Molecular and Cellular Probes
(1992) 6, 381-388
Chlamydia trachomatis confirmatory testing of PCR-positive genitourinary specimens using a second set of plasmid primers James B . Mahony, 1,2 * Kathleen E . Luinstra,' Dan Jang,' John Sellors 4 and Max A . Chernesky'-
2,3
'McMaster University Regional Virology and Chlamydiology Laboratory, St . Joseph's Hospital, Departments of 2Pathology, 3 Pediatrics and 4Family Medicine, McMaster University, Hamilton, Ontario, Canada L8N 4A6 (Received 4 November 1991, Accepted 9 January 1992)
A plasmid-based PCR for the detection of Chlamydia trachomatis was evaluated with a confirmatory PCR employing a second set of plasmid primers . A total of 258 genitourinary specimens including 134 female endocervical and urethral specimens and 124 male urethral specimens were tested by culture, blocked EIA and PCR . Fifty-four specimens were positive by culture, 50 were positive by EIA and 71 were positive by PCR . Fourteen specimens that were PCR-positive but culture- and EIA-negative were confirmed positive by the confirmatory PCR . Two of the 187 specimens which were negative by culture and EIA were positive by PCR but failed to confirm with the second set of primers . Using an expanded gold standard of culture, blocked EIA and confirmed PCR, the overall sensitivities for culture, blocked EIA and confirmed PCR were 76 . 0% (54/71), 70 . 4% (50/71) and 100% (71/71) and the specificities were 100% (187/187), 100% (187/187), respectively. These results demonstrated that a confirmatory PCR was useful for sorting out discordant specimens and establishing the true specificity of PCR . Furthermore, these results demonstrate that PCR is more sensitive than culture and EIA and suggest that a confirmed PCR test should be included in the gold standard for the evaluation of new tests for diagnosing Chlamydia trachomatis infections .
KEYWORDS : Chlamydia trachomatis, polymerase chain reaction, confirmatory testing .
INTRODUCTION culture when culture is set as the gold standard .' -' Recent studies have shown that when the standard is expanded to include a combination of culture, confirmed EIA or EIA and IF then the sensitivity of culture and EIA are 80-90% .2-5 The development of nucleic acid probe-based tests for detecting C . trachomatis in
Genital tract infections due to Chlamydia trachomatis have been diagnosed by sampling the male urethra, female cervix and urethra, and more recently first-void urine (FVU) specimens from both men and women . Although the development of non-culture techniques has played a significant role in our attempts to control the epidemic spread of Chlamydia trachomatis infections, evaluations of immunofluorescence (IF) and enzyme immunoassay (EIA) have revealed that these tests are less sensitive than
clinical specimens has been disappointing since DNA probes have proven to be less sensitive than culture8,9 The first reports on the use of PCR for the detection
* Author to whom correspondence should be addressed . 0890-8508/92/050381 + 08 $08 .00/0
381
© 1992 Academic Press Limited
382
J . B . Mahony et al.
of C. trachomatis in clinical specimens appeared in 1989 . 10-11 These studies used either plasmid or major outer membrane protein (MOMP)-based amplification . More recently, additional PCR assays employing plasmid, MOMP or rRNA targets have been described ."" Most of these reports indicated that PCR had excellent sensitivity and specificity, however, none of these studies confirmed PCR-positive specimens . We report here an evaluation of a plasmidbased PCR which included a confirmatory PCR and show that PCR was more sensitive than culture and EIA .
METHODS AND MATERIALS Specimens Specimens were collected from two groups of patients. Group I was composed of 134 women attending a birth control or planned parenthood clinic for either a check-up, birth control information or diagnosis and treatment of an STD . Group II consisted of 124 men attending a hospital-based STD clinic who were either symptomatic or asymptomatic and were attending for a check-up or because they were a contact of a partner with an STD . The prevalence of C . trachomatis infection in Group I and 11 was 7% and 15%, respectively 6,' Specimens were collected, from individuals giving informed consent, under a study protocol approved by a hospital ethics review committee . Endocervical and urethral swabs were collected as described previously .' Specimens for isolation of C . trachomatis were collected in chlamydia transport medium using cotton-tipped swabs and were transported to the laboratory at 4 ° C and inoculated within 24 h . Specimens for chlamydial antigen detection by EIA were collected using manufacturers' collection kits.
Laboratory propagated organisms
Chlamydia trachomatis serovars A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, L3 were grown in embryonated eggs and supplied as yolk sac preparations by Dr Julius Schachter (University of California, San Francisco) . Laboratory isolates of Lactobacillus spp ., Escherichia
coli, Staphylococcus aureus, Gardnerella vaginalis, Neisseria gonorrhoea, Acinetobacter spp ., Klebsiella pneumoniae, were grown on semi-solid agar using conventional media . Herpes simplex type I, KOS strain, was propagated in human diploid fibroblast cell culture .
Isolation of C . trachomatis
Chlamydia trachomatis was isolated in McCoy cell cultures using 96-well microculture plates, iodine staining, and a blind passage as described .'
EIA Specimens for antigen detection were collected with the manufacturer's collection kit and tested by either Chlamydiazyme or Chlamydia Testpack (Abbott Diagnostics, N . Chicago), according to the manufacturer's instructions . Chlamydiazyme-positive specimens were confirmed using the manufacturer's blocking reagent . Some discordant specimens were tested by IDEIA (NovoBiolabs, England) or MagicLite (Ciba Corning, Medfield, MA) .
PCR A 241 by sequence of the cryptic plasmid extending from 727-967 base pairs (bp) downstream from the unique Bam HI restriction site was amplified by PCR using the KL1/KL2 primers as described previously ." Amplification with Taq polymerase (Cetus Ampli Taq) and a thermal cycler (Cetus Perkin-Elmer, Norwalk, CT) was performed using 35 cycles of denaturation (1 min at 94 ° C), annealing (1 min at 55 ° C), and extension (2 min at 72 ° C) . Culture specimens (0 .2 ml) were centrifuged at 12,000 g for 20 min in a microfuge and the pellets were lysed for 15 min at 55 ° C and 1 h at 37 ° C with 200 tg ml -1 proteinase K and 0 . 7% SDS in a Tris-EDTA buffer pH 8 . 0 . DNA was purified by phenol/chloroform extraction and ethanol precipitation . One hundred nanograms of DNA was used for PCR . Discordant specimens were tested by a confirmatory PCR using a second set of plasmid primers (T1/T2) which amplified a 517 by fragment as described by Claas et al."
DNA hybridization The specificity of amplification was verified by Southern blotting analysis ." PCR reaction products (10 µl) were electrophoresed in 2% agarose gels, transblotted to nylon membranes (zetaprobe, BioRad) then probed with a "-P-labelled oligonucleotide probe KL3 17 or cloned C . trachomatis plasmid DNA .
383
C. trachomatis confirmatory PCR PCR control procedures Several precautions were taken to minimize contamination of specimens by pre-amplified product . These included the use of: 1 . separate biosafety containment hoods for preparation of specimens, setting up PCR reactions and analysing products ; 2 . plugged pipette tips or positive displacement pipettors ; 3 . several negative controls interspersed with clinical specimens; 4. periodic swabbing of work areas to detect amplified DNA ; and 5 . the limited use of confirmatory primers in resolving discordants .
RESULTS Using plasmid primer pair KL1/KL2 PCR amplified DNA from all 15 serovars of Chlamydia trachomatis (Fig. 1) . PCR amplification followed by DNA hybridization with a 32 P-labelled oligonucleotide probe detected C . trachomatis at a sensitivity of 1 fg equivalent to one elementary body assuming 10 plasmid copies per bacterial cell ."' The specificity of PCR was verified by Hin dIll restriction endonuclease digestion of the 241 by product which yielded predicted fragments of 167 by and 74 by (Fig . 2) . DNA from other micro-organisms found in the genital tract including E . coli, Lactobacillus spp ., Acinetobacter spp ., Staphylococcus aureus, G . vaginalis, N. gonorrhoea, K. pneu-
moniae, Herpes simplex virus, or human leukocytes or McCoy cells were not amplified with this primer pair (Fig . 3) . A total of 258 specimens including 134 from Group I women and 124 from Group II men were tested by culture, EIA and PCR. Fifty-four specimens were positive by culture including two on blind passage and 50 were positive by blocked EIA . A total of 187 specimens were negative by culture and EIA; two of these (Fig. 4a, lanes 3 and 9) were PCR-positive but failed to confirm using the confirmatory PCR . An additional 14 culture-negative, EIA-negative specimens (8 endocervical swabs and 6 male urethral swabs) were PCR positive; all 14 of these were confirmed PCR-positive with a second primer pair (Fig . 4). Using an expanded gold standard of culture, blocked EIA, and confirmed PCR, the overall sensitivities of culture, EIA and PCR were 76.0% (54/71), 70. 4% (50/71) and 100% (71/71) and specificities were 100% (187/187), 100% (187/ 187) and 100% (187/187), respectively (Table 1) . The performance of culture, EIA and PCR were similar for male and female specimens . When the results were analysed according to specimen type, PCR was consistently more sensitive than culture and EIA for male urethral and female endocervical and urethral specimens (Table 2) . The performance of PCR was similar for specimens from symptomatic and asymptomatic individuals .
(b)
(a) A B Ba C D E F G H I
241 by
241 by
241 by
J K LI L2 L3
Fig. 1 . PCR amplification of C . trachomatis serovars . DNA, 100 ng, from C . trachomatis serovars A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, and L3 were amplified using KL1/KL2 primers . The product, 10 ml, was electrophoresed on a 2% agarose gel (panel a), transblotted to zetaprobe and probed with "P-labelled KL3 oligonucleotide probe (panel b) . The outside lanes (in panel a) contain 1 kb DNA size markers .
3 84
J. B . Mahony et al.
2
4
5
6
7
8
241 by 167 by
74 by
Fig. 2 . Hin dill restriction of C . trachomatis DNA amplified by PCR . An endocervical specimen, a urethral specimen and C . trachomatis serovar L2 were amplified using KL1/KL2 primers . PCR products, 20 ml, were digested with Hin dill and electrophoresed on a 5% polyacrylamide gel together with undigested product . Lane 1 contains 1 kb DNA size markers ; lane 2, C, trachomatis L2 undigested ; lane 3, C . trachomatis digested ; lane 4, endocervical specimen undigested; lane 5, endocervical specimen digested ; lane 6, urethral specimen undigested ; lane 7, urethral specimen digested ; lane 8, DNA size markers, pBR322 Hae III digest .
( b )
3
4
5
6
7
8
9
10
11
Fig . 3 . Specificity of C. trachomatis PCR amplification . Bacterial, viral and human DNA was amplified with C . trachomatis KL1/KL2 primers . Lane 1 contains 1 kb DNA size markers ; lane 2, 10 fg C . trachomatis DNA serovar L2 ; lane 3, 100 ng DNA from E . coli; lane 4, G . vaginalis; lane 5, N . gonorrhorea; lane 6, Lactobacillus spp . lane 7, Staphylococcus spp ; lane 8, Acinetobacter spp . ; lane 9, Herpes simplex virus type I (KOS strain) ; lane 10, McCoy cells; lane 11, human leukocytes were amplified by PCR and the products analyzed by agarose gel electrophoresis (panel a) and hybridization with "P-labelled KL3 probe (panel b) .
DISCUSSION
standard provides an improved evaluation of tests since some specimens contain non-viable organisms
This is the first report of a C . trachomatis PCR assay
or free LPS with no elementary bodies .^- 5 Our plas-
which uses a second PCR to confirm positive genito-
mid-based PCR amplified a 241 by fragment of the
urinary tract specimens . In a comparison of PCR,
genetically conserved cryptic plasmid in all 15 sero-
culture and EIA using 258 specimens, confirmed PCR
vars of C . trachomatis ." The confirmatory PCR used a
had an overall sensitivity of 100% (71/71) for endo-
second set of plasmid primers to amplify a 517 by
cervical and urethral specimens compared with
target remotely located from the first amplicon . Bobo
76.0% for culture and 70 .4% for EIA . Other studies
et a1 . 20
using an expanded gold standard (culture and
PCR for confirming positive ocular specimens in the
blocked EIA) have shown that culture and EIA have
diagnosis of trachoma in Tanzania . Their first PCR
sensitivities of 80-90% . 2-5 Use of an expanded gold
used primers targeted at conserved regions of the
recently reported the use of a confirmatory
C . trachomatis confirmatory PCR
385
517 by
Fig. 4 . Confirmatory PCR testing of discordant specimens. Sixteen PCR-positive, culture- and EIA-negative specimens were tested by confirmatory PCR using a second primary pair T1/T2 and analysed by Southern blotting . The 517 by PCR product was detected using 32P-labelled C . trachomatis serovar L2 plasmid DNA . Panel a, lane 1, positive control ; lane 2, negative control ; lanes 3-12, female specimens . Panel b, lane 1, positive control ; lane 2, negative control ; lanes 3-8, male specimens.
Table 1 . Performance of culture, blocked EIA and confirmed PCR for the detection of Chlamydia trachomatis in 258 male and female genital tract specimens Test/sex
Sensitivity (%)
Specificity (%)
Positive PV (%)
Culture Male Female Total
79 .4 (27/34) 73 .0 (27/37) 76 .0 (54/71)
100(90/90) 100(97/97) 100 (187/187)
100(27/27) 100(27/27) 100(54/54)
92 .8 (90/97) 90 .7 (97/107) 91 .7 (187/204)
EIA Male Female Total
70 .6 (24/34) 70 . 3 (26/37) 70 . 4 (50/71)
100(90/90) 100(97/97) 100 (187/187)
100(24/24) 100(26/26) 100(50/50)
90 .0 (90/100) 89 .8 (97/108) 89 .9 (187/208)
100(90/90) 100(97/97) 100 (187/187)
100(34/34) 100(37/37) 100(71/71)
PCR Male Female Total
100(34/34) 100(37/37) 100(71/71)
Negative PV (%)
100(90/90) 100(97/97) 100 (187/187)
386
J . B. Mahony et al . Table 2. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of culture, blocked EIA and confirmed PCR for male urethral and female endocervical and urethral specimens Specimen
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
Male Urethra Culture EIA PCR
79 . 4 (27/34) 70 . 6 (24/34) 100(34/34)
100(90/90) 100(90/90) 100(90/90)
100(27/27) 100(24/24) 100(34/34)
92 . 8 (90/97) 90 . 0 (90/100) 100(90/90)
Female Cervix Culture EIA PCR
78 . 3 (18/23) 78 . 3 (18/23) 100(23/23)
100(49/49) 100(49/49) 100(49/49)
100(18/18) 100(18/18) 100(23/23)
90. 7 (49/54) 90 . 7 (49/54) 100(49/49)
Urethra Culture EIA PCR
64 .3(9/14) 57 . 1 (8/14) 100(14/14)
100(48/48) 100(48/48) 100(48/48)
100(9/9) 100(8/8) 100(14/14)
90. 6 (48/53) 88 . 9 (48/54) 100(48/48)
MOMP gene while the second PCR used primers
plasmid and showed that it had a sensitivity of 100%
spanning the hypervariable region .
(26/26). EIA was not done on all specimens and the
The specificity of PCR was improved by adding the
sensitivity of culture could not be calculated . In our
confirmatory PCR . The first PCR detected two positive
study, there were 71 positive specimens and all were
specimens which were negative in the confirmatory
detected by PCR . To date we have tested over 150
PCR . These two false-positive specimens from asymp-
culture or EIA positive endocervical or urethral speci-
tomatic individuals may have been the result of
mens and PCR has been positive in each case, despite
laboratory contamination or alternatively DNA muta-
one report of C. trachomatis lacking the cryptic
tion in the primer regions and emphasize the need for
plasmid . 21 In another study involving 131 specimens
a confirmatory PCR . When an expanded gold stan-
Quinn
dard of culture, blocked EIA and confirmed PCR was
100% and 98 . 5%, respectively, for PCR compared to
et al .
reported a sensitivity and specificity of
used the specificity of PCR increased from 98 . 9 to
culture when a MOMP gene fragment was ampli-
100% . In a separate evaluation using over 500 first
fied ." Their study included endocervical specimens,
void urine specimens obtained from asymptomatic men with a low prevalence (< 2%) of C . trachomatis
of which 64 were culture-positive . PCR products were authenticated by hybridization using a biotinylated
infection the specificity of PCR has remained at 100%
RNA probe and detection of RNA/DNA hybrids by
(Mahony et
al.
manuscript in preparation) . In the
EIA ." This group reported in a separate retrospective
absence of a confirmatory PCR discordant specimens
analysis a PCR sensitivity of 100% (46/46) using
(PCR-positive and culture- or EIA-negative) should be
culture-positive specimens ." Collectively these stud-
verified by testing either follow-up specimens or
ies indicate that PCR is more sensitive than both
implementing other tests including EIAs that measure different chlamydial antigens' or IF that detects ele-
that culture is only 80-90% sensitive ." One contrib-
mentary bodies. Despite these efforts, the increased
uting factor to the low sensitivity of culture is the
culture and EIA and confirm other reports showing
sensitivity of PCR will continue to identify true posit-
lability of C. trachomatis and the need to refrigerate
ive specimens that are negative by other tests.
specimens ." Furthermore, these studies indicate that the low sensitivity (76 .0%) of culture would have resulted in some C . trachomatis infections going
Detection of C. trachomatis by PCR has been reported by others to be more sensitive than culture . Ostergaarde et al. compared PCR to culture in a study of 223 specimens and performed EIA on the discordants (PCR-positive, culture-negative) .' $ They used PCR to amplify a 473 by fragment of the cryptic
undiagnosed . The increasing incidence of upper genital tract infection in women leading to pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility may be due in part to false-negative culture
C. trachomatis confirmatory PCR
results . The use of PCR with improved sensitivity may help detect early infections in women and decrease the late sequelae of C . trachomatis infection . In this study we have taken every possible precaution to avoid false-positive PCR results ." We have used separate specimen processing, PCR set-up and product analysis areas in the laboratory to eliminate carry-over and prevent false-positive results . In addition we have confirmed all positives with a second PCR using a different primer pair . The increased sensitivity of PCR compared to culture and EIA together with the improved specificity make PCR an important test for reference laboratories to consider when faced with difficult diagnostic challenges, such as discordant culture or EIA results . Our results suggest that PCR should be included in the gold standard for the evaluation of new tests for diagnosing C . trachomatis infections . In the future, multiplex PCR involving co-amplification of multiple DNA targets such as chromosomal and plasmid DNA may replace the need for a separate confirmatory PCR .
ACKNOWLEDGEMENTS We gratefully acknowledge Julius Schachter for providing C. trachomatis serovars, Anna Pietruszkiewicz for growing C. trachomatis and Lisa Rizzo and Ann Costie for typing the manuscript . Presented in part at the Eighth I .S .S.T .D .R . Meeting, Copenhagen, 1989 and the Seventh International Symposium on Human Chlamydial Infections, Harrison Hot Springs, 1990 .
REFERENCES 1 . Chernesky, M . A., Mahony, J . B ., Castriciano, S. et al . (1986) . Detection of Chlamydia trachomatis antigens by enzyme immunoassay and immunofluorescence in genital specimens from symptomatic and asymptomatic men and women . Journal of Infectious Diseases 154, 141-8 . 2 . Chernesky, M ., Castriciano, S ., Sellors, J . et al. (1990) . Detection of Chlamydia trachomatis antigens in urine as an alternative to swabs and cultures. Journal of Infectious Diseases 161, 124-6. 3 . Mahony, J. B ., Castriciano, S ., Sellors, J . et al . (1989) . Diagnosis of Chlamydia trachomatis genital infection by cell culture and two enzyme immunoassays detecting different chlamydial antigens . Journal of Clinical Microbiology 27, 1934-8, 4. Moncada, J ., Schachter, J ., Bolan, G . et al. (1990) . Confirmatory assay increases specificity of the Chlamydiazyme test for Chlamydia trachomatis infection of the cervix . Journal of Clinical Microbiology 28, 1770-3. 5 . Schachter, J. (1986) . Rapid diagnosis of sexually transmitted diseases : speed has a price . Diagnostic Microbiology and Infectious Disease 4, 185-9 .
387
6 . Sellors, J ., Mahony, J ., Jang, D . et al . (1991) . Rapid, onsite diagnosis of chlamydial urethritis in men by detection of antigens in urethral swabs and urine . Journal of Clinical Microbiology 29, 407-9. 7 . Sellors, J . W., Mahony, J . B ., Jang, D . et al. (1991) . Comparison of cervical, urethral and urine specimens for the detection of Chlamydia trachomatis in women . Journal of Infectious Diseases 164, 205-8 . 8. LeBar, W., Herschman, B ., jemal, C . & Pierzchala, J . (1989) . Comparison of DNA probe, monoclonal antibody enzyme immunoassay and cell culture for the detection of Chlamydia trachomatis . journal of Clinical Microbiology 27, 826-8 . 9 . Mercer, L . J ., Robinson, D . C., Sahm, D . F ., Lawrie, M . J . & Hajj, S . N . (1990) . Comparison of Chemiluminescent DNA probe to cell culture for the screening of Chlamydia trachomatis in a gynecology clinic population . Obstetrics and Gynecology 72, 803-9 . 10 . Mahony, J . B ., Luinstra, K . E . & Chernesky, M . A . (1989) . Detection of Chlamydia trachomatis DNA in culture positive and culture negative specimens by polymerase chain reaction (PCR) . Eighth I .S .S .T.D .R . Meeting, Copenhagen, Denmark, Abstract *20. 11 . Ostergaarde, L., Bickelund, S . & Christiansen, G. (1989) . The polymerase chain reaction (PCR) used in the diagnosis of genital Chlamydia trachomatis infections . Eighth I .S .S .T.D.R . Meeting, Copenhagen, Denmark . Abstract #k 130. 12 . Dutilh, B ., Bebear, C ., Rodriguez, P ., Vekris, A ., Bonnet, J & Garret, M . (1989). Detection of Chlamydia trachomatis using the polymerase chain reaction . Eighth I .S .S .T.D .R . Meeting, Copenhagen, Denmark . Abstract # 129 . 13 . Bobo, L ., Coultee, F ., Yolken, R ., Quinn, T . & Viscid, R. P . (1990). Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay . Journal of Clinical Microbiology 28,1968-73 . 14 . Claas, H . C., Melchers, W . J ., de Bruijn, I . H . et al. (1990) . Detection of Chlamydia trachomatis in Clinical Specimens by the polymerase chain reaction . European Journal of Clinical Microbiology and Infectious Diseases 9, 864-8 . 15 . Ostergaarde, L ., Birkelund, S . & Christiansen, G . (1990). Use of polymerase chain reaction for detection of Chlamydia trachomatis . Journal of Clinical Microbiology 28, 1254-60 . 16 . Quinn, T . C ., Bobo, L., Holland, S . M ., Gaydos, C . A., Hook, E . & Viscidi, R . P . (1990). Diagnosis of Chlamydia trachomatis cervical infections by Polymerase Chain Reaction . In Chlamydial Infections . (Bowie, W . R ., Caldwell, H . D ., Jones, R . P ., Mardh, P . A ., Ridgway, G . L., Schachter, J . & Stamm, W . E., eds) pp . 491-4. Cambridge, UK: Cambridge University Press . 17 . Mahony, J . B ., Luinstra, K . E ., Sellors, J. W. & Chernesky, M. A. (1990) . Comparison of Polymerase Chain Reaction (PCR), Enzyme Immunoassay and Culture for the Diagnosis of C . trachomatis Infections in Symptomatic and Asymptomatic Males and Females . In Chlamydial Infections . (Bowie, W . R., Caldwell, H . D ., Jones, R . P., Mardh, P. A ., Ridgeway, G . L., Shachter, J., Stamm, W . E. & Ward, M . E ., eds) pp. 487-90. Cambridge, UK Cambridge University Press. 18. Sambrook, J ., Fritsch, E . F . & Maniatis . (1989) . Molecular Cloning : A Laboratory Manual . New York: Cold Spring Harbor Laboratory .
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J . 8. Mahony et al .
19 . Palmer, L . & Falkow, 5 . (1986) . A common plasmid of Chlamydia trachomatis . Plasmid 16, 52-62 . 20 . Bobo, L ., Munoz, B ., Viscidi, R ., Quinn, T ., Mkocha, H . & West, S . (1991) . Diagnosis of Chlamydia trachomatis eye infection in Tanzania by polymerase chain reaction/enzyme immunoassay . Lancet 338, 847-50 . 21 . Peterson, E . M., Markoff, B . A ., Schachter, J . & de la Maza, L. M . (1990) . The 7 .5 Kb plasmid present in
Chlamydia trachomatis is not essential for the growth of this microorganism . Plasmid 23, 144-8 . 22 . Mahony, J . B . & Chernesky, M . A . (1985) . Effect of swab type and storage temperature on the isolation of Chlamydia trachomatis from clinical specimens . Journal of Clinical Microbiology 22, 856-7 . 23. Kwok, S . & Higuchi, R . (1989) . Avoiding false positives with PCR . Nature 339, 237-8 .
(1992) 6, 381-388
Chlamydia trachomatis confirmatory testing of PCR-positive genitourinary specimens using a second set of plasmid primers James B . Mahony, 1,2 * Kathleen E . Luinstra,' Dan Jang,' John Sellors 4 and Max A . Chernesky'-
2,3
'McMaster University Regional Virology and Chlamydiology Laboratory, St . Joseph's Hospital, Departments of 2Pathology, 3 Pediatrics and 4Family Medicine, McMaster University, Hamilton, Ontario, Canada L8N 4A6 (Received 4 November 1991, Accepted 9 January 1992)
A plasmid-based PCR for the detection of Chlamydia trachomatis was evaluated with a confirmatory PCR employing a second set of plasmid primers . A total of 258 genitourinary specimens including 134 female endocervical and urethral specimens and 124 male urethral specimens were tested by culture, blocked EIA and PCR . Fifty-four specimens were positive by culture, 50 were positive by EIA and 71 were positive by PCR . Fourteen specimens that were PCR-positive but culture- and EIA-negative were confirmed positive by the confirmatory PCR . Two of the 187 specimens which were negative by culture and EIA were positive by PCR but failed to confirm with the second set of primers . Using an expanded gold standard of culture, blocked EIA and confirmed PCR, the overall sensitivities for culture, blocked EIA and confirmed PCR were 76 . 0% (54/71), 70 . 4% (50/71) and 100% (71/71) and the specificities were 100% (187/187), 100% (187/187), respectively. These results demonstrated that a confirmatory PCR was useful for sorting out discordant specimens and establishing the true specificity of PCR . Furthermore, these results demonstrate that PCR is more sensitive than culture and EIA and suggest that a confirmed PCR test should be included in the gold standard for the evaluation of new tests for diagnosing Chlamydia trachomatis infections .
KEYWORDS : Chlamydia trachomatis, polymerase chain reaction, confirmatory testing .
INTRODUCTION culture when culture is set as the gold standard .' -' Recent studies have shown that when the standard is expanded to include a combination of culture, confirmed EIA or EIA and IF then the sensitivity of culture and EIA are 80-90% .2-5 The development of nucleic acid probe-based tests for detecting C . trachomatis in
Genital tract infections due to Chlamydia trachomatis have been diagnosed by sampling the male urethra, female cervix and urethra, and more recently first-void urine (FVU) specimens from both men and women . Although the development of non-culture techniques has played a significant role in our attempts to control the epidemic spread of Chlamydia trachomatis infections, evaluations of immunofluorescence (IF) and enzyme immunoassay (EIA) have revealed that these tests are less sensitive than
clinical specimens has been disappointing since DNA probes have proven to be less sensitive than culture8,9 The first reports on the use of PCR for the detection
* Author to whom correspondence should be addressed . 0890-8508/92/050381 + 08 $08 .00/0
381
© 1992 Academic Press Limited
382
J . B . Mahony et al.
of C. trachomatis in clinical specimens appeared in 1989 . 10-11 These studies used either plasmid or major outer membrane protein (MOMP)-based amplification . More recently, additional PCR assays employing plasmid, MOMP or rRNA targets have been described ."" Most of these reports indicated that PCR had excellent sensitivity and specificity, however, none of these studies confirmed PCR-positive specimens . We report here an evaluation of a plasmidbased PCR which included a confirmatory PCR and show that PCR was more sensitive than culture and EIA .
METHODS AND MATERIALS Specimens Specimens were collected from two groups of patients. Group I was composed of 134 women attending a birth control or planned parenthood clinic for either a check-up, birth control information or diagnosis and treatment of an STD . Group II consisted of 124 men attending a hospital-based STD clinic who were either symptomatic or asymptomatic and were attending for a check-up or because they were a contact of a partner with an STD . The prevalence of C . trachomatis infection in Group I and 11 was 7% and 15%, respectively 6,' Specimens were collected, from individuals giving informed consent, under a study protocol approved by a hospital ethics review committee . Endocervical and urethral swabs were collected as described previously .' Specimens for isolation of C . trachomatis were collected in chlamydia transport medium using cotton-tipped swabs and were transported to the laboratory at 4 ° C and inoculated within 24 h . Specimens for chlamydial antigen detection by EIA were collected using manufacturers' collection kits.
Laboratory propagated organisms
Chlamydia trachomatis serovars A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, L3 were grown in embryonated eggs and supplied as yolk sac preparations by Dr Julius Schachter (University of California, San Francisco) . Laboratory isolates of Lactobacillus spp ., Escherichia
coli, Staphylococcus aureus, Gardnerella vaginalis, Neisseria gonorrhoea, Acinetobacter spp ., Klebsiella pneumoniae, were grown on semi-solid agar using conventional media . Herpes simplex type I, KOS strain, was propagated in human diploid fibroblast cell culture .
Isolation of C . trachomatis
Chlamydia trachomatis was isolated in McCoy cell cultures using 96-well microculture plates, iodine staining, and a blind passage as described .'
EIA Specimens for antigen detection were collected with the manufacturer's collection kit and tested by either Chlamydiazyme or Chlamydia Testpack (Abbott Diagnostics, N . Chicago), according to the manufacturer's instructions . Chlamydiazyme-positive specimens were confirmed using the manufacturer's blocking reagent . Some discordant specimens were tested by IDEIA (NovoBiolabs, England) or MagicLite (Ciba Corning, Medfield, MA) .
PCR A 241 by sequence of the cryptic plasmid extending from 727-967 base pairs (bp) downstream from the unique Bam HI restriction site was amplified by PCR using the KL1/KL2 primers as described previously ." Amplification with Taq polymerase (Cetus Ampli Taq) and a thermal cycler (Cetus Perkin-Elmer, Norwalk, CT) was performed using 35 cycles of denaturation (1 min at 94 ° C), annealing (1 min at 55 ° C), and extension (2 min at 72 ° C) . Culture specimens (0 .2 ml) were centrifuged at 12,000 g for 20 min in a microfuge and the pellets were lysed for 15 min at 55 ° C and 1 h at 37 ° C with 200 tg ml -1 proteinase K and 0 . 7% SDS in a Tris-EDTA buffer pH 8 . 0 . DNA was purified by phenol/chloroform extraction and ethanol precipitation . One hundred nanograms of DNA was used for PCR . Discordant specimens were tested by a confirmatory PCR using a second set of plasmid primers (T1/T2) which amplified a 517 by fragment as described by Claas et al."
DNA hybridization The specificity of amplification was verified by Southern blotting analysis ." PCR reaction products (10 µl) were electrophoresed in 2% agarose gels, transblotted to nylon membranes (zetaprobe, BioRad) then probed with a "-P-labelled oligonucleotide probe KL3 17 or cloned C . trachomatis plasmid DNA .
383
C. trachomatis confirmatory PCR PCR control procedures Several precautions were taken to minimize contamination of specimens by pre-amplified product . These included the use of: 1 . separate biosafety containment hoods for preparation of specimens, setting up PCR reactions and analysing products ; 2 . plugged pipette tips or positive displacement pipettors ; 3 . several negative controls interspersed with clinical specimens; 4. periodic swabbing of work areas to detect amplified DNA ; and 5 . the limited use of confirmatory primers in resolving discordants .
RESULTS Using plasmid primer pair KL1/KL2 PCR amplified DNA from all 15 serovars of Chlamydia trachomatis (Fig. 1) . PCR amplification followed by DNA hybridization with a 32 P-labelled oligonucleotide probe detected C . trachomatis at a sensitivity of 1 fg equivalent to one elementary body assuming 10 plasmid copies per bacterial cell ."' The specificity of PCR was verified by Hin dIll restriction endonuclease digestion of the 241 by product which yielded predicted fragments of 167 by and 74 by (Fig . 2) . DNA from other micro-organisms found in the genital tract including E . coli, Lactobacillus spp ., Acinetobacter spp ., Staphylococcus aureus, G . vaginalis, N. gonorrhoea, K. pneu-
moniae, Herpes simplex virus, or human leukocytes or McCoy cells were not amplified with this primer pair (Fig . 3) . A total of 258 specimens including 134 from Group I women and 124 from Group II men were tested by culture, EIA and PCR. Fifty-four specimens were positive by culture including two on blind passage and 50 were positive by blocked EIA . A total of 187 specimens were negative by culture and EIA; two of these (Fig. 4a, lanes 3 and 9) were PCR-positive but failed to confirm using the confirmatory PCR . An additional 14 culture-negative, EIA-negative specimens (8 endocervical swabs and 6 male urethral swabs) were PCR positive; all 14 of these were confirmed PCR-positive with a second primer pair (Fig . 4). Using an expanded gold standard of culture, blocked EIA, and confirmed PCR, the overall sensitivities of culture, EIA and PCR were 76.0% (54/71), 70. 4% (50/71) and 100% (71/71) and specificities were 100% (187/187), 100% (187/ 187) and 100% (187/187), respectively (Table 1) . The performance of culture, EIA and PCR were similar for male and female specimens . When the results were analysed according to specimen type, PCR was consistently more sensitive than culture and EIA for male urethral and female endocervical and urethral specimens (Table 2) . The performance of PCR was similar for specimens from symptomatic and asymptomatic individuals .
(b)
(a) A B Ba C D E F G H I
241 by
241 by
241 by
J K LI L2 L3
Fig. 1 . PCR amplification of C . trachomatis serovars . DNA, 100 ng, from C . trachomatis serovars A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, and L3 were amplified using KL1/KL2 primers . The product, 10 ml, was electrophoresed on a 2% agarose gel (panel a), transblotted to zetaprobe and probed with "P-labelled KL3 oligonucleotide probe (panel b) . The outside lanes (in panel a) contain 1 kb DNA size markers .
3 84
J. B . Mahony et al.
2
4
5
6
7
8
241 by 167 by
74 by
Fig. 2 . Hin dill restriction of C . trachomatis DNA amplified by PCR . An endocervical specimen, a urethral specimen and C . trachomatis serovar L2 were amplified using KL1/KL2 primers . PCR products, 20 ml, were digested with Hin dill and electrophoresed on a 5% polyacrylamide gel together with undigested product . Lane 1 contains 1 kb DNA size markers ; lane 2, C, trachomatis L2 undigested ; lane 3, C . trachomatis digested ; lane 4, endocervical specimen undigested; lane 5, endocervical specimen digested ; lane 6, urethral specimen undigested ; lane 7, urethral specimen digested ; lane 8, DNA size markers, pBR322 Hae III digest .
( b )
3
4
5
6
7
8
9
10
11
Fig . 3 . Specificity of C. trachomatis PCR amplification . Bacterial, viral and human DNA was amplified with C . trachomatis KL1/KL2 primers . Lane 1 contains 1 kb DNA size markers ; lane 2, 10 fg C . trachomatis DNA serovar L2 ; lane 3, 100 ng DNA from E . coli; lane 4, G . vaginalis; lane 5, N . gonorrhorea; lane 6, Lactobacillus spp . lane 7, Staphylococcus spp ; lane 8, Acinetobacter spp . ; lane 9, Herpes simplex virus type I (KOS strain) ; lane 10, McCoy cells; lane 11, human leukocytes were amplified by PCR and the products analyzed by agarose gel electrophoresis (panel a) and hybridization with "P-labelled KL3 probe (panel b) .
DISCUSSION
standard provides an improved evaluation of tests since some specimens contain non-viable organisms
This is the first report of a C . trachomatis PCR assay
or free LPS with no elementary bodies .^- 5 Our plas-
which uses a second PCR to confirm positive genito-
mid-based PCR amplified a 241 by fragment of the
urinary tract specimens . In a comparison of PCR,
genetically conserved cryptic plasmid in all 15 sero-
culture and EIA using 258 specimens, confirmed PCR
vars of C . trachomatis ." The confirmatory PCR used a
had an overall sensitivity of 100% (71/71) for endo-
second set of plasmid primers to amplify a 517 by
cervical and urethral specimens compared with
target remotely located from the first amplicon . Bobo
76.0% for culture and 70 .4% for EIA . Other studies
et a1 . 20
using an expanded gold standard (culture and
PCR for confirming positive ocular specimens in the
blocked EIA) have shown that culture and EIA have
diagnosis of trachoma in Tanzania . Their first PCR
sensitivities of 80-90% . 2-5 Use of an expanded gold
used primers targeted at conserved regions of the
recently reported the use of a confirmatory
C . trachomatis confirmatory PCR
385
517 by
Fig. 4 . Confirmatory PCR testing of discordant specimens. Sixteen PCR-positive, culture- and EIA-negative specimens were tested by confirmatory PCR using a second primary pair T1/T2 and analysed by Southern blotting . The 517 by PCR product was detected using 32P-labelled C . trachomatis serovar L2 plasmid DNA . Panel a, lane 1, positive control ; lane 2, negative control ; lanes 3-12, female specimens . Panel b, lane 1, positive control ; lane 2, negative control ; lanes 3-8, male specimens.
Table 1 . Performance of culture, blocked EIA and confirmed PCR for the detection of Chlamydia trachomatis in 258 male and female genital tract specimens Test/sex
Sensitivity (%)
Specificity (%)
Positive PV (%)
Culture Male Female Total
79 .4 (27/34) 73 .0 (27/37) 76 .0 (54/71)
100(90/90) 100(97/97) 100 (187/187)
100(27/27) 100(27/27) 100(54/54)
92 .8 (90/97) 90 .7 (97/107) 91 .7 (187/204)
EIA Male Female Total
70 .6 (24/34) 70 . 3 (26/37) 70 . 4 (50/71)
100(90/90) 100(97/97) 100 (187/187)
100(24/24) 100(26/26) 100(50/50)
90 .0 (90/100) 89 .8 (97/108) 89 .9 (187/208)
100(90/90) 100(97/97) 100 (187/187)
100(34/34) 100(37/37) 100(71/71)
PCR Male Female Total
100(34/34) 100(37/37) 100(71/71)
Negative PV (%)
100(90/90) 100(97/97) 100 (187/187)
386
J . B. Mahony et al . Table 2. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of culture, blocked EIA and confirmed PCR for male urethral and female endocervical and urethral specimens Specimen
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
Male Urethra Culture EIA PCR
79 . 4 (27/34) 70 . 6 (24/34) 100(34/34)
100(90/90) 100(90/90) 100(90/90)
100(27/27) 100(24/24) 100(34/34)
92 . 8 (90/97) 90 . 0 (90/100) 100(90/90)
Female Cervix Culture EIA PCR
78 . 3 (18/23) 78 . 3 (18/23) 100(23/23)
100(49/49) 100(49/49) 100(49/49)
100(18/18) 100(18/18) 100(23/23)
90. 7 (49/54) 90 . 7 (49/54) 100(49/49)
Urethra Culture EIA PCR
64 .3(9/14) 57 . 1 (8/14) 100(14/14)
100(48/48) 100(48/48) 100(48/48)
100(9/9) 100(8/8) 100(14/14)
90. 6 (48/53) 88 . 9 (48/54) 100(48/48)
MOMP gene while the second PCR used primers
plasmid and showed that it had a sensitivity of 100%
spanning the hypervariable region .
(26/26). EIA was not done on all specimens and the
The specificity of PCR was improved by adding the
sensitivity of culture could not be calculated . In our
confirmatory PCR . The first PCR detected two positive
study, there were 71 positive specimens and all were
specimens which were negative in the confirmatory
detected by PCR . To date we have tested over 150
PCR . These two false-positive specimens from asymp-
culture or EIA positive endocervical or urethral speci-
tomatic individuals may have been the result of
mens and PCR has been positive in each case, despite
laboratory contamination or alternatively DNA muta-
one report of C. trachomatis lacking the cryptic
tion in the primer regions and emphasize the need for
plasmid . 21 In another study involving 131 specimens
a confirmatory PCR . When an expanded gold stan-
Quinn
dard of culture, blocked EIA and confirmed PCR was
100% and 98 . 5%, respectively, for PCR compared to
et al .
reported a sensitivity and specificity of
used the specificity of PCR increased from 98 . 9 to
culture when a MOMP gene fragment was ampli-
100% . In a separate evaluation using over 500 first
fied ." Their study included endocervical specimens,
void urine specimens obtained from asymptomatic men with a low prevalence (< 2%) of C . trachomatis
of which 64 were culture-positive . PCR products were authenticated by hybridization using a biotinylated
infection the specificity of PCR has remained at 100%
RNA probe and detection of RNA/DNA hybrids by
(Mahony et
al.
manuscript in preparation) . In the
EIA ." This group reported in a separate retrospective
absence of a confirmatory PCR discordant specimens
analysis a PCR sensitivity of 100% (46/46) using
(PCR-positive and culture- or EIA-negative) should be
culture-positive specimens ." Collectively these stud-
verified by testing either follow-up specimens or
ies indicate that PCR is more sensitive than both
implementing other tests including EIAs that measure different chlamydial antigens' or IF that detects ele-
that culture is only 80-90% sensitive ." One contrib-
mentary bodies. Despite these efforts, the increased
uting factor to the low sensitivity of culture is the
culture and EIA and confirm other reports showing
sensitivity of PCR will continue to identify true posit-
lability of C. trachomatis and the need to refrigerate
ive specimens that are negative by other tests.
specimens ." Furthermore, these studies indicate that the low sensitivity (76 .0%) of culture would have resulted in some C . trachomatis infections going
Detection of C. trachomatis by PCR has been reported by others to be more sensitive than culture . Ostergaarde et al. compared PCR to culture in a study of 223 specimens and performed EIA on the discordants (PCR-positive, culture-negative) .' $ They used PCR to amplify a 473 by fragment of the cryptic
undiagnosed . The increasing incidence of upper genital tract infection in women leading to pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility may be due in part to false-negative culture
C. trachomatis confirmatory PCR
results . The use of PCR with improved sensitivity may help detect early infections in women and decrease the late sequelae of C . trachomatis infection . In this study we have taken every possible precaution to avoid false-positive PCR results ." We have used separate specimen processing, PCR set-up and product analysis areas in the laboratory to eliminate carry-over and prevent false-positive results . In addition we have confirmed all positives with a second PCR using a different primer pair . The increased sensitivity of PCR compared to culture and EIA together with the improved specificity make PCR an important test for reference laboratories to consider when faced with difficult diagnostic challenges, such as discordant culture or EIA results . Our results suggest that PCR should be included in the gold standard for the evaluation of new tests for diagnosing C . trachomatis infections . In the future, multiplex PCR involving co-amplification of multiple DNA targets such as chromosomal and plasmid DNA may replace the need for a separate confirmatory PCR .
ACKNOWLEDGEMENTS We gratefully acknowledge Julius Schachter for providing C. trachomatis serovars, Anna Pietruszkiewicz for growing C. trachomatis and Lisa Rizzo and Ann Costie for typing the manuscript . Presented in part at the Eighth I .S .S.T .D .R . Meeting, Copenhagen, 1989 and the Seventh International Symposium on Human Chlamydial Infections, Harrison Hot Springs, 1990 .
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6 . Sellors, J ., Mahony, J ., Jang, D . et al . (1991) . Rapid, onsite diagnosis of chlamydial urethritis in men by detection of antigens in urethral swabs and urine . Journal of Clinical Microbiology 29, 407-9. 7 . Sellors, J . W., Mahony, J . B ., Jang, D . et al. (1991) . Comparison of cervical, urethral and urine specimens for the detection of Chlamydia trachomatis in women . Journal of Infectious Diseases 164, 205-8 . 8. LeBar, W., Herschman, B ., jemal, C . & Pierzchala, J . (1989) . Comparison of DNA probe, monoclonal antibody enzyme immunoassay and cell culture for the detection of Chlamydia trachomatis . journal of Clinical Microbiology 27, 826-8 . 9 . Mercer, L . J ., Robinson, D . C., Sahm, D . F ., Lawrie, M . J . & Hajj, S . N . (1990) . Comparison of Chemiluminescent DNA probe to cell culture for the screening of Chlamydia trachomatis in a gynecology clinic population . Obstetrics and Gynecology 72, 803-9 . 10 . Mahony, J . B ., Luinstra, K . E . & Chernesky, M . A . (1989) . Detection of Chlamydia trachomatis DNA in culture positive and culture negative specimens by polymerase chain reaction (PCR) . Eighth I .S .S .T.D .R . Meeting, Copenhagen, Denmark, Abstract *20. 11 . Ostergaarde, L., Bickelund, S . & Christiansen, G. (1989) . The polymerase chain reaction (PCR) used in the diagnosis of genital Chlamydia trachomatis infections . Eighth I .S .S .T.D.R . Meeting, Copenhagen, Denmark . Abstract #k 130. 12 . Dutilh, B ., Bebear, C ., Rodriguez, P ., Vekris, A ., Bonnet, J & Garret, M . (1989). Detection of Chlamydia trachomatis using the polymerase chain reaction . Eighth I .S .S .T.D .R . Meeting, Copenhagen, Denmark . Abstract # 129 . 13 . Bobo, L ., Coultee, F ., Yolken, R ., Quinn, T . & Viscid, R. P . (1990). Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay . Journal of Clinical Microbiology 28,1968-73 . 14 . Claas, H . C., Melchers, W . J ., de Bruijn, I . H . et al. (1990) . Detection of Chlamydia trachomatis in Clinical Specimens by the polymerase chain reaction . European Journal of Clinical Microbiology and Infectious Diseases 9, 864-8 . 15 . Ostergaarde, L ., Birkelund, S . & Christiansen, G . (1990). Use of polymerase chain reaction for detection of Chlamydia trachomatis . Journal of Clinical Microbiology 28, 1254-60 . 16 . Quinn, T . C ., Bobo, L., Holland, S . M ., Gaydos, C . A., Hook, E . & Viscidi, R . P . (1990). Diagnosis of Chlamydia trachomatis cervical infections by Polymerase Chain Reaction . In Chlamydial Infections . (Bowie, W . R ., Caldwell, H . D ., Jones, R . P ., Mardh, P . A ., Ridgway, G . L., Schachter, J . & Stamm, W . E., eds) pp . 491-4. Cambridge, UK: Cambridge University Press . 17 . Mahony, J . B ., Luinstra, K . E ., Sellors, J. W. & Chernesky, M. A. (1990) . Comparison of Polymerase Chain Reaction (PCR), Enzyme Immunoassay and Culture for the Diagnosis of C . trachomatis Infections in Symptomatic and Asymptomatic Males and Females . In Chlamydial Infections . (Bowie, W . R., Caldwell, H . D ., Jones, R . P., Mardh, P. A ., Ridgeway, G . L., Shachter, J., Stamm, W . E. & Ward, M . E ., eds) pp. 487-90. Cambridge, UK Cambridge University Press. 18. Sambrook, J ., Fritsch, E . F . & Maniatis . (1989) . Molecular Cloning : A Laboratory Manual . New York: Cold Spring Harbor Laboratory .
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