INTRODUCTION

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Genitourinary tract infections are a major cause of morbidity in sexually active individuals worldwide, with an estimated 330 million new curable sexually transmitted diseases (STDs) diagnosed per year worldwide and between 5 and 12 million cases diagnosed annually in North America (1, 4, 11). The annual cost of treatment of chlamydial infections and their sequelae was estimated at $2.2 billion in 1990 for North America (29). Although the number of reported cases of Neisseria gonorrhoeae has declined substantially in many industrialized countries, this STD is still important worldwide, with an estimated 78 million new infections occurring annually (30).

Over the past 15 years, the diagnosis of STDs has been largely dependent on traditional methods, such as culture, enzyme immunoassay, and direct fluorescent-antibody staining for Chlamydia trachomatis and culture for N. gonorrhoeae. In the last 5 years there have been major improvements in our ability to detect these STDs, first with the advent of nucleic acid amplification technologies and then with the introduction of testing noninvasively obtained urine specimens (5-7, 9, 10, 17, 18, 22, 23, 25). Commercially available PCR, ligase chain reaction (LCR), and transcription-mediated amplification tests for C. trachomatis have been developed, and comparative studies have been reported (19, 25, 26). Fewer nucleic acid amplification tests exist for N. gonorrhoeae (23), and some coamplification assays have been developed for C. trachomatis and N. gonorrhoeae.

Despite the early successes of these commercial amplification tests, they are not without problems. The sensitivity of amplification assays can be reduced by the presence of amplification inhibitors in clinical specimens, and different assays may be affected differently by inhibitors (6, 16, 28). The utility of first-generation PCR and LCR assays has been limited by the number of specimens that can be tested in a single run. Batch sizes for both the Amplicor and LCx assays are limited by the small number of specimens that can be tested in a single run, 24 on two wheels for Amplicor and 22 on one carousel for LCx. The handling of a small number of specimens has hampered the installation of these tests in large-volume laboratories. Specimen extraction has also been problematic, and the need for automation for large-volume testing has resulted in the production of automated nucleic acid extractors and robotic pipetting stations. Front-end robotic extraction stations have therefore become the "holy grail" of diagnostic companies, especially for applications involving bloodborne viruses, for which large-volume testing is necessary for protection of the blood supply.

We evaluated a new RNA amplification and detection kit, called the NucliSens Basic Kit, which uses isothermal nucleic acid sequence-based amplification (NASBA) for the detection of C. trachomatis and N. gonorrhoeae 16S rRNAs in clinical specimens. We compared the Basic Kit to PCR for C. trachomatis and to culture for N. gonorrhoeae, using genitourinary tract specimens.

(These results were presented in part at the 39th Annual ICAAC meeting in San Francisco, CA, September 1999.)

	MATERIALS AND METHODS

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Specimens. Two hundred fifty randomly collected urethral or cervical swab specimens from 121 women and 129 men, submitted for culture of N. gonorrhoeae, were used in this study. Specimens were obtained from Gamma-Dynacare Medical Laboratories in Brampton, Ontario, and St. Joseph's Hospital in Hamilton, Ontario, Canada. N. gonorrhoeae culture was performed using modified New York City medium (13). The plates were incubated for 48 h at 35°C in an atmosphere of 5% CO₂-95% air. After incubation, the plates were examined for the presence of N. gonorrhoeae; presumptive positives were confirmed by oxidase testing (Vitek), Gram stain, and carbohydrate utilization assays (Quadferm; API). Following culture, the charcoal swabs were frozen at 70°C and shipped to the Regional Virology and Chlamydiology Laboratory at St. Joseph's Hospital for molecular testing, which was performed in a blinded fashion, without knowledge of culture results.

Nucleic acid isolation. Nucleic acids were isolated from swab specimens and from laboratory strains of N. gonorrhoeae (ATCC 43069) and C. trachomatis (L1/LGV434) by using the isolation reagents, based on the method of Boom et al. (2), from the NucliSens Basic Kit (Organon Teknika, Boxtel, The Netherlands). Frozen charcoal swabs were thawed at room temperature and swirled in 1 ml of phosphate-buffered saline. The tubes were vortexed and left for 10 min to allow the charcoal to settle to the bottom; then 800 µl of each supernatant was transferred to a fresh tube, and any remaining charcoal particles were allowed to settle. Finally, 700 µl of supernatant was transferred to a tube containing 9 ml of guanidinium isothiocyanate (GuSCN)-based lysis buffer; this was followed by the addition of 50 µl of activated silica. Silica particles carrying adsorbed nucleic acids were washed twice with 1 ml of GuSCN-based wash buffer, twice with 1 ml of 70% ethanol, and once with 1 ml of acetone. After the silica was dried at 56°C for 10 min, the nucleic acid was eluted in 50 µl of elution buffer and stored at 70°C prior to testing, which usually took place the next day.

PCR. PCR amplifications of 16S rRNA gene fragments were performed as described previously (14, 15), with slight modifications. Primers for N. gonorrhoeae (reverse primer, 5'-GGCGGTCAATTTCACGCG; forward primer, 5'-ACTGCGTTCTGAACTGGGTG) amplified a 281-bp fragment, while primers for C. trachomatis (reverse, 5'-CACATAGACTCTCCCTTAAC; forward, 5'-AGCAATTGTTTCGGCAATTG) amplified a 205-bp fragment of the 16S rRNA gene. Oligonucleotides were synthesized at McMaster University's Central Molecular Biology Facility. PCR was performed using AmpliTaq Gold (Perkin-Elmer, Branchburg, N.J.) in a total volume of 25 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, 0.2 mM each deoxynucleoside triphosphate, 1 µM each primer, 1.25 U of Taq polymerase, and 2.5 µl of purified nucleic acid. Amplification consisted of denaturation for 10 min at 95°C followed by 30 cycles of amplification. Each cycle consisted of 30 s at 94°C, 30 s at 60°C (for N. gonorrhoeae) or 50°C (for C. trachomatis), and 1 min at 72°C. The final elongation step was extended for another 8 min. After amplification, the amplified DNA was analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. Positive and negative amplification controls and blank contamination controls were incorporated into each run.

Cloning of C. trachomatis and N. gonorrhoeae 16S rRNA gene fragments. 16S rRNA gene fragments were cloned into the vector pGEM-T in order to generate in vitro-transcribed RNA. Briefly, C. trachomatis and N. gonorrhoeae DNAs were amplified by using oligonucleotide primers that targeted 16S rRNA gene fragments of 180 bp for C. trachomatis and and of 606 bp for N. gonorrhoeae. C. trachomatis primers were the same as those used for NASBA (CTP1 and CTP2) (Table 1) but without the T7 promoter sequence on the reverse primer. Primers for N. gonorrhoeae were 5'-CTGTTGCCAATATCGGCGGC-3' (forward) and 5'-ACAGCCATGCAGCACCTGTG-3' (reverse). PCR was performed in 50-µl reaction volumes consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, 0.2 mM each deoxynucleoside triphosphate, 0.6 µM each primer, and 1 U of AmpliTaq Gold polymerase (Perkin-Elmer; Roche Molecular Systems, Branchburg, N.J.) and a thermal cycling program of 1 min at 95°C, 1 min at 56°C, and 2 min at 72°C for 40 cycles. Amplified fragments were resolved by 2% agarose gel electrophoresis and visualized by staining with ethidium bromide. Single bands for C. trachomatis and N. gonorrhoeae were excised and eluted from the gel by using the GeneClean II DNA gel purification system (Bio101, La Jolla, Calif.) according to the manufacturer's instructions. Cloning of the fragments was carried out with the pGEM-T cloning vector system (Promega Corp., Madison, Wis.) according to the manufacturer's instructions. The vectors containing the 16S rRNA genes of C. trachomatis and N. gonorrhoeae were designated CT180-pGEM and NG606-pGEM, respectively.

Generation of in vitro transcripts. In vitro-transcribed C. trachomatis and N. gonorrhoeae 16S rRNAs were synthesized using a CT180-pGEM or NG606-pGEM clone that generated sense(+)RNA for C. trachomatis and N. gonorrhoeae, respectively, by standard protocols. One microgram of linearized CT180-pGEM or NG606-pGEM was used as a template for in vitro transcription in a 100-µl reaction volume containing 20 mM MgCl₂, 40 mM Tris-HCl (pH 8.1), 1 mM spermidine, 0.01% Triton X-100, 20 mM dithiothreitol, 4 mM each nucleoside triphosphate (Amersham Pharmacia Biotech, Arlington Heights, Ill.), and 50 U of T7 RNA polymerase (United States Biochemical Corp., Cleveland, Ohio). Reactions were allowed to proceed for 4 h at 37°C prior to addition of 1 U of RQ1 RNase-free DNase to degrade the DNA template. RNA transcripts were purified by two rounds of phenol-chloroform-isoamyl alcohol (24:1:1 [vol/vol/vol]) extraction followed by one chloroform extraction and precipitation with 2.5 volumes of absolute ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2). Purified RNA transcripts suspended in RNase-free distilled water were quantified by spectrophotometry and visualized by formaldehyde-formamide (2.4 M) agarose gel electrophoresis, after which ethidium bromide staining was performed in order to verify the sizes and integrity of the transcripts.

NASBA. NASBA was performed, as described in the Basic Kit application manual, by the method of Kievits et al. (12) with some modifications (24). Primers and probes specific for N. gonorrhoeae or C. trachomatis 16S rRNA were synthesized and purified by high-performance liquid chromatography (Central Facility, Institute for Molecular Biology and Biotechnology, McMaster University) (Table 1). The primers for NASBA targeted the same region of the 16S rRNA as did the PCR primers for both bacteria. P1 primers contained the T7 promoter sequences plus a sequence complementary to the target RNA, and P2 primers contained a sequence identical to the target RNA plus a 5' electrochemiluminescent (ECL) tag (20 nucleotides [nt] in length) as a detector molecule.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Relative positions of NASBA primers and probes specific for N. gonorrhoeae and C. trachomatis. Nucleotide sequences for N. gonorrhoeae and C. trachomatis are from Rossau et al. (21) and Pudjiatmoko et al. (20), respectively. The antisense P1 primers for both N. gonorrhoeae and C. trachomatis (NGP1 and CTP1, respectively) include the T7 promoter sequence at the 5' end. The sense P2 primers contain sequence complimentary to the ECL universal detector probe.

Northern blotting. Northern blot analysis was performed as described previously, using a fluorescein-labeled oligonucleotide probe (24).

	RESULTS

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We evaluated the NucliSens Basic Kit for its ability to detect C. trachomatis and N. gonorrhoeae in genitourinary tract specimens by developing separate NASBA assays for C. trachomatis and N. gonorrhoeae 16S rRNAs and determining (i) the analytical sensitivity for each assay with in vitro transcripts and pretitrated bacterial stocks and (ii) the clinical performance by comparing the results obtained using the Basic Kit to those obtained by culture for N. gonorrhoeae and by PCR for trachomatis. The Basic Kit contains all the reagents, with the exception of probes and primers, necessary for performing NASBA, including reagents for RNA extraction, amplification, and ECL detection. RNA is first extracted with the isolation reagents by the Boom method, using GuSCN and silica, and then amplified by isothermal NASBA and detected by ECL using a biotinylated capture probe immobilized on streptavidin magnetic beads and a generic detector probe. ECL readouts in RLU are provided by the NucliSens Reader. Target-specific primers for each NASBA, including a P1 primer containing a 5'-terminal T7 polymerase promoter sequence and a P2 primer containing a 5'-terminal ECL sequence complimentary to the ruthenium-labeled detector probe, as well as a biotinylated capture probe were synthesized separately, as shown in Table 1. The locations of the primers and probes for amplification of N. gonorrhoeae and C. trachomatis 16S rRNAs are shown in Fig. 1. The sizes of the amplified N. gonorrheae and C. trachomatis RNAs were 280 and 205 nt, respectively.

Both NASBA assays, for C. trachomatis and for N. gonorrhoeae, were optimized for KCl concentration. For C. trachomatis, the level of amplified product increased as the concentration of KCl was increased from 50 to 90 mM and then decreased at 100 mM KCl (Fig. 2). For N. gonorrhoeae, however, amplification was strongest at 90 mM KCl and there was less of a difference between the amplifications at 70 (the standard concentration for NASBA) and 100 mM than for C. trachomatis.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Optimal KCl concentration for NASBA amplification of N. gonorrhoeae and C. trachomatis 16S rRNAs. NT, no template; PC, positive control.

The analytical sensitivity of NASBA was determined by testing serial dilutions of freshly prepared stock cultures of C. trachomatis and N. gonorrhoeae and by testing in vitro-generated transcripts. Aliquots of freshly prepared stocks were titrated to determine the number of viable C. trachomatis (in inclusion-forming units [IFU] per milliliter) and N. gonorrhoeae (in CFU per milliliter) organisms and then were used for extraction of total nucleic acids with the Basic Kit guanidinium/silica extraction protocol, which extracts both DNA and RNA. Serial dilutions of nucleic acids were then tested by PCR and NASBA. RNA products were analyzed in parallel by ECL using the Basic Kit and by Northern blot analysis. For N. gonorrhoeae, the PCR reached an endpoint at a dilution of 10⁴ while the NASBA reached an endpoint at 10⁵ with both ECL and Northern blot detection (Fig. 3). For C. trachomatis, NASBA with Northern blot detection reached an endpoint at a dilution of 10⁵ while ECL and PCR reached endpoints at dilutions of 10⁷ and 10⁵, respectively (Fig. 4). Based on the infectious titers of freshly prepared bacterial stock cultures, NASBA could detect 1 IFU of C. trachomatis and 1 CFU of N. gonorrhoeae. The sensitivity of each NASBA was further determined by using serial dilutions of in vitro-generated transcripts; both assays consistently detected 100 copies of 16S rRNA, and occasionally 10 copies were detectable (data not shown).

View larger version (52K): [in this window] [in a new window]   FIG. 3.   Sensitivity of NASBA and PCR for detecting N. gonorrhoeae. Serial dilutions of N. gonorrhoeae nucleic acid (extracted by the Boom silica method) were amplified by NASBA and PCR. Aliquots of NASBA reaction products were analyzed for a specific product by hybridization with a biotinylated capture probe and a ruthenium-labeled detector probe followed by ECL detection in the NucliSens Reader. PCR products were analyzed by agarose gel electrophoresis. NT, no template.

View larger version (58K): [in this window] [in a new window]   FIG. 4.   Sensitivity of NASBA and PCR for detecting C. trachomatis. Serial dilutions of C. trachomatis nucleic acid were amplified by NASBA and PCR. NASBA products were detected by ECL using the Basic Kit and by Northern blot analysis. PCR products were analyzed by agarose gel electrophoresis. NT, no template.

The Basic Kit was further evaluated by testing clinical specimens collected from symptomatic individuals. Two hundred and sixteen swab specimens, including 142 culture-positive swab samples and 74 culture-negative swab specimens collected from 121 women and 129 men, were tested for N. gonorrhoeae by culture and by NASBA using the Basic Kit. The Basic Kit detected 139 of 142 culture-positive swabs and was negative for 73 of 74 culture-negative swabs, for a sensitivity and specificity of 97.9 and 98.7%, respectively (Table 2). The four discordant specimens were tested by PCR; one of the three culture-positive, Basic Kit-negative specimens and one culture-negative, Basic Kit-positive specimen was positive by PCR. Ninety-six genital swab specimens collected for detection of N. gonorrohoea were randomly selected and tested for C. trachomatis DNA by PCR and for 16S rRNA by using the Basic Kit. C. trachomatis DNA was detected in 24 of 96 swab samples by PCR. The Basic Kit detected all 24 PCR-positive specimens and gave negative results for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively (Table 2).

The Basic Kit was next evaluated for its ability to detect both C. trachomatis and N. gonorrhoeae in the same specimen. Four swabs positive for C. trachomatis, four swabs positive for N. gonorrhoeae, and four swabs spiked with C. trachomatis and N. gonorrhoeae to simulate dually positive specimens were tested with the Basic Kit, using a multiplex NASBA assay with primers for both organisms. Amplification products were then tested independently with C. trachomatis- and N. gonorrhoeae-specific probes. Specimens 1 to 4 were positive for C. trachomatis and had signals ranging from 36,000 to 70,000 RLU using the C. trachomatis-specific probe and <300 RLU using the N. gonorrhoeae-specific probe (Fig. 5). Specimens 5 to 8 were N. gonorrhoeae positive and had signals of 29,000 to 154,000 RLU using the N. gonorrhoeae probe and <300 using the C. trachomatis probe. Specimens 9 to 12 were positive for both organisms and had mean RLU values of 4,799 for C. trachomatis and 74,434 for N. gonorrhoeae.

View larger version (35K): [in this window] [in a new window]   FIG. 5.   Detection of C. trachomatis and N. gonorrhoeae in genital swab specimens by NASBA and ECL detection with the Basic Kit. Twelve specimensfour positive for N. gonorrhoeae (NG1 to NG4), four positive for C. trachomatis (CT1 to CT4), and four positive for both N. gonorrhoeae and C. trachomatis (NG-CT1 through NG-CT4)and four controls (NT, no template; NGPC, N. gonorrhoeae positive; CTPC, C. trachomatis positive; and NG-CTPC, N. gonorrhoeae and C. trachomatis positive) were amplified and probed separately with a C. trachomatis-specific probe and an N. gonorrhoeae-specific probe. Dotted line represents cutoffs of positivity. y-axis, RLU.

	DISCUSSION

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We evaluated the NucliSens Basic Kit for the detection of C. trachomatis and N. gonorrohoeae 16S rRNAs in genital tract specimens. Under optimal conditions of amplification, the Basic Kit consistently detected 100 molecules of 16S rRNA for each bacterium and 1 IFU of C. trachomatis and 1 CFU of N. gonorrhoeae. For gentital swab specimens, the Basic Kit had a sensitivity comparable to that of culture for detecting N. gonorrhoeae and equivalent to that of PCR for detecting C. trachomatis (Table 2). The specificity of the Basic Kit for detecting either C. trachomatis or N. gonorrhoeae in genital swab samples exceeded 98.5%. Using a multiplex NASBA assay, the Basic Kit was able to detect both bacteria when they were present in the same clinical specimen.

The Basic Kit provides an open platform for RNA amplification and detection and includes all reagents necessary for detecting RNA by NASBA with the exception of specific primers and probes. The kit includes reagents for RNA extraction, enzymes and buffers for amplification, and a generic probe for detection in the NucliSens Reader. RNA extraction is performed using the well-established Boom method of GuSCN stabilization followed by adsorption to and elution from silica. The Basic Kit software installed in the Reader enables the user to enter different cutoff levels, which allows validation for various specimen types. We have used these isolation reagents to extract RNA from a variety of clinical specimens, including genital swab samples, whole blood or peripheral blood mononuclear cells, nasopharyngeal swab specimens, cultured cells, and brain tissue. Following amplification, a specific product is detected by using a ruthenium-labeled generic probe which binds to the complimentary ECL tag of the antisense RNA product. The NucliSens Reader provides readouts in RLU which can be easily used for establishing cutoffs for qualitative assays or for quantitation of RNA analytes (8, 24).

NASBA with the Basic Kit was easy to perform and offered some advantages over PCR. NASBA involves an isothermal reaction but does not require a thermal cycler. The amplification conditions for NASBA are generally constant, and optimization of conditions for each new assay can be simpler than optimization of PCR. The concentrations of enzymes and primers used for NASBA are standardized and do not differ from assay to assay. For PCR, the optimal annealing conditions, including temperature and primer concentration, must by determined to prevent nonspecific priming events. Because NASBA uses an isothermal amplification at 41°C, the optimal annealing temperature for primers does not have to be determined emperically. Nonspecific annealing of primers to the target nucleic acid is controlled for at the detection level by using a specific capture probe for ECL. The only variable that has to be optimized with NASBA is the KCl concentration, and this is easily done in a single experiment using a KCl concentration range of 50 to 120 mM. Most targets will have optimal KCl concentrations between 70 and 90 mM. In our hands, NASBA for both C. trachomatis and N. gonorrhoeae had an optimal KCl concentration of 90 mM, which facilitated coamplification of both targets in a multiplex format using both sets of NASBA primers. We have used PCR primers for NASBA on several occasions with good results by simply adding the T7 promoter sequence to one primer. This was the case in the present study, in which the same primers were used for both PCR and NASBA for both C. trachomatis and N. gonorrhoeae.

The Basic Kit assays for C. trachomatis and N. gonorrhoeae had excellent analytical sensitivity and clinical performance. As mentioned above, both assays detected one viable bacterial cell. This may be an underestimation of the sensitivity since chlamydial counts determined by immunofluorescent staining with a monoclonal antibody often exceed IFU counts due to the presence of nonviable bacteria; furthermore, nonviable cells contain 16S rRNA. The Basic Kit detected 100 molecules of 16S rRNA, and in some experiments a sensitivity of 10 copies was achieved. In the only other published report on NASBA for C. trachomatis, Morré et al. compared the sensitivities of three different targets of amplification. The cryptic plasmid and omp1 targets had a detection limit of 1 IFU of C. trachomatis, while NASBA for 16S rRNA was 10-fold more sensitive than either the plasmid or the omp1 assay and 10-fold more sensitive than PCR (17). In their hands, NASBA detected 10 to 100 molecules of 16S rRNA, which is the same sensitivity that we obtained. A level of sensitivity of one bacterium for NASBA compares favorably with that of other amplification methods, including PCR and LCR (14). Based on results obtained with 216 genital swab specimens, the Basic Kit had sensitivities of 97.9% for N. gonorrhoeae and 100% for C. trachomatis. Several studies evaluating commercially available PCR, LCR, and transcription-mediated amplification tests for C. trachomatis reported sensitivities between 90 and 98% when an expanded reference standard was used (3, 6, 7, 9, 10). Using the Basic Kit, we were able to detect C. trachomatis and N. gonorrhoeae with similar sensitivities. The Basic Kit also detected C. trachomatis and N. gonorrhoeae present in the same specimen with the use of a multiplex NASBA employing two sets of primers. Recently developed coamplification assays employing PCR and LCR have been evaluated for the detection of both organisms. Commercially available coamplification assays have been evaluated in symptomatic men and women with moderate to high prevalence of C. trachomatis infection who attended a sexually transmitted disease clinic, and these assays have performed with good sensitivity and specificity (3, 10, 27).

We have used the Basic Kit to detect bacteria other than C. trachomatis and N. gonorrhoeae such as Chlamydia pneumoniae and Mycoplasma pneumoniae, as well as viruses such as human papillomavirus. We have used NASBA to both detect and quantitate C. trachomatis 16S rRNA (24) in clinical specimens and C. pneumoniae ompA mRNA levels during the chlamydial replication cycle (8). Quantitation of ompA and hsp60 mRNAs by using the Basic Kit platform may provide a useful molecular tool for studying latent chlamydial infections in those with chronic diseases.

In summary, the NucliSens Basic Kit offers a convenient platform for the development of sensitive assays for detection of sexually transmitted infections by such organisms as C. trachomatis and N. gonorrhoeae. The excellent sensitivity and specificity obtainable with the Basic Kit assays and the availability of over 60 different assay protocols on the Basic Kit website indicate that this kit is suitable for widespread usage in both research and clinical settings.