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Male accessory gland infection and subfertility: a diagnostic challenge
Trum, J.W.
Publication date
1999
Link to publication
Citation for published version (APA):
Trum, J. W. (1999). Male accessory gland infection and subfertility: a diagnostic challenge.
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JWTrum, Y Pannekoek, L Spanjaard, OP Bleker, F van der Veen.
Abstract
The accuracy of the PACE2 DNA hybridization assay relative to a polymerase chain reac-tion in the diagnosis of Chlamydia trachomatis infecreac-tion was assessed in a male populareac-tion.
C. trachomatis infection was diagnosed in 12.9% of men. Sensitivity of the DNA
hybridi-zation assay was 100% and specificity was 99%. Although the PCR on semen or urine is nowadays the reference standard, the PACE2 assay is a highly specific test thus represen-ting a good screening test when high numbers of tests are processed and particularly when cost is a driving factor.
Introduction
The traditional laboratory test to detect Chlamydia trachomatis has consisted of cell cultu-res of inocula prepared from urogenital specimens (Black, 1997). Nowadays the polymerase chain reaction (PCR) on semen or urine is the reference standard (vanden Brule et
al.,1993 ; Witkin et al.,1993; Black,1997). Preliminary studies using PCR for detection of
C. trachomatis infections among healthy sperm donors and male members of infertile
couples reported prevalences of 16% to 39.3% (vanden Brule et al.,1993 ; Witkin et al.,1993). In both studies however, very few men,16 and 28 respectively were included. In a large study among male infertility patients using the PACE2 DNA hybridization assay, we found a prevalence of only 0.5% of C. trachomatis infection (Trum et al.,l998). The question therefore arises whether this percentage reflects a genuine low prevalence among the study population or points to differences in accuracy between the PCR and DNA hybridization assay detection methods. The accuracy of a DNA hybridization assay relati-ve to PCR has been evaluated in just two studies. In these studies the sensitivity was 77.3% and the specificity 100% (Kluytmans et ai, 1991; Altwegg et al., 1994). In the first study the performance of a DNA hybridization assay was compared with PCR on urethral specimens in only 16 male patients in whom test results of cell culture and DNA hybridi-zation assay were discordant (Kluytmans et ai, 1991). In the second study the DNA hybridization assay and PCR were compared using cervical specimens in women (Altwegg
et ai, 1994).
This study was therefore undertaken to assess study the accuracy of the DNA hybridiza-tion assay of an urethral swab sample with the PCR for C. trachomatis in semen as the reference strategy.
Materials and methods
Between July 1996 and September 1996 we asked a cohort of 83 subfertile men of 18 years and older who consecutively presented at the Center for Reproductive Medicine of the Academic Medical Center in Amsterdam to participate in the study. This study was carried out as part of a study in the detection of sexually transmitted diseases (STD's) as a cause of male infertility. Approval by the Institutional Review Board of our hospital was obtained. To obtain an adequate number of men to test positive, we also asked male part-ners of women who presented the department of gynecology and who tested positive for
C. trachomatis to participate in the study. Sixteen women tested positive for C. trachomatis.
Four women were routinely screened for the presence of C. trachomatis during pregnancy, two women were screened prior to insertion of an intra uterine device, and eight women were screened before a hysteroscopy was scheduled. Two women with abnormal PAP sme-ars tested positive. None of these women or their partners were known to be subfertile. All men underwent digital prostatic massage after which a urethral swab specimen was
taken. The cotton swab was examined for C. trachomatis using a hybridization fluorescence assay (Gen-Probe, San Diego, Ca.). The assay was performed according to the manufactu-rer's instruction. After RNA-DNA hybridization, the chemiluminescence signals were recorded by a luminometer (Leader-50,Gen-Probe). The cutoff level was set relative to the mean chemiluminescence value of the negative controls as instructed.
From all men semen fluid was collected by masturbation for detection of C. trachomatis by means of Amplicor PCR (Roche Diagnostics Systems, New Jersey). The PCR was perfor-med on DNA that was extracted from 1 OOul of the semen plasma samples, using protocol Y/D (Boom et al., 1990). After extraction, DNA was dissolved in 50ul H 2 0 .
Country) and urine diluent (UD) was added to 25ul DNA, mixed, and used for the PCR which was accomplished by using a Perkin Elmer Thermocycler T C 9600 (Perkin Elmer, USA). After amplification, amplicons were directly detected by the enzyme immuno assay as described by the manufacturer. Specimens giving an absorbency value equal or greater than 0.25 were considered positive.
Sensitivity and specificity of the DNA hybridization assay were calculated together with their 95% confidence intervals with the PCR as reference standard.
Results
The mean age of the studied men was 34.7 years (standard deviation 6.7). None of them could recall having previously suffered urogenital symptoms. Their female partners were asymptomatic as well.
A positive test result for C. trachomatis was found in 13 men (12.9%). Twelve of these were partners of women who presented at the department of gynecology and who had tested positive for C. trachomatis. Three men of women who tested positive, had negative test results both with the PCR on semen and the PACE2 DNA hybridization assay. Three men who tested positive for C. trachomatis and one men who tested negative while his wife tested positive, reported multiple sexual partners; the remaining men and their partners claimed to have been in a monogamous sexual relationship for at least two years prior to examination.
The discriminative capacity of the DNA hybridization assay to detect a C. trachomatis infection in these men is shown in Table I. Sensitivity was 100% (95% CI : 0.75 tol) and specificity was 99% (95% CI : 0.94 tol).
Table I :The accuracy of PCR on semen D N A versus D N A hybridization assay of an urethral swab specimen. PCR on semen D N A No: POS PCR on semen D N A No: NEG TOTAL No: D N A hybridization assay No : POS 13 1 14 D N A hybridization assay No : N E G 0 85 85 13 86 99 Sensitivity : 100% (95% CI : 0.75 to 1) Specificity : 99% (95% CI : 0.94 to 1)
Discussion
This study shows the widely used PACE2 DNA hybridization assay to be an excellent test for the detection of C. trachomatis with a sensitivity of 100% and a specificity of 99%. The DNA hybridization assay can detect approximately 103 chlamydial elementary bodies, whereas the PCR is more sensitive and can detect as little as one elementary body
(Black, 1997). However, in asymptomatic men like our patients, apparently the number of elementary bodies is sufficiently high to be reliably detected with the DNA hybridization assay. A C. trachomatis infection may be present in the urethra with or without ascending infection. A positive PCR of semen reflects infection of the urethra, and/or prostate, and/or the seminal vesicles and/or epididymides. Sampling the urethra with a cotton swab for the DNA hybridization assay, was performed after digital prostatic massage thereby obtaining also epithelial cells from the upper genital tract. The DNA hybridization assay can thus reflect an urethral infection and/or an ascending C. trachomatis infection.
The prevalence of C. trachomatis infection (1.5%) in a large male infertility population when using the DNA hybridization assay was lower than that among healthy sperm donors (16%) using the PCR (Trum et al.,l998 ; vanden Bruhle et al.,l993). We found that this discre-pancy could not be explained by the test we used. Population bias may account for this dif-ference.
Although the sequelae of a C. trachomatis infection in men are not yet fully understood, it is a sexually transmitted disease that may cause severe damage in the female genital tract lea-ding to tubal infertility (Mol et al., 1997; Paavonen and Wolner-Hansen, 1989; Sellors et al.,1988). A challenge to the control of chlamydial disease is the fact that up to 50% of men who are infected do not experience any symptoms (Zelin et al.,1995). This results in a large reservoir of unrecognized, infected men who are capable of transmitting the infection to
sexual partners. To what extend a C. trachomatis infection is sexually transmitted is not known. In this study three women tested positive with the PCR, while their partners tested negative with both tests (19%). Due to the number of men tested (99) and the
12.9% prevalence of C. trachomatis infection,the 9 5 % CI of the sensitivity is varying from 0.75 t o l . However, when choosing a test for screening asymptomatic persons in a popula-tion with a low prevalence of infecpopula-tion, the positive predictive value of the test will be high when the specificity of the test is good. The PACE2 DNA hybridization assay, with a spe-cificity of 99% and a small 9 5 % CI (0.94 tol), is therefore, a good screening test.
Furthermore, when large volumes have to be tested, costs become an important issue. The cost of the DNA hybridization assay is about half of the cost of PCR.
The DNA hybridization assay can be used in conjunction with a probe for detection of
Neisseria gonorrhoeae, so that a single swab specimen can be used to test the presence of
both pathogens. However, the urethral swab test after digital prostatic massage for DNA hybridization is experienced negatively by most men. In that event a PCR or LCR on semen or on urine which show a sensitivity of 94 to 99% and a specificity of 100%, are preferable (Chernesky et al.,1994 ; Lee et ai, 1995). This is an important issue when it comes to screening men without clinical signs of an infection in a high risk population and when sampling of the urethra is difficult to obtain.
In conclusion: the PACE2 DNA hybridization assay is highly specific thus representing a good screening test when high numbers of tests are processed in a population with a low prevalence of C. trachomatis infection particularly when cost is a driving factor. However, commercial PCR kits will be soon available at a lower price. Furthermore, PCR testing on urine or semen is a more sensitive - and less disincentive test to men and therefore the screening test of choice.
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