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Performance of Anyplex™ II multiplex real-time PCR for the diagnosis of seven sexually transmitted infections: comparison with currently available methods
Department of Obstetrics and Gynecology, St. Vincent's Hospital, The Catholic University of Korea College of Medicine, 93-6 Ji-dong, Paldal-gu, Suwon, 442-723, Korea
The real-time PCR assay is the most sensitive test for screening and diagnosing sexually transmitted infections (STIs) and has made diagnosing these infections easier for clinicians. The aim of this study was to investigate the reliability, accuracy, and usefulness of the real-time multiplex PCR assay for the detection of seven sexually transmitted microorganisms in clinical samples.
Methods
A total of 897 specimens from 365 symptomatic patients and 532 asymptomatic volunteers were collected over a 10-month period. A total of 696 subjects provided 50 ml of first-voided urine as samples, and 201 female patients provided endocervical swab specimens. Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum were tested for using five diagnostic methods: multiplex real-time PCR (Anyplex™ II), multiplex PCR (Seeplex®), strand displacement amplification (SDA, BD ProbeTec™ ET), PCR (AmpliSens®), and a commercially available Mycoplasma IST 2 Kit.
Results
Multiplex real-time PCR (Anyplex™ II) showed outstanding results in all fields, particularly sensitivity and specificity, compared with other diagnostic tools. This method yielded 100% sensitivity and high specificity for the detection of C. trachomatis, N. gonorrhoeae, T. vaginalis, M. genitalium, and M. hominis. It was also useful for discriminating between U. urealyticum and U. parvum.
Conclusions
Multiplex real-time PCR was found to be an equivalent or superior modality for the diagnosis of STIs. It could be a cost-effective and rapid diagnostic tool for the simultaneous detection of multiple STI microorganisms.
Sexually transmitted infections (STIs) are important individual, medical, social, and economic issues. The World Health Organization (WHO) estimates that 498.9 million new cases of syphilis, gonorrhea, chlamydia, and trichomoniasis occurred worldwide in 2008.
World Health Organization, Department of Reproductive Health and Research. Global incidence and prevalence of selected curable sexually transmitted infections—2008. Geneva: WHO; 2012. Available at: http://apps.who.int/iris/bitstream/10665/75181/1/9789241503839_eng.pdf (accessed 24 05 2013).
The reported prevalences of syphilis, gonorrhea, chlamydia, and trichomoniasis are 0.37%, 0.43%, 5.58%, and 2.4%, respectively, in the Republic of Korea.
Prevalence of sexually transmitted infections and sexual behavior of young adults and middle-aged people presenting to health examination centers in Korea.
It is important to increase vigilance and to recognize that STIs are often asymptomatic or cause nonspecific symptoms, that STIs can increase the infectiousness of HIV, and that regular testing for STIs is crucial.
Nucleic acid amplification tests (NAATs) are the most sensitive tests for STI screening and diagnosis. NAATs are more sensitive than the previously available diagnostic tests (e.g., culture, antigen detection, and nucleic acid hybridization) by approximately 20–30%.
Performance of the APTIMA Combo 2 assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in female urine and endocervical swab specimens.
because they can be used with noninvasively collected specimens such as first-voided urine samples and self- or clinician-collected vaginal swabs. More recently, the multiplex PCR assay has made it convenient for clinicians in many clinical fields to test for multiple causative organisms simultaneously. The multiplex PCR assay is a cost-effective diagnostic test because it allows for faster detection and a reduction in labor and reagent costs.
Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis.
Real-time PCR comprises amplification and fluorescence detection of an amplified DNA target in the same step. Compared with conventional PCR, real-time PCR is cost-effective because it improves the detection sensitivity, decreases the amplification time, and simplifies downstream processing.
Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples.
There are possible limitations in multiplexing. One possible limitation is PCR drift as a result of stochastic fluctuation in the interactions of PCR reagents, particularly in the early cycles, which could arise in the presence of very low template concentrations or through the assay design.
The reliability and accuracy of multiplex PCR for STIs have not been evaluated thoroughly. The aim of this study was to evaluate the performance and usefulness of real-time PCR assays for the detection of seven sexually transmitted microorganisms in clinical samples.
2. Patients and methods
2.1 Study population and clinical specimens
The study population consisted of two groups: symptomatic patients and asymptomatic volunteers. Three hundred sixty-five patients who were suspected of having an STI and who visited urology or gynecology hospitals in metropolitan areas were enrolled in the symptomatic patients group. Five hundred thirty-two volunteers who visited the Korean Industrial Health Association for a health examination and signed the informed consent form for this study were enrolled in the asymptomatic volunteers group. Over a period of 10 months, 897 specimens (from 365 symptomatic patients and 532 asymptomatic volunteers) were collected and tested. The specimens included 510 first-voided urine samples from men, 186 first-voided urine samples from women, and 201 endocervical swabs from women. The male participants and female volunteers were asked to provide 50 ml of first-voided urine (at least 2 h after previous urination) in a sterile 50-ml screw-cap plastic bottle. The endocervical swab specimens from female participants were collected by doctors, who followed the regular procedures for speculum examination and used manufactured collection kits. The specimens were immediately placed in a cooler and transported to the Central Diagnostic Laboratory in Seoul (Department of Laboratory Medicine, Chung-Ang University Hospital). The specimens were transported without added transport medium and were refrigerated and examined within 48 h.
2.2 Laboratory tests
Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum were detected by four NAATs: multiplex real-time PCR (Anyplex™ II, Seegene, Seoul, Korea), multiplex PCR (Seeplex®, Seegene, Seoul, Korea), strand displacement amplification (SDA) (BD ProbeTec™ ET, Becton–Dickinson Microbiology System, Sparks, MD, USA), and PCR (AmpliSens®, PCR Kit, InterLabService Ltd, Moscow, Russia), and using a commercially available Mycoplasma IST 2 Kit (bioMérieux, Marcy l’Etoile, France) (Figure 1).
Figure 1Flow chart showing the population and samples in the study.
2.3.1 Pretreatment of clinical specimens and DNA extraction
The swab specimens (approximately 2–3 ml) in the collection tubes were equilibrated to room temperature and mixed by vortexing. The caps from the specimen tubes were removed carefully to avoid contamination; 1-ml mixed specimens were transferred to 1.5-ml microcentrifuge tubes and the tubes were centrifuged at 15 000 × g (13 000 rpm). The supernatant was discarded and the pellet was resuspended in 1 ml of 1 × phosphate-buffered saline (PBS) by vortexing thoroughly to re-dissolve and disperse the sample. The urine specimens (approximately 10 ml) were equilibrated to room temperature and centrifuged at 5000 × g for 15 min. The supernatant was discarded and the pellet was resuspended in 1 ml 1 × PBS before DNA extraction. The DNA was extracted from the pretreated specimens (swab or urine) using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), in accordance with the manufacturer's instructions.
2.3.2 Multiplex real-time PCR (Anyplex™ II STI-7 Detection Kit)
Real-time PCR amplification for seven microorganisms (C. trachomatis, N. gonorrhoeae, T. vaginalis, M. genitalium, M. hominis, U. urealyticum, and U. parvum) was performed using the Anyplex™ II STI-7 Detection Kit (Seegene, Seoul, Korea), in accordance with the manufacturer's protocol, in a CFX96 real-time thermocycler (Bio-Rad, Hercules, CA, USA). Each PCR was performed in 5 μl of extracted DNA, 4x STI-7 TOM, and Anyplex PCR Mix in a 20-μl reaction. The thermal cycle conditions consisted of an initial incubation at 50 °C for 4 min to activate the UDG system and prevent contamination, and pre-denaturation at 95 °C for 15 min, followed by 50 cycles of alternating incubations: 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 30 s. The melting temperature was analyzed by increasing the reaction temperature from 55 °C to 85 °C (5 s/0.5 °C). The whole process control was added to the samples immediately before the DNA extraction to confirm the DNA extraction and PCR inhibition.
PCR amplification was performed using the Seeplex® STD6 ACE Detection Kit (Seegene, Seoul, Korea), in accordance with the manufacturer's protocol. The kit contains six pairs of primers that are specifically designed from highly conserved regions of genetic sequences for six microorganisms (N. gonorrhoeae, C. trachomatis, T. vaginalis, M. genitalium, M. hominis, and U. urealyticum) using Seegene DPO technology.
Performance of the APTIMA Combo 2 assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in female urine and endocervical swab specimens.
An internal control is present in the PCR mix for the detection of PCR inhibiting conditions (981 bp). For the negative control, sterile deionized water was used instead of DNA as the PCR template. To verify the integrity of the primers used in the PCR assay, positive DNA controls from the kit were assayed in the presence of all primer pairs. The amplified fragments were separated by agarose gel electrophoresis and identified by ethidium bromide staining.
The BD ProbeTec™ (Becton–Dickinson Microbiology System, Sparks, MD, USA) utilizes SDA technology as the amplification method and fluorescent energy transfer (ET) as the detection method to test for C. trachomatis and N. gonorrhoeae. This semi-automated system is based on the simultaneous amplification and detection of the target DNA, using amplification primers directed against the plasmid DNA and a fluorescently labeled detector probe. Positive and negative controls for specimen processing are included in the kit, with an amplification control to monitor assay inhibition. The assay was performed in accordance with the manufacturer's instructions. The presence or absence of C. trachomatis and N. gonorrhoeae is determined by relating the method-other-than-acceleration (MOTA) scores for the specimen to pre-determined cutoff values. The results with a MOTA value between 2000 and 20 000 (low positive) were rechecked, and the specimens that showed a final score of less than 2000 were determined to be negative in this study.
Direct comparison of the BD ProbeTec ET system with in-house LightCycler PCR assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae from clinical specimens.
For the PCR amplification of T. vaginalis and M. genitalium, each PCR kit (AmpliSens®Trichomonas vaginalis-EPH PCR Kit and AmpliSens®Mycoplasma genitalium-EPH PCR Kit; InterLabService Ltd, Moscow, Russia) was used in accordance with the manufacturer's protocol. A total of 10 μl of the DNA samples was added to the PCR pre-mix in a 25-μl reaction. One drop (25 μl) of mineral oil was added to each PCR reaction mixture to prevent contamination during the PCR reaction. The analysis of each amplification product was performed by separating the DNA fragment in agarose gel.
2.4 Mycoplasma IST 2 Kit
The identification of M. hominis and U. urealyticum was performed with a commercially available Mycoplasma IST 2 Kit (bioMérieux, Marcy l’Etoile, France). The Mycoplasma IST 2 Kit combines a selective culture broth with a strip containing 22 tests. A positive test is indicated by a change of broth color from yellow to red as a result of the phenol red indicator in the broth (urea for U. urealyticum and arginine for M. hominis). Approximately 500 μl of urine and 100 μl of an endocervical swab specimen (and two micro bank beads) were inoculated into the R1 solution, which was combined with a vial of R2 and vortexed. Fifty-five microliters of the solution was dispensed into each of the 22 test wells on the strip. Two drops of mineral oil were added to each well. The strips were incubated at 37 °C for 24–48 h. Colony forming units of ≥104/ml were considered a positive result for M. hominis and U. urealyticum infection.
2.5 Analysis of results
All specimens were tested in parallel using four NAATs and one Mycoplasma detection kit (Figure 1). An expanded gold standard, which was defined for the determination of true-positive specimens, was established to evaluate the new multiplex real-time PCR assay.
The results were considered true positives for C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium if they were positive according to any of the two NAATs. For M. hominis and U. urealyticum, any two positive results (a positive for either of the detection kits and a positive for one of the NAATs, or two positive for the NAATs) were considered a true-positive. The results of U. parvum were not analyzed because only one NAAT was available for U. parvum and the Mycoplasma IST 2 Kit is not able to differentiate between U. parvum and U. urealyticum.
The sensitivity and specificity of each test method for each microorganism were calculated for the specimens tested in parallel. The positive predictive value (PPV) and negative predictive value (NPV) were calculated for all specimens.
2.6 Quality control
Published guidelines were followed to guard against nuclease and nucleic acid contamination,
including the maintenance of a strict physical separation between the PCR setup and analysis, the use of sterile filter tips, and the regular decontamination of all surfaces with hypochlorite.
The assay controls included a negative extraction control, which was derived from pooled negative specimens, and a negative amplification control comprised of RNase–DNase-free sterile deionized water. All the tests were performed blind at the central laboratory. Results were recorded on pre-designed results sheets, scanned, and returned to the organizing researchers for comparison and analysis.
2.7 Institutional Review Board regulation
The Institutional Review Board of The Catholic University of Korea College of Medicine approved the study protocol, and all subjects provided written informed consent to participate in this study (XC12OIMI0003 V).
3. Results
3.1 Clinical incidence of seven sexually transmitted infections in 897 subjects
The distribution of the microorganisms in the clinical specimens by source is shown in Table 1. The total group of 897 patients consisted of 445 patients who had negative test results, 257 patients who were single microorganism positive, and 195 patients who were positive for multiple microorganisms. One-third of the female patients had all negative results (vaginal swab 35.3%, urine 33.3%), whereas 61.2% of the male patients had all negative results. The most prevalent microorganism was U. parvum (296 cases). U. urealyticum, C. trachomatis, and M. hominis were detected, in that order. U. parvum was the most frequently detected microorganism in the female patients (50%), while it was detected in only 17.1% of the male patients. C. trachomatis and N. gonorrhoeae were more frequently detected in the male patients than in the female patients.
Table 1Distribution of organisms in the positive clinical samples. The summation of microorganism distribution (%) exceeds 100% because multiple microorganism positivity was counted individually
3.2 Clinical sensitivity, specificity, PPV, and NPV
The sensitivity, specificity, PPV, and NPV of each diagnostic assay using the expanded gold standard as reference are shown in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7. When the expanded gold standard was applied, Anyplex™ II had 100% sensitivity and specificity for C. trachomatis. The two other assays had greater than 90% PPV and NPV. For N. gonorrhoeae, Anyplex™ II and Seeplex® showed 100% sensitivity, and BD ProbeTec™ showed 80% sensitivity. This difference in the sensitivity is likely attributable to very low positivity being considered negativity in the BD ProbeTec™ because the BD ProbeTec™ analyzes the results using the MOTA score (Table 2, Table 3).
Table 2Numbers of positive and negative results for Chlamydia trachomatis and comparisons with the expanded standard
In the diagnostic assay for T. vaginalis and M. genitalium using the expanded gold standard, the three assays showed greater than 90% sensitivity and specificity. Anyplex™ II and AmpliSens® showed low PPV for T. vaginalis (75.0% and 37.5%, respectively), which is likely attributable to errors that occur because of the small number of positive cases used when diagnosing a disease that has very low prevalence (Table 4, Table 5).
In the diagnostic assay of M. hominis and U. urealyticum using (1) a detection kit positive and one of the NAATs positive, or (2) two of the NAATs positive, as the expanded gold standard, the sensitivity and specificity were shown to be lower for the Mycoplasma IST 2 Kit than for Anyplex™ II and Seeplex® (Table 6, Table 7). A direct comparison is difficult due to the differences between the diagnostic methods and principles in the NAATs and those in the commercial culture kit. The aforementioned difference is likely to be attributable to weak positivity being considered negativity in the Mycoplasma IST 2 Kit, in which ≥104 colony-forming units/ml is considered a positive result.
In the diagnostic assay for U. urealyticum, the PPV was shown to be 29.0% for the Mycoplasma IST 2 Kit. It is likely that a significant portion of microorganisms detected as U. parvum by Anyplex™ II were detected as U. urealyticum in the Mycoplasma IST 2 Kit. This explanation is reasonable because 96 (35.1%) of the cases determined to be U. urealyticum-negative and U. parvum-positive using Anyplex™ II, were determined to be U. urealyticum-positive using the Mycoplasma IST 2 Kit (Table 8).
Table 8Distribution of Mycoplasma IST 2 Kit results
This study was conducted to compare the diagnostic performance of a recently developed real-time multiplex PCR to that of the conventional methods used for the multiple detection of bacterial STIs. We analyzed the sensitivity and specificity of the methods available for the diagnosis of seven sexually transmitted microorganisms comparatively. A combination test was performed on the specimens, and the expanded gold standard was used to assess the new diagnostic test. Although this assessment method increases the workload and costs because more tests are conducted on the specimens, it has been known to reduce data bias if no perfect gold standard is available.
In the detection of C. trachomatis, due to the technical difficulty resulting from the necessity of immediately inoculating specimens into cell lines after specimen collection, the culture test has a low sensitivity of 50% and is no longer used clinically; as a result NAATs have been used as standard tests.
In the detection of N. gonorrhoeae, the culture test using Thayer–Martin agar has been used as a standard method. Due to difficulties in specimen collection, transfer, and storage, this test has a low sensitivity and is unsuitable for screening.
Except for special circumstances (e.g., measuring antimicrobial sensitivity), NAATs have become the tests of choice and are used instead of the culture test. In the detection of C. trachomatis and N. gonorrhoeae, the two most common pathogens of bacterial STIs, NAATs have been applied predominantly in the form of combination-type commercial kits that can detect pathogens simultaneously, or multiplex-type commercial kits that can detect additional pathogens. In this study, real-time PCR and PCR had 100% and 96.8% sensitivity, respectively, for C. trachomatis, and 100% sensitivity for N. gonorrhoeae. SDA had 83.9% and 88.9% sensitivity for C. trachomatis and N. gonorrhoeae, respectively, which is relatively low. This difference in the sensitivity is likely attributable to the fact that very low positivity is considered negativity in SDA because SDA (BD ProbeTec™ ET) analyzes the results using the MOTA score. No definite reason for the low sensitivity has been presented. Other studies have shown similar results, confirming that the NAATs do not always produce the same results for the same specimens.
Performance of three nucleic acid amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by use of self-collected vaginal swabs obtained via an Internet-based screening program.
In this study, Anyplex™ II and AmpliSens® had 75.0% and 37.5% PPV, respectively, for T. vaginalis. This bias should be considered when diagnosing T. vaginalis in regions with a low prevalence.
With the recent accumulation of considerable evidence on M. genitalium, this organism has been recognized as a pathogen of acute and chronic urethritis in males.
Proactive diagnosis and treatment of M. genitalium is required. The detection of M. genitalium via culture is no longer used in routine clinical practice because it takes many weeks and presents technical difficulties.
Due to cross-reactivity with M. pneumoniae, it is not appropriate to use a serological test to diagnose M. genitalium. NAATs are currently recognized as the tools available for detecting M. genitalium. Several real-time PCRs have been introduced to detect M. genitalium. In such methods, the quantification of M. genitalium DNA has been reported to be useful in judging the treatment efficacy and for assessing the DNA load of clinical specimens.
Use of TaqMan 5′ nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic.
This method could provide vital information in the detection of M. genitalium, which has no established therapeutic agent and has non-distinct symptoms. In this study, the three NAATs had high sensitivity and specificity. Real-time PCR had 100% sensitivity and specificity and showed robustness in various specimens.
In addition to M. genitalium, M. hominis and Ureaplasma species are included in genital mycoplasmas. In a study conducted in the USA, M. hominis and Ureaplasma species were detected in 21–53% and 40–80% of asymptomatic and sexually active women, respectively, and their prevalences were slightly lower in men than in women.
The results of this study are consistent with those of a previous study conducted in the USA (Table 1). As the high detection rates of M. hominis and Ureaplasma species might have been caused by commensalism and infection, their roles as pathogens is controversial. Much evidence shows that M. hominis detected in urethral specimens is not associated with male urethritis. M. hominis was shown to act as a pathogen in 5% of upper urinary tracts of patients with acute pyelonephritis,
Ureaplasma species, which were formerly known as U. urealyticum biovars 1 and 2, were identified as separate species – U. urealyticum (biovar 2) and U. parvum (biovar 1) – by PCR. U. urealyticum has been reported to be a pathogen of male urethritis
but its role as an STI pathogen is unclear. The discrimination of U. urealyticum and U. parvum is important in the diagnosis of STIs because unnecessary treatment might be carried out if U. parvum, most of which are colonized as commensals, is mistakenly considered to be U. urealyticum. In this study, it was impossible to differentiate between U. urealyticum and U. parvum using the Mycoplasma IST 2 Kit. A significant segment of the U. urealyticum positivity identified via the Mycoplasma IST 2 Kit was revealed to be U. parvum positivity via PCR (Table 8). It is necessary to conduct more analytical research on the detection and discrimination of Ureaplasma species.
One of the most important features of real-time PCR is quantification. Unlike bacterial culture, in which only viable bacteria can be quantified, real-time PCR can quantify both viable and nonviable bacteria. The quantification of the bacterial load in clinical specimens can provide a critical clue in discriminating between infection and commensalism, which leads to an evaluation of the patient's prognosis by determining disease severity and assessing treatment efficacy.
The determination of the bacterial load using real-time PCR could be used as a useful marker in the diagnosis of STIs. Further study is required to explore this possibility.
In conclusion, the performance of the multiplex real-time PCR was equal or superior to that of the NAATs and detection kits that are currently used for STI diagnoses. The multiplex real-time PCR is cost-saving because it can rapidly detect multiple microorganisms simultaneously via its multiplex function, and it is expected that it will be used as a standard diagnostic test for STIs in the near future.
Acknowledgements
This study is an investigator-initiated trial supported by Seegene Inc., Seoul, South Korea. This funding source had no involvement in the study design, conduct, analysis, or publication. We declare that we have no financial conflicts of interest. The authors would like to thank Eun-Sil Jang for assistance with data collection.
Ethical considerations: The study was reviewed and approved by the Institutional Review Board of The Catholic University of Korea College of Medicine (XC12OIMI0003 V). All subjects provided written informed consent to participate in this study.
Conflict of interest: No conflict of interest to declare.
References
World Health Organization, Department of Reproductive Health and Research. Global incidence and prevalence of selected curable sexually transmitted infections—2008. Geneva: WHO; 2012. Available at: http://apps.who.int/iris/bitstream/10665/75181/1/9789241503839_eng.pdf (accessed 24 05 2013).
Prevalence of sexually transmitted infections and sexual behavior of young adults and middle-aged people presenting to health examination centers in Korea.
Performance of the APTIMA Combo 2 assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in female urine and endocervical swab specimens.
Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis.
Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples.
Direct comparison of the BD ProbeTec ET system with in-house LightCycler PCR assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae from clinical specimens.
Performance of three nucleic acid amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by use of self-collected vaginal swabs obtained via an Internet-based screening program.
Use of TaqMan 5′ nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic.
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