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Deoxyribonucleic acid hybridization analysis for the detection of urogenital Chlamydia trachomatis infections in women
Deoxyribonucleic acid hybridization analysis for the detection of urogenital Chlamydia trachomatis infections in women Chia C. Pao, Ph.D., Shyh~Shyan Lin, M.S., Tsyr-En Yang, M.S., Yung-Kuei Soong, M.D., Pai-Shun Lee, R.N., and Jung-Yaw Lin, Ph.D. Taipei, Taiwan, Republic of China The presence of Chlamydia trachomatis-reiated deoxyribonucleic acid sequences in endocerilical specimens of 317 women was analyzed by deoxyribonucleic acid hybridization techniques with deoxyribonucleic acid from C. trachomatis used as probes. Samples from 56 of 172 high-risk patients (32.6%) and 16 of 145 low-risk patients (11.0%) contained C. trachomatis-reiated deoxyribonucleic ac_id sequences. Direct detection of chlamydia! antigen with enzyme-linked immunoassay on the same patients yielded positive rates of 26.3% and 7.3% for the high- and low-risk patients, respectively. C. frachomatis culture confirmed 86.3% of deoxyribonucleic acid:..positive results and 84.0% of antigen-positive results. The overall sensitivities of chlamydia! deoxyribonucleic acid and antigen assays were 91.7% and 68.8%, respectively, whereas the specificities were 95.3% and 94.7%. Results also suggested that the test of the C. trachomatis deoxyribonucleic acid correlated better with tlle female urogenital chlamydia! infections than did the antigen test of C. trachomatis. The combined results of higher sensitivity in detecting the microorganism and better correlation with disease activity may make the deoxyribonucleic acid hybridization test a useful tool for the early and accurate diagnosis of C. trachomatis infections in female patients. (AM J 0BSTET GYNECOL 1987;156:195-9.)
Key words: Chlamydia trachomatis, female urogenital infection, deoxyribonucleic acid hybridization, deoxyribonucleic acid probe
Infections caused by Chlamydia trachomatis, now recognized as the most prevalent of all sexually transmitted diseases, are very damaging and c~n result in a wide range of diseases including trachoma and lymphogranuloma venereum. '- 3 Men, women, and infants are all affected, but worhen bear a disproportionately heavy burden because of their increased risk for other adverse reproductive consequences. In women, C. trachomatis may cause mucopurulent cervicitis and urethral syndrome in the lower urogenital tract and endometritis and salpingitis in the upper urogenital tract. These infections can also result in sterility and other complications during pregnancy. 3 -5 Although chlamydia! infections can now be effectively treated with antibiotics, one of the most difficult aspects of controlling chlamydia! infection in women is that unrecognized and misdiagnosed chlamydia! infections in women are common. it has been suggested tha:t a substantial portion of women infected with chlamydiae may be asymptomatic. 2 ' 3 From the Departments of Biochemistry and Obstetrics and Gynecology, Chang Gung Memorial Hospital, and the Department of Biochemistry, National Taiwan University School of Medicine. This work was supported entirely by Research Grant MRP-131 awarded to C. C. P. from Chang Gung Memorial Hospital, Taipei, Taiwan, Republic of China. Received for publication May 30, 1986; accepted August 14, 1986. Reprint requests: Professor Chia C. Pao, Department of Biochemistry, Chang Gung Memorial Hospital, 199, Tun Hwa North Road, Taipei, Taiwan 10560, Republic of China.
Chlamydiae possess a uriique development cycle. Although they are now classified as bacteria, they share many properties with viruses. chlamydiae are obligatory intracellular parasites and depend on the energy supply of the host cells to grow." For this reason, culture of chlamydiae is laborious and requires tissue culture techniques and facilities. Time and expenses are increased further when subculturing is needed, which is quite often. Therefore the culture method has not become routine procedure in the laboratory diagnosis of chlamydia! infection. Direct cytologic staining is available,but it has only limited applications because of its lacking of specificity and it requires a considerable amount of experience if false positive and false negative results are to be avoided. in recent years, antigen detection methods, .such as direct-smear immunofluorescence staining tests 7 and enzyme-linked lmmunoassays," have improved rapidly; these offer an alternative to the conventional culture and cytologic staining methods and open the door to further investigation of the broad spectrum of diseases caused by this microorganism. In the following text, we report the use of deoxyribonucleic acid (DNA) fragments from the human biovar strain of C. trachomatis in a DNA hybridization test on clinical specimens to determine its value as a reliable culture-independent diagnostic test in the detection of chlamydia! infection. The DNA hybridization test was found to have compared favorably with the conventional culture and antigen results.
195
196 Pao et al.
January 1987 Am J Obstet Gyneco]
...
......
·
'Ill•··
•
Fig. L Autoradiographs of the chlamydia! DNA slot blot hybridization test of endocervical samples. Shown here are the DNA results of 60 patients participating in this study. Specimens were treated and handled according to the procedures outlined in Material and methods. The actual size of each slot was 0.75 by 8 mm, or 6 mm 2 • Slots A and B were positive controls of 20 and 2 pg of chlamydia! DNA, respectively.
Material and methods
Study protocol. Individuals visiting the outpatient clinic of the Department of Obstetrics and Gynecology of the Chang Gung Memorial Hospital in Taipei, Taiwan, were recruited for this study. A total of I72 patients who had signs or complaints of one or more of the following symptoms were included in the high-risk group: primary infertility (65 patients), urethritis or urethral syndrome (29 patients), cervicitis (10 patients), cervical erosion (nine patients), pelvic inflammatory disease (53 patients), tubal disorders (15 patients; I2
confirmed by laparoscopy and three suspected), previous ectopic pregnancy (six patients), recurrent or habitual a,bortions (24 patients), and previous stillbirth or neonatal death (three patients). Another I45 patients who were matched in age and in marital and socioeconomic status and who visited the clinic for reasons other than those listed above were selected as the lowrisk group. Informed consent was obtained from all patients. Laboratory procedures. Two endocervical and/or urethral swabs were collected with the STD-EZE swab (Abbott Laboratories, Inc., North Chicago, Illinois). Extracts from the swabs were pooled and divided into three equal portions for chlamydia! DNA, antigen, and c;ulture analysis. Blood samples were also drawn for antichlamydial antibody determination. Chlamydia! antigen was determined by the enzyme-linked immunoassay kit Chlamydiazyme, commercially obtained from Abbott. 8 Antichlamydial antibody was assayed with an enzyme-linked immunoassay kit obtained from Exdevelop Phartna, Taby, Sweden. Chlamydia! DNA in the endocervical swab was determined by a modified DNA: DNA hybridization technique that we have previously used for the quantitation of hepatitis type B virus DNA, 9 which was based on methods initially described by others. 10 Briefly, a purified cloned chlamydia! plasmid DNA of 6.7 kilobases, pLGVI25,'' consisting only of the chlamydia! plasmid genome was used as probe. This DNA was originally cloned by Dr. Steven H. Larsen oflndiana University School of Medicine and was kindly. made available to us. Alternatively, a cloned DNA coded for the major outer membrane protein of C. trachomatis serovar L2, pFEN207, 12 obtained from Dr. F. E. Nano of Rocky Mountain Laboratories was used as a probe. The DNA probe was labeled with [alpha- 32 P]-deoxycytidine 5'-triphosphate by nick translation of the purified chlamydia! DNA to approximately I X 108 cpm per microgram of DNA. DNA from endocervical material was extracted with phenol and denatured with 40 f.Ll of sodium hydroxide, 2 mol/L, and sodium chloride, 2 mol/L, for 10 minutes at room temperature. Then 75 f.Ll of Tris hydrochloride, I mol!L, pH 7.4, and sodium chloride, 4 mol/L, was added to neutralize the reaction mixture. Each specimen was deposited on a 0.45 f.Lm pore size nitrocellulose membrane filtersheet through wells of a microsample filtration manifold under vacuum (BASS and Minifold II, both from Schleicher and Schuell, Inc., Keene, New Hampshire). After loading, the nitrocellulose filter was gently removed from the manifold under continued vacuum, blotted, air dried, and baked at 70° C for 2 hours in vacuo. DNA hybridization was performed at 65° C with about 2 x 10 7 cpin of the labeled DNA used as probe for each filter of 63 by 228 mm in size. After prehybridization and . hybridization, the filters
Chlamydia! DNA in female urogenital infections
Volume 156 Number l
197
Table I. Prevalence of chlamydia! DNA, antigen, culture, and antichlamydial antibody in high- and low-risk women High risk Type of assay
w
Chlamydia DNA Chlamydia antigen Chlamydia culture Antichlamydia antibody
561172 351133 391106 29/152
I
Low risk
%
n
32.6* 26.3t 36.8:1: 19.1§
16/145 91124 9/92 181117
I
Overall
%
n
11.0* 7.3t 9.8:j: 15.4§
72/317 44/257 48/198 47/269
I
% 22.7 17.1 24.2 17.5
Percentages represent prevalence, which is equal to number of patients with positive results per number tested in each assay and patient group. *x' = 20.68, p < 0.001. tx' = 16.35, p < 0.001. +x' = 19.55, p < o.oo1. §x' = 0.61, p < o.5.
Table II. Sensitivity and specificity of Chlamydia trachmatis DNA and antigen assays Specijicit~t
Sensitivity* Type of assay
DNA assay Antigen assay
Risk group
High Low Overall High Low Overall
% 89.7 100.0 91.7:1: 74.4 44.4 68.8:j:
I
n
%
35/39 9/9 44/48 29/39 4/9 33/48
97.0 94.0 95.3 95.5 94.0 94.7
I
n 65/67 78/83 1431150 64/67 78/83 142/150
*Sensitivity = number of samples that were DNA and culture positive or antigen and culture positive per number of samples that were culture positive. tSpecificity = number of samples that were DNA and culture negative or antigen and culture negative per number of samples that were culture negative. +x' = 7.94, p < O.oi.
were washed sequentially in 2X SSC, 0.1% sodium dodecyl sulfate, 30 minutes twice at room temperature (IX SSC = sodium citrate, 15 mmol/L, and sodium chloride, 150 mmol/L, pH 7.0); 0.1XSSC, 0.1% sodium dodecyl sulfate, also 30 minutes twice at room temperature; and 0.1X SSC, 0.5% sodium dodecyl sulfate, 30 minutes twice at 65° C. Filters were then air dried and autoradiographed for 24 to 48 hours. After autoradiography, the filters were cut out and counted in a liquid scintillation counter. The amount of chlamydia! DNA in the swab samples was calculated according to the chromic phosphate P 32 counts obtained from a series of dilutions of purified chlamydia! DNA prepared from a stock of known concentration and hybridized to the probe DNA on the same filters. The sensitivity of detection of our method was determined to be approximately 1.0 pg of chlamydia! DNA. For the purpose of analyzing the results, a DNA concentration readout of ~2.0 pg was interpreted as a positive result in this study (Fig. 1). Culture for chlamydia was performed with the use of cycloheximide-treated McCoy cell monolayers as described in detail elsewhere. 13
Results
The incidence of endocervical chlamydia with the DNA, antigen, and culture techniques as well as the finding of serum antichlamydial antibody is shown in Table I. The incidences displayed by the high-risk patients were significantly higher than those from the lowrisk group in three of four categories, excepting antichlamydial antibody, where the difference between the high- and low-risk groups was not significant. The overall positive rates for DNA and culture were close, and both were significantly higher than the positive rate for chlamydia! antigen (Table 1). The two DNA probes, pLGV125 and pFEN207, gave essentially the same results. The mean age of the infected participants, based on either the DNA, antigen, or culture findings, did not differ significantly from the mean age of those who were not infected. A comparison of sensitivity and specificity of the DNA and antigen tests is presented in Table II. The overall sensitivity was 91.7% for the DNA test and 68.8% for the antigen test (p < 0.0 I). On the other hand, the DNA and antigen tests showed equally high degrees of specificity. The overall specificity was 95.3%
198 Pao et al.
for the DNA test and 94.7% for the antigen test. On the basis of an average incidence of 22.7% for the DNA test and I7 .I% for the antigen test, the predictive values of positive results were 86.3% and 84.0% for the DNA and antigen tests, respectively. For negative results, the predictive values were 97.3% and 90.4%. For I98 patients with a complete set of DNA, antigen, and culture test results available, the discordance between culture results and those of antigen and DNA was analyzed. (I) Of the I 50 culture-negative patients, eight were found to be antigen positive and seven were DNA positive. The chlamydia! DNA concentrations in the endocervical specimens of these seven patients were below 20% of the mean DNA concentrations of all patients tested, and four of these seven patients responded to treatment. The endocervical chlamydia! DNA tests became negative 2 weeks after the patients were treated with Vibramycin. If these four patients were infected, the resolved sensitivity of the DNA test would be 92.3% instead of91.7% (Table II). (2) Among the 48 culture-positive patients, as many as I5 were not recognized by the chlamydia! antigen test, and four were negative for the DNA test. Of these I5 culturepositive/antigen-negative patients, I4 were found to have chlamydia on DNA testing. (3) Fourteen of 21 patients who were antigen negative but had chlamydia! DNA were confirmed to have chlamydia! infection on the basis of culture results. On the other hand, only three of II patients who were antigen positive but with no DNA in the endocervical specimens had confirmation of chlamydiae by culture. Comment
Several lines of evidence indicated that the higher sensitivity of the DNA method was accompanied by a better reflection of the actual disease status: (I) Chlamydia! DNA sequences could be detected in none of the specimens from the eight patients with false positive antigen results; (2) specimens from I4 of the I5 patients with false negative antigen results (93.3%) contained chlamydia! DNA; (3) the respective culture confirmation rates for the patients with chlamydia antigen-negative/DNA-positive and antigen-positive/DNA-negative results were 66.7% (14 of 21) and 27.3% (3 of 11) (X 2 = 4.36, p < 0.05). The possibility of using the DNA probe to detect chlamydiae in clinical specimens such as endocervical materials was further supported by the report that the chlamydia! plasmid DNA used here as a probe, pLGV125, had extensive homologous DNA sequences in all 15 C. trachomatis serovars. 11 In addition, little if any DNA homology could be detected with bacteria of other genera that were likely to be present in locations where chlamydiae might be found. 14 The 68.8% overall sensitivity achieved by the enzyme-linked immunoassay (Chlamydiazyme, Abbott)
January 1987 Am J Obstet Gynecol
that detects an antigen of C. trachomatis was lower than what has been reported by others." We could not rule out the possibility that this discrepancy was due to the presence of a different but very closely related antigen in our patient population. However, this discrepancy did not seem to be related to the reported cross"reactivity between Chlamydiazyme and organisms other than chlamydiae." Historically, time-consuming and expensive tissue culture techniques were required to definitively diagnose most C. trachomatis infections. Despite recent rapid and profound improvements in the antigen and antibody detection systems, these tests are not ideal, mainly because of their sensitivity and their lack of complete correlation with the disease activity. The usefulness of DNA: DNA hybridization has been recognized recently in the identification of a number of microorganisms and is particularly helpful in cases of infectious agents that are traditionally difficult to culture. The DNA hybridization system used here seemed to have higher sensitivity, without compromising specificity, in the detection of chlamydia! infection when compared with the commercially available enzyme-linked immunoassays. Assuming a plasmid DNA length of 6. 7 kilobases and 10 copies of plasmid molecules per chlamydia genome equivalent," a sensitivity of 2 pg represents a level of detection of about 30,000 chlamydia genome equivalents. When the number of elementary and reticulate bodies in each infected cell is considered, the minimal number of infected cells needed for detection by the DNA probe system will actually be even lower. Such higher sensitivity could offer earlier detection and treatment of subclinical or latent chlamydia! infections in women, many of whom were asymptomatic, thus reducing the incidence of serious complications and sequelae. It also opened new doors for the detection of chlamydia! infections under other clinical conditions, such as during pregnancy. Chlamydia! infection during pregnancy may lead to both serious sequelae in the mother and infections such as pneumonia or eye infection in infants. In summary, results of the chlamydia! DNA hybridization test showed a higher sensitivity than the antigen detection method in the diagnosis of chlamydia! infections and correlated highly with the disease activity in our patient population. The results of our study indicated that the DNA hybridization test will contribute significantly to the early and accurate diagnosis of urogenital chlamydia! infections in women. We thank Dr. Delon Wu and Dr. Chau-Hsiung Chang for their interest and encouragement during this study. Appreciation is also expressed to Miss Wen-Ling Yang and Miss Ruey Ken for their technical assistance.
Volume 156 Number l
REFERENCES 1. Chlamydia trachomatis infections: policy guidelines for prevention and control. MMWR 1985;34(3S):53S-74S. 2. Thompson SE, Washington AE. Epidemiology of sexually transmitted Chlamydia trachomatis infections. Epidemiol Rev 1983;5:96. 3. Sweet RL, Schachter J, Landers DV. Chlamydia! infections in obstetrics and gynecology. Clin Obstet Gynecol 1983; 26:143. 4. Martin DH, Koutsky L, Eschenbach DA, eta!. Prematurity and perinatal mortality in pregnancies complicated by maternal Chlamydia trachomatis infections. JAMA 1982;247: 1585. 5. Johannisson G, Low hagen GB, Lycke E. Genital Chlamydia trachomatis infection in women. Obstet Gynecol 1980;56: 671. 6. Schachter J, Caldwell HD. Chlamydiae. Annu Rev Microbioi 1980;34:285. 7. Tam MR, Stamm WE, Handsfield HH, et a!. Cultureindependent diagnosis of Chlamydia trachomatis using monoclonal antibodies. N Eng!J Med 1984;310:1146. 8. Howard LV, Coleman PF, England BJ, Herrmann JE. Evaluation of Chlamydiazyme for the detection of genital infections caused by Chlamydia trachomatis. J Clin Micro bioi 1986;23:329.
Chlamydia! DNA in female urogenital infections
9. Liaw YF, Tai Dl, Chu CM, et a!. Early detection of hepatocellular carcinoma in patients with chronic type B hepatitis. Gastroenterology 1986;90:263. 10. Lieberman HM, LaBrecque DR, Kew MC, Hadziyannis SJ, Shafritz DA. Detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/Anti-HBe status in HBsAg carriers. Hepatology 1983;3:285. 11. Hyypia T, Larsen SH, Stahlberg T, Terho P. Analysis and detection of Chlamydia! DNA. J Gen Microbial 1984; 130:3159. 12. Nano FE, Caldwell HD. Expression of the chlamydia! genus-specific lipopolysaccharide epitope in Escherichia coli. Science 1985;228:742. 13. Clyde WA, Kenny GE, Schachter]. Laboratory diagnosis of chlamydia! and mycoplasmal infections. In: Drew WL, ed. Cumulative techniques and procedures in clinical microbiology, Cumitech 19. Washington DC: American Society for Microbiology, 1984. 14. Kingsbury DT, Weiss E. Lack of deoxyribonucleic acid homology between species of the genus Chlamydia. J Bacterial 1968;96: 1421. 15. Saikku P, Puolakkainen M, Leinonen M, Nurminen M, Nissinen A. Cross-reactivity between Chlamydiazyme and acinetobacter strains. N Eng! J Med 1986;314:922.
Divergent effects of two low-dose oral contraceptives on sex hormone-binding globulin and free testosterone Claudia Jung-Hoffmann, M.D., and Herbert Kuhl, Ph.D. Frankfurt am Main, Federal Republic of Germany The effect of a triphasic combination of ethinyl estradiol and levonorgestrel on the serum concentrations of total testosterone, free testosterone, and sex hormone-binding globulin as measured directly by radioimmunoassay and on the binding capacity of sex hormone-binding globulin was compared with that of a preparation containing ethinyl estradiol and desogestrel. Blood samples were taken on days 6, 11, 21, and 28 of a control cycle, the third cycle of treatment with either ethinyl estradiol-levonorgestrel or ethinyl estradiol-desogestrel (11 volunteers each), the third cycle of a 3-month washout period, and the third treatment cycle after crossover change of the preparations. There was a significant reduction in total testosterone by 16% during treatment with both preparations. Ethinyl estradiol-desogestrel increased the concentration ( + 175%) and bindig capacity ( + 330%) of sex hormone-binding globulin to a much greater extent than with ethinyl estradiol-levonorgestrel ( + 92% and + 160%). Contrary to this, a significant suppression of non-protein-bound testosterone by 35% was found during treatment with both oral contraceptives. The results demonstrate that an excessive elevation of the levels of sex hormone-binding globulin above the normal range does not cause a corresponding suppression of free testosterone. It is assumed that the decrease in the apparent binding affinity of high sex hormone-binding globulin concentrations to testosterone may be due to protein-protein interactions. (AM J OssTET GvNECOL 1987;156:199-203.)
Key words: Low-dose oral contraceptives, sex hormone-binding globulin, testosterone, free testosterone
From the Division of Gynecological Endocrinology, Department of Obstetrics and Gynecology,]. W. Goethe University. Received for publication March 3, 1986; revised July 1, 1986; accepted August 14, 1986.
Reprint requests: Dr. Herbert Kuhl, Division of Gynecological Endocrinology, Department of Obstetrics and Gynecology,]. W. Goethe University, Theodor-Stern-Kai 7, D-6000 Frankfurt am Main 70, Federal Republic of Germany.
199
Key words: Chlamydia trachomatis, female urogenital infection, deoxyribonucleic acid hybridization, deoxyribonucleic acid probe
Infections caused by Chlamydia trachomatis, now recognized as the most prevalent of all sexually transmitted diseases, are very damaging and c~n result in a wide range of diseases including trachoma and lymphogranuloma venereum. '- 3 Men, women, and infants are all affected, but worhen bear a disproportionately heavy burden because of their increased risk for other adverse reproductive consequences. In women, C. trachomatis may cause mucopurulent cervicitis and urethral syndrome in the lower urogenital tract and endometritis and salpingitis in the upper urogenital tract. These infections can also result in sterility and other complications during pregnancy. 3 -5 Although chlamydia! infections can now be effectively treated with antibiotics, one of the most difficult aspects of controlling chlamydia! infection in women is that unrecognized and misdiagnosed chlamydia! infections in women are common. it has been suggested tha:t a substantial portion of women infected with chlamydiae may be asymptomatic. 2 ' 3 From the Departments of Biochemistry and Obstetrics and Gynecology, Chang Gung Memorial Hospital, and the Department of Biochemistry, National Taiwan University School of Medicine. This work was supported entirely by Research Grant MRP-131 awarded to C. C. P. from Chang Gung Memorial Hospital, Taipei, Taiwan, Republic of China. Received for publication May 30, 1986; accepted August 14, 1986. Reprint requests: Professor Chia C. Pao, Department of Biochemistry, Chang Gung Memorial Hospital, 199, Tun Hwa North Road, Taipei, Taiwan 10560, Republic of China.
Chlamydiae possess a uriique development cycle. Although they are now classified as bacteria, they share many properties with viruses. chlamydiae are obligatory intracellular parasites and depend on the energy supply of the host cells to grow." For this reason, culture of chlamydiae is laborious and requires tissue culture techniques and facilities. Time and expenses are increased further when subculturing is needed, which is quite often. Therefore the culture method has not become routine procedure in the laboratory diagnosis of chlamydia! infection. Direct cytologic staining is available,but it has only limited applications because of its lacking of specificity and it requires a considerable amount of experience if false positive and false negative results are to be avoided. in recent years, antigen detection methods, .such as direct-smear immunofluorescence staining tests 7 and enzyme-linked lmmunoassays," have improved rapidly; these offer an alternative to the conventional culture and cytologic staining methods and open the door to further investigation of the broad spectrum of diseases caused by this microorganism. In the following text, we report the use of deoxyribonucleic acid (DNA) fragments from the human biovar strain of C. trachomatis in a DNA hybridization test on clinical specimens to determine its value as a reliable culture-independent diagnostic test in the detection of chlamydia! infection. The DNA hybridization test was found to have compared favorably with the conventional culture and antigen results.
195
196 Pao et al.
January 1987 Am J Obstet Gyneco]
...
......
·
'Ill•··
•
Fig. L Autoradiographs of the chlamydia! DNA slot blot hybridization test of endocervical samples. Shown here are the DNA results of 60 patients participating in this study. Specimens were treated and handled according to the procedures outlined in Material and methods. The actual size of each slot was 0.75 by 8 mm, or 6 mm 2 • Slots A and B were positive controls of 20 and 2 pg of chlamydia! DNA, respectively.
Material and methods
Study protocol. Individuals visiting the outpatient clinic of the Department of Obstetrics and Gynecology of the Chang Gung Memorial Hospital in Taipei, Taiwan, were recruited for this study. A total of I72 patients who had signs or complaints of one or more of the following symptoms were included in the high-risk group: primary infertility (65 patients), urethritis or urethral syndrome (29 patients), cervicitis (10 patients), cervical erosion (nine patients), pelvic inflammatory disease (53 patients), tubal disorders (15 patients; I2
confirmed by laparoscopy and three suspected), previous ectopic pregnancy (six patients), recurrent or habitual a,bortions (24 patients), and previous stillbirth or neonatal death (three patients). Another I45 patients who were matched in age and in marital and socioeconomic status and who visited the clinic for reasons other than those listed above were selected as the lowrisk group. Informed consent was obtained from all patients. Laboratory procedures. Two endocervical and/or urethral swabs were collected with the STD-EZE swab (Abbott Laboratories, Inc., North Chicago, Illinois). Extracts from the swabs were pooled and divided into three equal portions for chlamydia! DNA, antigen, and c;ulture analysis. Blood samples were also drawn for antichlamydial antibody determination. Chlamydia! antigen was determined by the enzyme-linked immunoassay kit Chlamydiazyme, commercially obtained from Abbott. 8 Antichlamydial antibody was assayed with an enzyme-linked immunoassay kit obtained from Exdevelop Phartna, Taby, Sweden. Chlamydia! DNA in the endocervical swab was determined by a modified DNA: DNA hybridization technique that we have previously used for the quantitation of hepatitis type B virus DNA, 9 which was based on methods initially described by others. 10 Briefly, a purified cloned chlamydia! plasmid DNA of 6.7 kilobases, pLGVI25,'' consisting only of the chlamydia! plasmid genome was used as probe. This DNA was originally cloned by Dr. Steven H. Larsen oflndiana University School of Medicine and was kindly. made available to us. Alternatively, a cloned DNA coded for the major outer membrane protein of C. trachomatis serovar L2, pFEN207, 12 obtained from Dr. F. E. Nano of Rocky Mountain Laboratories was used as a probe. The DNA probe was labeled with [alpha- 32 P]-deoxycytidine 5'-triphosphate by nick translation of the purified chlamydia! DNA to approximately I X 108 cpm per microgram of DNA. DNA from endocervical material was extracted with phenol and denatured with 40 f.Ll of sodium hydroxide, 2 mol/L, and sodium chloride, 2 mol/L, for 10 minutes at room temperature. Then 75 f.Ll of Tris hydrochloride, I mol!L, pH 7.4, and sodium chloride, 4 mol/L, was added to neutralize the reaction mixture. Each specimen was deposited on a 0.45 f.Lm pore size nitrocellulose membrane filtersheet through wells of a microsample filtration manifold under vacuum (BASS and Minifold II, both from Schleicher and Schuell, Inc., Keene, New Hampshire). After loading, the nitrocellulose filter was gently removed from the manifold under continued vacuum, blotted, air dried, and baked at 70° C for 2 hours in vacuo. DNA hybridization was performed at 65° C with about 2 x 10 7 cpin of the labeled DNA used as probe for each filter of 63 by 228 mm in size. After prehybridization and . hybridization, the filters
Chlamydia! DNA in female urogenital infections
Volume 156 Number l
197
Table I. Prevalence of chlamydia! DNA, antigen, culture, and antichlamydial antibody in high- and low-risk women High risk Type of assay
w
Chlamydia DNA Chlamydia antigen Chlamydia culture Antichlamydia antibody
561172 351133 391106 29/152
I
Low risk
%
n
32.6* 26.3t 36.8:1: 19.1§
16/145 91124 9/92 181117
I
Overall
%
n
11.0* 7.3t 9.8:j: 15.4§
72/317 44/257 48/198 47/269
I
% 22.7 17.1 24.2 17.5
Percentages represent prevalence, which is equal to number of patients with positive results per number tested in each assay and patient group. *x' = 20.68, p < 0.001. tx' = 16.35, p < 0.001. +x' = 19.55, p < o.oo1. §x' = 0.61, p < o.5.
Table II. Sensitivity and specificity of Chlamydia trachmatis DNA and antigen assays Specijicit~t
Sensitivity* Type of assay
DNA assay Antigen assay
Risk group
High Low Overall High Low Overall
% 89.7 100.0 91.7:1: 74.4 44.4 68.8:j:
I
n
%
35/39 9/9 44/48 29/39 4/9 33/48
97.0 94.0 95.3 95.5 94.0 94.7
I
n 65/67 78/83 1431150 64/67 78/83 142/150
*Sensitivity = number of samples that were DNA and culture positive or antigen and culture positive per number of samples that were culture positive. tSpecificity = number of samples that were DNA and culture negative or antigen and culture negative per number of samples that were culture negative. +x' = 7.94, p < O.oi.
were washed sequentially in 2X SSC, 0.1% sodium dodecyl sulfate, 30 minutes twice at room temperature (IX SSC = sodium citrate, 15 mmol/L, and sodium chloride, 150 mmol/L, pH 7.0); 0.1XSSC, 0.1% sodium dodecyl sulfate, also 30 minutes twice at room temperature; and 0.1X SSC, 0.5% sodium dodecyl sulfate, 30 minutes twice at 65° C. Filters were then air dried and autoradiographed for 24 to 48 hours. After autoradiography, the filters were cut out and counted in a liquid scintillation counter. The amount of chlamydia! DNA in the swab samples was calculated according to the chromic phosphate P 32 counts obtained from a series of dilutions of purified chlamydia! DNA prepared from a stock of known concentration and hybridized to the probe DNA on the same filters. The sensitivity of detection of our method was determined to be approximately 1.0 pg of chlamydia! DNA. For the purpose of analyzing the results, a DNA concentration readout of ~2.0 pg was interpreted as a positive result in this study (Fig. 1). Culture for chlamydia was performed with the use of cycloheximide-treated McCoy cell monolayers as described in detail elsewhere. 13
Results
The incidence of endocervical chlamydia with the DNA, antigen, and culture techniques as well as the finding of serum antichlamydial antibody is shown in Table I. The incidences displayed by the high-risk patients were significantly higher than those from the lowrisk group in three of four categories, excepting antichlamydial antibody, where the difference between the high- and low-risk groups was not significant. The overall positive rates for DNA and culture were close, and both were significantly higher than the positive rate for chlamydia! antigen (Table 1). The two DNA probes, pLGV125 and pFEN207, gave essentially the same results. The mean age of the infected participants, based on either the DNA, antigen, or culture findings, did not differ significantly from the mean age of those who were not infected. A comparison of sensitivity and specificity of the DNA and antigen tests is presented in Table II. The overall sensitivity was 91.7% for the DNA test and 68.8% for the antigen test (p < 0.0 I). On the other hand, the DNA and antigen tests showed equally high degrees of specificity. The overall specificity was 95.3%
198 Pao et al.
for the DNA test and 94.7% for the antigen test. On the basis of an average incidence of 22.7% for the DNA test and I7 .I% for the antigen test, the predictive values of positive results were 86.3% and 84.0% for the DNA and antigen tests, respectively. For negative results, the predictive values were 97.3% and 90.4%. For I98 patients with a complete set of DNA, antigen, and culture test results available, the discordance between culture results and those of antigen and DNA was analyzed. (I) Of the I 50 culture-negative patients, eight were found to be antigen positive and seven were DNA positive. The chlamydia! DNA concentrations in the endocervical specimens of these seven patients were below 20% of the mean DNA concentrations of all patients tested, and four of these seven patients responded to treatment. The endocervical chlamydia! DNA tests became negative 2 weeks after the patients were treated with Vibramycin. If these four patients were infected, the resolved sensitivity of the DNA test would be 92.3% instead of91.7% (Table II). (2) Among the 48 culture-positive patients, as many as I5 were not recognized by the chlamydia! antigen test, and four were negative for the DNA test. Of these I5 culturepositive/antigen-negative patients, I4 were found to have chlamydia on DNA testing. (3) Fourteen of 21 patients who were antigen negative but had chlamydia! DNA were confirmed to have chlamydia! infection on the basis of culture results. On the other hand, only three of II patients who were antigen positive but with no DNA in the endocervical specimens had confirmation of chlamydiae by culture. Comment
Several lines of evidence indicated that the higher sensitivity of the DNA method was accompanied by a better reflection of the actual disease status: (I) Chlamydia! DNA sequences could be detected in none of the specimens from the eight patients with false positive antigen results; (2) specimens from I4 of the I5 patients with false negative antigen results (93.3%) contained chlamydia! DNA; (3) the respective culture confirmation rates for the patients with chlamydia antigen-negative/DNA-positive and antigen-positive/DNA-negative results were 66.7% (14 of 21) and 27.3% (3 of 11) (X 2 = 4.36, p < 0.05). The possibility of using the DNA probe to detect chlamydiae in clinical specimens such as endocervical materials was further supported by the report that the chlamydia! plasmid DNA used here as a probe, pLGV125, had extensive homologous DNA sequences in all 15 C. trachomatis serovars. 11 In addition, little if any DNA homology could be detected with bacteria of other genera that were likely to be present in locations where chlamydiae might be found. 14 The 68.8% overall sensitivity achieved by the enzyme-linked immunoassay (Chlamydiazyme, Abbott)
January 1987 Am J Obstet Gynecol
that detects an antigen of C. trachomatis was lower than what has been reported by others." We could not rule out the possibility that this discrepancy was due to the presence of a different but very closely related antigen in our patient population. However, this discrepancy did not seem to be related to the reported cross"reactivity between Chlamydiazyme and organisms other than chlamydiae." Historically, time-consuming and expensive tissue culture techniques were required to definitively diagnose most C. trachomatis infections. Despite recent rapid and profound improvements in the antigen and antibody detection systems, these tests are not ideal, mainly because of their sensitivity and their lack of complete correlation with the disease activity. The usefulness of DNA: DNA hybridization has been recognized recently in the identification of a number of microorganisms and is particularly helpful in cases of infectious agents that are traditionally difficult to culture. The DNA hybridization system used here seemed to have higher sensitivity, without compromising specificity, in the detection of chlamydia! infection when compared with the commercially available enzyme-linked immunoassays. Assuming a plasmid DNA length of 6. 7 kilobases and 10 copies of plasmid molecules per chlamydia genome equivalent," a sensitivity of 2 pg represents a level of detection of about 30,000 chlamydia genome equivalents. When the number of elementary and reticulate bodies in each infected cell is considered, the minimal number of infected cells needed for detection by the DNA probe system will actually be even lower. Such higher sensitivity could offer earlier detection and treatment of subclinical or latent chlamydia! infections in women, many of whom were asymptomatic, thus reducing the incidence of serious complications and sequelae. It also opened new doors for the detection of chlamydia! infections under other clinical conditions, such as during pregnancy. Chlamydia! infection during pregnancy may lead to both serious sequelae in the mother and infections such as pneumonia or eye infection in infants. In summary, results of the chlamydia! DNA hybridization test showed a higher sensitivity than the antigen detection method in the diagnosis of chlamydia! infections and correlated highly with the disease activity in our patient population. The results of our study indicated that the DNA hybridization test will contribute significantly to the early and accurate diagnosis of urogenital chlamydia! infections in women. We thank Dr. Delon Wu and Dr. Chau-Hsiung Chang for their interest and encouragement during this study. Appreciation is also expressed to Miss Wen-Ling Yang and Miss Ruey Ken for their technical assistance.
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REFERENCES 1. Chlamydia trachomatis infections: policy guidelines for prevention and control. MMWR 1985;34(3S):53S-74S. 2. Thompson SE, Washington AE. Epidemiology of sexually transmitted Chlamydia trachomatis infections. Epidemiol Rev 1983;5:96. 3. Sweet RL, Schachter J, Landers DV. Chlamydia! infections in obstetrics and gynecology. Clin Obstet Gynecol 1983; 26:143. 4. Martin DH, Koutsky L, Eschenbach DA, eta!. Prematurity and perinatal mortality in pregnancies complicated by maternal Chlamydia trachomatis infections. JAMA 1982;247: 1585. 5. Johannisson G, Low hagen GB, Lycke E. Genital Chlamydia trachomatis infection in women. Obstet Gynecol 1980;56: 671. 6. Schachter J, Caldwell HD. Chlamydiae. Annu Rev Microbioi 1980;34:285. 7. Tam MR, Stamm WE, Handsfield HH, et a!. Cultureindependent diagnosis of Chlamydia trachomatis using monoclonal antibodies. N Eng!J Med 1984;310:1146. 8. Howard LV, Coleman PF, England BJ, Herrmann JE. Evaluation of Chlamydiazyme for the detection of genital infections caused by Chlamydia trachomatis. J Clin Micro bioi 1986;23:329.
Chlamydia! DNA in female urogenital infections
9. Liaw YF, Tai Dl, Chu CM, et a!. Early detection of hepatocellular carcinoma in patients with chronic type B hepatitis. Gastroenterology 1986;90:263. 10. Lieberman HM, LaBrecque DR, Kew MC, Hadziyannis SJ, Shafritz DA. Detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/Anti-HBe status in HBsAg carriers. Hepatology 1983;3:285. 11. Hyypia T, Larsen SH, Stahlberg T, Terho P. Analysis and detection of Chlamydia! DNA. J Gen Microbial 1984; 130:3159. 12. Nano FE, Caldwell HD. Expression of the chlamydia! genus-specific lipopolysaccharide epitope in Escherichia coli. Science 1985;228:742. 13. Clyde WA, Kenny GE, Schachter]. Laboratory diagnosis of chlamydia! and mycoplasmal infections. In: Drew WL, ed. Cumulative techniques and procedures in clinical microbiology, Cumitech 19. Washington DC: American Society for Microbiology, 1984. 14. Kingsbury DT, Weiss E. Lack of deoxyribonucleic acid homology between species of the genus Chlamydia. J Bacterial 1968;96: 1421. 15. Saikku P, Puolakkainen M, Leinonen M, Nurminen M, Nissinen A. Cross-reactivity between Chlamydiazyme and acinetobacter strains. N Eng! J Med 1986;314:922.
Divergent effects of two low-dose oral contraceptives on sex hormone-binding globulin and free testosterone Claudia Jung-Hoffmann, M.D., and Herbert Kuhl, Ph.D. Frankfurt am Main, Federal Republic of Germany The effect of a triphasic combination of ethinyl estradiol and levonorgestrel on the serum concentrations of total testosterone, free testosterone, and sex hormone-binding globulin as measured directly by radioimmunoassay and on the binding capacity of sex hormone-binding globulin was compared with that of a preparation containing ethinyl estradiol and desogestrel. Blood samples were taken on days 6, 11, 21, and 28 of a control cycle, the third cycle of treatment with either ethinyl estradiol-levonorgestrel or ethinyl estradiol-desogestrel (11 volunteers each), the third cycle of a 3-month washout period, and the third treatment cycle after crossover change of the preparations. There was a significant reduction in total testosterone by 16% during treatment with both preparations. Ethinyl estradiol-desogestrel increased the concentration ( + 175%) and bindig capacity ( + 330%) of sex hormone-binding globulin to a much greater extent than with ethinyl estradiol-levonorgestrel ( + 92% and + 160%). Contrary to this, a significant suppression of non-protein-bound testosterone by 35% was found during treatment with both oral contraceptives. The results demonstrate that an excessive elevation of the levels of sex hormone-binding globulin above the normal range does not cause a corresponding suppression of free testosterone. It is assumed that the decrease in the apparent binding affinity of high sex hormone-binding globulin concentrations to testosterone may be due to protein-protein interactions. (AM J OssTET GvNECOL 1987;156:199-203.)
Key words: Low-dose oral contraceptives, sex hormone-binding globulin, testosterone, free testosterone
From the Division of Gynecological Endocrinology, Department of Obstetrics and Gynecology,]. W. Goethe University. Received for publication March 3, 1986; revised July 1, 1986; accepted August 14, 1986.
Reprint requests: Dr. Herbert Kuhl, Division of Gynecological Endocrinology, Department of Obstetrics and Gynecology,]. W. Goethe University, Theodor-Stern-Kai 7, D-6000 Frankfurt am Main 70, Federal Republic of Germany.
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