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

  • Ze-yu Wang1,

    Affiliated with

    • Guang-yu Fu1,

      Affiliated with

      • Shan-mei Wang2,

        Affiliated with

        • Dong-chun Qin3,

          Affiliated with

          • Zhong-quan Wang1 and

            Affiliated with

            • Jing Cui1Email author

              Affiliated with

              BMC Infectious Diseases201313:36

              DOI: 10.1186/1471-2334-13-36

              Received: 19 August 2012

              Accepted: 22 January 2013

              Published: 24 January 2013

              Abstract

              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.

              Keywords

              Chlamydia trachomatis α-mannosidase Activity Gold standard Marker

              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 [69], 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 [1215]. 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.

              Methods

              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.
              Table 1

              Reference strains and cell line

              Strains or cell line

              Accession numbers

              Organizations

              Acinetobacter baumannii

              ATCC19606

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Candida albicans

              ATCC10231

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Candida glabrata

              ATCC15126

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Candida guilliermondii

              ATCC6260

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Candida krusei

              ATCC14243

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Cryptococcus neoformans

              CMCC(F)D2q

              China Medical Microbiological Culture Collection Center (fungi)(Nanjing, China)

              Candida parapsilosis

              CGMCC2.1846

              China General Microbiological Culture Collection Center (Beijing, China)

              Candida tropicalis

              ATCC750

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Chlamydia trachomatis Serovar D

              VR-885

              American Type Culture Collection(Manassas, USA)

              Enterococcus faecalis

              ATCC29212

              Huankai Microbial Sci & Tech. Co., Ltd (Guangzhou, China)

              Enterococcus faecium

              ATCC700221

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Escherichia coli

              ATCC25922

              Henan Provincial Institute of Food and Drug Control (Zhengzhou, China)

              Gardnerella vaginalis

              ATCC14018

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Haemophilus influenzae

              ATCC10211

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Klebsiella pneumoniae

              CMCC46117

              Tianhe Microorganism Reagent Co., Ltd (Hangzhou, China)

              McCony

              CRL-1696

              American Type Culture Collection(Manassas, USA)

              Mycoplasma hominis

              ATCC15488

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Neisseria gonorrhoeae

              ATCC19424

              Henan Provincial Center for Disease Control and Prevention (Zhengzhou, China)

              Pseudomonas aeruginosa

              ATCC25619

              Land Bridge Biotechnology Co., Ltd (Beijing, China)

              Proteus mirabilis

              CMCC(B)49005

              Huankai Microbial Sci & Tech. Co., Ltd (Guangzhou, China)

              Salmonella enteritidis

              ATCC13076

              Land Bridge Biotechnology Co., Ltd (Beijing, China)

              Staphylococus aureus

              ATCC25923

              Henan Provincial Institute of Food and Drug Control (Zhengzhou, China)

              Staphylococcus aureus

              ATCC29213

              Capital Institute of Pediatrics (Beijing, China)

              Staphylococcus epidermidis

              ATCC12228

              Huankai Microbial Sci & Tech. Co., Ltd (Guangzhou, China)

              Staphylococus saprophyticus

              ATCC49453

              Land Bridge Biotechnology Co., Ltd (Beijing, China)

              Stenotrophomonas maltophilia

              ATCC17666

              Land Bridge Biotechnology Co., Ltd (Beijing, China)

              Streptococus agalactiae

              ATCC13813

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Streptococus pneumoniae

              ATCC49619

              National Center for Clinical Laboratory (Beijing, China)

              Streptococus pyogenes

              ATCC19615

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Trichomonas vaginalis

              ATCC30001

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              Ureaplasma urealyticum

              ATCC15531

              Harmony Biotechnology Co., Ltd (Shanghai, China)

              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 colour-changing unit (CCU) method, as previously reported [17]. Once the concentrations of mycoplasma reached 106 CCU/ml in LMB, the cultures of LMA should be immediately stored at 4°C.
              Table 2

              Composition of LMA for Mycoplasma (per liter) *

              Ingredient

              Concentration

              Ingredient

              Concentration

              NaCl

              6.4 g

              Beef heart extract power

              7.2 g

              CaCl2

              112 mg

              Yeast extract power

              2.72 g

              MgCl2·6H2O

              80 mg

              Peptone

              8.0 g

              MgSO4·7H2O

              80 mg

              Horse serum

              200 ml

              KCl

              320 mg

              Penicillin sodium

              1,000,000 units

              Na2HPO4·12H2O

              122 mg

              Ampicillin sodium

              375 mg

              KH2PO4

              48 mg

              Vancomycin

              40 mg

              Cysteine hydrochloride

              0.8 g

              Polymyxin B

              40,000 units

              Arginine hydrochloride

              4.0 g

              Nystatin

              15,000 USP units

              Urea

              4.0 g

                

              *Adjusted to pH 6.25.

              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.
              Table 3

              The media and culture methods for bacteria and candida

              Strains

              Media

              Temp.

              Time

              Bacteria

                 

              A. baumannii, E. faecalis,

              Blood agar base a

              37°C

              24 h

              E. faecium, E. coli, K. pneumoniae,

              P. mirabilis, P. aeruginosa,

              S. enteritidis, S. aureus,

              S. epidermidis, S. saprophyticus,

              S. maltophilia, S. agalactiae,

              S. pyogenes, S. pneumoniae

              G. vaginalis *

              Blood agar base

              37°C

              48 h

              H. influenzae *

              Thayer Martin media b

              24 h

              N. gonorrhoeae *

              48 h

              Candida

                 

              C. albicans, C. tropicalis,

              Sabouraud dextrose agar c

              37°C

              24 h

              C. glabrata, C. parapsilosis,

              C. guilliermondii, C. krusei,

              C. neoformans

              30°C

              48 h

              a,b,c Oxoid, Basingstoke, UK; a supplemented with 5% sheep blood; b supplemented with 5% horse blood; * cultured in a candle jar.

              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 (OD512 ≧ 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 (OD512 < 0.150). The suspensions containing bacteria or cells was centrifuged at 5,000 rpm for 5 min, and then OD512 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. Serial 10-fold dilutions of the chlamydial suspension were inoculated in six duplicate into wells (100 μl/well) of cycloheximide-treated McCoy cell monolayers that had been incubated with MEM medium (Gibco, Grand Island, NY, USA) in a 96-well flat-bottom microtiter plate (Nunc Inc., Roskilde, Denmark) at 37°C under 5% CO2 for 48 h. Inclusion body titers of the chlamydial supernatant were quantified by titrating the number of inclusion-forming units (IFU). The contents of each well were stained with a C. trachomatis direct fluorescent antibody kit (Academy of Military Medical Science, Beijing, China) and examined by microscopy for IFU counts. The average IFU of each dilution culture of three replicate wells was taken as the concentration of C. trachomatis in the corresponding dilution of the chlamydia suspension. Each dilution culture of the other three replicate wells was examined via the enzymatic method after collection to determine the LOD for C. trachomatis of the enzymatic method. 10-1 u/ml α-D-mannosidase (EC3.2.1.24; Sigma, USA) solution was 5-fold serially diluted to 10-4 u/ml. These enzyme solutions fold-diluted were detected via the enzymatic method to determine the LOD for α-D-mannosidase.

              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” [1820] 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 OD512 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 (OD512) as the cut-off 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 OD512 ranging from 0.161 to 1.955, and 421 samples were negative with OD512 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 4. The enzymatic method was least reliable when prostate specimens were used. The sensitivity of the enzymatic method was 91.5% (95% confidence interval [CI], 85.9% to 97.1%), and the specificity of this method was 90.0% (95% CI, 87.3% to 92.7%) (Table 5). The sensitivity and specificity of the LCR were 94.7% (95% CI, 90.2% to 99.2%) and 100% (95% CI, 100.0% to 100.0%), respectively. However, in those specimens other than prostate fluid samples, the sensitivity and specificity of the enzymatic method were 91.8% (95% CI, 86.0% to 97.6%) and 98.3% (95% CI, 97.1% to 99.5%), in the meantime the sensitivity and specificity of the LCR were 95.3% (95% CI, 90.8% to 99.8%) and 100% (95% CI, 100% to 100%) (Table 6), respectively. There were no significant differences in performance between the enzymatic method and the expanded gold standard (P > 0.05).
              Table 4

              C. trachomatis detection results of 553 cases with culture, LCR and enzymatic method used an expanded gold standard as the reference standard

              Method

              No. of true positives/positives

              No. of true negatives/negatives

              Enzymatic method

              86/132

              413/421

               

              Urethral discharge 22/24

              Urethral discharge 126/127

               

              Prostate massage liquid 8/47

              Prostate massage liquid 4/5

               

              Cervical secretion 31/33

              Cervical secretion 196/199

               

              Vaginal secretion 25/28

              Vaginal secretion 87/90

              Cell culture

              60/60

              459/493

               

              Urethral discharge 18/18

              Urethral discharge 128/133

               

              Prostate massage liquid 7/7

              Prostate massage liquid 43/45

               

              Cervical secretion 21/21

              Cervical secretion 198/211

               

              Vaginal secretion 14/14

              Vaginal secretion 90/104

              LCR

              85/89

              459/464

               

              Urethral discharge 22/22

              Urethral discharge 128/133

               

              Prostate massage liquid 8/8

              Prostate massage liquid 43/45

               

              Cervical secretion 32/32

              Cervical secretion 198/211

               

              Vaginal secretion 27/27

              Vaginal secretion 90/104

              Expanded gold standard

              94

              459

               

              Urethral discharge 23

              Urethral discharge 128

               

              Prostate massage liquid 9

              Prostate massage liquid 43

               

              Cervical secretion 34

              Cervical secretion 198

               

              Vaginal secretion 28

              Vaginal secretion 90

              Table 5

              Clinical performances of three assays for C. trachomatis using specimens included prostate massage liquid

              Methods

              % Sensitivity (95% CI)

              % Specificity (95% CI)

              Enzymatic methoda

              91.5 (85.9, 97.1)

              90.0 (87.3, 92.7)

              Cell cultureb

              63.8 (54.1, 73.5)

              100.0 (100.0, 100.0)

              LCRc

              94.7 (90.2, 99.2)

              100.0 (100.0, 100.0)

              a Χ2 = 8.030, P = 0.005, P < 0.05; b Χ2 = 8.721, P = 0.003, P < 0.05; c Χ2 = 0.164, P = 0.685, P > 0.05.

              Table 6

              Clinical performances of three assays for C. trachomatis using specimens excluded prostate massage liquid

              Methods

              % Sensitivity (95% CI)

              % Specificity (95% CI)

              Enzymatic methoda

              91.8 (86.0, 97.6)

              98.3 (97.1, 99.5)

              Cell cultureb

              64.6 (54.3, 74.9)

              100.0 (100.0, 100.0)

              LCRc

              95.3 (90.8, 99.8)

              100.0 (100.0, 100.0)

              a Χ2 = 0, P = 1, P > 0.05; b Χ2 = 8.605, P = 0.003, P < 0.05; c Χ2 = 0.116, P = 0.734, P > 0.05.

              The result of the chlamydial suspension quantified with 617 IFU/ml was light pink, and might be considered positive (OD512 = 0.162). Meanwhile, the result of the chlamydial suspension was colourless if quantified with 126 IFU/ml, and might be considered negative (OD512 = 0.098). Therefore, the LOD was 617 IFU/ml for C. trachomatis. In addition, the LOD was 10-3 u/ml (OD512 = 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 [2530]. 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, 3335] 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.

              Conclusions

              The present study demonstrated that there were no significant differences between the enzymatic method and the reference method when prostate specimens were excluded. Therefore, α-D-mannosidase activity may be a useful marker for C. trachomatis in urogenital tract specimens, with many advantages, such as its speed, ease of use, convenience, and need for no special equipment. Taken together, these data show that the enzymatic method has great potential as a clinical method for C. trachomatis screening.

              Declarations

              Authors’ Affiliations

              (1)
              Department of Pathogen Biology, School of Basic Medicine, Zhengzhou University
              (2)
              Department of Clinical Laboratory, Henan Provincial People’s Hospital
              (3)
              Department of Clinical Laboratory, First Affiliated Hospital, Zhengzhou University

              References

              1. Centers for Disease Control and Prevention: Sexually transmitted disease surveillance, 2009. Atlanta, GA: Centers for Disease Control and Prevention; 2011.
              2. Madeleine MM, Anttila T, Schwartz SM, Saikku P, Leinonen M, Carter JJ, Wurscher M, Johnson LG, Galloway DA, Daling JR: Risk of cervical cancer associated with Chlamydia trachomatis antibodies by histology, HPV type and HPV cofactors. Int J Cancer 2007, 120:650–655.PubMedView Article
              3. Smith JS, Bosetti C, Muñoz N, Herrero R, Bosch FX, Eluf-Neto J, Meijer CJ, Van den Brule AJ, Franceschi S, Peeling RW: Chlamydia trachomatis and invasive cervical cancer: A pooled analysis of the IARC multicentric case–control study. Int J Cancer 2004, 111:431–439.PubMedView Article
              4. Joki-Korpela P, Sahrakorpi N, Halttunen M, Surcel HM, Paavonen J, Tiitinen A: The role of Chlamydia trachomatis infection in male infertility. Fertil Steril 2009, 91:1448–1450.PubMedView Article
              5. Malik A, Jain S, Rizvi M, Shukla I, Hakim S: Chlamydia trachomatis infection in women with secondary infertility. Fertil Steril 2009, 91:91–95.PubMedView Article
              6. Blas MM, Canchihuaman FA, Alva IE, Hawes SE: Pregnancy outcomes in women infected with Chlamydia trachomatis: a population-based cohort study in Washington State. Sex Transm Infect 2007, 83:314–318.PubMedView Article
              7. Medina M, Moya M, Hidalgo L, Calle A, Terán Eui P: Molecular identification of endocervical Chlamydia trachomatis infectionamong gestations at risk for preterm birth in Ecuador. Arch Gynecol Obstet 2009, 279:9–10.PubMedView Article
              8. Silveira MF, Ghanem KG, Erbelding EJ, Burke AE, Johnson HL, Singh RH, Zenilman JM: Chlamydia trachomatis in fection during pregnancy and the risk of preterm birth:a case–control study. Int J STD AIDS 2009, 20:465–469.PubMedView Article
              9. Wilkowska-Trojniel M, Zdrodowska-Stefanow B, Ostaszewska-Puchalska I, Redźko S, Przepieść J, Zdrodowski M: The influence of Chlamydia trachomatis infection on spontaneous abortions. Adv Med Sci 2009, 54:86–90.PubMed
              10. Rours IG, Hammerschlag MR, Ott A, De Faber TJ, Verbrugh HA, de Groot R, Verkooyen RP: Chlamydia trachomatis as a cause of neonatal conjunctivitis in Dutch infants. Pediatrics 2008, 21:e321-e326.View Article
              11. Black CM: Current methods of laboratory diagnosis of Chlamydia trachomatis infections. Clin Microbiol Rev 1997, 10:160–184.PubMed
              12. Manafi M: Fluorogenic and chromogenic enzyme substrates in culture media and identification tests. Int J Food Microbiol 1996, 31:45–58.PubMedView Article
              13. Orenga S, James AL, Manafi M, Perry JD, Pincus DH: Enzymatic substrates in microbiology. J Microbiol Methods 2009, 79:139–155.PubMedView Article
              14. Van Winkelhoff AJ, Clement M, De Graaff J: Rapid characterization of oral and nonoral pigmented Bacteroides species with the ATB anaerobes ID system. J Clin Microbiol 1988, 26:1063–1065.PubMed
              15. Wohlsen TD: Comparative evaluation of chromogenic agar CM1046 and mFC agar for detection of E. coli and thermotolerant coliform bacteria from water samples. Lett Appl Microbiol 2011, 53:155–160.PubMedView Article
              16. Wang ZY, Kou J, Cui J, Wang ZQ: Chlamydia diagnosis method and reagent. China: CN patent 102286608A; 2011.
              17. Taylor-Robinson D, Thomas M, Dawson PL: The isolation of T-mycoplasmas from the urogenital tract of bulls. J Med Microbiol 1969, 2:527–533.PubMedView Article
              18. Berg ES, Anestad G, Moi H, Størvold G, Skaug K: False- negative results of a ligase chain reaction assay to detect Chlamydia trachomatis due to inhibitors in urine. Eur J Clin Microbiol Infect Dis 1997, 16:727–731.PubMedView Article
              19. Jang D, Sellors JW, Mahony JB, Pickard L, Chernesky MA: Effects of broadening the gold standard on the performance of a chemiluminometric immunoassay to detect Chlamydia trachomatis antigens in centrifuged first void urine and urethral swab samples from men. Sex Transmitted Dis 1992, 19:315–319.View Article
              20. Lee HH, Chernesky MA, Schachter J, Burczak JD, Andrews WW, Muldoon S, Leckie G, Stamm WE: Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine. Lancet 1995, 345:213–216.PubMedView Article
              21. Mahony JB, Chernesky MA: Effect of swab type and storage temperature on the isolation of Chlamydia trachomatis from clinical specimens. J Clin Microbiol 1985, 22:865–867.PubMed
              22. Mårdh P, Zeeberg B: Toxic effect of sampling swabs and transportation test tubes on the formation of intracytoplasmic inclusions of Chlamydia trachomatis in McCoy cell cultures. Br J Vener Dis 1981, 57:268–272.PubMed
              23. Ngeow YF: Limitations of serodiagnosis in chlamydial genital tract infections. Ann Acad Med Singapore 1996, 25:300–304.PubMed
              24. Wang ZY, Fu GY, Chen RF, Qin Y: Fast diagnosis reagent for genital tract Chlamydia trachomatis. China: CN patent 1217004C; 2005.
              25. Cheng A, Qian Q, Kirby JE: Evaluation of the Abbott RealTime CT/NG assay in comparison to the Roche cobas amplicor CT/NG assay. J Clin Microbiol 2011, 49:1294–1300.PubMedView Article
              26. Cosentino LA, Landers DV, Hillier SL: Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by strand displacement amplification and relevance of the amplification control for use with vaginal swab specimens. J Clin Microbiol 2003, 41:3592–3596.PubMedView Article
              27. Jalal H, Al-Suwaine A, Stephen H, Carne C, Sonnex C: Comparative performance of the Roche COBAS Amplicor assay and in-house real-time PCR assay for diagnosis of Chlamydia trachomatis infection. J Med Microbiol 2007, 56:320–322.PubMedView Article
              28. Kerndt P, Ferrero DV, Aynalem G, Monga D, Wang S, Zhang N, Wong C, Liggins M, Meng Q: First report of performance of the versant CT/GC DNA 1.0 Assay (kPCR) for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. J Clin Microbiol 2011, 49:1347–1353.PubMedView Article
              29. Moller JK, Pedersen LN, Persson K: Comparison of the Abbott RealTime CT new formulation assay with two other commercial assays for detection of wild-type and new variant strains of Chlamydia trachomatis. J Clin Microbiol 2010, 48:440–443.PubMedView Article
              30. Pedersen LN, Pødenphant L, Møller JK: Highly discriminative genotyping of Chlamydia trachomatis using omp1 and a set of variable number tandem repeats. Clin Microbiol Infect 2008, 14:644–652.PubMedView Article
              31. Greenwell P, Kakourou G, Raghooputh S: Analysis of glycosidases activity in Chlamydia trachomatis L2 Serotype. Int J med Update 2006, 1:25–32.
              32. Siriwardena A, Strachan H, El-Daher S, Way G, Bryan W, Glushka J, Moremen K, Boons GJ: Potent and selective inhibition of class II α-D-mannosidase activity by a bicyclic sulfonium salt. ChemBioChem 2005, 6:845–848.PubMedView Article
              33. Skudlarek MD, Orgebin-Crist MC: Effect of swainsonine on rat epididymal glycosidases. J Reprod Fert 1988, 84:611–617.View Article
              34. Tulsiani DR: Glycan-modifying enzymes in luminal fluid of the mammalian epididymis: an overview of their potential role in sperm maturation. Mol Cell Endocrinol 2006, 250:58–65.PubMedView Article
              35. Vázquez-Reyna AB, Balcázar-Orozco R, Flores-Carreón A: Biosynthesis of glycoproteins in Candida albicans: biochemical characterization of a soluble alpha-mannosidase. FEMS Microbiol Lett 1993, 106:321–325.PubMedView Article
              36. Tulsiani DR, Skudlarek MD, Orgebin-Crist MC: Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity. Biol Reprod 1990, 42:843–858.PubMedView Article
              37. Gao X, Chen XS, Yin YP, Zhong MY, Shi MQ, Wei WH, Chen Q, Peeling RW, Mabey D: Distribution study of Chlamydia trachomatis serovars among high-risk women in china performed using PCR-restriction fragment length polymorphism genotyping. J Clin Microbiol 2007, 45:1085–1089.View Article
              38. Pre-publication history

                1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2334/​13/​36/​prepub

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