Document Type : Original Article
Authors
1 Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran
2 Faulty of Nursing and Midwifery, Birjand University of Medical Sciences, Birjand, Iran
Abstract
Keywords
Chlamydial infection in women can cause urogenital inflammations
including urethritis, cervicitis and salpingitis
(
The co-infection of CT with other sexually transmitted
pathogens may have complicated consequences.
Genital CT infection may increase human immunodeficiency
virus (HIV) viral shedding, therefore, identifying
and treating patients with CT infection may reduce
the genital transmission of HIV (
There are several diagnostic methods for CT, such as
isolation in cell lines, immunofluorescence, serologic assays
and molecular testing methods such as polymerase
chain reaction (PCR) (
Although CT infection has been proven to be the most
prevalent sexually transmitted infection (STI) (
This was a cross-sectional study performed in Birjand,
South Khorasan province of Iran from May 2015 to October
2016. The age of women ranged from 17 to 45 years,
all of which were referred for routine Pap smear test. Those
who had taken antibiotics within 3 weeks prior to their
visit were excluded from the study. All patients signed an
informed consent. This work was approved by the Ethics
Committee of the Vice-chancellor for Research of Birjand
University of Medical Sciences (#1393-12-07). Data collection
and recording were performed based on questionnaires
and forms. Total endocervical epithelial cells were
collected from 248 women visiting different gynecologists
in Birjand. These samples had been previously checked for
HPV-DNA and their results were used in this study (
On the same day of cytological examination, the residual
fluids containing endocervical cells were processed for
DNA isolation. A Bioneer DNA extraction kit (Bioneer Co,
South Korea) was used according to the manufacturer’s
instructions with minor alterations including the preheating
of samples and an additional round of centrifugation.
The extracted DNA was checked using a Nanodrop Biophotometer
(Eppendorf D30, Germany) at 260/280 nm,
and samples with a ratio between 1.8- 2 were selected. An
internal control gene (
The isolated DNA was subjected to PCR using primers
beta 1 (5.-TCAACCCTACAGTCACCCAT-3.) and
beta 2 (5.-CTAACAATTACGAACAGCAATGAG- 3.),
as previously described, to assess its integrity (
The products of nested PCR were sequenced bidirectionally using the same forward and reverse primers at Bioneer Company, South Korea (run on an ABI 3730XL DNA Analyzer). The obtained sequences were aligned using Mega BLAST to determine genotypes. The phylogenetic analysis in the given region of Omp1 was performed using MEGA6; the Jukes-Cantor model was selected for nucleotide substitution with Gamma distributed rates among sites. Selected codon positions were 1st, 2nd, 3rd and noncoding sites. To assess the reliability of the phylogeny, 1,000 bootstrap replications were performed. The accession numbers of reference sequences of CT genotypes used in this study were KM369934 (E), X62918 (D), KM369939 (G), DQ064292 (J), KM369936 (F), AF202456 (Ia), DQ064282 (B), DQ064295 (L2), AF063204 (K), X16007 (H), FM872306 (A) and CP006945 (C).
The type of distribution was checked, and skewness and
kurtosis were in the range of (
The demographic and clinical data of the 248 women screened are shown in Table 1. The mean age of patients was 37.54 ± 5.21 years. Most participants had a normal cytology (214 cases; 86.3%), however, 34 cases (13.7%) had an abnormal cytology result. In the abnormal group, there were 20 cases (58.82%) with atypical squamous cell of undetermined significance (ASCUS) and 14 (41.17%) with low-grade squamous intraepithelial lesions (LSIL). There were no cases with high-grade squamous intraepithelial lesions (HSIL) and/or cervical neoplasia. Based on cytological examination, observation of inflammatory cells and clinical data, 38 of all cases (15.32%) were found to have cervicitis.
The results of PCR for the
The forward and reverse sequences obtained from eight
samples were assembled to a consensus using CLC software
(CLC Genomics Workbench 7,
Demographic and clinical characteristics of these genotypes are in Table 3. The cases positive for genotype D were younger (28.45 ± 3.26 years), although it was not statistically significant. Interestingly, in the cases positive for genotype E, the co-infection of HPV and LISL were found more frequently.
In isolates of genotype E, there were no mutations at the amino acid level; however, there was a missense mutation in a case with genotype D (i.e. thr326ala). There were also two silent mutations, C915T in two cases of genotype E and T956A in two cases, with genotypes D and E. Overall, the nucleotide region 900-1000, a part of the variable domain- .V, was more prone to have a mutation.
The prevalence of CT among different cytological groups, as well as co-infection with HPV
Cytology | n (%) | Age (Y) Mean ± SD | HPV DNA n (%) | CT DNA n (%) | HPV/CT co-infection | P value |
---|---|---|---|---|---|---|
Normal cytology | 214 (86.29) | 35.55 ± 4.66 | 33 (15.42) | 20 (9.34) | 11 (5.14) | |
Total abnormal | 34 (13.7) | 38.45 ± 4.21 | 12 (35.29) | 11 (32.35) | 3 (8.82) | 0.022* |
ASCUS | 20 (58.82) | 37.1 ± 3.35 | 8 (40) | 5 (25) | 2 (10) | |
LSIL | 14 (41.17) | 35.3 ± 5.6 | 4 (28.57) | 6 (42.85) | 1 (7.14) | 0.056** |
Total | 248 | 37.54 ± 5.21 | 45 (18.14) | 31(12.5) | 14 (5.64) | |
CT;
The prevalence of
Age ranges (Y) | Total number (%) | CT+/each group n (%) | CT+/total (%) | Cervicitis+/each group n (%) | CT+and cervicitis+/each group n (%) |
---|---|---|---|---|---|
≤20 | 11 (4.43) | 2 (18.18) | 0.8 | 3 (27.27) | 1 (9.09) |
21-30 | 130 (52.42) | 16 (12.3) | 6.45 | 20 (15.38) | 4 (3.07) |
31-40 | 81 (32.66) | 9 (11.11) | 3.62 | 12 (14.81) | 2 (2.46) |
40˃ | 26 (10.48) | 4 (15.32) | 1.6 | 3 (11.5) | 0 |
Total | 248 | 31 (12.5) | 31 (12.5) | 38 (15.32) | 7 (2.82) |
Demographic and clinical characteristics of individuals with genotypes D and E identified in this study
Genotype | n | Mean age ± SD | Cervicitis n (%) | HPV n (%) | ASCUS n (%) | LISL n (%) |
---|---|---|---|---|---|---|
D | 4 | 28.45 ± 3.26 | 2 (50) | 1 (25) | 3 (75) | 0 |
E | 4 | 34.51 ± 2.52 | 0 | 3 (75) | 1 (25) | 4 (100) |
Total | 8 | 31.48 ± 2.55 | 2 (25) | 4 (50) | 4 (50) | 4 (50) |
HPV; Human papillomavirus, ASCUS; Atypical squamous cell of undetermined significance, and LSIL; Low-grade squamous intraepithelial lesions.
Agarose gel electrophoresis of polymerase chain reaction (PCR)
products. A. The positive samples for amplification of human
The phylogenic tree constructed based on the maximum likelihood
method. Accession numbers and genotypes were given, and those in circle
shape are the sequences reported here.
The Pap smear test is approved for screening cervical abnormalities
and is performed routinely around the world.
Therefore, a large and continuous sampling is in progress
and is accessible. This study showed the capacity of the
liquid Pap smear to enhance the molecular detection of
genital CT infection, as other studies have also indicated
(
The large variance observed in the reported data may
be due to sampling size, sample source, experimental test,
socio-economic state of the population and other factors.
The CT prevalence has been reported at variable rates in
other parts of the world such as 6.2% in Australia (
We found that the incidence of CT infection was higher
among patients with abnormal cytology (32.35%). Interestingly,
this figure was 42.85% for the LISL group,
25% for the ASCUS group 9.34% for the normal group,
indicating an ascending pattern toward malignancy. In a
case-control serological survey in Iran, a strong association
between CIN and CT was identified. In specific, in
the CIN and healthy group, there were 45 and 12.9% seropositive
individuals respectively (
This result confirmed the shared risk of CT and HPV
infection in the development of cervical cancer. The co-
infection rate of HPV and CT was 14/248 (5.64%) in the
total sample set and 14/31 (45.16%) among CT-infected
patients reported here. A study in Italy showed that 58%
of CT-infected women were also positive for HPV (
HPV and CT share similar transmission routes, and since CT may enhance the rate of other STI infections, it may have a role in the progression of cervical cancer. It may, however, be an independent co-risk factor of CIN with an unknown mechanism.
In the current study, the CT genotypes D and E were
equally identified. These genotypes were also prominent
in other genotyping surveys from Iran. Genotyping of CT
from endocervical specimens in Shiraz identified genotype
F (46.6%), E (33.3%) and D (13.3%) along with a
singleton G (
The results of this study revealed a relatively high prevalence of genital CT in East Iran and underscore the benefit of liquid Pap smear samples for molecular assays. The association between the rate of CT and CIN grade merits further investigation. Determining the prevalence and genotypes can provide important epidemiologic knowledge for transmission patterns, prevention, and treatment programs for controlling STI infections. Further investigations in this region are also needed to obtain a more reliable prevalence of CT and to determine its relevance to any other genital infections or cervical carcinoma.