1. health and fitness
2. disease

Journal of Andrology, Vol. 32, No. 1, January/February 2011
Copyright E American Society of Andrology
How to Detect Chlamydia
trachomatis in Males?
Review
ADRIAN ELEY
From the Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, United Kingdom.
ABSTRACT: Much is known about the role of Chlamydia
trachomatis in female infertility, although the same cannot be said
about the organism’s role in male infertility. Recently a number of
researchers have provided a possible explanation of the pathogenesis of C trachomatis in male infertility and have suggested further
studies. Unfortunately, current screening recommendations for C
trachomatis in an infertile couple are vague and unhelpful, and many
do not even mention this type of screening in the male. To enable
any progress to be made in this field, it is essential that investigators
know how best to detect C trachomatis, especially in the male. It is
important, therefore, to know which specimen is best for C
trachomatis detection, with respective strengths and weaknesses
of each specimen. Similarly, it is equally important to have
knowledge of which test is appropriate for the type of specimen
being examined. First void urine is currently the specimen of choice
for the routine detection of C trachomatis in males. Moreover, the
best detection protocols in the developed world are based on
molecular diagnosis of first void urine. These methods provide the
best combination of sensitivity and specificity that is currently
available on a clinical sample that can be self-taken. Interestingly,
because semen is routinely collected for analysis in men of infertile
couples, it has been suggested that protocols be developed for the
optimal detection of C trachomatis in this specimen. Semen might
provide additional information on infection of the upper genital tract,
which may not be detected in first void urine. Finally, the importance
of comparing tests for C trachomatis detection in updating our
knowledge has been highlighted by the inability of some molecular
methods to detect the new variant strain of C trachomatis.
Key words: First void urine, semen, molecular diagnostics.
J Androl 2011;32:15–22
C
There are different serovars or serotypes of the
organism, with those commonly causing genital infections belonging to serovars D through K and with
serovars D through G being the most common in the
Western world (Donati et al, 2009). A new variant of C
trachomatis serovar E (nvCT) with a 377-bp deletion in
the cryptic plasmid was recently detected in Sweden
(Ripa and Nilsson, 2006, 2007). Interestingly, because
some of the commercially available C trachomatis assays
based on a plasmid target were unable to detect strains
with the deletion in the plasmid, a large number of falsenegative results were discovered (Soderblom et al, 2006;
Unemo et al, 2007). This resulted in the affected
companies having to develop new molecular tests for
C trachomatis diagnostics.
Current screening recommendations for C trachomatis in an infertile couple are vague and unhelpful. In the
United Kingdom, the Royal College of Obstetricians
and Gynaecologists (1999) and the National Institute
for Health and Clinical Excellence recommend that
before undergoing uterine instrumentation, women
(presumably in an attempt to prevent spread of
infection) should be offered screening for C trachomatis
using an appropriately sensitive technique. If the result
of a test for C trachomatis is positive, women and their
sexual partners should be referred for appropriate
management with treatment and contact tracing. One
hlamydia trachomatis is a bacterium with a unique
developmental cycle comprising infectious, metabolically inert elementary bodies (EBs) and noninfectious, metabolically active reticulate bodies. It is
responsible for the most common sexually transmitted
bacterial infection worldwide, affecting more than 90
million people (World Health Organization, 2001), and
has been known for some time to have a significant
impact on human reproduction (Paavonen and EggertKruse, 1999), although its role in male infertility is still
controversial (Ochsendorf, 2008). Nevertheless, our in
vitro experiments have revealed that exposure of
spermatozoa to chlamydial EBs can result in premature
sperm death and stimulate an apoptosis-like response in
sperm (Hosseinzadeh et al, 2001; Eley et al, 2005a,b).
Interestingly, similar findings, which led to increased
levels of sperm DNA fragmentation, were also found in
in vivo studies (Satta et al, 2006; Gallegos et al, 2008),
therefore providing a possible explanation of the
pathogenesis of the organism in male infertility.
Correspondence to: Dr Adrian Eley, Department of Infection and
Immunity, University of Sheffield Medical School, Beech Hill Road,
Sheffield, S10 2RX, United Kingdom (e-mail: a.r.eley@sheffield.
ac.uk).
Received for publication March 5, 2010; accepted for publication
August 5, 2010.
DOI: 10.2164/jandrol.110.010363
15
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January ÙFebruary 2011
Table. Strengths and weaknesses of different clinical specimens used for detecting Chlamydia trachomatis
Specimen
Strengths
Urethral swab
Urine
Semen
Serum
Quality of specimen can be assured
Easy to collect
Routinely collected for semen analysis
Relatively easy to collect
source (Institute for Clinical Systems Improvement) has
described symptoms of a possible genital tract infection
in the male, although no further information was
provided to help investigate the cause. A number of
sources have commented on the finding of significant
numbers of leukocytes in a urethral smear, first void
urine, or semen as indicative of a possible genital tract
infection but gave no additional information as to how
that should be investigated further (Male Infertility Best
Practice Policy Committee of the American Urological
Association, 2006; European Association of Urology).
The only more specific advice is found in the European
Association of Urology guidelines and is from an older
reference of Taylor-Robinson (1997), who stated that
the ideal diagnostic test for C trachomatis in semen has
not yet been established despite modern DNA detection
techniques.
Interestingly, in spite of the above United Kingdom
guidelines, Sowerby and Parsons (2004) found that 53%
of in vitro fertilization clinics in the United Kingdom
neither screen the woman nor give antibiotic prophylaxis, and only 4% screen the male partner. In the
screening of sperm, egg, and embryo donors, recent
guidance (Association of Biomedical Andrologists,
2008) has suggested that all donors be screened for C
trachomatis before and after donation, according to the
strategy developed by the British Association for Sexual
Health and HIV (2006). However, it is not clear how
best to detect C trachomatis in the male because many
different methods have been employed over the years.
The aim of this review is to assess established methods
of detection in the light of newer technical developments.
Which Specimen?
Essentially, there are 4 possible specimens that may be
examined, each with its respective strengths and
weaknesses (Table).
Historically, urethral swabs were collected as part of
the genital examination within the genitourinary medicine clinic and were required for chlamydial culture.
Unfortunately, in males, the taking of urethral swabs
may be painful because it requires deep insertion, and
trying to limit the procedural pain can have a negative
effect on specimen quality. The idea of using self-taken,
noninvasive specimens such as urine, especially when
Weaknesses
Not easy to collect and painful
Cannot culture the organism
Inhibiting to cell culture and sometimes to NAATs
Not from the infection site
combined with antigen detection tests such as the
enzyme immunoassay (EIA), has resulted in a move
away from routine use of urethral swabs. Comparative
studies have shown that although urethral swabs gave
higher rates of detection than first void urine for both
sensitivity and specificity (81.0% vs 76.2% and 99.0% vs
95.5%, respectively) when an EIA test was used (Sellors
et al, 1991), the difference between the specimens was
reduced when a nucleic acid amplification test (NAAT)
was used (96.2% vs 92.5% and 100% vs 100%,
respectively) (Carroll et al, 1998). A recent systematic
review has confirmed the latter finding (Cook et al,
2005). Since the early 1990s, first void or first catch urine
(usually the first 15–50 mL) has been shown to be an
acceptable specimen for the detection of C trachomatis
genital infection in men (Chernesky et al, 1990).
Apart from the previously described changes in
methodology for improved C trachomatis detection in
urine, another factor has emphasized the advantages of
using urine. A recent study has shown that there is no
significant difference in organism load between first void
urine and urethral swabs in men when quantitative
polymerase chain reaction (PCR) is used for assessment
(Michel et al, 2007). However, it has been known for a
while that NAAT inhibitors can be present in many
clinical specimens, and there is some controversy on the
extent of NAAT inhibition in urine (Mahony et al, 1998;
Toye et al, 1998; Van der Pol et al, 2001).
Therefore, detection of C trachomatis in urine is
advantageous because the sample is self-taken, noninvasive, and contains a high organism load (although
there can be NAAT inhibitors present). In routine
diagnosis of chlamydial infection in the male, a semen
specimen would not normally be requested, but semen
specimens are routinely collected in infertile men to test
semen quality. Moreover, such a specimen might
provide additional information as to whether there is a
chlamydial infection of the upper genital tract. Unfortunately, there is no approved methodology for testing
of semen for C trachomatis (Chernesky, 2005; Peeling
and Embree, 2005), and this poses a serious problem.
The question as to whether semen is a suitable sample
for detection of C trachomatis in infertile men has yet to
be answered.
The identification of chlamydial antibodies is clearly
an indirect approach to detect C trachomatis in any
Eley
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C trachomatis and Male Infertility
clinical specimen (usually serum but can be semen).
Moreover, there is no single sensitive and specific C
trachomatis antibody test that has been consistently used
to investigate chlamydial serology (Johnson and Horner, 2008), and this deficiency makes it difficult to
compare results from different studies. Among commercially available assays, there is often variable
sensitivity and specificity because there may be crossreactivity with Chlamydophila (Chlamydia) pneumoniae
(Gijsen et al, 2001), a common respiratory pathogen. It
is obvious, therefore, that there are limitations with the
use of chlamydial serology (in whatever test specimen) in
general, to detect the presence of C trachomatis.
Which Test?
There are a number of laboratory methods to detect C
trachomatis that range from cell culture to NAATs, as
discussed next. Other research methods that are not
generally available in a diagnostic laboratory, such as
electron microscopy, will not be discussed.
Culture—In the early days of C trachomatis screening,
the bacterium was grown on cell monolayers such as
McCoy and inoculated with the clinical specimen such
as a urethral swab (Mardh et al, 1980). The characteristic chlamydial inclusion body can be detected when
stained with a fluorescent antibody that binds to either
the chlamydial major outer membrane protein or
lipopolysaccharide (LPS). Older nonspecific stains,
including Giemsa or iodine, have lower sensitivities
and are not recommended for routine use (Stamm et al,
1983). However, with the introduction of noninvasive
samples such as urine, cell culture proved too insensitive
(Taylor-Robinson and Thomas, 1991), with sensitivities
of less than 70% compared with direct immunofluorescence (DIF) (Taylor-Robinson, 1997). Culture sensitivity can be enhanced by blind passaging, which increases
the number of infected cells that can be detected by
staining. It was also discovered that components of
semen were toxic to the growth and maintenance of the
monolayer, and a solution to this toxicity problem was
the dilution of the semen to decrease the toxic effect;
however, this decrease in toxicity was achieved at the
expense of sensitivity to detect C trachomatis (Tjiam et
al, 1987).
Culture is now regarded as slow, labor intensive, and
hence quite costly. Nevertheless, it has excellent
specificity and allows for antibiotic susceptibility testing
and serovar determination, which may be advantageous
in epidemiologic studies.
Antigen Detection—There are 2 commercial approaches to antigen detection, and these are EIA and
DIF. EIA tests are usually based on the ability to detect
chlamydial LPS with an anti-LPS antibody. The
17
chlamydial LPS extract from the clinical sample is
added to the anti-LPS antibody, which coats wells of a
microtiter plate. Upon binding of LPS to the antibody, a
colorimetric reaction occurs that can be measured in a
microplate reader. The test is easy to perform but only
has a sensitivity of 105 to 107 organisms/mL. It also can
be cross-reactive with other bacteria such as staphylococci, Bacteroides species, and Escherichia coli (EggertKruse et al, 1995; Ivanov et al, 2009). The test specificity
can be improved by retesting any positive reactions with
a confirmatory blocking antibody or by using the DIF
or direct fluorescence antibody test. The ability to
automate these tests has led to a possibility of highthroughput testing.
The DIF or direct fluorescence antibody test uses an
antibody that is tagged with a fluorescent label and is
able to detect chlamydial EBs directly from smears made
from clinical specimens. Smears are then examined
under a fluorescence microscope. Cross-reactions are
not usually a problem because the morphology of EBs
is distinctive. It is the only method that allows for
confirmation of specimen quality. The sensitivity of the
method is higher than that of the EIA test (and can be
increased further by low-speed centrifugation of specimens before staining) (Taylor-Robinson and Thomas,
1991) and is related to the minimum number of EBs that
are counted (which can be in the range of 1–10) and the
proficiency of the person reading the smear.
Unfortunately, a skilled and experienced microscopist
is required for optimal performance. DIF has a
sensitivity of 80% to 90% and specificity of 98% to
99% relative to the culture method when both are
performed optimally (Black, 1997).
Molecular Testing—These tests that detect either
DNA or RNA can be divided into those that amplify
or do not amplify the target. A test not amplifying the
target typically uses a probe to detect ribosomal RNA
(Gen-Probe PACE 2 assay) and has a sensitivity of
approximately 104 organisms/mL. Although it has a
similar sensitivity level as culture, this test has the
advantage of good specimen stability characteristics,
whereas culture requires strict transport and handling
criteria. However, amplified molecular tests such as
PCR show much greater sensitivity with a theoretic
single nucleic acid sequence that can be detected. A
systematic review in 2002 established that both PCR and
ligase chain reaction (LCR) showed better sensitivities
than nonmolecular methods (Watson et al, 2002). The
authors also concluded that these NAATs used with
noninvasive samples such as urine were more effective at
detecting asymptomatic chlamydial infection and that
these tests performed well in low-prevalence populations. Skidmore et al (2006) documented the 2 NAATs
currently in widespread use in the United Kingdom: the
18
Becton Dickinson ProbeTec, which uses strand displacement amplification (SDA) technology (Spears et al,
1997), and the Roche Cobas Amplicor PCR (which is
now being superseded by a real-time PCR method, the
Roche Cobas TaqMan CT). A third NAAT currently
available in the United Kingdom is transcriptionmediated amplification by Gen-Probe (Pasternack et
al, 1997). Such methods are widely available in the
developed world, including Europe and the United
States. It is now generally accepted that in C trachomatis
detection, NAATs have become the method of choice
(Hamdad and Orfila, 2005; Gaydos et al, 2008)—so
much so that they must be used in the United Kingdom
National Chlamydia Screening Programme (United
Kingdom Department of Health).
The first NAAT to be used successfully for the
detection of C trachomatis in males was PCR using inhouse methodology. This test has been used subsequently in a number of studies involving men of infertile
couples (Ochsendorf et al, 1999; Hosseinzadeh et al,
2004; Gdoura et al, 2008). Since then, PCR has been
developed commercially so that the method is standardized (eg, the Roche Cobas Amplicor PCR has an built-in
control for specimen inhibitors [as does LCR, as
described below]) (Hamdad-Daoudi et al, 2004; de
Barbeyrac et al, 2006). A more recent development has
been the introduction of real-time or quantitative PCR,
which allows detection of C trachomatis DNA copy
numbers (Al-Mously et al, 2009). In the 1990s, a
commercial system for LCR (Abbott), similar to
Amplicor, also proved to be successful at detecting C
trachomatis in men of infertile couples (Eggert-Kruse et
al, 1997; Fujisawa et al, 1999; Eggert-Kruse et al,
2002a,b). However, the LCR commercial system was
discontinued several years ago and is no longer
available.
Serology—Until recently, there was no consensus
about the detection of chlamydial IgG and IgA in serum
and the presence of C trachomatis in the male genital
tract. It was generally believed that detection of serum
IgG and IgA was of no diagnostic value in male
infertility (Wolff et al, 1994; Dieterle et al, 1995). Studies
by Radouani et al (1996), Weidner et al (1996), and Levy
et al (1999) confirmed a lack of correlation between
serologic results and direct detection of C trachomatis in
semen. However, recent studies (Idahl et al, 2004, 2007)
have shown that the presence of C trachomatis IgG and
IgA antibodies in serum from the male partner of an
infertile couple was correlated with pregnancy (as
confirmed by routine ultrasound at gestation weeks
15–17) and associated with subtle negative changes in
semen characteristics. In particular, C trachomatis
serum IgA in men correlated with reduced chances of
achieving pregnancy (P 5 .021; relative risk [RR] 5
Journal of Andrology
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January ÙFebruary 2011
0.65; 95% confidence intervals [CI], 0.42–1.005), and the
chance was further reduced in combination with C
trachomatis IgG (P 5 .001; RR 5 0.35; 95% CI, 0.15–
0.84) (Idahl et al, 2007).
The role of chlamydial IgG and IgA antibodies in
semen and their relation to semen quality is perhaps
more controversial. Many studies have shown no
association (Eggert-Kruse et al, 1997, 1998; Habermann
and Krause, 1999; Penna Videau et al, 2001; Liu and
Zhu, 2003), whereas others studies have shown an
association (Wolff et al, 1991; Ochsendorf et al, 1999). A
recent study also showed no relationship between past
or present C trachomatis infection, defined by positive
direct and serologic markers (in serum and/or semen),
and quality of semen defined according to World Health
Organization parameters such as sperm count, motility,
and morphology (de Barbeyrac et al, 2006). Perhaps the
fundamental problem is that the presence of chlamydial
IgG or IgA antibodies in serum or semen does not allow
a distinction between past or present genital infection
(Dieterle et al, 1995). Therefore, an appropriate current
summary suggests that the determination of C trachomatis antibodies in serum or seminal plasma seems to be
of limited diagnostic value in male infertility work-up
(de Barbeyrac et al, 2006).
Comparisons Between the Specimens and Tests
Not all of the 4 specimens discussed previously are
currently suitable and/or recommended for testing.
Urethral swabs are probably the best samples to
examine and are required for culture. However, for the
preceding reasons, first void urine samples are now more
commonly examined, although urethral swabs are still
an alternative specimen.
Semen is rarely tested, and there are no recommended
methods to test it. Serum and/or semen antibodies both
provide limited information and are controversial.
Therefore, for the purpose of this review, our comparisons of different tests will be limited to the examination
of urine.
Although urine is probably not quite as good a
specimen as a urethral swab in terms of sensitivity,
testing a centrifuged deposit of urine has been shown to
be as sensitive as testing a urethral swab if the same test
is applied to each (Thomas et al, 1991). Nevertheless,
testing of urine in cell cultures is too insensitive, with a
positivity rate of only 23.7% in men (Chernesky et al,
1990). Although EIA is easy to perform, sensitivity
levels can be low, varying between 62% and 97%
(Taylor-Robinson and Thomas, 1991). Specificity can
also be an issue. In studies of men of infertile couples,
prevalence rates using EIA were high—25.19% and
33.33%, respectively (Bornman et al, 1998; Mania-
Eley
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C trachomatis and Male Infertility
Pramanik et al, 2001). DIF shows improved sensitivity
when compared with EIA and has value in looking at
nonviable chlamydiae in specimens, owing to prolonged
transport and suboptimal storage; however, the technique requires an experienced microscopist. Therefore,
amplified testing of urine is considered the most effective
way for diagnosing infections in men, and the effectiveness has been shown when making comparisons with
Gen-Probe PACE 2 and EIA (Chernesky et al, 2003).
Although specificity was high for both tests, Gen-Probe
PACE 2 only had a sensitivity of 65.9% in comparison
with LCR, which had a sensitivity of 90.1% (Carroll et
al, 1998).
When LCR was applied to testing of men of infertile
couples, detection rates were low at 0% and 4.54%,
respectively (Eggert-Kruse et al, 1997; Bollmann et al,
2001). An advantage of LCR was that it was only
available commercially so that studies using this test
were comparable. However, many studies with PCR
have used in-house methodologies that are not
comparable. A recent example of such methodology
applied to men of infertile couples in Tunisia gave a
prevalence rate of 39.4% (Gdoura et al, 2008).
Amplicor, which is only available commercially, is a
PCR-based method.
When this test was applied to men of infertile couples,
low prevalence rates of 5.4% and 0%, respectively, were
found (Hamdad-Daoudi et al, 2004; Rosemond et al,
2006). More recently, ProbeTec SDA has been tested in
the same population group, and a prevalence rate of
3.52% was found (Kokab et al, 2010).
A relatively early comparison of amplified methods
then available (Amplicor, SDA, and transcriptionmediated amplification) showed that they performed
equally well (Templeton et al, 2001). Since then, a
number of new real-time methods, including Roche
COBAS TaqMan CT (Hadad et al, 2009) and Abbott
real-time CT assay (Walsh et al, 2009), have come on the
market. Comparative studies of new real-time methods
and existing NAATs have been made but with
limitations. For example, Abbott real-time CT compared with ProbeTec SDA showed increased sensitivity
(Walsh et al, 2009). In a more comprehensive study,
comparisons were made for Abbott real-time CT, Roche
COBAS TaqMan CT, and Gen-Probe tests (Aptima
Combo 2 and Aptima C trachomatis assay, which target
23S rRNA and 16S rRNA, respectively). Although
patient numbers were small, the Abbott real-time CT
showed better sensitivity (59/83 vs 57/83) (Moller et al,
2008). However, both tests underperformed in comparison with the Gen-Probe tests because the latter tests
were able to detect the new variant C trachomatis strain
(Magbanua et al, 2007), whereas the other tests could
not.
19
Conclusions and Future Directions
For the routine detection of C trachomatis in males, first
void or first catch urine is currently the specimen of
choice because these methods provide the best combination of sensitivity and specificity that are currently
available on a clinical sample that can be self-taken.
Nevertheless, urethral swabs are a useful, alternative
specimen and are essential if culture of the organism is
required. Semen might provide additional information
on detection, but there are still no approved methods for
detecting C trachomatis in semen. Therefore, until
comparative studies are performed with, for example,
first void urine, no recommendations can be made.
Current serologic methods cannot be recommended
mostly on grounds of poor specificity.
Unless culture is required, the best detection protocols
in the developed world are based upon molecular
diagnosis of first void urine as discussed earlier, although
some may not be able to detect the new variant strain. In
countries that do not have access to molecular techniques, the best test would be to use EIA on first void
urine. However, choice of specimen is often influenced by
a number of local factors, including the population size of
the group to be screened, prevalence rate in the
population, predictive values of the tests being used,
and whether testing is performed in a laboratory. All of
these factors will contribute to the final decision.
Because semen is routinely collected for analysis in men
of infertile couples, it is suggested that protocols be
developed for the optimal detection of C trachomatis in
this specimen. There are suggestions that semen might
provide additional information on infections of the upper
genital tract that may not be detected in first void urine
(Eggert-Kruse et al, 1997; Bornman et al, 1998; Fujisawa et
al, 1999; Rosemond et al, 2006; Gdoura et al, 2008;
Gallegos-Avila et al, 2009; Kokab et al, 2010; Mazzoli et al,
2010). Moreover, recent recommendations for future
testing of semen for C trachomatis have been proposed
(Eley and Pacey, 2010). They include making a comparison
of commercially available NAATs as well as comparing
tests of semen with testing of first void urine, the use of an
internal control for inhibitors, and confirmation of any
positive results using the minimum testing algorithm.
Finally, the importance of comparing tests for C
trachomatis detection in updating our knowledge has
been highlighted by the inability of some molecular
methods to detect the new variant strain.
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