Herpes simplex virus type 2 and Chlamydia trachomatis in adenocarcinoma of the uterine cervix

Herpes simplex virus type 2 and Chlamydia trachomatis in adenocarcinoma of the uterine cervix

8 of 209 (3.8%) female patients attending a venereal-diseases clinic were found on cytological and virological examination to have an inapparent infection of the cervix uteri with Herpesvirus hominis, and a significant correlation was found between this and gonococcal infection. (“GETTY IMAGES”). However, there was no such association with the grade and stage of the disease. Both sites contained occasional epithelial and stromal cells with nuclear inclusions consistent with HSV infection. Samples were first screened for β-globin DNA sequences, and 67 cases were considered adequate to further analysis. In a previous analysis, DNA of HPV was identified in 79.4% of specimens included in this series (51% HPV 18 and 34% HPV 16).The local ethical committee approved the study. The Licensed Material may only be used in materials for personal, noncommercial use and test or sample use, including comps and layouts.

Cervical cancer (CC) is an important public health problem worldwide. Following skin and breast carcinoma, CC is the third most common cancer among Brazilian women [1], ranking first in some developing countries [2]. Adenocarcinoma of the uterine cervix (AC) constitutes a relatively rare histologic form of cervical cancer and occurs in 15–20% of primary cervical neoplasias [3] and [4], but in spite of effective cytological screening programs, the incidence rate of AC is increasing [3], [5], [6], [7] and [8]. Persistent viral infection with the high-risk types of HPV is established as a necessary cause for the development of squamous cell carcinoma (SCC) and the majority of cases of adenocarcinoma of the uterine cervix [9], [10] and [19]. If Licensed Material featuring a person is used (i) in a manner that implies endorsement, use of or a connection to a product or service by that model; or (ii) in connection with a potentially unflattering or controversial subject, you must print a statement that indicates that the person is a model and is used for illustrative purposes only. The natural history studies of HPV infection have shown that, different from those that progress to cancer, most infections are transient and not associated with detectable cytological abnormalities [12]. The reasons for this variable natural history are poorly understood but it has been generally assumed that other causes or co-factors must be important for the development of neoplasia in HPV-infected women.

Although several co-factors have been associated with the risk of SCC [13], [14], [15], [16] and [17], their impact on the progression of HPV-infected cervical cells to adenocarcinoma remains unclear. Since 1989, when Schmauz et al. Payment of the comp service fee relates solely to comping use during the 30-day comp licence period and does not entitle you to make any additional use of the Licensed Material either before or after expiry of the 30 days. Among other STD, herpes simplex virus-2 (HSV-2) and Chlamydia trachomatis (CT) infection has been investigated as HPV co-factor for cervical carcinogenesis [15] and [16]. To examine the prevalence of the HSV-2 and CT infection and its influence as a co-factor of HPV in the etiology of adenocarcinoma of the uterine cervix, we performed a retrospective study using PCR to detect the presence of HSV-2 DNA and CT DNA in paraffin-embedded cancer tissue. From January 1995 to December 2003, 206 cases of AC were diagnosed in a public hospital in the South of Brazil. In a previous study conducted by Cambruzzi et al.

Additional Rights Available. Paraffin-embedded tissue was selected and DNA extraction was performed. The biopsy specimens for DNA analysis were deparaffinise with xylene followed by ethanol, and the DNA was isolated using the QIAamp Tissue Kit (QIAGEN Inc., Santa Clara, CA, USA) according to the manufacturer’s instructions. The DNA was eluted in 100 μl of elution buffer and 10 μl was used for the amplification reaction. Samples were first screened for β-globin DNA sequences, and 67 were considered adequate to further analysis. The sole and exclusive remedy for a breach of the foregoing warranty is the replacement of the digital copy of the Licensed Material. To detect HSV-2 DNA using polymerase chain reaction, a pair of primers that bracket 391 base pair segment of DNA polymerase gene were used as described by Madhavan et al.
Herpes simplex virus type 2 and Chlamydia trachomatis in adenocarcinoma of the uterine cervix

[20]. The sequences are an upstream primer HSV-5′-ATG GTG AAC ATC GAC ATG TAC GG-3′ and one type specific downstream primer HSV-2–5′-CCT CCT TGT CGA GGC CCC GAA AC-3′ (Invitrogen Custom Primers, São Paulo, Brazil). The reaction conditions contained 1 ìg sample of DNA. General. One microlitre of DNA was added as the template. The thermal profile consisted of denaturation at 94 °C for 2 min followed by 40 cycles with 45 s of denaturation (94 °C), annealing (60 °C) and extension (72 °C). Negative and positive HSV-2 controls as well as water as a negative contamination PCR control were included in each assay.

The amplified products were analyzed by gel electrophoresis using 2% agarose containing 0.5 μg/ml of ethidium bromide. The results were considered positive when the product that was equivalent to the fragment described was found. PCR primer sets specific for the CT gene were used. The primer pairs CT05 and CT06 were designed by Bobo et al. [21]. These primers: CT05 (5′-GAT AGC GAG CAC AAA GAG AGC TAA-3′) and CT06 (5′-TTC ACA TCT GTT TGC AAA ACA CGG TCG AAA ACA AAG-3′) amplify a 281 bp product from the CT major outer membrane protein (MOMP) gene. To confirm CT identification, another PCR test, with primers annealing to the Chlamydia spp., was performed.

The primers employed, CH1 5′-ATG TCC AAA CTC ATC AGA CGA G-3′ and CH2 5′-CCT TCT TTA AGA GGT TTT ACC CA-3′ amplify a 603 bp product from omp2 gene, designed by Hartley et al. [22]. PCR reactions were performed in a volume of 50 μl containing 20 nmol/L of Tris–HCl (pH 8.4), 50 mmol/L of KCl, 3.0 (for CH1 and CH2) and 2.5 mmol/L of MgCl2 (for CT05 and CT06), 200 μmol/L of deoxynucleoside triphophase, 2.5 U of Platinum Taq DNA polymerase (INVITROGEN, Grand Island, NY, USA) and 25 pmol of both forward and reverse primers. PCR was performed in a PTC-1196 DNA thermocycler (MJ Research Inc., Watertown, MA, USA) under the following conditions: 5-minute preincubation at 94 °C, followed by 35 cycles of 30 s at 94  °C, 30 s at 60 °C and 20 s at 72 °C. Final extension was performed for 4 min at 72 °C. The amplimers were examined by electrophoresis on 2% agarose gels according to standard procedures. Negative and positive C.

trachomatis controls as well as water as a negative contamination PCR control were included in each assay. PCR amplification was performed in duplicate for each DNA sample. The results were considered positive when the product that was equivalent to the fragment described was found. Overall, 67 of the 206 paraffin-embedded cases of cervical adenocarcinoma were β-globin positive and were further analyzed for the presence of HSV-2 and CT DNA. None of the 67 cervical adenocarcinoma cases included in our analysis contained HSV-2 DNA or CT DNA. HPV infection is the main etiologic factor for invasive and pre-invasive cervical neoplasia [9] and [10]. However, from women infected with high-risk types of HPV, only a small subset will develop invasive cervical cancer, suggesting that other co-factors must be present for the development of malignancy [10] and [23].

Although cervical screening offers some protection against invasive carcinoma [24], due to early detection of pre-malignant lesions, the identification of co-factors for HPV improves the understanding of etiology of AC and may also be useful from a prevention standpoint.

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