1. Number of specimens tested
Evaluation of Real-Time PCR and Transcription-Mediated Amplification for Detection of Trichomonas vaginalis in Urine
Marcia M. Hobbs, Kimberly D. Rich, E. Byrd Quinlivan, Rebecca Zeitlin,
John L. Schmitz and Melissa B. Miller
The University of North Carolina at Chapel Hill, Chapel Hill, NC
Contact Information
Marcia M. Hobbs
Infectious Diseases Research
Campus Box #7031
Chapel Hill, NC 27599
Phone: (919)
Fax: (919)
Abstract
Assay Outputs
Comparisons of Real Time PCR and Gen-Probe TMA for detection of T. vaginalis in urine
Background: Infection with the protozoan parasite Trichomonas vaginalis (TV) is the most common non-viral sexually transmitted infection (STI) with prevalence estimates frequently surpassing those for gonorrhea (GC) and chlamydia (CT). Yet, the development of nucleic acid amplification tests (NAATs) for TV has not kept pace with improvements in GC and CT detection. This study was designed to evaluate and compare real-time PCR (rtPCR) and transcription-mediated amplification (TMA) for detection of TV in urine from a southern US cohort of people living with HIV. Methods: First-void urine was collected from patients enrolled in a clinical study to monitor STIs in patients attending an HIV clinic in North Carolina from September 2004 through December Specimens were tested by rtPCR using primers specifically targeting the TV beta-tubulin genes (Hardick et al. 2003, J Clin Micro 41:5619) and by TMA using Gen-Probe APTIMA® TV analyte specific reagents targeting TV rRNA. All specimens were prepared for rtPCR within 4 days of collection. Results from rtPCR were considered negative, low positive or high positive based on the threshold cycle number at which the PCR product accumulated significantly over baseline levels. TMA results with >30,000 relative light units were considered positive. We tested 136 specimens that had been promptly transferred to APTIMA urine transport tubes, and 88 frozen urines that were processed up to 220 days after storage at -20°C. Results: TMA results for 29 specimens with both fresh preps and frozen urine aliquots were 100% concordant. For comparison with rtPCR, APTIMA results from fresh (N=136) and frozen (N=59) samples were included. Results from rtPCR and TMA were concordant for 97% of specimens. Ninety-six percent of specimens with high positive rtPCR results and 57% of those with low positive results were positive by TMA. Conclusions: Urine-based NAATs are valuable screening assays for detection of TV in HIV+ patients in a hospital clinic setting. T. vaginalis was detected in 16.4% of specimens by rtPCR and in 15.4% of specimens by APTIMA TMA.
Hi Pos
Lo Pos
Negative
Real-Time PCR
Gen-Probe TV ASR
Time (Seconds)
Relative Light Unites (RLU)
0.0
0.5
1.0
1.5
2.0
Figure 1. Real-Time PCR and Gen-Probe TV ASR assay outputs. Left panel. Actual trace from LightCycler Fit Points Report showing accumulation of fluorescence from R-T PCR probes with positive control TV total nucleic acid (gray line), 3 high positive (Hi Pos), 2 low positive (Lo Pos) and 3 negative clinical urine specimens. Threshold cycle number (Ct) corresponds to the point where the signal accumulates significantly above background levels, indicated here by the horizontal red line. Right panel. Simulated trace of signal from hybridized acridinium ester labeled probe in APTIMA hybridization protection assay following transcription-mediated amplification. Relative light units (RLU) are calculated from the peak height.
Gen-Probe Result (RLU)
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
Real-Time PCR Result
Hi Pos
Lo Pos
Neg
Table 1. Test Results by Real-Time PCR and Gen-Probe TMA
neg
POS
Hi Pos 1 23
Lo Pos 15 3 4
Alternate TMA Result
Initial TMA Result
Final RT PCR Result
Initial RT PCR Result
Number of specimens tested
Figure 5. Comparison of Real-Time PCR and Gen-Probe TV ASR results. Among 483 urine specimens, 25 were high positives, 7 were low positives and 451 were R-T PCR negative. The distribution of Gen-Probe TV ASR results in relative light units (RLU) for each R-T PCR category is shown along the X axis. TV ASR was positive (> 30,000 RLU) for 24/25 Hi Pos, 4/7 Lo Pos and 5/483 negative R-T PCR results.
Methods
Table 2. Sensitivity and Specificity Estimates based on test results from 75 specimens shown in Table 1.
*Specificities of all tests were probably higher than indicated since most concordant negative specimens were not tested for this analysis.
Specimens. First-void urine (median volume: 35 mL, range: mL) was collected from 288 patients (men and women) enrolled in a clinical study to monitor STIs in patients attending an HIV clinic in North Carolina from September 2004 through December For Real-Time PCR, 483 specimens were processed using a MagNA Pure LC instrument with reagents from the MagNA Pure LC DNA Isolation kit according to the manufacturer’s instructions (Roche Diagnostics). For TMA testing, urine specimens were handled in one of 3 ways: (1) For all specimens, urine was stored at -20 °C for up to 482 days. Thawed specimens were processed by transferring 2mL urine to Gen-Probe urine transport tubes. Processed specimens were stored at 2-8 °C for up to 21 days before testing. (2) For a subset of 73 specimens, 2 mL of urine was processed within 24h of collection, and processed specimens were stored at 2-8 °C for up to 56 days before testing. (3) For 162 specimens, 2 mL of urine was processed within 24h of collection, and processed specimens were stored at -20 °C for up to 217 days before testing.
Real-Time PCR. Primers and probes, specific for the T. vaginalis b-tubulin genes, were from Hardick et al. (2003, J Clin Micro 41: ). PCR was performed with the LC FastStart master hybridization probe kit (Roche) in a LightCycler (Roche) as previously described. LC analysis was performed using Fit Points Analysis and Arithmetic Baseline Adjustment of the F2/F1 channel. Positives were defined by a threshold cycle number (Ct) less than 40 and significant fluorescence above baseline. Specimens with Ct <30 were considered high positives (Hi Pos), and specimens with >30 Ct <40 were considered low positives (Lo Pos). Specimens with initial Hi Pos or Lo Pos results were retested using the same assay and were considered final positives if the repeat result was also positive. Positive controls were purified T. vaginalis total nucleic acid or urine spiked with T. vaginalis from in vitro cultures; sterile water was used as a negative control.
Transcription-Mediated Amplification (TMA). Specimens were tested on a Gen-Probe DTS 400 instrumentation system using T. vaginalis analyte-specific reagents (TV ASR) with the APTIMA General Purpose Reagent (GPR) kit (Gen-Probe, Inc.). The APTIMA TV ASR procedure includes target capture, TMA and a hybridization protection assay. Primers and probes for this assay specifically target T. vaginalis rRNA. The APTIMA GPR kit contains Target Capture, Amplification, Enzyme, Probe and Selection reagents. Reagents were reconstituted as described in the APTIMA GPR product insert. 50mL of each TV ASR oligonucleotides (1 target capture oligo, 3 amplification oligos, 1 probe oligo) was added to the appropriate GPR component, except for TV ASR amplification oligo #3, which was diluted 1:10,000 with nuclease-free water before 50mL was added to the reconstituted Amplification reagent. Positive controls were purified T. vaginalis total nucleic acid or urine spiked with T. vaginalis from in vitro cultures; sterile water was used as a negative control. TMA testing was performed as described in the APITMA Combo 2 kit product insert (Gen-Probe, Inc.). We used a cutoff value of 30,000 relative light units (RLU) to define a positive TMA result. All specimens with a positive R-T PCR or TV ASR result, and a portion of specimens with negative results from both assays, were also tested using an alternate TV TMA assay (ALT TV). The ALT TV primers and probes target a different region of the T. vaginalis rRNA than those for TV ASR. ALT TV TMA was performed using the same technology as the TV ASR.
Comparative Analyses. For specimens with all 3 test results, we assessed sensitivity and specificity in 2 ways: (1) Each test was compared to a rotating standard defined by concordant positive results with the other 2 tests. (2) Each test was compared to a single standard defined as 2 positive results by any 2 tests.
Limits of Detection
Real-Time PCR
T. vaginalis concentration
(organisms/mL urine)
0.1
1.0 10
100
1000
10,000
Real-Time PCR threshold cycle number 30 35 40 45 50
50 trichomonads/mL urine (0.2 organisms/PCR)
Gen-Probe TV ASR
0.001
0.01 1
10000
Gen-Probe TV ASR results
(mean RLU + sem)
1e+3
1e+4
1e+5
1e+6
1e+7
0.05 trichomonads/mL urine (0.01 organisms/test)
Figure 2. Limits of detection for Real-Time PCR and Gen-Probe TV ASR. Urine was spiked with laboratory-grown T. vaginalis parasites at concentrations ranging from .005 to 5000 organisms/mL. Left panel. The lowest concentration yielding positive R-T PCR results was 50 trichomonads/mL urine, corresponding to 0.2 organisms/PCR. Right panel. The lowest concentration yielding positive Gen-Probe TV ASR results was 0.05 trichomonads /mL urine, corresponding to 0.01 organisms/test.
97.6
( )
97.0
( )
Any 2 Pos tests
TV ASR
92.9
( )
87.9
( )
R-T PCR
87.2
( )
96.4
( )
R-T PCR + ALT TV
88.6
( )
87.1
( )
TV ASR + ALT TV
% Specificity (95% CI)
% Sensitivity (95% CI)
Reference Standard
Assay
100
( )
ALT TV
89.4
( )
R-T PCR + TV ASR
Gen-Probe performance
on Frozen Urine
Gen-Probe Fresh Urine Results (RLU)
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
Gen-Probe Frozen Urine Results (RLU)
Figure 4. Specimen storage conditions did not substantially affect Gen-Probe TV ASR results. For TV ASR testing, 235 urine specimens were processed within 24h of collection (Fresh Urine Results); 73 of those preps were stored at 2-8 °C up to 56 days before testing (Red symbols) and 162 preps were stored at -20 (Black symbols). The graph compares these results with preps made from urine aliquots of the same specimens that had been stored at -20 °C for up to 16 months (Frozen Urine Results). Points above the diagonal line in the graph represent specimens with higher RLU values from frozen urine; points below the line represent specimens with lower RLU values from frozen urine, compared to the results from fresh urine. The distribution of results from fresh urines was similar for frozen preps and those stored at 2-8 °C.
Summary and Conclusions
Real-Time PCR and Gen-Probe APTIMA-based TMA assays performed similarly for T. vaginalis detection in urine from patients with HIV.
Sensitivities ranged from 87-97% and specificities ranged from %.
Performance of the Gen-Probe TV ASR and ALT TV was slightly higher than R-T PCR, but the differences were not statistically significant.
Urine volume and storage conditions did not affect the performance of the Gen-Probe TV ASR assay.
Support for this study was provided by Gen-Probe, Inc. and by the Microbiology Core Laboratory of the North Carolina Sexually
Transmitted Infections and Topical Microbicides Cooperative Research Center (National Institutes of Health Grant U19-
AI31498).
Gen-Probe, APTIMA, Combo 2, and TMA are trademarks of Gen-Probe Incorporated; LightCycler and MagNA Pure are trademarks of Roche Diagnostics