ARTICLE |
Correspondence to: Thilo Schlott, Dept. of Pathology, Div. of Cytopathology, Georg-August-University, Robert-Koch-Str. 40, D-37075 Goettingen, Germany..
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The in situ polymerase chain reaction (PCR) is a technique that has important applications in the diagnosis of viral and bacterial diseases. This study investigated an in situ PCR assay established to detect the presence of Chlamydia trachomatis in endocervical swabs. In addition, histological sections of endocervical squamous cell carcinoma were analyzed because previous studies had revealed a significant association with C. trachomatis. A total of 20 cervical neoplasms (squamous cell carcinoma in situ; n = 10; invasive squamous cell carcinoma; n = 10) and endocervical smears taken from five patients with and without inflammatory changes were analyzed by conventional PCR. Chlamydial DNA was found in 10 histological samples (six carcinomas in situ, four invasive carcinomas) and in one endocervical swab from a patient with known C. trachomatis infection. Positive specimens were used for establishing an in situ PCR assay (IS-PCR). After IS-PCR, these samples showed dense cytoplasmic staining of endocervical cells (smears) and non-neoplastic epithelial cells (cervical neoplasms). The other tumor samples and smears did not demonstrate positive PCR reaction. The results indicate that in situ PCR is an effective technique for localizing C. trachomatis in target cells because IS-PCR detection of chlamydial DNA correlated with histological and cytological features. (J Histochem Cytochem 46:10171023, 1998)
Key Words: PCR, in situ PCR, electron microscopy, Chlamydia trachomatis, endocervical swabs, endocervical squamous cell carcinoma
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chlamydia trachomatis is a pleomorphic nonmotile organism about 0.21.5 µm in length. This obligate intracellular bacterium depends on host cell metabolites and shows a unique growth cycle characterized by formation of reticulate bodies, intermediate bodies, and elementary bodies (
For a long time, the cell culture method established 30 years ago was the technique of choice to detect the intracellular bacteria (
In situ PCR is a novel method that has been already used to detect low copy number DNA sequences of viral HIV, HPV, CMV, and HBV in tissue (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sample Collection
Paraffin-embedded squamous cell carcinomas of the cervix were collected from 20 patients aged 2136 years. The tumors were histologically classified as squamous cell carcinoma in situ (n = 10) and invasive squamous cell carcinoma (n = 10) and were obtained from 1995 to 1996 by cold-knife conization. In addition, cervical smears from five patients with or without inflammatory cellular changes were analyzed. Two of the five smears were obtained from patients with C. trachomatis infection. The remaining swabs were from patients who were not infected. The endocervical samples were obtained by inserting a cotton swab, rotating it 360° for 10 sec, and transferring the cells to a glass slide. The sample was fixed with 96% ethanol (v/v) before transport. Positive controls were McCoy mouse fibroblasts infected with C. trachomatis L2. A melanoma cell line served as the negative control.
PCR
DNA preparation.
Histological sections and cervical smears were scraped into sterile Eppendorf microfuge tubes using a clean razor blade. The histological samples were incubated twice at 37C for 5 min with xylene to remove the residual paraffin. The resulting pellets were used for DNA isolation by applying the QIAamp Tissue Kit (QIAGEN; Hilden, Germany). The eluted DNA was concentrated by vacuum centrifugation, suspended in 3 µl H2O, and stored at -20C.
Amplification. For amplification of the C. trachomatis plasmid sequence, the PCR primers were 5'-GGACAAATCGTATCTCGG-3' (T1, sense primer) and 5'-GAAACCAACTCTACGCTG-3' (T2, antisense primer) (Mahony et al. 1992). The combination yielded a 517-BP fragment. Reaction tubes were heated to 94C for 7 min followed by 40 cycles at 95C for 1 min, 55C for 1 min, and 72C for 1 min and a final extension at 72C for 7 min.
The standard PCR mix consisted of 2.75 mM MgCl2 (PerkinElmer; Weiterstadt, Germany), 10x PCR buffer [PerkinElmer; contains 500 mM KCl, 100 mM Tris-HCl, pH 8.3, at 25C, 0.3 µg primer, 10 µM of each dNTP (Pharmacia; Freiburg, Germany), 1 U of Taq DNA polymerase (PerkinElmer)]. One µl of extracted DNA was used for PCR. The final volume was 50 µl. The mixture was overlaid with mineral oil (Sigma; Munich, Germany). Thermal cycling was performed on a DNA thermal cycler 480 (PerkinElmer) using thin-walled reaction tubes. Standard precautions against cross-contamination were followed (
In Situ PCR
Fixation and Digestion of Material.
The paraffin-embedded cervix carcinoma specimens were cut in 4-µm sections, mounted on 3-aminopropyl-triethoxysilane-coated glass slides (PerkinElmer), and incubated overnight at 60C in an incubator. The sections were dewaxed by washing twice in xylol at 37C for 5 min. Residual xylol was extracted by placing the samples in 80, 90, and 100% ethanol for 1 min. Tissue was air-dried and rehydrated by placement in 100, 90, and 80% ethanol for 1 min. The slides were washed with 1x PBS buffer (150 mM NaCl, 10 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.1) for 5 min and transferred into TEN/protease A buffer (1 M Tris-HCl, pH 8.0, 0.5 ml/0.5 M EDTA, pH 8.0 0.1 ml/5 M NaCl 1.0 ml/ 6 µg protease/ml final concentration) for 10 min. The specimens were washed with 1x PBS for 5 min and air-dried.
The cervical smears placed on coated glass slides were fixed in 100% ethanol and air-dried. The swabs were fixed overnight in 1x PBS/formaldehyde 4% (v/v), washed with 1x PBS for 5 min, and air-dried. TEN/protease A buffer (1 M Tris-HCl, pH 8.0, 0.5 ml/0.5 M EDTA, pH 8.0, 0.1 ml/5 M NaCl 1.0 ml/800 ng protease/ml final concentration) was pipetted onto the cells. The cells were incubated at room temperature (RT) for 5 min, washed with 1x PBS, and air-dried.
Amplification. PCR amplification was conducted on an in situ thermal cycler (PerkinElmer). The PCR mix was trapped over the air-dried samples by silicon diaphragms using mounting clips and an assembly tool (PerkinElmer).
The selection of cycling conditions depended on the type of material. For amplification of DNA isolated from histological sections, reaction tubes were heated to 94C for 7 min followed by 40 cycles at 95C for 1 min, 60C for 1 min, and 72C for 1.5 min and a final extension at 72C for 7 min. For amplification of DNA isolated from cytological smears, reaction tubes were heated to 94C for 7 min followed by 40 cycles at 95C for 1 min, 55C for 45 sec, and 72C for 1 min and a final extension at 72C for 7 min.
The PCR mix consisted of 2.75 mM MgCl2 (PerkinElmer) 10x PCR buffer [PerkinElmer; contains 500 mM KCl, 100 mM Tris-HCl, pH 8.3, at 25C, 0.3 µg primer, 10 µM of each dNTP (Pharmacia), 0.5 nM biotin-16-dUTP (Boehringer; Mannheim, Germany), 5 U Taq DNA (PerkinElmer)]. Polymerase PCR was performed on a final volume of 50 µl. The mix contained 0.3 µg of the primers T1 and T2 for a 517-BP sequence of the endogenous plasmid. It was necessary to add BSA (Sigma) in a final concentration of 0.5% (w/v) for successful amplification of DNA on histological material.
Detection of Biotin-labeled PCR Fragments
Labeled DNA fragments were detected with primary antibody, anti-biotin monoclonal antibody immunoglobulin (Boehringer) at 1:200 dilution and using the Universal immunostaining kit streptavidinperoxidase with ACE (Immunotech; Coulter, Marseille, France) according to the manufacturer's instructions, with the following modification. Before adding the primary antibody, sections and smears were incubated with drug-free serum (Bio-Rad; Munich, Germany) at RT for 2 hr. The recommended incubation time with primary antibody was reduced from 1 hr to 30 min.
Controls
Appropriate controls were performed as recommended by Komminoth and co-workers (1992). These included the use of (a) irrelevant primers for amplification (primers directed against the outer surface protein OspA of the bacterium Borrelia burgdorferi; /
positive controls (an antibody solution provided in the kit). The test specimens were used as an internal control. The antibody reacted with plasma cells in the sections. Alternatively, dewaxed sections of lymph nodes from patients with centroblastic lymphoma (Kiel classification) served as positive controls that were stained with the solution. In each run, diffuse cytoplasmic staining of target cells proved that the test was done correctly and that the fixation was at least sufficient and appropriate.
Sequence Analysis of C. trachomatis DNA Fragment Obtained by IS-PCR
Several histological sections (15 µm each) were cut from a cervical sample that had shown C. trachomatis positivity according to the results of conventional and IS-PCR. The sections were used for IS-PCR. After amplification, the sections were pooled and washed with 1x PBS for 5 min. The DNA was isolated as described above, concentrated in a vacuum concentrator (Eppendorf; Hamburg, Germany), and separated on 3% low melting point agarose (Biozym). A cube of agarose containing a 517-BP product was cut from the gel. The amplification product was purified with the QIAEX II Gel Extraction Kit (QIAGEN) and suspended in 3 µl H2O. Total DNA was labeled with the PRISM Ready Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems; Weiterstadt, Germany) according to the manufacturer's instructions and was analyzed in an Applied Biosystems DNA sequencer (model 373A).
Electron Microscopy
McCoy cells infected with C. trachomatis and cells from fresh cervical smears were harvested by centrifugation at 1000 x g for 10 min and fixed in a solution of glutaraldehyde 0.3% (v/v) (grade I; Sigma) in PBS at 4C for 1.5 hr. The cells were embedded in the low-temperature resin Lowicryl K4M (Chem Werke Lowi; Waldkraiburg, Germany) according to
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Detection of C. trachomatis by Conventional PCR
In preceding work, conventional PCR was used to select C. trachomatis-positive carcinoma samples that were later used to establish an in situ PCR assay. Paraffin-embedded material from 20 young women with endocervical carcinomas (10 squamous cell carcinomas in situ, 10 invasive squamous cell carcinomas) and fresh endocervical smears from five patients with and without cellular changes were analyzed for C. trachomatis DNA. By amplifying a 517-BP sequence of the cryptic plasmid (
|
Establishment of a C. trachomatis-specific IS-PCR
The IS-PCR assay established was based on amplification of a 517-BP DNA fragment of the cryptic plasmid. We used biotin-labeled nucleotides that were incorporated into the product during PCR. The voluminous biotin structure and the length of the PCR fragment reduced the formation of "diffusion" artifacts and the loss of PCR products during the washing steps. Both effects were observed in preceding IS-PCR experiments performed with other primer pairs flanking C. trachomatis plasmid sequences shorter than 500 BP. We used positive McCoy cells and the endocervical specimens that had given positive results in conventional PCR for establishing the IS-PCR assay. The remaining samples and a melanoma cell line served as negative controls. Typically, dense staining was observed in columnar endothelial cells, in squamous metaplastic epithelial cells, in a few superficial squamous cells, and even in some basal cells of the positive controls (Figure 2). In contrast, malignant neoplastic cells found in histological sections did not show positive staining. Other samples, such as the endocervical sections and smears of noninfected women and the melanoma cell line, gave negative results, i.e., the cytoplasm of target cells lacked staining.
|
The best IS-PCR results for smears were generally achieved with specimens that had been fixed with 96% ethanol (v/v) before transport and that contained enough epithelial cells with preserved morphology. Applying the TEN/protease A assay resulted in data that are easy to reproduce. In addition, other digestion techniques were performed, e.g., treating specimens with (a) trypsin (2 mg/ml, 37C) for 20, 30, 40, 50 min and (b) proteinase K (10 µg/ml) for 10 min followed by 0.2% (w/v) glycine for 5 min. However, with these techniques either unspecific background staining or lack of staining was observed.
Detection of C. trachomatis by Electron Microscopy (EM)
To quantify the number of bacteria in an infected cell, the positive control McCoy cells were examined by EM. The cells were embedded in resin and ultrathin sections were cut from the samples. EM indicated that the Lowicryl K4M sections of the McCoy cells contained large numbers of chlamydial inclusion bodies (Figure 3). EM was also performed with cervical smears from a patient with known C. trachomatis infection. In the ultrathin sections, however, the few cells infected could not be detected among other noninfected cells. The paraffin-embedded material was not suited for EM because the morphology of dewaxed cells was easily destroyed by the EM techniques used for embedding.
|
Specificity of the Direct IS-PCR Assay Applied on Sections
After the IS-PCR procedure and in lieu of staining biotin-labeled PCR fragments, we isolated total DNA from the sections and smears that had shown significant staining of target cells in preceding IS-PCR experiments. In all cases, a weak DNA band of 517 BP was visualized on agarose gels. DNA contaminations that would result in faint smears or fragment ladders were not observed. The specificity of the IS-PCR fragment was proved by automatic sequencing and sequence alignment with the published plasmid sequence. The sequence alignment revealed 100% homology between the sequence of the amplified fragment and the published sequence of the cryptic plasmid (data not shown).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intracellular bacteria represent model organisms for the application of new genetic technologies such as in situ PCR, which allows visualization of amplified target DNA under the light microscope (
C. trachomatis infections are difficult to detect in conventional cytological smears because swabs of infected women often reveal normal leukocyte populations and show no inflammatory changes in epithelial cells or only nonspecific cytological features. Such smears contain very low numbers of C. trachomatis bacteria. This is why techniques such as time-consuming cultivation fail, because the transport conditions limit the survival time of the sensitive bacteria outside the human body. Therefore, other genetic techniques, such as sensitive non-IS-PCR and IS-PCR, appear more suited for cytological specimens. In the second part of our study, we used our IS-PCR to localize C. trachomatis in fresh endocervical swabs. We observed fine granular staining of chlamydial DNA in infected cells, which might be attributable to complete proteolytic destruction of the bacterial inclusion bodies. Because the three-layered cell wall of the inclusion bodies most likely consists of protein, as reported for the inclusion membrane of C. psittaci (
Direct IS-PCR is a difficult technique that requires intensive optimization work. The data published to date on this type of IS-PCR technique are still contradictory. Indeed, several working groups have demonstrated that direct IS-PCR on archival histological material leads to false-positive results (
The C. trachomatis-specific IS-PCR established in this study has important applications. First, this technique enhances the histology- and cytology-based diagnosis of Chlamydia infections because the cell structure is preserved and PCR data can be easily correlated to morphological features. Second, the assay may prove a useful diagnostic adjunct for testing the outcome of antibiotic therapy, an increasingly important factor in light of the fact that chronic infections give rise to nonproliferative as well as antibiotic-resistant forms of bacteria.
![]() |
Acknowledgments |
---|
We thank Dr Marianne Scriba of the Division of Medical Microbiology, Department of Human Genetics and Hygiene, University of Goettingen, for providing us with the C. trachomatis-infected McCoy cells. This study is part of the doctoral dissertation of Cand. Med. Götz Ruda.
Received for publication January 6, 1998; accepted April 18, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barbour AG, Garon CF (1988) The genes encoding major surface outer proteins of Borrelia burgdorferi are located on a plasmid. Ann NY Acad Sci 539:144-152[Medline]
Brunham RC, McLean IW, Binns B, Peeling R (1985) Chlamydia trachomatis: its role in tubal infertility. J Infect Dis 152:1275-1282[Medline]
Caldwell HD, Kuo CC (1977) Serological diagnosis of Lymphogranuloma venerum by counter immunoelectrophorese with a Chlamydia trachomatis protein antigen. J Immunol 118:442-445[Abstract]
Carlemalm E, Garavito RN, Villiger W (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J Microsc 126:123-143
DeSanjose S, Munoz N, Bosch FX, Reimann K, Pedersen NS, Orfila J, Ascunce N, Gonzalez LC, Tafur L, Gilli M, Lette I, Viladiu P, Tormo MJ, Moreo P, Shah K, Wahren B (1994) Sexually transmitted agents and cervical neoplasia in Colombia and Spain. Int J Cancer 56:358-363[Medline]
Domeika MA, Bassiri M, Mardh PA (1994) Enzyme immunoassay and direct immunofluorescence for detection of Chlamydia trachomatis in male first-void urine. Acta Dermatol Venereol Scand 74:323-326
Eley A, Oxley KM, Spencer RC, Kinghorn GR, Ben-Ahmeida ET, Potter CW (1992) Detection of Chlamydia trachomatis by the polymerase chain reaction in young patients with acute epididymitis. Eur J Clin Microbiol Infect Dis 11:620-623[Medline]
Frost EH, Deslandes S, Veilleux S, BourgauxRamoisy D (1991) Typing of Chlamydia trachomatis by detection of restriction fragment length polymorphism in the gene encoding the major outer membrane protein. J Infect Dis 163:1103-1107[Medline]
Hodinka RT, Davis CH, Choong J, Wyrick PB (1988) Ultrastructural study of endocytosis of Chlamydia trachomatis by McCoy cells. Infect Immun 56:1456-1463[Medline]
Honig JF, Becker HJ, Brinck U, Korabiowska M (1995) Detection of human papillomavirus DNA sequences in leucocytes: a new approach to identify hematological markers of HPV infection in patients with oral SCC. Bull Group Int Rech Sci Stomatol Odontol 38:25-31[Medline]
Hoyme UB (1989) Chlamydieninfektionen. Zentralbl Gynäkol 111:65-77[Medline]
Komminoth P, Long AA (1993) In situ polymerase chain reaction: an overview of methods, applications and limitations of a new molecular technique. Virchows Arch [B] 64:67-73[Medline]
Komminoth P, Long AA, Ray R, Wolfe HJ (1992) In situ polymerase chain reaction detection of viral DNA, single copy genes and gene rearrangements in cell suspensions and cytospins. Diag Mol Pathol 1:85-97[Medline]
Kwok S, Higuchi R (1989) Avoiding false positives with PCR. Nature 339:237[Medline]
Lampe MF, Suchland RJ, Stamm WE (1993) Nucleotide sequence of the variable domains within the major outer membrane protein from serovariants of Chlamydia trachomatis. Infect Immun 61:213-219[Abstract]
Lan J, Melgers I, Meijer CJL, Walboomers JMM, Rosendaal R, Burger C, Bleker OP, Van den Brule AJC (1995) Prevalence and serovar distribution of asymptomatic cervical Chlamydia trachomatis infections as determined by highly sensitive PCR. J Clin Microbiol 33:3194-3197[Abstract]
Long AA, Komminoth P, Lee E, Wolfe HF (1993) Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Histochemistry 99:151-162[Medline]
Long AA, Komminoth P, Wolfe HJ (1992) Detection of HIV provirus by in situ polymerase chain reaction. N Engl J Med 327:1529[Medline]
Mahoney JB, Luinstra KE, Sellors JW, Jang D, Chernesky MA (1992) Confirmatory polymerase chain reaction testing for Chlamydia trachomatis in first-void urine from asymptomatic and symptomatic men. J Clin Microbiol 30:2241-2245[Abstract]
Mardh PA, Holmes KK, Oriel JD, Piot P, Schachter J (1982) Chlamydial Infections. Amsterdam, Elsevier Biomedical Press
Martinez A, Miller MJ, Quinn K, Unsworth EJ, Ebina M, Cuttitta F (1995) Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections. J Histochem Cytochem 43:739-747
NunezTroconis JT (1995) Cervical intraepithelial neoplasia: Chlamydia trachomatis and other cofactors. Invest Clin 36:101-116[Medline]
Oriel DD (1992) Male genital Chlamydia trachomatis infections. J Infect 25:35-37[Medline]
Paavonen J, Vesterinen B, Meyer B, Saikku P, Suni J, Purola E, Saksela E (1979) Genital Chlamydia trachomatis infections in patients with cervical atypia. Obstet Gynecol 54:289-291[Abstract]
Palmer L, Falkow S (1986) A common plasmid of Chalmydia trachomatis. Plasmid 16:52-62[Medline]
Rice PA, Schachter J (1991) Pathogenesis of pelvic inflammatory disease. What are the questions? JAMA 266:2587-2591[Abstract]
Ripa KT (1982) Microbiology diagnosis of Chlamydia trachomatis infection. Infection 10:S19-S24[Medline]
Rockey DD, Heinzen RA, Hackstadt T (1995) Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells. Mol Microbiol 15:617-626[Medline]
Sriprakash KS, Macavoy ES (1987) Characterization and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis. Plasmid 18:205-214[Medline]
Su H, Raymond L, Rockey DD, Fischer E, Hackstadt T, Caldwell HD (1996) A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells. Proc Natl Acad Sci USA 93:11143-11148
TerMeulen J, Yu X, Mgaya HN, ChangClaude J, Naher H, Meinhard G, Mkiwa M, Pawlita M (1995) Prevalence of transformation zone Chlamydia trachomatis DNA and serum antibodies in Tanzanian gynaecological in-patients. J Trop Med Hyg 98:89-94[Medline]
Wilson O, Jacobs AL, Stewart S, Carson DD (1990) Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells. J Cell Physiol 142:60-67