Rapid screening for Chlamydia trachomatis infection by detecting α-mannosidase activity in urogenital tract specimens

Background Chlamydia trachomatis may cause multiple different urogenital tract disorders, but current non-culture assays for rapid screening of C. trachomatis typically use immunochromatography-based methods. We established another new rapid non-culture method for detection of C. trachomatis based on the measurement of α-mannosidase enzymatic activity in urogenital tract specimens. Method To evaluate the performance of this method, α-mannosidase activities of C. trachomatis serotype D strain 、 and 29 standard strains related to clinical urogenital pathogens were investigated. Furthermore, 553 urogenital tract specimens were used for clinical assays via cell culture method and ligase chain reaction method (LCR), adopting an expanded gold standard. Results Only C. trachomatis was positive for α-mannosidase activity among different types of microbes tested in the research. When prostate fluid specimens, which have some interfering activity, were excluded, the sensitivity and specificity of the enzymatic method were 91.8% (78/85) and 98.3% (409/416), respectively. There were no significant differences (P > 0.05). Conclusions These results showed that α-mannosidase activity could be utilised as a screening marker of C. trachomatis infection.


Background
C. trachomatis infection is the most common sexually transmitted disease (STD) in the United States [1]. Mounting evidence has indicated that it not only evokes nongonococcal urethritis (NGU), cervicitis, pelvic inflammatory disease (PID), salpingitis, orchitis, and epididymitis, but also increases risk of invasive cervical cancer [2,3] and gives rise to serious reproductive disorders such as infertility [4,5], miscarriage/premature birth/missed miscarriage [6][7][8][9], and neonatal conjunctivitis [10]. A large number of methods have been established for screening and diagnosis of C. trachomatis infection [11]. Nevertheless, few of these assays meet the requirements of outpatient diagnosis, especially in terms of sensitivity, specificity, time, and simplicity of operation. Currently, non-culture assays for C. trachomatis screening typically adopt immunochromatography-based methods. Technologies based on chromogenic reactions of specific microbial enzymes have been widely applied in bacterial identification systems and chromogenic media [12][13][14][15]. However, no chromogenic assay for detecting C. trachomatis has been made available to date. Our previous findings [16] suggested that C. trachomatis might have very high α-mannosidase activity. The purpose of the study was to establish a novel screening method for C. trachomatis infection without culture that would be rapid and convenient for use in outpatient clinics.

Ethics statement
All patients were treated in accordance with the Helsinki Declaration on the participation of human subjects in medical research. Ethics approval for the study was obtained from the First Affiliated Hospital Ethics Committee of Zhengzhou University (Approved No. 20100802) and Henan Provincial People's Hospital Ethics Committee (Approved No. 20100901).

Organisms
Reference strains and cell lines were obtained from the organisations shown in Table 1.

Specimens
This study evaluated 553 specimens from clinical patients attending the STD (257, 46.47%) and Gynaecology (296, 53.53%) clinics at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) and the Henan Provincial People's Hospital (Zhengzhou, China), respectively. For the 203 male cases, three urethral discharge specimens (151 outpatients, 74.38%) or three prostate massage liquid specimens (52 outpatients, 25.62%) were collected with sterile rayon swabs (Copan, Brescia, Italy). Meanwhile, for the 350 female cases, three cervical secretion specimens (232 outpatients, 66.29%) or three vaginal secretion specimens (118 outpatients, 33.71%) were collected with sterile rayon swabs using vaginal forceps. Three swabs collected for each specimen, were no significant differences in sampling link and randomly used with three methods. None of the patients received any antibiotics one week before sample collection, when samples were taken before diagnosis.

Media, culture and inoculation
Liquid media A (LMA) and liquid media B (LMB) were prepared for mycoplasma culture. The components of LMA were shown in Table 2, but LMB consisted of 50 mg/l phenol red besides LMA components. The media were inoculated with both Ureaplasma urealyticum and Mycoplasma hominis and analyzed by using the colourchanging unit (CCU) method, as previously reported [17].
Once the concentrations of mycoplasma reached 10 6 CCU/ml in LMB, the cultures of LMA should be immediately stored at 4°C. Trichomonas vaginalis was obtained after 24 h incubation at 37°C in Trichomonas medium (Oxoid, Basingstoke, UK) supplemented with 8% horse blood and 1,000 units/ml penicillin sodium and 500 mg/ml streptomycin. The collection was stored at 4°C. All other micro-organisms except C. trachomatis used in the study were inoculated and cultured as described in Table 3. The collections described in Table 3 were resuspended to 0.5 McFarland standards in a sterile solution of 0.9% NaCl and then stored at 4°C.

Enzymatic method
The enzymatic method was based on the substrate of α-D-mannosidase. The substrate solution contained 1.5 mg/ml 6-chloro-3-indolyl-α-D-mannoside (J&K, Shanghai, China), 100 mM citrate buffer (pH 4.0), and 1% Triton X-100. The sample diluent was 0.9% NaCl. The chromogenic reagent contained 0.08% fast violet B salt (J&K, Shanghai, China). Aliquots (50 μl) of substrate solution and chromogenic reagent were added sequentially into sample solutions (extracted from every swab sample with 500 μl sample diluent) or the aforementioned microbial suspensions as well as the chlamydial suspension mentioned in the section called limit of detection (LOD) of the enzymatic method. The result was considered to be positive (OD 512 ≧ 0.150) if the colour changed from colourless to red or brown after 15 min of incubation at 37°C; otherwise, the result was considered to be negative (OD 512 < 0.150). The suspensions containing bacteria or cells was centrifuged at 5,000 rpm for 5 min, and then OD 512 value of the supernatants were measured.

LOD of the enzymatic method
McCoy cells infected with C. trachomatis serovar D were stored at −80°C, and frozen-thawed twice to obtain chlamydial suspension before use.

Reference method
Cell culture and LCR method were used to evaluate the clinical performance of the enzymatic method. The swabs used for culture were dipped directly into incidental transport medium (Copan, Brescia, Italy) and cultured according to the aforementioned method. The cultures were tested using a C. trachomatis direct fluorescent antibody kit. LCR was carried out using the LCx C. trachomatis assay (Abbott Laboratories, Abbott Park, Israel) according to the manufacturer's instructions. Although culture method has a good specificity, its sensitivity may be influenced by various factors. Therefore, the research adopted an "expanded gold standard" [18][19][20] described as follows: any positive by either culture or LCR was classified as a true positive, whatever the result of the enzymatic method.

Results
In our developed assay, only C. trachomatis samples tested was positive for α-D-mannosidase activity; but OD 512 of both other organisms and cell cultures which were not inoculated with C. trachomatis used in the study was all below 0.100, which fell into the range of negative results negative with 0.150 (OD 512 ) as the cutoff value, even if the reactions were allowed to proceed for 1 h at 37°C. Of the 553 clinical samples, 132 samples were positive with OD 512 ranging from 0.161 to 1.955, and 421 samples were negative with OD 512 ranging from 0.013 to 0.142. C. trachomatis detection results of 553 cases with culture, LCR and enzymatic method used an expanded gold standard as the reference standard were showed in  (Table 6), respectively. There were no significant differences in performance between the enzymatic method and the expanded gold standard (P > 0.05). The result of the chlamydial suspension quantified with 617 IFU/ml was light pink, and might be considered positive (OD 512 = 0.162). Meanwhile, the result of the chlamydial suspension was colourless if quantified with 126 IFU/ml, and might be considered negative (OD 512 = 0.098). Therefore, the LOD was 617 IFU/ml for C. trachomatis. In addition, the LOD was 10 -3 u/ml (OD 512 = 0.155) for α-D-mannosidase.

Discussion
There are various well-known methods for detecting C. trachomatis, including cell culture-, immunology-, molecular biology-, and biochemistry-based methods. Cell culture is complicated to perform and requires experience to produce accurate results, and it also has more stringent requirements for the sampling swabs and transporting before inoculation [21,22]. Therefore, cell culture is rarely used in clinics. Among the available immunological methods, serological tests for the C. trachomatis antibody have significant limitations [23], but methods for C. trachomatis antigen detection (mainly referring to the lipopolysaccharide, LPS), especially immunochromatography-based methods, have been widely used due to their simplicity of operation. There are two ways to extract the LPS antigen for C. trachomatis immunochromatographic assays: heat extraction and acid extraction. Neither of these methods guarantees the full extraction of LPS as an intact antigen, which influences the sensitivity of this method. The biochemical detection of glycogen in C. trachomatis inclusions [24] is greatly affected by Candida spp. that often exist in these specimens and are especially common during the female menstrual cycle and pregnancy.
With the increasing glycogen in vaginal epithelial cells, this method may also cause false positives. The detection of C. trachomatis in the United States and Europe has mainly focused on molecular biology methods [25][26][27][28][29][30]. Although these methods are both high sensitivity and high specificity, it can be challenging for molecular biology methods to meet the requirements of the actual application in clinical screening.
Enzymatic studies of C. trachomatis, especially for enzymes with diagnostic significance, have not been reported in the literature. Previous studies [31] and our research have shown that C. trachomatis secretes extracellular enzymes with high α-D-mannosidase activity.
Although some organisms used in the study such as C. albicans have genes encoding α-1,2-mannosidase [32], α-D-mannosidase activity was invisible by naked eyes with 6-chloro-3-indolyl-α-D-mannoside as substrate. This may be because the extracellular α-D-mannosidase from these organisms is much less or the enzyme activity is relatively low. Previous records [31,[33][34][35] on substrates of α-D-mannosidase mainly involved p-nitrophenol-α-D-mannoside and 4-methylumbelliferyl-α-D-mannoside. However, 6-chloro-3-indolyl-α-D-mannoside is a novel chromogenic substrate, and colour reaction of its chromogen is much more sensitive especially in the case of the presence of an azo reagent such as fast violet B salt. Our results showed that clinical specimens such as urethral discharge, cervical secretions, and vaginal secretions did not interfere with the chromogenic detection of α-D-mannosidase activity to screen for C. trachomatis, although prostate massage liquid produced more false positive results. Some human sperm surface proteins possess α-D-mannosidase activity [36], which may be the reason that prostate specimens produce less reliable results. Serotype D was only one of the most prevalent (11.1%), and no serovar L2 was found in China [37].
Although the results of this study suggested that other serotypes, such as serotypes E, F, G, K, H, J, I, and Ba, at least most of them, might have α-D-mannosidase activity, there seems to be some limitation of tests on C. trachomatis cultures based on only one strain of serotype D. Further studies and more comprehensive clinical evaluations should be conducted due to little research on α-D-mannosidase activity of C. trachomatis. In addition, our studies did not evaluate C. pneumoniae or C. psittaci; the α-D-mannosidase activities of these species should be studied as well.