gonorrhoeae, there is currently no FDA-cleared assay
available, and the development of such an assay does
not appear to be on the near horizon. A NucliSens
Basic Kit is available that contains all quality-
controlled reagents and standardized procedures for
RNA release, isolation, amplification, and detection.
Users can develop their own assays by designing
primers and probes for targets of interest such as C.
trachomatis and N. gonorrhoeae.
Multiplex Testing and Microarrays
Testing for multiple organisms from a single sample
type, or multiplexing, is growing in popularity in
clinical laboratories. Detection of C. trachomatis and
N. gonorrhoeae from a single sample makes sense
because the organisms share a mode of transmission,
have similar risk profiles, and infect similar body sites.
These facts have led to NAATs that combine testing
for both organisms. Additional sexually transmitted
disease agents may be detectable from these same
samples, although care must be taken to ensure that
the infecting organism’s nucleic acid can be found in
the same specimen collection site. For example, test-
ing of endocervical swabs for Trichomonas vaginalis
may not be appropriate since the organism infects
primarily the vagina and ectocervix. One sample may
not fit all situations.
Through innovations such as microarray technol-
ogy, multiplexing is achievable for the simultaneous
detection of numerous microorganisms (21, 27, 29,
62, 191, 213). A microarray is a solid support, such as
a glass slide, that is used as a platform for immobi-
lization of nucleotide sequences. These nucleotides act
as probes for complementary nucleic acid sequences
in the specimen. Many hundreds of probes can be
deposited on one slide with great precision with
proper instrumentation. Further development is
needed before microarray technology is useful in the
routine clinical laboratory for diagnosis of disease.
This so-called “lab on a chip” technology may dra-
matically change the way clinical microbiology is
performed by allowing a rapid and comprehensive
screening approach to testing for infectious diseases.
CUMITECH 44
INTERPRETATION AND
REPORTING OF RESULTS
Current commercially manufactured amplified meth-
ods for detecting C. trachomatis and N. gonorrhoeae
nucleic acid generate qualitative results; the result is
positive, negative, and, in some cases, equivocal. The
package inserts for these tests contain instructions
for the interpretation of results and serve as the guide-
lines for reporting. Laboratories should consider
reporting results as “detected” or “not detected” for
the organism’s nucleic acid instead of positive or neg-
ative (i.e., C. trachomatis DNA detected) (157) along
with indicating, either in the test name or in the
result fields, that a NAAT was used to generate
results. This emphasizes that a negative result does
not rule out infection and may be due to limitations
in assay sensitivity or the presence of inhibitors in the
sample that led to a falsely negative result. Also, with
a report of “detected,” the presence of the organism’s
nucleic acid versus viable organisms is emphasized
and must be correlated with the clinical picture to
determine whether active disease or infection is pres-
ent. For assays with an internal amplification control,
the presence of inhibitors can be detected and thus
prevents reporting of false-negative results. Inclusion
of additional information on the report is also useful
to aid clinicians in interpreting tests. See “Reporting
Comments” below.
When they are used as a screening test, all positive
tests for C. trachomatis and N. gonorrhoeae NAATs
should be considered presumptive evidence of infec-
tion (33). Because positive results can have signifi-
cant adverse consequences and psychological effects,
consideration must be given to the specificity of the
methodology and the prevalence of disease in the
population. The PPV of the result for the individual
patient is influenced by both factors. As is discussed
later in this Cumitech, in low-prevalence populations
a positive result for C. trachomatis and/or N. gonor-
rhoeae may not reflect infection. For N. gonorrhoeae
cross-reactivity between the pathogenic and non-
pathogenic Neisseria species has been demonstrated
for some NAATs that can affect specificity (145, 206)
(Becton Dickinson, BD ProbeTecET C. trachomatis
and N. gonorrhoeae amplified DNA assays, package
insert L000203, 1999). However, these nonpatho-
genic species are rarely isolated from urogenital sites.
Cross-reactivity among chlamydia species in NAATs
has not been noted. Because of concerns about speci-
ficity and PPV, additional testing after a positive
screening test may be necessary in certain popula-
tions to ensure the reliability of a positive result (33).
Equivocal results mean that the presence or absence
of the organism’s nucleic acid cannot be determined.
Testing of a second sample is recommended.
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Test of Cure
After initial diagnosis of chlamydial and gonococcal
infections, test of cure is not recommended for most
patients treated with recommended first-line antimi-
crobials. Exceptions include cases of suspected treat-
ment failure, noncompliance or reinfection, and preg-
nant women treated for C. trachomatis infection (35).
If NAAT is done for test of cure for C. trachomatis
infection, specimens should be collected
3 weeks
following the end of treatment. NAAT done before 3
weeks may be falsely positive due to continued shed-
ding of nonviable organisms (79, 154, 225). Culture
should be used for test of cure following treatment of
N. gonorrhoeae infections (35). This practice allows
susceptibility testing in cases where agents with
known resistance are used to treat gonorrhea.
Children and Sexual Abuse or Assault Cases
The populations used to evaluate NAATs for C. tra-
chomatis and N. gonorrhoeae for FDA clearance
include mostly sexually active adolescents and adults
(
13 years of age). These tests are not approved for
rectogenital testing in children, and their perfor-
mance is not clearly established in this population
(94). Because the prevalence of infections in children
is expected to be lower than that in adults, the pre-
dictive value of positive results may be very low.
To date, culture of C. trachomatis and N. gonor-
rhoeae remains necessary for medicolegal purposes.
With specificities of nearly 100%, culture is still con-
sidered the gold standard for issues such as child sex-
ual abuse and rape (94, 95). Protocols need to be
written and distributed to personnel where specimens
from cases of sexual abuse may be collected. Use of
nonamplified methods may miss some cases of sexual
abuse; however, the implications of false-positive
reports are paramount. In some instances, a NAAT
may be requested in addition to culture to ensure
maximal sensitivity. Further studies are needed to
establish the utility of the amplified tests in the set-
ting of child sexual abuse.
Reporting Comments
Suggested reporting comments for C. trachomatis
and N. gonorrhoeae testing are given in Table 12.
These comments are designed to communicate reasons
for the rejection of specimens and special considera-
tion for unusual circumstances. In most situations,
specimens not collected in the manner described in
the manufacturer’s package insert should be rejected
unless these sample types have been validated by the
testing laboratory. This also applies to sample types
not included in the package insert. Federal regula-
tions require all clinical testing to be performed only
after the performance characteristics of the test have
been validated, either by a kit manufacturer (and ver-
31
ified by the clinical laboratory) or by the clinical lab-
oratory itself in the case of a user-developed assay or
a modification of a marketed assay (50).
QUALITY CONTROL
Quality control is important for all laboratory tests,
but especially for NAATs due to their exquisite sensi-
tivity and the risk of contamination. Because of the
possible social and psychological ramifications of
reporting a positive N. gonorrhoeae or C. trachoma-
tis result, and the requirement to report results to state
health departments and partners of infected individ-
uals, care needs to be taken to ensure the accuracy of
results at all times. A study of five laboratories per-
forming C. trachomatis NAATs on urine samples
from asymptomatic men has shown variance within
and between laboratories in the performance of sim-
ilar assays. The authors recommend that strict adher-
ence to quality control, proper training, and contin-
uous monitoring of test performance characteristics
are essential (112). Several key points about quality
control for C. trachomatis and N. gonorrhoeae
NAATs are discussed below. The reader is referred to
the recent National Committee for Clinical Labora-
tory Standards (now named Clinical and Laboratory
Standards Institute, Wayne, Pa.) document MM3-A
for a useful and complete review of guidelines for
quality control in molecular infectious disease diag-
nostics (157).
Kit Controls
The manufacturer’s directions in the package insert
should be followed for the number and frequency of
controls necessary for each of the NAAT assays. In
general, positive and negative controls are included
with each batch of specimens that is being tested for
each of the two pathogens. All controls must perform
as expected for the results to be acceptable for re-
porting.
Roche COBAS AMPLICOR: a positive and negative
control is run with each A-ring. The C. trachomatis
and N. gonorrhoeae positive control materials are
noninfectious plasmid DNA containing sequences
specific for each organism. The C. trachomatis-
positive control serves as the N. gonorrhoeae-negative
control; the N. gonorrhoeae-positive control serves as
the C. trachomatis-negative control. An internal inhi-
bition control is provided, which is plasmid DNA
containing the C. trachomatis primer-binding se-
quences and a unique probe-binding region.
BD ProbeTec ET: a positive and negative control is
included with each test run. The positive control con-
tains C. trachomatis and N. gonorrhoeae linearized
plasmid DNA; the negative control contains salmon
testes DNA. An amplification control is provided,
32 Leber et al. CUMITECH 44

Table 12. Suggested reporting comments for C. trachomatis and N. gonorrhoeae NAAT results

Sample Action Report comment References

type/situation

C. trachomatis or Report result: Additional testing is recommended 33

N. gonorrhoeae detected after an initial positive screening test if
nucleic acid a low positive predictive value can be
detected expected, such as in low-prevalence
populations, or if a false-positive result
would have serious psychosocial or
legal consequences.
C. trachomatis or Report result: A result of not detected does not
N. gonorrhoeae not detected eliminate the possibility of infection.
nucleic acid not
detected
C. trachomatis or Report result: Results are inconclusive. Please submit a
N. gonorrhoeae equivocal new specimen for repeat or alternate
nucleic acid testing if clinically indicated.
equivocal
Inhibition present Report result: A valid result cannot be determined. Roche Diagnostics, package insert,
inhibition Inhibition may be due to lubricants, COBAS AMPLICOR CT/NG Test for
noted mucus, blood, or other substances. Chlamydia trachomatis, Revision 1.0,
Please submit a new specimen for 1999; Roche Diagnostics, package
repeat or alternate testing if clinically insert, COBAS AMPLICOR CT/NG Test
indicated. for Neisseria gonorrhoeae, Revision 3,
1999; Becton Dickinson, BD ProbeTec
ET Chlamydia trachomatis and Neisse-
ria gonorrhoeae amplified DNA assays,
package insert L000203, 1999
Sample not collected Reject sample Testing not performed. Specimen was not Alternate specimen collection procedures
according to manu- collected properly. (Additional descrip- must be verified by laboratory if not
facturer’s directions tion of inappropriate collection may be included in FDA-cleared package insert
added such as “Large cleaning swab (50).
submitted.”)
Sample type other Reject sample Testing not performed. Sample type is not Sample types must be validated by lab-
than those included acceptable for testing. (Recommend oratory if not included in FDA-cleared
in manufacturer’s alternate testing, such as culture, for the package insert (50).
package insert specimen if appropriate.)
Vaginal sample from Reject sample or Interpret results with caution. This sample Women with hysterectomies are capable
hysterectomized consult with type has not been validated by the of acquiring and transmitting STDs (39,
patient clinician before manufacturer or the laboratory. 122).
testing
Sexual assault or Reject sample or Culture is the recommended method Data, experience, and court cases are
abuse consult with for detecting C. trachomatis and N. insufficient to assess the applicability of
counsel or gonorrhoeae in urogenital, pharyngeal, NAATs to detect C. trachomatis or N.
clinician before and rectal specimens in cases of sexual gonorrhoeae in investigating sexual
testing abuse or assault. assault and abuse (33).
Patient age less than Reject sample Nucleic acid amplification testing for C. 33, 94
13 years or trachomatis (or N. gonorrhoeae) has not
prepubescent/ been validated for testing and is not
menarche recommended for prepubertal children.
Culture is the recommended test for
this population.

which functions as the inhibition control, and con-
sists of the same linearized plasmid used for the N.
gonorrhoeae-positive control.
Gen-Probe APTIMA: a positive and negative control
is included with each test run. The C. trachomatis- and
N. gonorrhoeae-positive control materials are nonin-
fectious RNA containing sequences specific for each
organism. The C. trachomatis-positive control serves
as the N. gonorrhoeae-negative control; the N.
gonorrhoeae-positive control serves as the C.
trachomatis-negative control. No inhibition control
is provided due to use of target capture for sample
processing.
Digene Hybrid Capture 2: a negative control and
positive control is included in each test run. The C.
trachomatis and N. gonorrhoeae controls are cloned
C. trachomatis and N. gonorrhoeae DNA targets,
composed of the same plasmid construct for each
individual organism. The negative control contains
carrier DNA. No inhibition control is provided.
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
The IC, if provided, should be employed as directed.
If the IC is negative (i.e., suggesting that inhibitory
substances are present in the sample), and the sample
is negative for one or both analytes, the negative
results should not be reported. Rather, the specimen
should be rerun after being frozen, refrigerated, or
diluted. If, after the second run, the amplification
control is positive, indicating no inhibition, then re-
sults can be reported as positive or negative for the
test sample and the analyte in question. If there is
noted inhibition and the analytes are positive, the
positive sample results can be reported without re-
peat testing. Consideration can be given to omitting
the IC if the laboratory demonstrates an acceptably
low rate of inhibition in a given sample type (157).
External Controls
For all NAATs, it is advisable to test external control
material to monitor assay performance. The controls
provided in commercial kits are nucleic acid
sequences and do not represent whole organisms (see
above). Therefore, they do not represent specimen
matrices, such as urine or cervical cells, containing
infectious organisms. To adequately control for all
steps of the NAAT, including specimen processing
and extraction, an additional whole organism con-
trol in a suitable matrix is advisable. These controls
should be taken through all steps of the procedure
(157). A number of companies manufacture whole
organism controls for use as positive external con-
trols. Such controls should be at concentrations in the
low range of assay to challenge assay sensitivity and
not serve as a source of contamination (157).
Positivity Rate
To avoid false-positive results due to contamination,
it is advisable to set up a system in the laboratory
whereby data are collected to determine the preva-
lence of each pathogen and hence the number of pos-
itive results to be expected per day, per run, or by
some other appropriate parameter. Once the ex-
pected positivity rate is determined, results should be
checked each day to determine if that limit has been
exceeded. If it has, steps should be taken to investi-
gate and correct any problems that are uncovered.
For example, if there are more male genital samples
on a particular day, one might expect a higher num-
ber of positive results due to the higher rate of symp-
tomatic infections in this population. Also, the tally
system should exclude results for repeat testing of
positives. Such results are included for counting only
in the initial run. If an obvious explanation is not
found for an elevated positivity rate, other methods
of tracing could involve repeat testing, making physi-
cian calls to determine the likelihood of a positive
result, and within the laboratory, checking on where
33
the positives were found in relationship to each other
and to the positive control on the testing run. Return-
ing to the initial specimen and reprocessing would be
appropriate, but the possibility of contamination of
the original specimen must be considered. Request-
ing additional samples from the patient is always an
option, although in many cases, this is not possible or
causes hardships on both patient and physician. If
any repeat testing is required because of possible lab-
oratory error, the patients should not be charged for
the additional assay(s).
Proficiency Testing
Proficiency testing is required for all laboratories and
determines what testing can be performed (50). Pro-
ficiency programs should be designed to make certain
reliable and reproducible results are obtained and to
evaluate the skill of the personnel performing testing.
Comparison to other laboratories also participating
in proficiency programs allows the credibility of
results to be determined. External proficiency testing
is available via agencies such as the College of Amer-
ican Pathology (CAP, Northfield, Ill.). A CAP survey
is available for C. trachomatis and N. gonorrhoeae
amplified testing, and all laboratories performing
this testing should participate. For further details, see
“Considerations for Laboratories.” Additionally, an
internal blind testing program may be considered in
addition to external programs. Blind retesting of
specimens allows assessment of competency for indi-
vidual technicians as well as the analytical perfor-
mance of the test.
Contamination Concerns
Use of any of the NAATs demands strict control
procedures to prevent contamination of specimens
(see “Considerations for Laboratories Performing
NAATs” below). This can include the use of various
chemical or physical controls. An example of chemi-
cal control is uracil-N-glycosylase (AmpErase) in the
reaction as is found in the Roche AMPLICOR prod-
ucts, and closed amplification systems such as real-
time amplification represent physical control mea-
sures. In addition, designated areas for specimen
processing, amplification setup, and amplification/
detection and strict adherence to unidirectional work-
flow are mandatory (148).
CONSIDERATIONS FOR LABORATORIES
PERFORMING NAATs
A variety of technical and procedural issues must be
considered before implementing nucleic acid amplifi-
cation testing for C. trachomatis and N. gonorrhoeae
(Table 13). These issues include the physical limita-
tions of laboratory space, availability of equipment
34 Leber et al. CUMITECH 44

Table 13. Considerations for laboratories performing nucleic acid amplification testing for C. trachomatis and
N. gonorrhoeae

Patient and client Laboratory (continued)

Population of patients Contamination control
Type of populations to be tested Unidirectional workflow
High-risk/symptomatic Separation of pre- and postamplification area
Screening/asymptomatic Decontamination of equipment and materials
Prevalence Contamination monitoring
Acceptable specimen sources Result interpretation and reporting
Ease of sample collection Acceptance/rejection criteria
Invasive versus noninvasive collection procedures Improper collection
Client education Testing in children
Sample collection and transport Sexual abuse
Result interpretation Nonvalidated sample types
Turnaround time requirements Result and rejection messages
Laboratory Confirmatory testing for positive samples
Physical facilities Quality control
Space requirements Verification and validation of assay performance
Electrical and other requirements Tracking of positivity rates
Location within the laboratory Monitoring for contamination (wipe testing)
Air flow External control material
Storage of reagents Proficiency testing
Storage of tested specimens External programs (CAP)
Waste disposable Internal programs
Equipment Financial considerations
Manufacturer provided equipment Cost per test
Additional equipment and material requirements Labor costs
Pipetters Reimbursement
Barrier pipette tips FDA-cleared kit
Incubators Laboratory-developed (home brew) assay
Refrigerator/freezers Research use only
Preventive maintenance Investigational use only reagents
Instrument throughput Payer mix
Interface between instrument and lab information system Cost-versus-benefit analysis
Personnel
Level of expertise
Training
Competency assessment

for assay performance, training and maintaining staff
competence, selection of an assay platform(s), and
client-related issues. Education of clients concerning
proper specimen selection, collection, handling, and
transport is important. Choice of a particular NAAT
may also be influenced by patient population; clini-
cal performance is influenced by prevalence of dis-
ease. In addition, ease of collection and patient pref-
erence as to acceptable specimen types should be
considered.
The sensitivity of NAATs dictates that contamina-
tion control be a top priority when designing or
restructuring existing laboratory space. The perfor-
mance of NAATs can be separated into four steps con-
sisting of reagent preparation, sample preparation,
amplification, and detection. In a perfect setting, these
steps would be carried out in separate rooms, but in
laboratories with limited space, configuration of these
areas as individual stations within one or two rooms
is practical. Each laboratory area should be labeled,
and each step of assay performance from specimen
handling to personnel movement should provide for
unidirectional workflow from preamplification to
detection areas. Physical separation of preamplifica-
tion and postamplification areas and control of air-
flow are primary mechanisms of protecting against
product carryover from aerosol contamination (148).
Other methods to prevent contamination include en-
vironmental monitoring for amplicon and regular de-
contamination of work surfaces. Decontamination
can be accomplished with bleach solutions, enzy-
matic inactivation of amplified target by uracil-N-
glycosylase, or surface irradiation with UV light (148).
Each NAAT approved for C. trachomatis and N.
gonorrhoeae testing has its own specific instrumenta-
tion provided by the manufacturer. Instrumentation
unique to each assay must have regular preventive
maintenance and a quality control program estab-
lished to ensure proper instrument function. All
instrument activities involved in assay performance
must be monitored as prescribed by the manufacturer.
Temperature-dependent equipment should be moni-
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
tored and values recorded daily. Pipettors should be
calibrated at specified intervals to ensure precision
and accuracy. Use of aerosol barrier pipette tips is
mandatory to prevent cross-contamination between
specimens. Biological safety cabinets must be certified
and calibrated at least annually. Cabinets as well as
dead air boxes should be decontaminated at the start
and end of each working day or immediately when a
spill occurs.
To prevent amplicon carryover, dedicated equip-
ment should be available for each work area (148).
Examples include water baths, incubators, heating
blocks, and pipettors. Equipment or materials that
can be moved should be clearly marked so that if they
do get moved, it is obvious and appropriate decon-
tamination can be performed. Items such as pens,
pencils, and radios are often overlooked but should
also be properly handled to prevent contamination.
Although NAATs for the diagnosis of C. tracho-
matis and N. gonorrhoeae have been available for
some time, they remain largely manual procedures.
The skill of the individual performing the testing is
critical to the quality of the results. Personnel chosen
to perform this type of testing should have good orga-
nizational skills, be detail oriented in their approach
to work, and be well versed in aseptic and molecular
technique. It is the laboratory director’s responsibil-
ity to ensure that all personnel have the appropriate
training, experience, and education for the type and
complexity of testing performed (50). Because NAATs
are classified as highly complex procedures, the com-
petency of individuals performing these tests must be
verified semiannually for the first year and docu-
mented annually thereafter (50, 68). The CAP also
requires that laboratories have an adequate training
program for new technologists and should have a con-
tinuing medical laboratory education program for the
technical staff. These requirements are listed in the
CAP molecular pathology checklist and outlined else-
where (13, 52, 146, 203, 217, 228). All laboratories
should participate in a formal proficiency test pro-
gram. The CAP program provides challenges for both
C. trachomatis and N. gonorrhoeae NAATs. The sur-
vey, HC6, contains five challenges per shipment and
is issued three times per year. Samples representing
both swab and urine samples are included. Internal
proficiency testing with previously characterized pos-
itive and negative patient samples is useful in demon-
strating consistency of results among laboratory staff
members. Internal and external proficiency testing
should be rotated among all staff members and be
handled in the same manner as clinical specimens.
New-hire training, proficiency testing, annual compe-
tency assessment, and ongoing continuing education
programs ensure that quality results are consistently
obtained.
35
Once the decision has been made to implement
NAATs for the diagnosis of C. trachomatis and N.
gonorrhoeae, the laboratory must choose a platform.
Laboratories must acknowledge that the cost of
NAATs will be greater than that of culture or noncul-
ture tests for either of these organisms. Depending on
the payer mix, reimbursement may offset the higher
cost per test. NAAT manufacturers have different
requirements for laboratory space, separation of
work areas, and other equipment. The type of testing
performed (e.g., screening asymptomatic individuals,
confirmatory testing, use in epidemiologic studies, or
strictly diagnostic purposes) should be considered
before selection (33). The sensitivity and specificity
of all of the NAATs described are comparable; how-
ever, their performance with different specimen types
may vary. A laboratory must also consider the pre-
dominant specimen types to be tested to adjust spec-
imen preparation time and workflow accordingly.
Throughput of the various NAATs will differ from
system to system based on the instrumentation’s use of
amplification controls. Laboratories must also con-
sider client turnaround time requirements, technician
time, and maintenance.
All clients must be instructed in proper specimen
collection and transport techniques. Laboratories
should supply clients with informational materials
concerning specimen selection, specimen collection,
the use of specific transport devices for a particular
assay, and the appropriate transport conditions.
Clients must be informed that strict adherence to
these requirements will ensure optimal assay per-
formance.
VERIFICATION OF A TEST METHOD
When a laboratory has selected a test to replace an
existing method, or implements a new method, for
diagnosing C. trachomatis and N. gonorrhoeae infec-
tions, it must demonstrate that it can meet the ana-
lytic system requirements described by Centers for
Medicare and Medicaid Services (50). Before report-
ing patient results with an FDA-cleared NAAT, the
laboratory must demonstrate that it can obtain
results comparable to those described in the manu-
facturer’s package insert. The performance character-
istics compared include accuracy, precision, and
reportable range (50). Results should be compared to
the manufacturer’s package insert claims and evalu-
ated accordingly (180). These guidelines do not apply
to any test system used in a laboratory before 24
April 2003. Recommendations for conducting stud-
ies to evaluate C. trachomatis and N. gonorrhoeae
tests have been reviewed previously (8, 33).
If a laboratory chooses to modify an FDA-cleared
test system or uses a system for which performance
36 Leber et al.
specifications are not provided by the manufacturer,
before reporting results they must establish and main-
tain documentation of the performance characteris-
tics for the following: accuracy, precision, analytical
sensitivity, analytical specificity to include interfering
substances, reportable range, reference intervals, and
any performance characteristic required in test per-
formance (50).
The traditional reference test for determining C.
trachomatis and N. gonorrhoeae infection is culture.
There are many inherent variables in the perfor-
mance of culture for these agents. The necessity for
maintaining viability, transport issues, and lack of
standardization affect culture results for each of these
organisms. Unless a culture has been contaminated,
it is assumed that culture is 100% specific. Demon-
stration of chlamydial inclusions stained with mono-
clonal anti-C. trachomatis antibodies or isolation of
N. gonorrhoeae from an infected site with confirma-
tion by biochemical testing is a fairly clear demon-
stration of infection. Culture for these agents is less
than a perfect standard as the sensitivity is generally
less than 100% because of the variables mentioned
above. The specificity of many of the early noncul-
ture tests was underestimated as these methods were
compared against less sensitive culture techniques.
Likewise, increased sensitivity of nonculture methods
may be expected if the new procedure is more likely
to detect culture-positive rather than culture-negative
specimens (16).
There are many evaluations of NAAT performance
characteristics in the literature. Many of these use a
sample that determines whether a subject is infected
at a single anatomic site rather than true infection sta-
tus. The use of highly sensitive methods allows the
laboratory to more accurately define infection status
by testing specimens from different anatomic sites,
e.g., endocervical and urine. Estimates of test sensi-
tivity are probably overestimated when the standard
for infection is based on a positive test from a single
specimen type rather than multiple sites because use
of multiple samples identifies more infections (33,
179, 205, 207). The infected patient is a more appro-
priate standard for evaluation of the performance of
NAATs rather than a specimen-based standard.
It is expected that NAAT sensitivity will exceed
that of culture or other nonamplified nonculture pro-
cedures. These apparent false-positive NAATs may
be subjected to supplemental testing to determine
their true status. This discrepant analysis is applied
to identify true positive infections that were missed
by a less sensitive standard. Discrepant analysis has
been used to arbitrate chlamydia laboratory tests for
many years (181, 182). Proponents of discrepancy
testing argue that estimates of sensitivity and speci-
ficity based on discrepant analysis are less biased
CUMITECH 44
than those based on traditional gold standards. How-
ever, use of discrepant analysis has been criticized as
biased in favor of new tests (91–93).
Cumitech 31 describes the protocol for verifica-
tion of tests that are performed as described in the
manufacturer’s package insert (67). The Clinical
Laboratory Improvement Amendments mandate 30
comparisons in order to validate a new test method-
ology. It is suggested that the minimum number of
paired specimens that should be run in parallel with
the existing test or reference method is that number
that would give at least 20 positives and 50 negatives
(67). For endocervical specimens, a swab should be
obtained for each test and placed in the transport
medium approved by the manufacturer. For urine,
the appropriate number of specimens from patients
of each gender should be obtained for at least 10 pos-
itives from each gender. It may be appropriate to
obtain fewer positives if the test has been verified for
other specimen types (50). For test reproducibility, it
is suggested that at a minimum 10 to 20 specimens
should be repeated. The tests selected should repre-
sent all possible types of results, both positive and
negative, and include specimens near the assay cutoff.
A laboratory’s comparison of commercially avail-
able NAATs may be complicated by the necessity to
collect multiple patient samples, which might require
approval from an internal review board and in-
formed consent. In addition, each of these assays has
its own approved transport. Currently, the user of any
amplification system is locked into using the manu-
facturer’s specific transport device. Use of a transport
device specific for a particular NAAT in another test
system represents an off-label use and must be subject
to the verification procedure described above. Once
the transport is validated, ongoing quality control
should be performed. Each new lot of the new trans-
port type should be tested with positive and negative
controls to ensure the consistency of the procedure.
The use of a universal type of nucleic acid transport
device would be more appropriate as laboratories,
particularly those serving a large diverse client base,
may receive more than one transport device for the
particular NAAT that is offered in-house. Transport
media such as M4 and M4RT (Remel, Lenexa,
Kans.) and 2-SP and FlexTrans (Bartels) wet and dry
transports have been validated with different ampli-
fication systems for C. trachomatis and N. gonor-
rhoeae (7, 65, 76, 80, 120, 150; J. Harrison, R. Saut-
ter, and W. LeBar, Abstr. 98th Gen. Meet. Am. Soc.
Microbiol., abstr. C-48, p. 139, 1998; R. Kendrick,
R. Sautter, W. LeBar, and T. Rudolph, Abstr. 15th
PASCV Meet., abstr. S34, 1999; J. Harrison, M.
Garassi, S. Wierzbicki, and W. LeBar, Abstr. 104th
Gen. Meet. Am. Soc. Microbiol., abstr. C-286, p. 176,
2004; J. Harrison, M. Garassi, S. Wierzbicki, and W.
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
LeBar, Abstr. 20th PASCV Meet., abstr. TM25, 2004;
G. S. Hall, J. Tuohy, T. Katanik, D. Wilson, and G. W.
Procop, Abstr. 102nd Gen. Meet., Am. Soc. Micro-
biol., abstr. C-162, p. 129, 2002). Recently, a device
called the Nucleic Acid Transport from Medical Pack-
aging Corporation (Camarillo, Calif.) was approved
for collection and transport of samples for testing for
C. trachomatis and N. gonorrhoeae in endocervical
and urethral samples with the BD ProbeTec ET,
Roche COBAS AMPLICOR, and Gen-Probe PACE 2.
GUIDELINES FOR SCREENING
AND TESTING FOR INFECTION
Symptomatic Testing
Current recommendations for patient management
include testing for C. trachomatis and N. gonor-
rhoeae in all symptomatic individuals with the most
sensitive and specific test available (35). For C. tra-
chomatis infections, NAATs are preferred for testing
urogenital specimens because of the high sensitivity
relative to other tests. The preferred test for N. gon-
orrhoeae is culture for female endocervical specimens
if collection and transport of the inoculated media
are adequate to maintain viability. Culture allows
monitoring for antimicrobial resistance if the patient
fails therapy. A Gram-stained smear for males with
urethral discharge with culture backup gives ade-
quate performance for the detection of N. gonor-
rhoeae in most instances (33).
Asymptomatic Screening
Annual screening of all sexually active women under
the age of 26 for C. trachomatis infection has been
recommended by the CDC, the U.S. Preventive Ser-
vices Task Force, and a variety of medical organiza-
tions (35, 53, 99, 159). Screening is also indicated for
all pregnant women and older women with risk fac-
tors, such as sexual contact with an untreated part-
ner or sex with a new or multiple partners. Follow-
up screening, at 3 to 4 months after antimicrobial
treatment, is recommended because of the high rate
of reinfection in women who have had a C. tra-
chomatis infection in the preceding several months
(35). This rescreening is distinct from test of cure, a
practice that is not routinely recommended.
Screening for N. gonorrhoeae infection is recom-
mended in women at high risk of sexually transmitted
diseases and in pregnant women with high risk or liv-
ing in areas with a high N. gonorrhoeae prevalence
(35). The U.S. Preventive Services Task Force recom-
mends screening all sexually active women, including
those who are pregnant, for gonorrhea infection if
they are at increased risk for infection. At-risk popu-
37
lations include the young (less than 25 years of age)
and those with individual or population risk factors
(202).
Presently there are no recommendations for screen-
ing for C. trachomatis or N. gonorrhoeae in men. As
many infections with these organisms are asympto-
matic in males, this population represents a major
reservoir for transmission of new infections and rein-
fection of treated partners. Further research is needed
to determine if screening young sexually active males
would be of benefit in reducing prevalence and pre-
venting infection in women (54). Currently, through
partner notification, some of the infected males are
identified and may receive treatment, but research
indicates that this is not an effective method to
reduce prevalence rates (86). Expedited partner ther-
apy, a process by which patients are given antimicro-
bials to give to their sex partners, is an alternative
approach that is gaining recognition. Several studies
have shown this approach to be effective in prevent-
ing reinfection with C. trachomatis and N. gonor-
rhoeae (87, 183).
Laboratory Guidelines for Screening Tests
In 2002, the CDC issued recommendations for the
selection, performance, and interpretation of screen-
ing tests for C. trachomatis and N. gonorrhoeae infec-
tions (33). This document is a useful reference for all
laboratories performing this testing, particularly
those using NAATs. A key point in this document is
that all screening tests should be considered presump-
tive evidence of infection. Additional testing should
be considered for persons with a positive screening
test result if a false-positive result would have a seri-
ous adverse consequence. Routine additional testing
should be considered when the prevalence of either C.
trachomatis or N. gonorrhoeae infection is low, re-
sulting in a low PPV (e.g., 90%). Additional testing
increases the specificity and can include retesting of
the specimen or collection and testing of a new speci-
men (Table 14).
A two-tiered testing scheme may be directed by
the ordering physician for individual patients or by
the laboratory for all positive specimens or for sam-
ples from select groups of patients with low preva-
lence. Either way, the interpretation of the combined
results must be clear. A positive result with the screen-
ing and an additional test is more likely to be a true
positive and represent infection. A positive screening
test and a negative additional test may mean the ini-
tial result was falsely positive and the patient is not
infected. Alternately, the additional test may be
falsely negative because of factors such as adminis-
tration of therapy based on the positive screening
test.
38 Leber et al.
Table 14. Considerations for additional testing after
a positive screening NAAT for C. trachomatis or
N. gonorrhoeaea
C. trachomatis
Only another NAAT test is appropriate for confirmation of a
positive screening test.
N. gonorrhoeae
Culture with confirmation is the preferred test after a posi-
tive NAAT, providing the culture specimen integrity can
be maintained.
Use of a NAAT after positive screen is acceptable but has
had limited evaluation
Samples for additional testing (in order of preference)
Test second specimen with a different test that uses a differ-
ent target or format.
Test the original specimen with a different test that uses a
different target or format.
Repeat the original test on the original specimen.
a
Adapted from reference 33.
Effectiveness of Screening
The goal of screening is to detect infection in asymp-
tomatic individuals and prevent subsequent adverse
health outcomes and the associated costs to society.
The economic impact of C. trachomatis and N. gon-
orrhoeae infections in young adults in the United
States is significant; the estimated direct medical cost
exceeds $325 billion annually (43).
Numerous publications have examined the effec-
tiveness of C. trachomatis screening programs (1,
100, 102–104, 143, 184). These programs have been
shown to reduce the prevalence of infection and to
reduce the number of cases of PID and other sequelae,
and they are cost-effective. In a study by Scholes et al.
(184) screening for C. trachomatis infection among
high-risk sexually active women reduced the inci-
dence of PID by 56%. This study used culture and
EIA for the detection of C. trachomatis. Other studies
supporting the effectiveness of C. trachomatis screen-
ing in sexually active women found that NAATs were
also cost-effective despite the increased cost of this
type of testing compared to nonamplified methods.
The NAATs are more sensitive and allow detection of
more infected individuals (103, 104, 128).
Despite the recommendations from multiple
groups and increasing coverage for testing by insur-
ance providers, the rate of C. trachomatis screening
in the United States remains low (54, 55, 188). In a
study by the CDC, data from the Health Plan
Employer Data and Information Set during 1999 to
2001 were examined to determine the rate of screen-
ing in commercial and Medicaid insurance plans (31).
Among sexually active females aged 16 to 26 years,
screening increased over the 3-year period, but by
2001 only 26% of these women were screened for C.
trachomatis in the commercial plans and 38% in
CUMITECH 44
Medicaid plans. Clearly screening needs to increase in
order to improve the quality of care and reduce the
costs associated with C. trachomatis infections
among young sexually active youth. The advent of
noninvasive sample types such as urine and vaginal
swabs for NAATs is hoped to increase the acceptance
of screening. Novel approaches such as NAATs
through pharmacy-based screening and mail-in test-
ing also warrant further study if the goal of sustained
and widespread reduction in C. trachomatis infection
is to be realized (19, 20, 204).
CODING AND REIMBURSEMENT ISSUES
The Current Procedural Terminology (CPT) codes
for amplified probe testing of C. trachomatis and
N. gonorrhoeae are 87491 and 87591, respectively
(5). Note that both target amplification (e.g., PCR,
TMA, and SDA) and signal amplification techniques
(e.g., Hybrid Capture) are accepted methodologies
for these codes. These codes are both method and
analyte specific so little interpretation is required for
accurate coding. Codes for molecular diagnostic pro-
cedures (83890–83901, 83912) should not be in-
cluded because the amplified probe technique is a
comprehensive procedure, which provides the rele-
vant information that is available through other tech-
niques such as molecular diagnostics. Therefore, pro-
cedures 83890–83901 and 83912 should not be
submitted for separate reimbursement when submit-
ted with procedures 87491 and 87591.
In most states, justification of medical necessity is
not required for these two CPT codes; no limited
coverage codes from the International Classification
of Diseases 9th Revision, Clinical Modification
(ICD-9-CM) are required for reimbursement in the
outpatient or inpatient settings. At the time of writ-
ing, reimbursement for 87491 and 87591 ranges
from approximately $22 for Medicaid to a range of
$40 to $70 for third-party payers. Each laboratory
must research reimbursement rates to determine if
internalization of C. trachomatis and N. gonor-
rhoeae NAAT will be financially feasible.
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