Sexually Transmitted Diseases:
Comparison of Penile Skin Swab with Intra-Urethral Swab and First Void Urine for Polymerase Chain Reaction-Based Diagnosis of Chlamydia trachomatis Urethritis in Male Patients
Pittaras, Theodore E. MD*†; Papaparaskevas, Joseph MD*; Houhoula, Dimitra P. PhD*; Legakis, Nicholas J. MD*; Frangouli, Efi MD†; Katsambas, Andreas MD‡; Tsakris, Athanassios MD*; Papadogeorgakis, Helen MD†
From the *Department of Microbiology, Medical School, University of Athens, Athens, Greece; †Department of Microbiology, Andreas Sygros Hospital for Skin and Venereal Diseases, Athens, Greece; and ‡Department of Dermatology, Medical School, University of Athens, Andreas Sygros Hospital for Skin and Venereal Diseases, Athens, Greece
Supported by the Kapodistrias grant 70/3/8156 of the National and Kapodistrian University of Athens, Greece.
Correspondence: Athanassios Tsakris, MD, PhD, MRCPath, Department of Microbiology, Medical School, National and Kapodistrian University of Athens, 75 Mikras Asias Street, 11527 Athens, Greece. E-mail: email@example.com.
Received for publication January 21, 2008, and accepted May 27, 2008.
Chlamydia trachomatis is considered the most frequent cause of male non-gonococcal urethritis.1 Nevertheless, the pathogen may be also detected in urethral specimens of men suffering from gonococcal urethritis and in asymptomatic cases.2–4 Nucleic acid amplification techniques (NAATs) have currently become the diagnostic methods of choice and have increased the overall diagnostic yield. Preferred specimens are considered either the intraurethral swab (IUS) or more recently the first void urine (FVU), a noninvasive sampling method, which facilitates large epidemiologic studies.1,4,5 In addition, there are anecdotal reports comparing alternative noninvasive sampling methods, such as the glans-penis swab for supplementing molecular diagnosis.6–9 In the current study we compared a noninvasive sampling method, the penile skin swab (PSS), with the IUS and FVU sampling procedures in the molecular diagnosis of male C. trachomatis urethritis.
Between September 2005 and September 2006, a total of 210 male patients who were examined at the outpatient sexually transmitted diseases clinic of “Andreas Sygros” Hospital, Athens, Greece, for acute nongonococcal urethritis, were enrolled in the study. Exclusion criteria were urination during the previous 2 hours before examination, antibiotic treatment during the previous 3 weeks, or genital anatomical abnormalities (e.g., phimosis).10
The following specimens were taken by the clinician in the following order: first the PSS using the large swabs of the COBAS Amplicor swab specimen collection set, vigorously rolled over the glans and collar of the penis to ensure collection of a high number of epithelial cells; second the IUS using the thin swabs of the same set from the posterior 2 to 4 cm of the urethra; third the FVU catch (10–50 mL of urine).2
IUS and PSS samples were divided in 2 parts. The first part was processed immediately using the COBAS Amplicor CT assay (Amplicor PCR; Roche Molecular Systems, Branchburg, NJ), according to the manufacturer's instructions. The polymerase chain reaction (PCR) amplification protocol targeted a 207 bp segment of the C. trachomatis conserved cryptic plasmid. The second half was put into 2-sucrose-phosphate transport medium, centrifuged at 4500 × g for 30 minutes and stored at −70°C. FVU samples were also divided in half, the first part was processed immediately using the COBAS Amplicor CT assay and the second half was stored at −70°C.
DNA extraction of the second half of the samples was performed using the NucleoSpin Tissue DNA extraction kit (Macherrey Nagel, Düren, Germany), according to the manufacturer's instructions. A previously described nested-PCR (nPCR) protocol11 was used for amplification of a 1320 basepair segment of the omp1 gene.12 All specimens were tested neat and diluted 10−1 for inhibition detection. PCR products were separated in a 1.5% agarose gel (Ultra Pure Agarose, GIBCO, PAisley, Scotland), stained with 0.5 μg/mL ethidium bromide, and documented under ultraviolet illumination.
Positive patients were considered those who yielded a positive result of at least one of the IUS, FVU, or PSS specimens with both PCR assays, together with clinical and microbiologic indications of acute nongonococcal urethritis. The protocol of the study was reviewed and approved by the hospital's Ethical Committee and a written consent was obtained from all patients.
Using the definition criteria, 47 of the 210 (22.4%) patients were diagnosed with C. trachomatis urethritis. The overall results of IUS, PSS, and FVU sampling methods are shown in Table 1. The sensitivities, specificities, PPVs, and NPVs of the sampling methods and their combinations using the Amplicor PCR alone, the nPCR alone, or both NAATs are shown in Table 2. No inhibition cases were detected with any one of the assays.
A positive IUS with both NAATs was identified in 40 of the 47 diagnosed patients, whereas discrepancies or a negative result were detected in 5 and 2 patients, respectively. Among the remaining 163 negative patients, one was identified as false-positive using only Amplicor PCR and one, using only nPCR (Table 1). Regarding the FVU, a positive result with both NAATs was identified in 41 of the 47 diagnosed patients, whereas discrepancies or a negative result were detected in 2 and 4 patients, respectively. Among the 163 negative patients, one was identified as false-positive using the Amplicor PCR and 4 different patients were identified as false-positive using nPCR (Table 1). Regarding the PSS samples, a positive result with both NAATs was obtained from 34 of the 47 diagnosed patients, whereas discrepancies or a negative result were obtained from 7 and 6 patients, respectively. Among the remaining 163 negative patients, one was identified as false-positive using only Amplicor PCR and one, using only nPCR (Table 1). The combined use of IUS and PSS with nPCR identified all 47 diagnosed patients (Table 2).
Our study showed that all 3 sampling techniques were <100% sensitive using a single NAAT assay at a time. In the literature, IUS is considered the standard sample for diagnosis of chlamydial urethritis.1 Nevertheless, as the procedure is uncomfortable and requires a visit to healthcare settings, noninvasive sampling techniques such as FVU have been alternatively proposed.1,2,13–18 In general, diagnostic sensitivity of IUS sampling has been considered superior in comparison with the FVU.10 The current study indicated that both techniques resulted in equal sensitivity when using the Amplicor PCR, whereas the IUS was slightly more sensitive when using the nPCR. Nevertheless, as these differences were only marginal, both methods were considered as equally suitable for diagnosis of chlamydial urethritis.
C. trachomatis tends to infect the columnar or the squamous-columnar cells of the epithelium of the urethra.4 Therefore, the glans penis skin, which is covered with squamus cells, may also be an adequate substrate for the C. trachomatis. In that respect, we tested the possible usage of PSS in the diagnosis of chlamydial male urethritis. It should be noted that PSS sampling was performed by the clinician and not the patient. As the method is not standardized yet, our intention was to have a fully reproducible collection procedure. Nevertheless, PSS was found less sensitive than FVU or IUS, when used alone with each one or in combination with both NAATs. Similar anecdotal reports6–9 yielded discrepant results for detection of C. trachomatis using penile swabs. These results may be attributed to the procedure, which requires collection of a high number of epithelial cells, from an anatomical site very close to, but not the actual infected area. In contrast to the sensitivity, the specificity of PSS sampling was equally high as the other samples. In addition, PSS seems not to depend on patient preparation, in contrast to FVU, where micturition within the previous 2 hours before sampling may decrease sensitivity.2,10
The combination of testing both IUS and PSS samples with nPCR yielded 100% sensitivity, increased from what was seen using 1 of the 2 sampling procedure at a time. The combined use of the 2 NAAT assays in any single sample at a time, also increased sensitivity, especially regarding PSS, and this was achieved without substantial loss of specificity. The suggestion, however, of incorporating a second NAAT in every day clinical practice cannot be easily considered because of additional cost and workload issues. The combination of 2 sampling methods with 1 NAAT is also a questionable approach and should be considered only when a significant increase in sensitivity occurs.
The 2 NAAT assays tested have different DNA targets. The Amplicor PCR is a commercially available, FDA approved, one-step method, targeting the conserved cryptic plasmid that exists in 4 to 10 copies in the genome of C. trachomatis, thus resulting in high sensitivity, and can be considered in every day clinical practice. The nPCR is an in-house established assay, targeting the single copy omp1 gene and its high sensitivity comes as a result of the nested procedure. In that respect, nPCR can be kept as a backup and confirmatory technique. As plasmid free strains19 or novel variants with deletions in the cryptic plasmid target sequence exist,20 a laboratory should be able to implement such alternative NAATs.
In conclusion, this study demonstrated that PSS, a noninvasive sampling method resulted in lower sensitivity when compared with traditional procedures and only marginally increased overall the diagnostic yield when used in combination with FVU or IUS for the molecular diagnosis of C. trachomatis urethritis. In that respect, its use should be further evaluated after optimization and in large epidemiologic studies.
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