Abstract
Background:
Nucleic acid amplification testing (NAAT) is the preferred method to detect Chlamydia trachomatis and Neisseria gonorrhoeae, but information regarding perfomance of currently available assays is needed. This study evaluated the perfomance of the Aptima Combo 2, GeneXpert, cobas4800, and ProbeTec Qx (CTQ/GCQ) to detect chlamydia and gonorrhea in pharyngeal, rectal, and urine specimen.
Methods:
Adult male patients seen at an urban human immunodeficiency virus clinic in Birmingham, Alabama who reported sex with men (men who have sex with men) and no antibiotic use in the past 30 days were enrolled between November 2014 and December 2016. Following a baseline survey, rectal and initial void urine specimens were self-collected. A composite infection standard was used, where 1 assay was compared with 3 others to determine sensitivity and specificity estimates for rectal and urine samples. Two pharyngeal samples were clinician-collected for chlamydia and gonorrhea testing and both had to be positive to be considered a true positive.
Results:
Among the 181 men enrolled into the study, 15.5% and 7.2% had at least 1 positive chlamydia and gonorrhea result at any site, respectively. Among all 4 assays, chlamydia sensitivity rates ranged from 82% to 96% among rectal samples. Rectal gonorrhea sensitivity estimates ranged from 67% to 99%. The GCQ assay was less sensitive in detecting rectal gonorrhea compared with the other assays (P = 0.02).
Conclusions:
More than 80% of chlamydia and gonorrhea infections would have been missed with urine-only screening, highlighting the importance in using NAATs to detect chlamydia and gonorrhea infections among men who have sex with men.
Chlamydia trachomatis and Neisseria gonorrhoeae are 2 of the most commonly reported sexually transmitted infections (STIs) in the United States.1 Infections are often asymptomatic, and many remain unaware of their infection status, increasing transmission risk to sexual partners, and providing few indicators to seek medical care. These STIs disproportionately affect people with human immunodeficiency virus (HIV) and men who have sex with men (MSM).1
Chlamydia and gonorrhea infections at extragenital (pharyngeal and rectal) sites are of increasing prevalence raising concerns about transmission and antibiotic resistance.1–3 Studies have shown that the vast majority of chlamydia and gonorrhea infections, respectively, would have been missed with urine-only testing over the course of a year among MSM.4 Early detection and treatment of active chlamydia and gonorrhea infection at all involved sites is critical in preventing transmission and reducing risk of HIV, especially among MSM.5
The United States Centers for Disease Control and Prevention (CDC) recommends annual screening for chlamydia and gonorrhea using nucleic acid amplification tests (NAATs) at genital and extragenital exposure sites among MSM and all sexually active adults with HIV.6 The NAATs are highly effective in detecting chlamydia and gonorrhea infections at extragenital sites and are much more sensitive in detecting extragenital infections compared with culture.7–10 Cornelisse et al8 found a 2-fold and 5-fold increase in the detection of rectal and pharyngeal gonorrhea when using NAATs compared with traditional methods. Despite testing and screening recommendations, extragenital screening rates are low and many infections are missed because of reliance on genital-only based screening.7,11,12
Low adoption of CDC recommended testing and screening practices to use NAATs for extragenital infections, is in part due to the challenges faced by many laboratories in validation of assays for off-label use. Until recently, no commercially available assays cleared by the Food and Drug Administration (FDA) had claims for detection of chlamydia and gonorrhea using rectal or pharyngeal sample types.13 Recent FDA approval of claims for rectal and oropharyngeal sample types for use with the Aptima Combo 2 (AC2) [Hologic, San Diego, CA] and GeneXpert (Xpert) [Cepheid Sunnyvale, CA] assays is an important advancement, but additional data regarding performance of other assays are needed. The objective of this study was to compare the performance of 4 commercially available NAAT assays for chlamydia and gonorrhea detection using samples from extragenital sites in sexually active MSM with HIV.
MATERIALS AND METHODS
Settings and Participants
Adult MSM living with HIV were prospectively enrolled into the study during routine HIV clinic visits at the 1917 clinic in Birmingham, Alabama between November 1, 2014, and December 31, 2016. Multiple participants enrolled into the study more than once. Men who have sex with men were eligible if they were 18 years and older, reported receptive anal intercourse and no antibiotic treatment (excluding trimethoprim-sulfamethoxazole) within the past 30 days.
Participants completed a baseline survey about current symptoms and recent sexual behaviors. Men received written instructions on how to self-collect 4 rectal swabs, with randomly assigned swab collection order, along with a first-catch urine sample. Because participants found collection of multiple oropharyngeal swabs uncomfortable, physicians collected 2 samples to assure sample validity. Men with positive CT and/or NG test results were treated with standard antibiotics in HIV clinic according to CDC guidelines. This study was approved by the Institutional Review Board at the University of Alabama at Birmingham, and all participants provided written informed consent before any study procedures.
Testing
Four assays were used in this study: Hologic APTIMA Combo 2 (AC2; Hologic, San Diego, CA),14–16 cobas4800 (c4800, Roche Molecular System, Indianapolis, IN),17 Cepheid GeneXpert CT/NG (Xpert; Cepheid, Sunnyvale, CA),18 and BD ProbeTec C. trachomatis Qx (CTQ) and N. gonorrhoeae QX (GCQ) amplified DNA assays on the BD Viper System with XTR Technology (Becton Dickinson Diagnostics, Sparks, MD).19,20 Testing was performed according to the manufacturer’s instructions for use for swab or urine specimens as appropriate. Pharyngeal samples were tested on the AC2 and CTQ/GCQ assays.
Composite Infection Standard
Rectal and genital sensitivity and specificity estimates were calculated relative to the composite infection standard (CIS). The CIS compares a result generated by 1 assay to results obtained from the other 3 assays. The CIS method can be used to estimate sensitivity and specificity and reduce bias when a true measure of infection does not exist.21,22 At least 2 positive results among the 3 comparator assays were required to define an infection. CTQ/GCQ and AC2 were used to test the 2 pharyngeal swabs with a head-to-head comparison. Since the performance of the assays is not known, we required both oropharyngeal samples to be positive for prevalence estimates.
Statistical Analysis
Descriptive statistics included frequencies and percentages for categorical variables and mean (± SD) or median (range) for continuous variables. Estimates of sensitivity and specificity for rectal and urine samples were obtained separately relative to the CIS defined as 2 or more positive NAATs. Comparison across platforms was performed using repeated measures analysis to test for within-subject effects. Sensitivity and specificity were calculated using the CIS method and McNemar χ2 tests were conducted to compare estimates. Ninety-five percent confidence intervals were estimated using the Clopper-Pearson method. To examine differences between symptom and infection status, Fisher exact and χ2 tests were conducted. Since only 2 oropharyngeal samples were collected, results were compared using McNemar χ2 and Kappa scores.23 SAS 9.4 (Cary, NC) was used for all analyses.
RESULTS
From November 2014 to December 2016, 181 sexually active MSM with HIV were enrolled. On average, participants were 37 years old (SD ± 10.4). Of the 181 men, 172 (95%) reported their sexual partner(s) as male only and 9 (5%) reported their sexual partners as male and female within the past 30 days, 69 (38%) reported receptive anal sex only, and 112 (62%) reported receptive and insertive anal sex. The median (range) of sexual partners reported 30 days before their baseline visit was 1 (1–60). Of the 181 participants, 139 (77%) reported a history of being diagnosed with any STI, 82 (46%) reported having gonorrhea, and 61 (34%) reported chlamydia. In total, 157, 179, and 181 men submitted pharyngeal, rectal, and urine samples. Participants enrolled in the study at a median (range) of 1 (1–6) times.
When enrolling into the study, 47 (26%) of the 181 men reported STI symptoms at any genital or extragenital sites. Of the 181 men 24 (13%), 16 (9%), and 16 (9%), reported throat, rectal and urogenital symptoms, respectively. Of the 47 men that experienced symptoms, 8 (17%) had a chlamydia infection and 3 (6%) had a gonorrhea infection. There was no significant difference in infection status between those with and without symptoms.
Additionally, among study participants, 28 (16%) had at least 1 positive chlamydia result and 13 (7%) had at least 1 positive gonorrhea result. Chlamydia was detected in none of the pharyngeal samples, 14% of rectal samples, and 3% of urine samples (Table 1). Although 156 men submitted all 3 sample types, none of the participants were positive for chlamydia or gonorrhea at all 3 sites. If STI screening had been exclusively performed on urine, 23 (82%) of chlamydia and 12 (92%) of gonorrhea infections would have been missed.
TABLE 1.
Distribution and Cumulative Prevalence of Gonorrhea and Chlamydia Infection by Anatomical Site, n (%)
| Anatomical Site | Specimens | Chlamydia | Gonorrhea | Coinfection (Chlamydia and Gonorrhea) | Any STI (Chlamydia or Gonorrhea) |
|---|---|---|---|---|---|
| Rectal | 179 | 25 (14.0) | 11 (6.2) | 6 (3.4) | 30 (16.8) |
| Urine | 181 | 5 (2.8) | 1 (0.6) | 1 (0.6) | 5 (2.8) |
| Pharynx | 157 | 0 (0) | 6 (4) | 0 (0) | 6 (4) |
| Rectal and Urine | 179 | 2 (1.1) | 0 (0) | 0 (0) | 2 (1.1) |
| Rectal and Pharynx | 156 | 0 (0) | 4 (2.6) | 0 (0) | 4 (2.6) |
| Urine and Pharynx | 158 | 0 (0) | 1 (0.6) | 0 (0) | 1 (0.6) |
Of the urine specimens tested for chlamydia, assays had a sensitivity and specificity above 80% and 99%, respectively (Table 2). Rectal samples tested for chlamydia had a wider range of sensitivity estimates with AC2, Cobas, Xpert, and CTQ detecting 96%, 82%, and 93%, and 92% of true positives, respectively. Specificity for rectal chlamydia was high, ranging from 98.6% to 99.5%. Differences among these sensitivity and specificity estimates were not statistically significant. Performance of AC2 and Cobas was not compared for chlamydia pharyngeal samples as no infections were detected within this group.
TABLE 2.
Clinical Performance for Chlamydia Detection Among HIV-Infected MSM by Sample Type
| Sensitivity (%, [n], (95% CI)) |
Specificity (%, [n], (95% CI)) |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| Sample Type | AC2 | CTQ | cobas4800 | Xpert | AC2 | CTQ | cobas4800 | Xpert | P* |
| Rectal CT | 95.8% [23/24] (78.9–99.9%) |
92.0% [23/25] (74.0–99.0%) |
81.5% [22/27] (61.9–93.7%) |
92.6% [25/27] (75.7–99.1%) |
98.6% [217/220] (96.1–99.7%) |
99.0% [203/205] (96.5–99.9%) |
99.5% [217/218] (97.5–100%) |
99.5% [213/214] (97.4–100%) |
0.55 |
| Urine CT | 100% [5/5] (47.8–100%) |
80.0% [4/5] (28.4–99.5%) |
80.0% [4/5] (28.4–99.5%) |
100% [5/5] (47.8–100%) |
100% [245/245] (98.5–100%) |
99.6% [244/245] (97.8–100%) |
99.2% [242/244] (97.1–100%) |
100% [245/245] (98.5–100%) |
0.90 |
P value from within assay platform repeated-measures analysis.
95% CI, 95% confidence interval.
Gonorrhea testing at the urogenital site was similar for all assays with 100% sensitivity and specificity ≥99% (Table 3). For rectal gonorrhea, testing was significantly less sensitive with the GCQ assay (67%) compared with AC2 (100%), Cobas (93%) and Xpert (93%) assays. Specificity for rectal gonorrhea was greater than 99% for all assays. There was no significant difference between the AC2 and GCQ assay (P = 0.41) in detecting pharyngeal gonorrhea infections.
TABLE 3.
Clinical Performance for Gonorrhea Detection Amona HIV-Infected MSM by Sample Type
| Sensitivity (%, [n], (95% CI)) |
Specificity (%, [n], (95% CI)) |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| Sample Type | AC2 | GCQ | cobas4800 | Xpert | AC2 | GCQ | cobas4800 | Xpert | P† |
| Rectal NG | 100% [12/12] (73.5–100%) |
66.7%* [10/15] (38.4–88.2%) |
92.9% [13/14] (66.1–99.8%) |
92.9% [13/14] (79.4–100%) |
99.1% [231/233] (96.9–99.9%) |
100% [215/215] (98.3–100%) |
98.7% [228/231] (96.3–99.7%) |
99.1% [225/227] (97.9–100%) |
0.02 |
| Urine NG | 100% [1/1] (2.50–100%) |
100% [1/1] (2.50–100%) |
100% [1/1] (2.50–100%) |
100% [1/1] (2.50–100%) |
99.2% [247/249] (97.1–99.9%) |
99.6% [248/249] (97.8–100%) |
99.2% [246/248] (97.1–99.9%) |
99.6% [248/249] (97.8–100%) |
0.88 |
Statistically different than the Rectal NG CIS.
P value from within assay platform repeated measures analysis.
DISCUSSION
The primary objective of this study was to compare the performance of 4 assays including AC2, Cobas, Xpert, and CTQ/GCQ for detection of chlamydia and gonorrhea infections from pharyngeal, rectal, and urine samples. Among our sample of sexually active MSM living with HIV, chlamydia and gonorrhea infections at genital and extragenital sites were common. Within our study, all 4 commercially available NAAT tests performed similarly well among pharyngeal, rectal, and urine samples for detection of chlamydia infections. Compared with previous studies, the CTQ assay had lower sensitivity estimates in detecting chlamydia using urine samples.24
Differences in assay performance was most pronounced in detecting rectal gonorrhea infections with GCQ sensitivity estimates being much lower than the other 3 assays. Additional studies are needed on the performance of the GCQ assay in detecting gonorrhea infections at the rectum. Additionally, sensitivity estimates among AC2 and GCQ assays were low in the detection of pharyngeal gonorrhea infections. AC2 sensitivity estimates differed from recent clinical trial results, which showed better performance in the detection of pharyngeal gonorrhea infections.25 The discrepancies in sensitivity estimates could be due to small sample sizes among pharyngeal swabs and low prevalence rates of chlamydia and gonorrhea at different anatomical sites. As men from this study were recruited from an HIV clinic, many of them were receiving regular sexual health care, which could also explain some of lower chlamydia and gonorrhea prevalence rates. Further, most men in this study did not report symptoms and may be expected to have lower organism loads.24
Uptake of extragenital NAAT testing has been slow in clinical settings and laboratories. This may be partly due to expense but also due to lack of published data about test validity for samples collected from extragenital sites. With the recent FDA clearance of extragenital specimen testing for the Hologic and Cepheid assays, and soon-to-be completed studies evaluating other assays in support of submissions to the FDA, the availability of NAATs for screening extragenital sites is expected to improve. As new assays for detection of genital infections are developed, it is unclear whether manufacturers will include data to seek additional claims for extragenital testing.
Further, in our study population, 82% of chlamydia and 92% of gonorrhea infections would have been missed with urine-only based screening. These results were higher than previous studies that showed 53% of chlamydia and 64% of gonorrhea infections would have been missed with urine-only testing,26 highlighting the importance of diagnostic tools that can detect infections at additional anatomical sites as STI rates continue to increase. We found an absence of symptoms in the presence of chlamydia and gonorrhea infection, similar to other studies, and highlighting the importance of screening independent from symptom status.9 It is critical to evaluate the performance of new assays at genital and extragenital sites in order to support clinical diagnostic laboratories that are not able to make wide scale comparisons using local data.
Acknowledgments
Sources of Funding: Reagents were supplied by BD Diagnostics, Cepheid, Hologic and Roche.
Conflict of Interest: B.VDP. receives research support, consulting fees and/or honorarium from the following: Abbott Molecular, BD Diagnostics, Binx Health, BioFire Diagnostics, Gen-Probe Hologic, Rheonix, Roche Diagnostics, and SpeeDx; and J.D-O. has received support from the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (K23 HD090993). For remaining authors none were declared.
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