Chlamydia trachomatis infection is the most prevalent bacterial sexually transmitted infection (STI) worldwide, including in Canada. According to the World Health Organization publication in 2012, the annual number of new cases of C. trachomatis detected in individuals is an estimated 105.7 million worldwide (http://apps.who.int/iris/bitstream/10665/75729/1/9789241504478_eng.pdf?ua=1). The prevalence of rectal chlamydia in men who have sex with men (MSM) has varied from 4% to 7%, using culture or direct immunofluorescence1–4 and rising to 14.1% using nucleic acid amplification tests (NAAT).4 The prevalence of rectal chlamydia in high-risk women has been reported to range from 6% to 14.6% in different jurisdictions using the APTIMA Combo 2 assay (AC2, Hologic Gen-Probe, San Diego, CA).5–7
Asymptomatic carriage of rectal C. trachomatis is believed to serve as an important reservoir for the ongoing transmission of infection and as such, there is a need for accurate detection of these cases. Although NAATs have generally been considered as more sensitive and specific compared with culture based methods,8,9 they are not U.S. Food and Drug Administration nor Health Canada approved for extra-genital sites (including the AC2 assay) and these assays require extensive validation.
Screening for C. trachomatis in Alberta, Canada, is performed on patients attending the STI clinics in Calgary and Edmonton, by sampling genitourinary sites and testing using the AC2 assay. In July 2012, 2 changes were made to the rectal screening criteria at the STI clinics. First, rectal screening criteria were expanded from exposure-based screening for men and women who reported receptive anal intercourse (RAI), to exposure-based screening for men but universal screening for women, where testing was performed regardless of history of RAI (in addition to genitourinary screening). Second, the testing methodology changed from cell culture to AC2. These changes resulted in a marked increase in rectal-only (rectal positive but urogenital negative) chlamydia cases from 2.0% (n = 25) before July to 20.0% (n = 242, P < 0.001) after July to December (unpublished data, Alberta Health Services). A previous publication by our group indicated that among women with rectal-only C. trachomatis, of all the rectal positive cases, only 13.5% reported RAI in the previous 2 months.7 This spike in the number of positive rectal-only samples for C. trachomatis was noted by clinicians and raised a concern that a proportion of these were false positives.
These findings coupled with the lack of Food and Drug Administration and Health Canada approval prompted us to conduct a re-evaluation and confirmation of the rectal positive specimens by other independent assays.
A total of 81 C. trachomatis rectal–positive specimens with a concurrently collected negative genitourinary tract specimen submitted to the Provincial Laboratory for Public Health, Alberta (ProvLab) from Alberta STI clinics between July and December 2012 were tested by the methods described below. Specimens of convenience stored at −70°C (for less than 1 year) in primary tubes that had sufficient volume to be tested by other independent assays were included in the study. Specimens were then split into volumes for further analysis by different approaches. Ethics was not required because the samples were tested as part of a laboratory validation study and did not impact patient care.
All rectal swab specimens included in this study initially tested positive by the AC2 assay that targets the 23S ribosomal RNA gene. The available specimens were then tested by the APTIMA single target assay for C. trachomatis (ACT) that targets the 16S ribosomal RNA. Sequencing of the ompA gene was performed using amplification and sequencing as previously described.10
To compare the results obtained by the different methodologies, the AC2 and ACT assays were considered as 2 independent assays given that the 2 assays target different regions of the genome. Sequencing assays targeting the ompA gene provided an alternative method to potentially resolve discordant results between the AC2 and ACT methods. A simple descriptive analysis was completed on characteristics of cases by sequence type.
Of the 81 specimens from patients with a positive rectal swab and a concurrent negative genitourinary swab for C. trachomatis tested, 53.1% (n = 43) were from men, more than three quarters (80.3%; n = 65) of the cases were asymptomatic.
All 81 specimens were retested by the ACT and sequencing. When an aggregate comparison method (2 of 3 tests positive on a specimen), 76 (93.8%) were positive by the ACT assay, and 65 (80.2%) of these specimens were also positive for C. trachomatis by ompA sequencing. Surprisingly, of 5 specimens that were negative for C. trachomatis by the ACT assay, 3 were positive for C. trachomatis by ompA sequencing. At first, this appears counterintuitive and explanations to why this occurred include partial inhibition and sampling variability/error due to an unequal distribution of low concentration nucleic acid within the specimens are possible.11
We then considered a rotating standards approach with each assay considered a reference standard. When the ACT was considered the reference standard, 76 specimens would be CT positive. However, this approach was problematic as an additional three AC2+/ACT− specimens were also able to be sequenced. Furthermore, use of the sequencing approach would be even more problematic because only 65 specimens would be considered true positive with the remaining 16 specimen (11 of which were AC2+/ACT+) being considered false positive.
Thus, a total of 79 (97.5%) rectal specimens could be confirmed by a second method, and 65 (80.3%) specimens could be sequenced to a corresponding serovar.
Of the 65 specimens that were typed, three quarters (76.9%; n = 50) of the cases self-identified as white, with 60% (n = 39) from men. Only 3 patients were known to be human immunodeficiency virus positive at the time of the clinic visit and only one patient reported rectal symptoms. The most common serovars detected were G (n = 18, 27.7%), J (n = 17, 26.2%), D (n = 14, 21.5%), E (n = 11, 16.9%), and F (n = 3, 4.6%). These 5 serovars were identified at both STI clinics, with serovar F identified only among female cases, and serovar J identified only among whites. In addition, 1 case of asymptomatic LGV (serovar L2b) was identified from a male client; and 1 case of serovar B which is often associated with trachoma, was identified in an asymptomatic female patient who reported local sexual partners. Due to potential biases in sampling specimens (eg, these were specimens of convenience), these proportions may not be truly representative of circulating serovars in our region and further statistical analysis was not carried out.
This study focused on high-risk patients, because the rectal specimens were collected from men reporting RAI and female STI clinic attendees. Other studies have identified the potential value of rectal screening for C. trachomatis in high-risk MSM populations.1–3,12,13 Rectal screening of MSM has been the subject of a previous review and was also a US Centers for Disease Control recommendation in 2010.14 Detection rates of C. trachomatis in rectal samples from MSM have been shown to be around 10%,1–3 with variable rates of rectal chlamydia being reported in female STI clinic attendees and female swingers.5,6,15
In conclusion, this study adds to the growing volume of data validating the use of NAATs in rectal screening for C. trachomatis. Such screening, as recommended by the US Centers for Disease Control,14 has the potential to accurately identify asymptomatic extragenital site infections in high risk populations, thereby decreasing transmission.
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