Sexually transmitted disease testing: evaluation of diagnostic tests and methods

Adolesc Med 15 (2004) 287 – 299

       Sexually transmitted disease testing:
    evaluation of diagnostic tests and methods
     Michael G. Spigarelli, MD, PhD, Frank M. Biro, MD*
Division of Adolescent Medicine, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue,
                                 Cincinnati, OH 45229-3039, USA

   Accurate diagnosis of sexually transmitted infections (STIs) remains a
cornerstone of the health care of adolescents and young adults, the two groups
with the highest rates of STI. Few medical care providers screen or test every
appropriate patient. Recent surveys have noted that relatively few physicians (one
third) screen asymptomatic adolescent girls during routine gynecologic exams [1]
and that female adolescents are screened more frequently than adolescent males
(67% versus 49%) [2]. Obstetrician-gynecologists are more likely than other
primary care physicians to screen women. For example, obstetrician-gynecolo-
gists screened 54.3% of nonpregnant women for chlamydia, compared with
34.7% of nonpregnant women screened by all physicians [3,4].
   From these surveys, several issues arose regarding screening practices. Female
physicians were more likely to screen than male physicians; physicians were less
likely to screen if they believed the prevalence of chlamydia to be low and more
likely to screen if they felt responsible for providing information about preven-
tion of STIs [1]. Nurse practitioners and physician assistants were more likely
than physicians to screen adolescents [2]. Many physicians believed that their
counseling regarding STIs was ineffective [5]. Physicians who tested for
gonorrhea or chlamydia were more likely to use a DNA probe or culture, rather
than urine-based DNA-amplification techniques [4].
   In addition to the newer testing methodologies, such as DNA-amplification
techniques, new testing strategies are helping to increase the number of patients
tested. Strategies such as urine-based testing for Chlamydia trachomatis and
Neisseria gonorrhoeae, self-obtained swabs, and use of specimens from liquid-
based Papanicolaou specimens are becoming accepted more widely.

   * Corresponding author.
   E-mail address: frank.biro@chmcc.org (F.M. Biro).

1547-3368/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.admecli.2004.02.006

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Specific infections and clinical presentations

Bacterial vaginosis

Biologic basis for infection
   Bacterial vaginosis (BV) results from an imbalance of vaginal flora, with a
decreased concentration of peroxide-producing Lactobacillus spp to an increased
concentration of several species, including Gardnerella vaginalis, Prevotella spp,
Bacteroides spp, Mobiluncus spp, Peptostreptococcus spp, and Mycoplasma
hominis [6]. The prevalence of BV is up to 64% in sexually transmitted disease
(STD) clinics, 24% in obstetrics clinics, and 15% in family planning clinics. At
least one half of cases are asymptomatic. Because BV represents a syndromic
diagnosis based on overgrowth of several organisms, the diagnosis and treatment
is difficult [7].

Traditional diagnostic tests
    BV is typically diagnosed by Amsel’s criteria, which require three of the
following four signs: (1) presence of clue cells, (2) homogeneous discharge that
adheres to the vaginal walls, (3) pH of vaginal fluid greater than 4.5, and (4) a
‘‘fishy’’ odor resulting from the presence of amines detected following the
addition of 10% potassium hydroxide (the whiff test) [8]. This methodology
relies on subjective tests for three of the four criteria. The objective criterion,
however, is limited by a lack of pH paper that changes color at 4.5, requiring
most practitioners to avoid this criterion or use pH paper that changes color at 4.7.
Thomason’s modification includes 20% or more clue cells with two of the other
three criteria [9]. The sensitivity and specificity of the clinical criteria for BV
were performed at a family planning clinic, as noted in Table 1. Office-based tests
are underused in the evaluation of BV, with lack of microscopy in 37% and no pH
or whiff test performed in 90% [10].
    Nugent’s method uses a standard scoring system for Gram-stained specimens
based on bacterial morphotypes. This system uses a total scoring system based on
weighted quantities of three morphotypes of bacteria (large gram-positive rods,
small gram-negative or gram-variable rods, and curved gram-negative or gram-
variable rods) from 1 through 10, with a score of 7 or higher diagnostic of BV

Table 1
Sensitivity and specificity of individual criteria for bacterial vaginosis
Criteria                        Sensitivity (%)      Specificity (%)         Positive predictive value (%)
Clue cells                      100                  91.6                    65.5
Whiff test                       99.2                92.6                    68.1
pH above 4.7                     97.0                85.8                    52.3
Homogenous discharge             92.4                95.1                    63.5
Whiff test, pH above 4.7         96.2                96.0                    79.3
Adapted from Hellberg D, Nilsson S, Mardh P-A. The diagnosis of bacterial vaginosis and vaginal
flora changes. Arch Gynecol Obstet 2001;265:11 – 5.

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[12]. This method removes much of the subjectivity of Amsel’s criteria. A score
of 6 or more yields sensitivity of 89.5% and specificity 97.1% [13].
   The culture of G vaginalis, although sensitive, does not correlate neces-
sarily with clinical symptomatology [11]. There are several other diagnostic
methods available for detection of Gardnerella products, as noted in the fol-
lowing discussion.

Molecular-based diagnostic testing
    The Centers for Disease Control and Prevention (CDC) noted ‘‘potential
clinical utility’’ for three new diagnostic methods: Affirm VP (Becton Dickinson
and Co., Franklin Lakes, New Jersey), FemExam (CooperSurgical, Trumbull,
Connecticut), and Proline IminoPeptidase (PIP) Activity Test (Quidel Corp., San
Diego, California) [14]. Affirm VP can detect and differentiate between DNA
from G vaginalis, Candida spp, and Trichomonas vaginalis through two, distinct,
single-stranded DNA probes for each of the three organisms that are coupled with
capture and color-development probes. In a comparison of Affirm VP with other
diagnostic methods, sensitivity was 90.5% and specificity 97.3% for presence of
clue cells, and 93.6% and 81.4%, respectively, when compared with Nugent
criteria [15]. FemExam detects an elevated vaginal pH and presence of trimethyl-
amine, and the PIP Activity Test measures the activity of amino peptidase.
    In practice, most practitioners seldom use pH or whiff testing, and nearly 40%
are not using a microscope [10], suggesting that a test such as FemExam, which
uses a color-changing method to detect pH and the presence or absence of amine
(the basis for the whiff test), would be clinically useful. The PIP Activity Test,
potentially more sensitive and specific than the whiff test, provides a diagnostic
test for only one of Amsel’s criteria, limiting its clinical utility. Although Affirm
VP provides good sensitivity and specificity for identification of Gardnerella spp,
it may have limited clinical utility because the syndrome of BV represents an
overgrowth of several organisms, not just the presence of Gardnerella spp.

Chlamydia trachomatis

Biologic basis for infection
   C trachomatis is a gram-negative, obligate, intracellular bacterium. The life
cycle of C trachomatis consists of an extracellular form (the elementary body) and
the intracellular form (the reticulate body). The elementary body attaches to and
penetrates columnar epithelial cells, where it transforms into the reticulate body,
the active reproductive form of the organism. The reticulate body forms large
inclusions within cells and then begins to reorganize into small elementary bodies.

Traditional diagnostic tests
   Culture media has been used to detect C trachomatis and has been considered
the criterion standard. This method relies on a time-consuming, labor-intensive
process that is further complicated by media and transport issues. Sensitivity has
been reported to range from 50% to 85% [16]. Culture is the only accepted

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Table 2
Sensitivity and specificity for detection of Chlamydia in cervical, urethral, and urine specimens from
asymptomatic women and men [17,25 – 35]
Test                Sensitivity             Specificity            Sensitivity             Specificity
Women               Cervical specimen                              Urine specimen
PCR                 92.8%                    99.7%                 85.5%                   99.5%
LCR                 88.6%                    99.7%                 95.6%                   98.6%
TMA                 98.4%                    98.8%                 96.8%                   99.0%
SDA                 96.8%                    97.9%                 83.9%                   98.3%
DFA                 77.8%                    97.3%                 —                       —
EIA                 73.2%                    99.9%                 —                       —

Men                 Urethral specimen                              Urine Specimen
PCR                 92.4%                    99.3%                 93.5%                   99.2%
LCR                 94.2%                    99.2%                 92.9%                   99.8%
TMA                 94.6%a                   98.4%a                96.3%a                  98.8%a
SDA                 85.7%                    97.6%                 94.0%                   98.8%
DFA                 80.0%a                  100.0%a                —                       —
EIA                 75.0%                    91.0%                 83.5%                   99.5%
Abbreviations: DFA, direct fluorescent antibody; EIA, enzyme immunoassay; LCR, ligase chain
reaction; PCR, polymerase chain reaction; SDA, strand displacement assay; TMA, transcription
medicated amplification.
    a
      Test manufacturer data rather than peer-reviewed literature sources (GenProbe, San
Diego, California).

procedure for medico-legal cases. Table 2 compares the clinical utility of various
diagnostic modalities in women and men.

Direct fluorescent antibody. Direct fluorescent antibody (DFA) uses a swab
specimen smeared on a glass slide, with direct application of a fluorescent-
labeled antibody. This antibody binds to the elementary body of the chlamydial
organism, and the slide is examined using a fluorescent microscope.
   DFA’s limitations include extensive time and expertise, the latter resulting in
interlaboratory variation. A relative advantage of the method is that it allows for
evaluation of sample adequacy, which is less crucial with the advent of DNA-
amplification techniques. DFA is useful in performing discrepant analyses where
the number of elementary bodies present in a given sample is counted.

Enzyme immunoassay. Enzyme immunoassay (EIA) uses an antibody raised
against the elementary body of the chlamydial organism. A second antibody is
added and reacts with the first antibody. This second antibody is linked to an
enzyme-based mechanism to produce a color change in the reagent, which can be
detected through a spectrophotometer.
   Several EIA assays are available commercially. In a review of studies
evaluating asymptomatic, young, sexually active subjects, the mean sensitivity
across all studies on cervical specimens was 65%, and on urine 38% [17].
Recently, an updated version of the EIA, using a polymer conjugate – enhanced

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EIA, has been introduced (IDEIA, DakoCytomation, Carpinteria, California).
This enhanced EIA has a sensitivity of 91.8% and a specificity of 98.2% [18].

Molecular-based diagnostic testing
DNA probe. Gen-Probe PACE 2 assay (Gen-Probe, San Diego, California) is a
DNA-RNA hybridization assay. The DNA probe, with an acridium ester label, is
hybridized to 16S chlamydial ribosomal RNA, with results expressed as relative
light units (RLUs). The difference is calculated between the sample RLU and
negative reference controls. Advantages include ease of transport, ability to batch
specimens, and cost. Disadvantages include lower sensitivity and specificity than
the nucleic-amplification techniques. In a large, multi-center study that used
independent reference standards, the sensitivity of PACE 2 was 60.8% to 71.6%
and specificity was 99.5% to 99.6% [19].

Nucleic-acid amplification tests. The development of methods to amplify the
amount of detectable signal has provided clinicians and laboratory directors with
numerous choices and strategies that allow accurate and reproducible testing
results. Nucleic-acid amplification tests (NAATs) provide a faster turnaround
time and a more automated process, in contrast to culture and DFA.

Polymerase chain reaction. Polymerase chain reaction (PCR) (Roche Molecu-
lar Systems, Branchburg, New Jersey) uses a three-step process to amplify the
chlamydial DNA. The first step is denaturation of the double-stranded DNA,
using heat to produce two complimentary DNA strands containing the target se-
quence, the cryptic plasmid of the chlamydial genome. Chlamydial-specific
primers, small portions of single-stranded DNA, anneal to the denatured DNA.
Using an enzyme known as Taq polymerase, the DNA is replicated using the
primer as an initiation point, then elongated, replicating the double-stranded
DNA. Once replicated, the newly formed amplicon undergoes a series of heat-
ing and cooling steps, exponentially increasing the amount of chlamydial
DNA. Following the amplification, the presence of chlamydial DNA is detected
through binding of a complimentary strand sequence attached to a colori-
metric probe. The major drawbacks to the method include price, specialized
equipment, and the necessity for repeated heating and cooling cycles, which can
allow cross-contamination.

Ligase chain reaction. Ligase chain reaction (LCR) (Abbott Laboratories,
Abbott Park, Illinois) is an assay that shares many of the same features of
PCR, with an additional step: ligation. Once the initial double-stranded DNA is
denatured, two pairs of primers are used that bind to the target sequence. The two
primers, which are separated by a few nucleotides, are joined by action of the
ligase enzyme, and then the polymerase enzyme extends the chain, forming a
substrate for the next round of replication. Postamplification, a colorimetric probe
is bound to the amplicons, allowing detection of the DNA.

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   As with PCR, major drawbacks of LCR include price, the necessity for specific
equipment, and repeated heating and cooling cycles, which can allow cross-
contamination. Recently, issues have arisen regarding availability of test substrate.

Transcription-mediated amplification. Transcription-mediated amplification
(TMA) (AMP CT, Gen-Probe, San Diego, California) uses RNA rather than
DNA as the starting substrate. The advantages of using single-stranded RNA
include elimination of the denaturation step and shorter persistence of nucleic
acid from nonviable organisms. A complementary primer, containing a specific
promoter sequence, and the enzyme reverse transcriptase create a complementary
DNA/messenger RNA (mRNA) hybrid. The enzyme then degrades the RNA
portion of the hybrid. A second primer, also with a promoter sequence, binds the
newly formed single-stranded DNA and creates a complementary DNA strand.
Through the incorporation of the promoter sequence in the primer, which then
undergoes replication, a mechanism is created for rapid, exponential transcription
of mRNA sequences. Once created, the mRNA amplicons can be detected by
binding a reporter sequence and assaying for colorimetric changes.

Strand displacement assay. The Strand displacement assay (SDA) (Becton
Dickinson, Sparks, Maryland) uses a target region on the cryptic plasmid of
C trachomatis. Initially, the double-stranded DNA is denatured, providing two
complimentary strands. In the target-generation phase, two separate primers are
bound to the target sequence, and then both primers are extended, with the
extension of one primer displacing the other extended primer. Once the extended
primer is displaced, it can hybridize with the opposite primer. After both primers
are extended, the exponential target-amplification phase begins, during which a
restriction enzyme makes a nick in a specific sequence of the hybridized DNA.
At this nick site, DNA polymerase can begin to synthesize another copy while
simultaneously displacing the downstream strand, and the process repeats. The
displaced strand then binds with the detector probe, which incorporates a
fluorescein label for detection. The detection probe has a mechanism to prevent
any fluorescence without binding to the displaced strand and subsequent cleavage
of the newly formed hybrid [20].

Urine-based testing
   Testing urine for chlamydia or gonorrhea using any of the NAATs has several
advantages, including eliminating the need for obtaining a cervical or urethral
swab, which improves the likelihood of screening. Urine specimens for NAAT
have slightly lower sensitivities, however, particularly in women. These tests are
further subject to decreased sensitivities associated with the order and method of
collection. For example, in a study involving male patients, initial (‘‘first-void’’)
urine specimen PCR was more sensitive than obtaining a second urethreal swab
[21]; the first-void specimen had a greater sensitivity than subsequent voids [22].
In a cost-minimization study in a juvenile detention facility, treating boys based
on results of urinary leukocyte esterase (sensitivity 75%, specificity 82%), and

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girls on the basis of NAAT on urine were the most cost-effective testing and
treatment strategies [23].
    Leukocyte esterase (LE) testing has been available for several years, and its
use as an STD-screening test has been the basis for several articles. From a
practical standpoint, LE testing is cost-effective in screening men, with a good
negative predictive value (97%) [23]. In female patients, leukocytes may be
present in vaginal secretions, whether or not accompanied by an infection, as well
as in urine specimens. Therefore, LE screening is helpful when screening men in
high-prevalence populations; it is a screen for urethritis, not a diagnostic test for a
specific organism.
    Because the NAATs measure nucleic acids rather than viable organisms,
concerns have arisen regarding the duration of persistence of chlamydia nucleic
acid. C trachomatis DNA was found to persist in most (21 of 25) specimens
1 week after treatment, and a substantial proportion of organisms at 3 weeks
posttreatment had persistent C trachomatis DNA (5 of 20 specimens) [24]. In this
study, C trachomatis RNA was unlikely to persist at 1 week (2 of 24 specimens),
and no specimens had persistent RNA at 2 weeks.
    Table 2 shows the sensitivity and specificity for detection of chlamydia in
asymptomatic women and men. Table 2 is an update of a previous study
evaluating asymptomatic women [17], providing additional studies using similar
criteria [25 – 34].
    A recent study examined discordant rates of chlamydia in asymptomatic
couples, and noted that screening men alone using a NAAT based on urine would
have identified more couples with at least one infected partner than screening
females alone (81% versus 54%) [63].

Self-testing
   Self-testing has been found to be a valid alternative to provider testing or urine
testing [36,37]. In recent studies, self-collected swabs analyzed by NAAT
identified at least as many participants infected with chlamydia or gonorrhea
compared with cervical specimens, and identified more infections than culture or
urine specimens [38,39].

Neisseria gonorrhoeae

Biologic basis for infection
   N gonorrhoeae is a small, gram-negative, obligate intracellular, diplococ-
cal bacterium.

Traditional diagnostic tests
   Culture media has been used for years to detect N gonorrhoeae and has long
been considered the criterion standard. This method relies on a labor-intensive
process. Sensitivity and specificity have been reported at 69.8% to 92.6% and
100%, respectively [40 –42].

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Table 3
Sensitivity and specificity for detection of gonorrhea from urine and provider-obtained specimens
Test                 Sensitivity            Specificity          Sensitivity            Specificity
Women                Cervical specimen                           Urine specimen
PCR                  96.1%                   99.1%               83.3%                  98.5%
LCR                  94.3%                   99.8%               99.0%                  99.9%
TMA                  99.2%                   98.7%               91.3%                  99.3%
SDA                  98.2%                   99.6%               83.7%                  99.4%
Culture              69.1%                   99.9%               —                      —

Men                  Urethral specimen                           Urine specimen
PCR                  99.0%                   99.2%               95.8%                  99.2%
LCR                  99.0%                   99.9%               96.4%                  99.9%
SDA                  98.4%                   96.5%               98.2%                  99.4%
Culture              85.7%                  100%                 —                      —
(See references [25 – 28,40,41,43 – 46]).

   Please see the discussion on chlamydia for a description of other diagnostic
methods. Table 3 shows the sensitivity and specificity of diagnostic tests for
gonorrhea in both men and women. The studies selected for inclusion had an
independent standard to reflect actual sensitivity and specificity as closely as
possible [25 –28,40,41,43– 46].

Trichomonas vaginalis

Biologic basis for infection
   Trichomoniasis is caused by the parasitic infestation of T vaginalis, a
flagellated protozoan that lives in the urogenital tract.

Traditional diagnostic tests
   The most common clinical diagnostic method involves examination of vaginal
secretions using direct microscopy. Samples obtained from the posterior fornix
are mixed with 0.9% saline in a traditional wet preparation method, placed upon a
microscope slide, and protected with a coverslip. This preparation is examined
for the presence of motile trichomonads. Although results are available imme-
diately, sensitivity of direct microscopy is limited (36% – 80% over five studies,
with a mean of 61.3%) [47 –51]. The sensitivity and specificity of results from a
routine Papanicolaou smear were 60.7% and 97.6%, respectively [52], and 61.4%
and 99.4%, respectively, from liquid-based Papanicolaou smear [53].
   Several culture techniques make results available in 2 to 7 days. The
sensitivity of InPouch TV (BioMed Diagnostics, White City, Oregon), Dia-
mond’s, and Trichosel media at 48 and 96 hours have been reported at 76% and
87%, 43% and 60%, and 14% and 75%, respectively [54].

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Molecular-based diagnostic testing
   The Affirm VP III (Becton Dickinson) is an automated DNA probe that de-
tects DNA from Gardnerella spp, Candida spp, and T vaginalis. A second probe
is coupled to an enzyme (horseradish peroxidase/streptavidin) to detect tricho-
monas RNA. The sensitivity and specificity of this test for trichomoniasis were
89.5% and 99.8%, respectively [55].
   There are several PCR-based tests for diagnosis of trichomoniasis using va-
ginal and urine specimens. In studies in women, sensitivity of PCR varied by
study and specimen site. PCR had sensitivity of 89% to 97% and specificity of
97% on vaginal specimens [56,57], and 64% to 91% and 93% to 100%, re-
spectively, on urine specimens [57,58], although one study noted that urine-based
PCR detected more infections than vaginal-based PCR [49]. In men, PCR testing
has greater sensitivity on a urine specimen compared with a urethral specimen
(100% versus 80%) or culture of a urethral specimen (73%) or urine (53%);
specificity of urine-based PCR is 88% [59].

Herpes simplex

Biologic basis for infection
   Genital herpes simplex infections result from infection from herpes simplex
virus type 1 or type 2 (HSV-1 or HSV-2). Approximately one third of first
episode cases are caused by HSV-1. It is important to distinguish between the two
types, because HSV-2 has a higher rate of relapse.

Diagnostic tests
   There are two broad categories of tests: virologic and serologic. The optimal
test in a patient with clinical disease (active genital ulcers) is cell culture, but the
sensitivity decreases with advancing age of ulcers. Unroofing a vesicle increases
the sensitivity of the culture. The process of looking under a microscope at
the stained contents of an ulcer base, know as a Tzanck prep, is neither sensi-
tive nor specific. Many preparations will appear negative because of a lack of
the characteristic, multinucleated, giant cells that are the hallmark for Herpes-
viridae infections.
   PCR assays have been available for the diagnosis of herpes simplex viruses for
several years, although these assays have been used to investigate suspected
herpes encephalitis [60]. A recent review of several published studies noted that
PCR had a greater sensitivity than cell culture [60], and a cost analysis revealed
that if serotyping was included in costs of positive cultures, total cost of
analyzing 100 clinical specimens was $753 with culture and serotyping, com-
pared to $820 with PCR [61].
   There are type-specific and nonspecific serologic tests for HSV. The CDC
recommends the glycoprotein G (gG) tests, the sensitivity of which is 80% to
98%, and specificity is 96% or more [14]. There are three gG assays approved by
the US Food and Drug Administration: POCkit HSV-2 (Diagnology, Research
Triangle Park, North Carolina), HerpeSelect-1 and -2 ELISA IgG (Focus

296               M.G. Spigarelli, F.M. Biro / Adolesc Med 15 (2004) 287–299

Technologies, Herndon, Virginia), and HerpeSelect 1 and 2 Immunoblot IgG
(Focus Technologies) [14]. Given the limitations of available serologic testing in
terms of specificity and the ability to distinguish current, recent, and old
infection, these tests have limited clinical applicability.

Summary
    Most STIs in adolescents are asymptomatic. Recent studies in adolescents
have documented relatively short periods of time until reinfection occurs (median
times 4.7 –7.6 months) [62 – 64], suggesting that sexually active adolescents
should be screened for STI every 6 months. Evidence-based practice is exceed-
ingly helpful in deciding when to test, whom to test, and which methodology to
use. In a recent critical analysis regarding screening women for chlamydia,
screening all women for chlamydia was more cost-effective than testing only
symptomatic women [65,66]. With the proliferation of highly sensitive and spe-
cific assays, and the usefulness of different specimen sources (such as urine or
self-swabs), health care providers of adolescents should screen all patients at risk
for STIs. Although these screening and diagnostic techniques provide greater
accuracy than previously attainable, screening should be part of a comprehensive
methodology designed to promote good health care decisions, such as encour-
aging abstinence, promoting safer sexual practices, and using the most appro-
priate methodology to detect and treat STIs.

Acknowledgments
   The authors would like to acknowledge Susan Cunningham for her cheerful
assistance in the preparation of this manuscript.

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