1
Department of Biology, Faculty of Sciences, Shahid Chamran University, Ahvaz, Iran
2
Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran
3
Shahid Beheshti Hospital, Isfahan, Iran
4
4Department of Pathobiology, School of Medicine, Yazd University of Medical Sciences, Yazd, Iran
Abstract
Background Chlamydia trachomatis (C. trachomatis) is the most prevalent cause of bacterial sexually transmitted infections (STI) recognized throughout the world. The aim of this study is to determine different genotypes of genital C. trachomatis and the association between the serological markers of inflammation and genotypes of C. trachomatis in sexually active women (n=80) attending Shahid Beheshti Hospital in Isfahan, Iran. Materials and Methods In this descriptive study, endocervical swabs were collected from 80 women. There were 17 endocervical samples that showed positivity for C. trachomatis by plasmid polymerase chain reaction (PCR) using KL1 and KL2 primers. The omp1 gene was directly amplified in 17 plasmid PCR positive samples and was used to differentiate the clinical genotypes by omp1 gene PCR-restriction fragment length polymorphism (PCR-RFLP). The levels of IgG and IgA specific to C. trachmatis and C-reactive protein (CRP) were evaluated. Results Based on restriction-digestion patterns, four genotypes were identified. Genotypes E (35.3%) and F (35.3%) were the most prevalent, followed by D/Da (23.5%) and K (5.9%). There was no significant association between genotypes and the presence of IgG and CRP. Patients infected with genotype E showed a serological marker of chronic inflammation, i.e. IgA seropositivity, significantly more than patients infected with other genotypes (p=0.042). Conclusion Nested PCR could increase the sensitivity of omp1 amplification. Based on the presence of IgA, chronic C. trachomatis infections were observed more frequently among genotype E-infected patients in our population.
Clamydia trachomatis (C. trachomatis) is the
most common bacterial cause of sexually transmitted
infections (STI). The World Health Organization
estimates that approximately 92 million new
cases of genital chlamydial infection occur worldwide
annually (1, 2). Because 50% of infections
in men and 80% in women are asymptomatic, the actual number of cases seems to be more than
the estimated number (1, 3). Currently, 19 human
genotypes and numerous variants including A, B/
Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a,
and L3 have been identified using polyclonal and
monoclonal antibodies against the major outer
membrane protein (MOMP) (4, 5). MOMP is a
predominant antigen, which is different in various
strains of C. trachomatis (6-8). Genotypes A-C
(the trachoma biovars) cause conjunctivitis and
lead to trachoma, the primary cause of preventable
blindness in third world countries (8). Genotypes
D-K are the most important cause of urogenital
and neonatal infections. They are the most common
cause of sexually transmitted genital infections,
eliciting local acute epithelial infections,
which can lead to pelvic inflammatory disease in
women. Untreated infection or chronic infection
can occasionally cause infertility, potentially fatal
ectopic pregnancy, and premature delivery (7-
9). Genotypes L1-L3 cause the invasive disease
known as lymphogranuloma venerum (7, 8).
Sequencing of the omp1 gene in C. trachomatis
showed a significant difference among genotypes
(10). Restriction fragment length polymorphism
(RFLP) analysis of omp1 gene has been used to
differentiate genotypes of C. trachomatis by using
different enzymes (11-14).
Determination of the epidemiological relationship
between the C. trachomatis genotype from
different areas could be a suitable guideline for designing
epidemiological programs for controlling
chlamydial infections, and consequently controlling
sexually transmitted diseases (STDs). Considering
the importance of this issue, the aim of
this study was to determine the prevalence of C.
trachomatis genotypes in symptomatic cervical infections
in Iranian women. A hypothesis currently
under investigation states that genetic variations in
the C. trachomatis genome may account for strain
(genotype) differences in the course and outcome
of infection with this bacteria (15-17). The chronic
status in the course of a C. trachomatis infection is
one of the most important aspects of this infection.
It is associated with the persistence of the bacteria
in the host cells that increases the risk of tubal factor
subfertility (18). IgA antibodies are assumed to
reflect chronic inflammation (19, 20). A level of
C-reactive protein (CRP) >10 mg/l usually occurs
in acute infections, and can be detected using common
tests for CRP. A CRP level <1 mg/l indicates
the absence of inflammation or infection (21). Because
CRP is a general serological marker of acute
inflammation, another object of this study is to detect
the serological markers of acute and chronic
infections in patients, with the intent to evaluate
the differences among different genotypes.
Materials and Methods
Study population and sample collection
This descriptive study was approved by the
Ethics Committee of Shahid Beheshti Hospital.
Samples were collected after obtaining written
informed consent from 80 patients who attended
the Gynecology Outpatient Department of Shahid
Beheshti Hospital in Isfahan, Iran in 2008.
An endocervical swab from each individual was
transferred to 5 ml of sterile phosphate buffered
saline (PBS) and stored at -70˚C until DNA extraction.
In addition, 5ml of peripheral blood was
collected from each patient for serological investigation
(22, 23).
DNA extraction
The endocervical swab sample was removed
from the vial and the PBS collection tube was centrifuged
at 2000 rpm for 15 minutes. The supernatant
was discarded and the bottom was vortexed
and transferred to a 1.5 ml microtube. This step
was followed by centrifugation at 2000 rpm for 15
minutes. The supernatant was then removed. Then,
400 μl of tris-base-EDTA (TE) solution that contained
10 mM tris–HCl, pH= 8.0 and 1 mM EDTA,
was added. 4 μl proteinase K (10 μg/ml) and 4 μl
triton 10% (v/v) were added and incubated at 55˚C
for 90 minutes, followed by 95˚C for 30 minutes.
These samples were maintained at -20˚C until
used (22-24).
Plasmid PCR
All 80 samples were examined by C. trachomatis
plasmid-based PCR using KL1 and KL2 primers.
Successful amplification of a 241 bp fragment
of the bacterial endogenous plasmid genome was
considered a positive result by polymerase chain
reaction (PCR). The primers used for the C. trachomatis
plasmid PCR were KL1 (5′-TCCGGAGCGAGTACGAAGA-3′) and KL2 (5′-AATCAATGCCCGGGATTGGT-3′; Metabion,
Germany) (9, 22, 24). The final reaction mix contained 5 μl of the extracted DNA sample, 16 pM
of each primers, 0.28 μM deoxynucleotide triphosphate
(Cinnagen, Iran), 3 mM MgCl2 (Cinnagen,
Iran), and 1 U of Taq polymerase (Cinnagen, Iran),
for a total volume of 25 μl (9, 22, 24). The amplification
protocol was 10 minutes of DNA denaturation
at 94˚C followed by 40 cycles of amplification,
with each cycle that consisted of denaturation
at 94˚C for 1 minute, annealing at 55˚C for 1
minute, and extension at 72˚C for 1 minute. The
final extension cycle at 72˚C was prolonged for 8
minutes (9, 22, 24). The PCR products were analyzed
by 1.5% agarose gel electrophoresis. C. trachomatis
genotype A was used as positive control
and a sample that contained only distilled water
was used as a negative control.
Omp1 PCR
We examined 17 plasmid PCR-positive samples
for omp1 PCR. An approximately 1.2 kb fragment
of the omp1 gene was amplified in the 17
plasmid-based PCR positive samples using three
primers: CT1 (forward strand: 5′- GCCGCTTTGAGTTCTGCTTCCTC-
3′), CT5 (reverse strand:
5′- ATTTACGTGAGCAGCTCTCTCAT-3′), and
PCTM3 (forward strand: 5′- TCCTTGCAAGCTCTGCCTGTGGGGAATCCT-
3′; Gene Fanavaran)
(14). Primary PCR was performed on 10
μl of the extracted DNA in a final reaction mixture
of 50 μl. The final reaction mixture contained 10
mM tris-HCl (pH= 8.3; Cinnagen), 50 mM KCl
(Cinnagen), 1.5 mM MgCl2 (Cinnagen), 200 μM
from each deoxynucleoside triphosphate (dATP,
dTTP, dGTP, and dCTP; Cinnagen), 25 ρmol
of each primer CT1, CT5, and 1U of Taq DNA
polymerase (Cinnagen). The amplification protocol
was 5 minutes of DNA denaturation at 95˚C
followed by 35 cycles of amplification, with each
cycle that consisted of denaturation at 95˚C for 1
minute, annealing at 55˚C for 1 minute, and extension
at 72˚C for 1.5 minutes. The final extension
cycle at 72˚C was prolonged for 4 minutes (14). In
this study, an Eppendorf Master Cycler Epgradient
was used. The semi-nested PCR was carried out
in the following manner: 1 μl of the primary PCR
product (as the DNA template) was added to a prepared
PCR mixture that contained primer PCTM3,
located 22 bp downstream of CT1 and the previous
primer, CT5. The position of CT1 is at 34-56
bp and PCTM3 is at 55-84 bp. The amplification
conditions of the semi-nested PCR were the same
as the conditions of the primary PCR. The PCR
products were analyzed by 1% agarose gel electrophoresis
(14). For a 1% agarose gel we added 1 g
of agarose to 100 ml of 1x electrophoresis buffer
and 0.5 μg/ml ethtidium bromide to the molten
agarose. In this study, C. trachomatis genotype E
was used as a positive control and a sample that
contained distilled water instead of DNA was used
as a negative control.
RFLP
The omp1 seminested PCR products were digested
with restriction enzymes according to the
previous study (12, 14). Restriction digestion was
performed in two manners: i. single digestion with
restriction enzyme AluI; ii. triple digestion with
three enzymes HpaII, EcoRI, and HinfI. The first
digestion was carried out with 10 μl of amplified
DNA on 4 U of AluI, using the assay buffer recommended
by the manufacturer, at 37˚C for 4 hours.
The second digestion was performed on 10 μl of
amplified DNA, first with 4 U of HpaII in 10 mM
tris HCl (pH=7.6) and 10 mM MgCl2 at 37˚C for
4 hours. HpaII was then added in a 10 minute incubation
at 60˚C. Next, 2 μl of 200 mM tris HCl
(pH=8) and 75 mM NaCl were added and samples
were incubated overnight with 4 U of EcoRI and
Hinfl at 37˚C (12). Rodriguez et al. reported the
single digestion differentiates between ten genotypes:
A, C, E, F, G, I, J, K, L1, and L2, while B,
Ba, D, H, and L3 show similar patterns. The triple
digestion differentiates 11 genotypes: D, E, F, G,
H, I, J, K, L1, L2, and L3 but genotypes A, C and
B, Ba have similar patterns (12,14). In our study,
a triple digestion was carried out to discriminate
genotypes D/Da from B/Ba. Genotype D was not
differentiated from Da, because D and Da have
similar patterns in both single and triple digestion
(12). CfoI digestion can be used to differentiate
genotype D from Da (25).
Digestion products were analyzed by a 6% polyacrylamide
gel stained with 15 μg of ethidium bromide
per ml. For identification of the clinical strains,
the RFLP pattern of each sample was compared with
the omp1 restriction fragment sizes (larger than 100
bp) presented in the previous study (12).
Serological tests
Blood samples were taken from 80 women and
the sera were used to determine the level of IgG and IgA antibodies against C. trachomatis using
p-ELISA kits (Medac, Germany). The p-ELISA
was based on a synthetic peptide from the immunodominant
region of the major outer membrane
protein. The ELISA kit used in this study was very
specific for detecting C. trachomatis without any
cross activity with other species of Chlamydia.
The level of CRP was determined by using a CRP
kit (Omega, UK). The sensitivity of the CRP test
was 6 mg/L. Both tests were performed according
to the manufacturers’ instructions.
Statistical analysis
Analysis of the association of genotypes and
the serological markers was carried out by a chisquare
test. Data analysis was performed with
SPSS statistical software version 15.0. P value less
than 0.05 was considered significant.
Results
The successful amplification of a 241 bp fragment
of the C. trachomatis plasmid genome was
considered a positive result by plasmid-PCR. A
total of 17 out of 80 samples (21.25%) were positive
for C. trachomatis by KL1 and KL2 primers.
omp1 was successfully amplified by CT1-CT5
PCR (primary-PCR) in 14 of 17 samples (82.3%).
In the three remaining samples, omp1 was amplified
after nested-PCR with PCTM3 and CT5 primers.
AluI digestion resulted in four genotypes: D/
Da (or B/Ba), E, F and K. Digestion with HpaII,
EcoRI, and HinfI were performed for distinguishing
the D/Da and B/Ba genotypes from each other.
The triple digestion patterns of these samples corresponded
with genotype D/Da (Fig 1). Restriction
patterns were compared with acquired restriction
patterns in reference 12. Of 17 samples, there were
4 (23.5%) genotype D/Da, 6 (35.3%) genotype E, 6
(35.3%) genotype F, and 1 (5.9%) genotype K. RFLP
patterns of omp1-seminested PCR products of four
genotypes in single digestion and of one genotype (D/
Da) in triple digestion manner are shown in figure 1.
Out of 17 patients, 5 (29.4%) showed positivity for
IgG, 3 (17.6%) for IgA, and 6 (35%) for CRP. Table 1
shows the relationship between the serological characteristics
of infections and genotypes.
Restriction patterns were compared with acquired restriction
patterns in reference 12. RFLP patterns of four clinical
genital C. trachomatis strains (D/Da, E, F, and K) obtained
by 6% polyacrylamide gel electrophoresis after restriction hydrolysis
of the omp1 gene-PCR products. Lane 1: AluI digestion
of genotype D/Da. Lane 2: HpaII-EcoRI-HinfI digestion
of genotype D/Da. Lanes E, F, and K indicate AluI digestion of
genotypes E, F, and K, respectively. Lane M: pBR322 digested
with HaeIII as the DNA size marker.
Serological features of infections in relation to C. trachomatis genotypes
Serological feature
Genotype D/Da
Genotype E
Genotype F
Genotype K
P value
IgG
1/4 (25%)
2/6 (33.3%)
2/6 (333%)
0(%)
0.42
IgA
0/4 (0%)
3/6 (50%)
0 (0%)
0(0%)
0.04
CRPa
2/4 (50%)
1/6 (16.7%)
3/6 (50%)
1/1 (100%)
0.18
Without immunological
1
2b
1
0
markers
Total
4
6
6
1
a; C-reactive protein.
b; Two samples of genotype E were IgG and IgA double-seropositive. Out of 6 genotype E samples, 2 samples were without immunological markers.
Discussion
C. trachomatis is a common sexually transmitted
infection with significant impact on public health.
Therefore, effective epidemiological control as
well as a correct and sensitive diagnostic method
for C. trachomatis are required (22, 23). In order to
develop epidemiological data and to detect various
genotype infections, accurate and specific typing
of C. trachomatis genotypes is necessary (12). Molecular
techniques, like PCR-RFLP, are more sensitive
and less time-consuming than immunofluorescence
(12). Also, since immunotyping has serious
limitations, a suitable and convenient tool that can
be used to type and survey epidemiological studies
is PCR-RFLP (12). In the current study, 17 out of
80 samples were positive with plasmid PCR. The
plasmid-PCR showed 10x higher sensitivity than
MOMP-PCR due to having ten copies of plasmid
in the elementary body compared to the MOMP
gene, which has only one (11, 24). Thus in this
study plasmid-PCR was used as a gold standard.
Results of omp1 PCR showed that the nested PCR
could increase the sensitivity of omp1 amplification, because
the omp1 fragment was amplified in 14 out of 17
samples using primary PCR with CT1 and CT5 primers
(82.3%), whereas omp1 was successfully amplified
in the 3 remaining samples by using nested PCR with
pCTM3 and CT5. This manner could be useful, particularly
for direct PCR on crude suspensions of samples
that contain low copy numbers of Chlamydia. RFLP patterns
with fragments larger than 100 bp produced from
the CT1-CT5 sequence of the omp1 gene for the 15 C.
trachomatis genotypes in the previous study were considered
as reference patterns in this study (12), but for
optimizing omp1-PCR in the present study, the pCTM3-
CT5 sequence was amplified and used for RFLP. Based
on BLAST searching at the NCBI, the first restriction
sites for AluI (AGCT) is located at 63-66 bp of the omp1
gene, and for HinfI (GAATC) at 78-82 bp of omp1 gene,
and both are apparent on the pCTM3 primer. Therefore,
RFLP patterns of products of CT1- CT5-PCR and
PCTM3-CT5-PCR in single digestion are different in
the 64 bp fragment and in triple digestion in a 78 bp fragme
nt, which both are smaller than 100 bp.
In this study, genotypes E and F were the most prevalent
genotypes, followed by genotype D/Da and K. The
first investigation on genotyping of C. trachomatis was
performed in Ahvaz, Iran. This study showed that the
most prevalent genotype was E (31.5 %), followed by F
(23.1 %), D/Da (13 %), K (9.2 %), I (8.3 %), G (7.5 %),
H (5.5 %), and J (1.9 %) (14). In other parts of the world,
genotypes E, F, and D were responsible for the most
genital C. trachomatis infections (26-29).
The acute phase protein CRP is a general serological
marker of inflammation. In this study, there was
no significant association between the level of CRP,
IgG antibodies, and genotypes (p=0.18 and 0.42, respectively).
Serum IgG antibodies against microorganisms
usually remain detectable for many years,
even after antibiotic treatment (30).
Detecting merely IgA antibody by p-ELISA, indicating
that C. trachomatis infection may be present
at an early stage of acute infection (31). The presence
of high titer of IgA antibodies is associated with
chronic inflammation (19, 20). In this study, we have
shown that genotype E samples were significantly related
to IgA seropositivity (p=0.042), which indicated
chronic C. trachomatis infections were observed
more frequently among genotype E. Molano et al.
reported that chronic C. trachomatis infections were
observed more frequently among D and E genotypes,
with a lower frequency among genotypes B, H, I, and
K (27). These researchers have also shown that in a
mouse model, the duration of lower genital tract infection
was longest with D and E genotypes, and improvement
in the upper genital tract occurred more
often in mice infected with D genotype compared to
mice infected with H genotype (27).
Our data and the above mentioned studies have
indicated that the course of a C. trachomatis infection
(such as whether the infection will be cleared or
chronic) may be influenced by differences among various
genotypes. However, there have not been enough
studies undertaken in these areas to prove this theory.
Serological responses to various genotypes might differ
in different areas and populations. They can be
justified with the role of genetic variations in immunologically
important host genes in the course and outcome
of infectious. To our knowledge, the evaluation
of the relationship between C. trachomatis genotypes
and features of the serological response has not been
carried out in Iran. Further studies need to be done on
larger populations in different parts of the world.
Conclusion
Our data showed that the nested PCR could increase
the sensitivity of omp1 amplification. Genotypes
E and F were the most prevalent genotypes
in this cohort. It was also shown that there was no
significant association between the levels of CRP,
IgG antibodies, and different genotypes. Furthermore,
genotype E samples were significantly related
to IgA seropositivity.
Taheri Beni, B., Jenab, A., Roghanian, R., Motamedi, H., Golbang, N., Golbang, P., & Yazdi, J. Z. (2012). Genotyping of Endocervical Chlamydia trachomatis Strains and Detection of Serological Markers of Acute and Chronic Inflammation in Their Host. International Journal of Fertility and Sterility, 6(2), 101-106.
MLA
Behrouz Taheri Beni; Anahita Jenab; Rasoul Roghanian; Hossein Motamedi; Naser Golbang; Pouran Golbang; Javad Zaeimi Yazdi. "Genotyping of Endocervical Chlamydia trachomatis Strains and Detection of Serological Markers of Acute and Chronic Inflammation in Their Host". International Journal of Fertility and Sterility, 6, 2, 2012, 101-106.
HARVARD
Taheri Beni, B., Jenab, A., Roghanian, R., Motamedi, H., Golbang, N., Golbang, P., Yazdi, J. Z. (2012). 'Genotyping of Endocervical Chlamydia trachomatis Strains and Detection of Serological Markers of Acute and Chronic Inflammation in Their Host', International Journal of Fertility and Sterility, 6(2), pp. 101-106.
VANCOUVER
Taheri Beni, B., Jenab, A., Roghanian, R., Motamedi, H., Golbang, N., Golbang, P., Yazdi, J. Z. Genotyping of Endocervical Chlamydia trachomatis Strains and Detection of Serological Markers of Acute and Chronic Inflammation in Their Host. International Journal of Fertility and Sterility, 2012; 6(2): 101-106.
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