Question
How do the results of urine and urethral swabs from men and urine, urethral swabs, and cervical swabs from women compare for the detection of M. genitalium and C. trachomatis by PCR?
Design
This study describes the evaluation of first void urine specimens and urogenital swab specimens from male and female STD clinic patients for detection of M. genitalium and C. trachomatis using in-house inhibitor-controlled PCR assays and confirmation of positive findings with independent PCR assays. The effect of freezing the urine before and after specimen preparation for PCR was also evaluated.
Participants
One thousand eight hundred fifty-two men and 753 women, each with complete specimen sets, attending the outpatient STD clinic at Huddinge University Hospital on their first visit for urethritis, cervicitis, N. gonorrhoeae, C. trachomatis, and M. genitalium screening were tested.
Description of Tests and Diagnostic Standard
Urethral (collected with cotton tipped aluminum swabs) and first void urine specimens were collected from men and women. Cervical swabs were also collected from women. The swabs were placed into 1.8 mL of SP4 mycoplasma broth medium for PCR analysis. Methylene blue stained smears prepared from urethral and cervical swabs were examined microscopically. Urethral smears showing 5 or more polymorphonuclear leukocytes per high power field (PMNL/hpf) in 5 or more fields were positive for urethritis; cervical smears showing 30 or more PMNL/hpf were positive for cervicitis. After the clinical examination, the patients collected 15 mL (men) or 4 mL (women) of first void urine. All specimens were sent by mail to the testing laboratory. For PCR analysis, 300 μL of a 20% w/v Chelex 100 slurry (BioRad) was added to 100 μL of the swab specimens in SP4 medium and to the pellet obtained by centrifuging 1.8 mL of urine. Specimens were incubated at 95oC for 10 minutes and centrifuged. Ten μL of supernatant (representing 3 μL and 60 μL of original swab and urine specimen, respectively) was used for PCR. M. genitalium PCR was performed using primers that targeted the 16S rRNA gene. An internal control was included to monitor for false-negative results caused by inhibition of the reaction. Positive results were confirmed with a second PCR using primers MgPa-1 and MgPa-3, which amplified a fragment of the MgPa adhesin gene. C. trachomatis PCR was performed using primers CP24 and CP27 that targeted the cryptic plasmid. Positive results were confirmed with an inhibitor controlled PCR detecting the 16S rRNA gene of chlamydia species. Both the swab and the urine specimens were tested in the confirmatory assays if any one of the specimens was positive. If the internal control did not amplify, the specimen was retested with 5 and 2 μL of DNA. Specimens that remained negative were classified as inhibitory. A patient was considered positive for M. genitalium or C. trachomatis if both the initial and the confirmatory PCR assays were positive for one or more specimens.
The effect of freezing before and after sample preparation was studied on a subset of male and female M. genitalium-positive urine specimens. One hundred two male and 22 female urine samples, which had been tested immediately after preparation, were tested after storage at =20oC for 1 to 18 months. Urine specimens that had also been stored at =20oC for 1 to 18 months from 68 male and 15 female patients in this same group were thawed, prepared, and tested.
Main Outcome Measures
The prevalences of M. genitalium and C. trachomatis in urine and urethral swabs from men and in urine, urethral swabs, and cervical swabs from women when detected by PCR were compared.
Main Results
The results of testing 1852 urine and urethral swab specimens from men and 753 urine, urethral swab, and cervical swab specimens from women by PCR for M. genitalium and C. trachomatis are shown in Table 1. The distribution of M. genitalium and C. trachomatis positive specimens by specimen type are shown in Table 2. In women, the sensitivity of the urine specimen for detection of M. genitalium was 88%. When the results of both the urine and cervical swab specimens from the same woman were combined, the sensitivity of M. genitalium detection increased to 96%. The sensitivity of C. trachomatis detection increased from 90% for urine specimens to 99% for both urine and cervical swab specimens. When the male urine specimens were tested after storage at =20oC, there was no difference in sensitivity compared to the results of testing immediately after collection and preparation. However, of the 15 female urine specimens stored and then prepared and tested, 11 (73%) remained M. genitalium positive.
Table 1. Results by specimen type of testing by PCR for M. genitalium and C. trachomatis 1852 men and 753 women attending an STD clinic
|
Specimen type
|
Number with result (%)
|
|
M. genitalium
|
C. trachomatis
|
|
Positive
|
Inhibitory
|
Positive
|
Inhibitory
|
|
Male urine
|
123 (6.6)
|
121 (0.5)
|
242 (13)
|
32 (0.2)
|
|
Male urethra
|
104 (5.6)
|
93 (0.5)
|
220 (11.9)
|
0 (0)
|
|
Female urine
|
45 (6.0)
|
2 (0.3)
|
66 (8.8)
|
0 (0)
|
|
Female urethra
|
29 (3.9)
|
2 (0.3)
|
57 (7.6)
|
0 (0)
|
|
Cervix
|
36 (4.9)
|
1 (0.1)
|
63 (8.4)
|
0 (0)
|
1 None of the corresponding urethral specimens were inhibitory
2 These 3 specimens were also inhibitory in the M. genitalium PCR assay
3 1 of 9 was positive for C. trachomatis
Table 2. Results by specimen type for 126 M. genitalium and 246 C. trachomatis PCR positive specimen sets from nen and 51 M. genitalium and 73 C. trachomatis PCR positive specimen sets from women
|
Gender
|
PCR result by specimen type
|
Number with PCR result for:
|
|
Urethra
|
Urine
|
Cervix
|
M. genitalium
|
C. trachomatis
|
|
Male
|
Pos
|
Pos
|
NA
|
101
|
216
|
|
Pos
|
Neg
|
NA
|
3
|
4
|
|
Neg
|
Pos
|
NA
|
22
|
26
|
|
Female
|
Pos
|
Pos
|
Pos
|
20
|
51
|
|
Pos
|
Neg
|
Pos
|
1
|
0
|
|
Neg
|
Pos
|
Pos
|
12
|
6
|
|
Neg
|
Pos
|
Neg
|
7
|
4
|
|
Pos
|
Pos
|
Neg
|
6
|
5
|
|
Neg
|
Neg
|
Pos
|
3
|
6
|
|
Pos
|
Neg
|
Neg
|
2
|
1
|
Authors
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