First evaluation of six nucleic acid amplification tests widely used in the diagnosis of Chlamydia trachomatis in Russia

Authors


Correspondence: M Unemo. E-mail: magnus.unemo@orebroll.se

Abstract

Abstract

Background  In Russia, nationally developed nucleic acid amplification tests (NAATs), which have never been validated to international commercially available NAATs, are mainly used in the diagnosis of Chlamydia trachomatis infection.

Objective  To evaluate the performance characteristics of six NAATs widely used to diagnose C. trachomatis infection in Russia.

Materials and methods   In total, 446 consecutive symptomatic patients (319 females and 127 males) were included. Five polymerase chain reaction (PCR) assays and one real-time nucleic acid sequence-based amplification (NASBA) assay were evaluated on cervical and vaginal samples from females and on urethral and first voided urine samples from males. As reference methods, the Cobas Amplicor PCR, as the main ‘gold standard’ method, and LightMix 480HT PCR were used.

Results  The overall prevalence of C. trachomatis infection was 12.6%. The Russian NAATs and the reference methods displayed a high level of concordance (97.9% to 99.2%). In comparison with the reference methods, the sensitivities, specificities, positive predictive values and negative predictive values of the Russian tests in different specimens ranged from 86.1% to 100%, 99.1% to 100%, 92.3% to 100% and 98.2% to 100%, respectively.

Conclusions  According to the reference methods, C. trachomatis NAATs developed and used in Russia have relatively good performance characteristics for both invasive and non-invasive samples. However, larger studies that include symptomatic and asymptomatic patients as well as genital and extra-genital samples, and in comparison with other internationally well-recognized, validated, and ideally Food and Drug Administration–approved C. trachomatis NAATs performed strictly according to the manufacturer's instructions, need to be conducted.

Conflicts of interest

None declared

Introduction

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection (STI) worldwide.1,2 In the past few decades, the introduction of nucleic acid amplification tests (NAATs), which have largely replaced cell culture, direct immunofluorescence (DIF) and other methods, has significantly improved the diagnosis of C. trachomatis in many countries. Presently, several international commercially available and strictly validated NAATs in the diagnosis of C. trachomatis exist, including Cobas Amplicor and Cobas TaqMan48 [conventional and real-time polymerase chain reaction (PCR), respectively; Roche Diagnostics, Branchburg, NJ, USA], m2000rt (real-time PCR; Abbott Molecular Diagnostics, Des Plaines, IL, USA), BD ProbeTec ET [strand displacement amplification (SDA); Becton Dickinson Diagnostic Systems, Sparks, MD, USA], AMP-CT and Aptima Combo 2 [transcription mediated amplification (TMA); Gen-Probe, Inc., San Diego, CA, USA]. The main advantages of the NAATs are that they combine a high sensitivity (for both symptomatic and asymptomatic individuals) with a high specificity. Furthermore, NAATs permit the use of non-invasive clinical samples, are rapid, are increasingly automated and have a high specimen throughput.3–5

In Russia, reliable figures regarding incidence and prevalence of most STIs, and in particular C. trachomatis infection, remain highly limited. This situation, which is common in many East European countries, primarily results from suboptimal diagnostics, case reporting and surveillance systems.6–12 However, in recent years, the introduction of NAATs in Russia has greatly advanced the diagnosis of C. trachomatis. Nevertheless, because of their substantially higher costs, none of the international commercially available NAATs mentioned above is used in the routine diagnosis of chlamydial infection in Russia, and only a few laboratories in Russia use some of these NAATs for rare discrepancy analysis. Instead, in recent years, several NAATs have been developed and widely used in Russia in the C. trachomatis diagnostics. Accordingly, evaluation and validation of all the Russian C. trachomatis NAATs to the international commercially available and highly validated diagnostic NAATs are crucial. The present study is the first ever comprehensive evaluation of the performance characteristics of any Russian C. trachomatis NAATs to internationally well-recognized and validated C. trachomatis NAATs, i.e. the Food and Drug Administration (FDA)–approved Roche Cobas Amplicor PCR, as well as to LightMix 480HT PCR (TIB MOLBIOL GmbH, Berlin, Germany). However, the FDA-approved protocol for the Roche Cobas Amplicor PCR was slightly modified; that is, (i) 2-SP buffer tubes, instead of the Amplicor sampling tubes (Roche Diagnostics), were used for collection of swab samples and (ii) DNA was isolated using automation, i.e. MagNA Pure LC (Roche Molecular Biochemicals, Mannheim, Germany), instead of manual preparation. Nevertheless, both these modifications have been effectively used and evaluated in previous studies and, especially, the latter one can certainly be an optimization that results in an increased sensitivity, substantially decreased rate of amplification inhibition, less labour intensive, etc.13–17

The aim of the present study was to evaluate the performance characteristics of five PCRs and a recently introduced nucleic acid sequence-based amplification (NASBA) assay currently used to diagnose C. trachomatis infection in Russia. The following NAATs were evaluated: conventional and real-time PCRs developed at the DNA Technology company, Moscow, Russia (cPCR-DT and rtPCR-DT); conventional PCR developed at the Lytech company, Moscow, Russia (cPCR-Ly); conventional and real-time PCRs developed at the Central Research Institute of Epidemiology, Moscow, Russia (cPCR-Ep and rtPCR-Ep); and real-time NASBA assay based on NucliSens Technology (bioMérieux, Boxtel, the Netherlands), with the target-specific oligonucleotides developed at the Central Research Institute of Epidemiology (Table 1). The Cobas Amplicor PCR (Roche Diagnostics), as the main ‘gold standard’ method, and LightMix 480HT PCR (TIB MOLBIOL GmbH) were used as reference tests.

Table 1.  Characteristics of five PCR assays and one real-time NASBA assay developed by three Russian companies and presently widely used in Russia for routine diagnosis of C. trachomatis
CharacteristicAssaya
cPCR-DTrtPCR-DTcPCR-LycPCR-EprtPCR-EpNASBA
  • a

    cPCR-DT and rtPCR-DT, conventional and real-time PCR manufactured by DNA Technology, Moscow, Russia; cPCR-Ly, conventional PCR manufactured by Lytech, Moscow, Russia; cPCR-Ep and rtPCR-Ep, conventional and real-time PCR manufactured by the Central Research Institute of Epidemiology, Moscow, Russia; NASBA assay developed by the Central Research Institute of Epidemiology, Moscow, Russia.

  • b

    Fluorescence reading steps.

  • c

    C. trachomatis specific probe/IC specific probe.

Genetic targetCryptic plasmid16S rRNA geneCryptic plasmidCryptic plasmidCryptic plasmid16S rRNA
Nucleic acid isolation technologyDNA-EXPRESS (Lytech)DNA-EXPRESS (Lytech)DNA-EXPRESS (Lytech)DNA-sorbA (Central Research Institute of Epidemiology)DNA-sorbA (Central Research Institute of Epidemiology)NucliSens Isolation Reagents (bioMérieux)
Internal Control (IC)Incorporated in the reaction mixtureIncorporated in the reaction mixtureIncorporated in the reaction mixtureAdded before DNA isolationAdded before DNA isolationAdded before nucleic acid isolation
Reaction volume (DNA/RNA template)35 (5) µL35 (5) µL25 (5) µL25 (10) µL25 (10) µL20 (5) µL
Amplification apparatusTercyc (DNA technology)iQ iCycler (Bio-Rad, USA)Tercyc (DNA technology)Tercyc (DNA technology)RotorGene (Corbett Research, Australia)NucliSens EasyQ (bioMérieux)
Amplification programHold:94 °C/90 sHold:80 °C/60 sHold:93 °C/30 sHold:95 °C/5 minHold:95 °C/5 minHold:41 °C/90 minb
 5 cycles:94 °C/20 sHold:94 °C/90 s35 cycles:93 °C/10 s42 cycles:95 °C/10 s10 cycles:95 °C/20 s  
  64 °C/5 s5 cycles:94 °C/30 s 60 °C/10 s 65 °C/10 s 65 °C/20 s  
  72 °C/5 s 64 °C/45 s 72 °C/10 s 72 °C/10 s 72 °C/20 s  
 40 cycles:94 °C/5 s2 cycles:80 °C/30 sbHold:72 °C/60 sHold:72 °C/60 s35 cycles:95 °C/20 s  
  64 °C/5 s45 cycles:94 °C/10 s     60 °C/30 sb  
  72 °C/5 s 64 °C/45 sb     72 °C/15 s  
Fluorophores in real-time NAATsc FAM/HEX  FAM/JOEFAM/JOE
Detection limit in PCRs (copies per reaction)2–44–64–62–42–4 

Materials and methods

Study population

Consecutive symptomatic female and male patients attending three youth centres in St. Petersburg, Russia from November 2006 to January 2007 were enrolled. All patients who had taken antibiotics within the past 4 weeks at the time of the study were excluded. Thus, the final sample included 446 patients (319 females and 127 males) who completed a questionnaire regarding age, gender and symptoms. All participating patients were clinically examined and had their laboratory samples taken. The mean age of the females was 20.7 years (range 15–30 years) and the mean age of the males was 20.8 years (range 15–27 years). From 21 of the female patients, only cervical swabs were obtained. Thus, a total of 871 samples were investigated.

Ethics

The present study was approved by the Ethics Committee at the D.O. Ott Research Institute of Obstetrics and Gynaecology, St. Petersburg, Russia.

Specimen collection and processing

Two cervical and two vaginal samples were randomly collected from each female patient and two urethral and one first void urine (FVU) sample from each male patient. All samples were collected; and DNA was extracted, analysed and interpreted according to the manufacturer's instructions. The cervical, vaginal and urethral samples to be analysed using cPCR-DT, rtPCR-DT and cPCR-Ly were collected with Dacron swabs that were directly placed in 200 µL of DNA-EXPRESS reagent (Lytech, Moscow, Russia). The genital samples to be analysed using cPCR-Ep, rtPCR-Ep, NASBA and Cobas Amplicor PCR were collected with Dacron swabs and placed in 1 mL of 2-SP buffer (0.2 m sucrose, 0.02 m phosphate). The FVU samples were collected in 25-mL containers and then shipped at ambient temperature within 4 h to the Laboratory of Microbiology, D.O. Ott Institute, St. Petersburg. From each cervical sample and FVU sample, representing ‘gold standard’ specimens for diagnosis, 500 µL aliquots were, after thorough vortexing, transported frozen to the Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden for analysis using Cobas Amplicor PCR and LightMix 480HT PCR. Furthermore, the vaginal and urethral samples requiring discrepancy analysis were also investigated in Sweden using the same two genetic assays. Discrepant vaginal and urethral samples referred to the samples that were positive in one or several Russian NAATs (but not all of them), or that were negative but the corresponding patients were positive in the ‘gold standard’ specimens.

Nucleic acid isolations

DNA-EXPRESS.  cPCR-DT, rtPCR-DT and cPCR-Ly were run on DNA templates obtained by DNA-EXPRESS technology (Lytech, Moscow, Russia), a technology based on thermocoagulation conditioning of DNA. In short, the tubes containing genital swab samples in the DNA-EXPRESS reagent were vortexed for 15 s, incubated at 98 °C for 10 min and finally centrifuged at 13 000 × g for 15 s. The supernatant was immediately used as a template in the PCRs.

FVU (10 mL) was centrifuged at 3000 × g for 15 min, the supernatant was discarded and 500 µL of the remaining pellet (urine, possible cells, etc.) were transferred to a 1.5 mL tube. After centrifugation of the concentrated FVU at 13 000 × g for 15 s, the supernatant was discarded and 200 µL of DNA-EXPRESS reagent were added. Subsequently, the sample was processed according to the procedure described above for genital swabs.

DNA-sorbA.  For cPCR-Ep and rtPCR-Ep, DNA was isolated using a silica-based kit (DNA-sorbA; the Central Research Institute of Epidemiology). Briefly, samples were vortexed, and then 100 µL of each sample were mixed with 300 µL of the lysis buffer and 10 µL of the internal control (IC). After vortexing and centrifugation of the mixture for several seconds, 20 µL of silica solution were added to each sample, and the mixtures were incubated at room temperature for 10 min, with vortexing every 2 min. Subsequently, the samples were centrifuged at 12 000 × g; the pellets were washed twice with 1 mL of 70% ethanol and dried at 65 °C for 10 min. Finally, DNA was eluted in 100 µL of Tris-EDTA buffer and immediately analysed in the PCRs.

FVU (1 mL) was centrifuged at 12 000 × g for 15 min; the supernatant was discarded; and 100 µL of the remaining pellet (urine, possible cells, etc.) were processed according to the procedure described above for genital swabs.

Nucleic acid isolation for NASBA.  Nucleic acid was isolated using the NucliSens Isolation Reagents (bioMérieux) according to the procedure described in the NucliSens Basic Kit application manual. In brief, samples were vortexed, and 100 µL of each sample were mixed with 900 µL of the lysis buffer and 10 µL of the IC. This solution was vortexed and briefly centrifuged, where 50 µL of the silica solution were added, and the mixture was incubated for 10 min, with vortexing every 2 min. The pellet was washed once with 1 mL of the wash buffer, twice with 1 mL of 70% ethanol and once with 1 mL of acetone. Finally, after drying the pellet at 56 °C for 10 min, the nucleic acid was eluted in 50 µL of the elution buffer and immediately analysed in the NASBA.

FVU (1 mL) was centrifuged at 12 000 × g for 15 min; the supernatant was discarded; and 100 µL of the remaining pellet (urine, possible cells, etc.) were processed in accordance with the procedure described above for genital swabs.

DNA Isolation for the Cobas Amplicor PCR and LightMix 480HT PCR.  After thorough vortexing, 200 µL of each sample was used for DNA isolation with magnetic silica particles in a robotized system (MagNA Pure LC DNA Isolation kit I in MagNA Pure LC instrument) using an elution in 100 µL of the elution buffer, which was subsequently diluted with 100 µL of the Amplicor specimen diluent before 50 µL of this mixture was used as template in the PCRs. This was performed in accordance with the instructions of the manufacturer (Roche Molecular Biochemicals), and as previously described and evaluated.13 The DNA preparations were stored at 4 °C prior to PCR, which was performed within one day.

NAATs

Russian PCRs.  The main characteristics of the Russian NAATs, which were all performed in strict accordance with the instructions of the manufacturer, are presented in Table 1. For the PCRs, optimized mastermixes, including Taq DNA polymerases, buffer, primers and dNTPs, were supplied by the manufacturers. A positive control (DNA isolated from the C. trachomatis reference strain D/UW-3/Cx [ATCC VR-885]) and a negative control (distilled water) were included in each PCR run.

The amplified products in the conventional PCRs were visualized after electrophoresis through a 1.5% agarose gel containing ethidium bromide. The Gene Ruler DNA Molecular Weight Marker (Fermentas, Kaunas, Lithuania) was included in each electrophoresis for determination of the sizes of the amplicons.

In cPCR-DT, rtPCR-DT and cPCR-Ly the IC was included in the mastermix in order to identify the presence of inhibitors. Samples were processed according to the manufacturer's instructions in case of inhibition. Accordingly, 100 µL of the initially processed inhibited sample were added to 200 µL of DNA-EXPRESS reagent. Thereafter, the mixture was vortexed for 15 s, incubated at 98 °C for 10 min, centrifuged at 13 000 × g for 15 s, and finally, the supernatant was used as a template in a new PCR.

In cPCR-Ep and rtPCR-Ep, the IC was added before DNA isolation in order to identify inhibition as well as to control DNA extraction efficiency. If the IC was not amplified, a new portion of the sample was re-tested after a 1 : 10 dilution in 2-SP buffer.

Real-time NASBA.  NASBA was performed using the NucliSens Basic kit (bioMérieux) and Russian-developed target-specific oligonucleotides (the Central Research Institute of Epidemiology) according to the procedure described in the NucliSens Basic kit application manual. Briefly, the reactions were run in a total volume of 20 µL containing 5 µL of purified nucleic acid, 5 µL of the enzyme mix and 10 µL of the amplification mix. The enzyme mix, containing T7 RNA polymerase, avian myeloblastosis virus reverse transcriptase, RNase H and bovine serum albumin, was added to the mixture during an incubation for 2 min at 41 °C, which was followed by a heat denaturation of the target RNA (2 min at 65 °C). The NASBA reactions with real-time detection of amplified products were run at 41 °C for 90 min in a NucliSens EasyQ Analyser (bioMérieux). Samples were considered positive if the relative fluorescence signal (RFS) of the C. trachomatis–specific probe displayed an increase of ≥ 1.18 as compared with the background. The results were considered invalid if the increase of the RFS with the C. trachomatis probe and IC specific probe was less than 1.18 and 1.11, respectively. Finally, samples were considered as negative if the increase of the RFS with the C. trachomatis probe was less than 1.18 but the RFS with the IC probe was ≥ 1.11, i.e. the sample was not inhibited. In this case a new aliquot of the sample was re-tested after diluted 1 : 10 in 2-SP buffer.

Reference methods: Cobas Amplicor PCR (Roche Diagnostics) and LightMix 480HT (TIB MOLBIOL GmbH).  For all cervical samples from females, all FVUs from males as well as all discrepant female vaginal and male urethral samples, the Cobas Amplicor PCR, targeting the cryptic plasmid, was performed as the main ‘gold standard method’, according to the manufacturer's instructions (Roche Diagnostics). Furthermore, all these samples were also analysed using the quantitative real-time PCR assay LightMix 480HT (TIB MOLBIOL), which targets a 136-bp fragment of the omp1 gene, on a LightCycler 480 Instrument (Roche Diagnostics), as previously described.18 This assay was used to (i) assure that none of the Cobas Amplicor–negative samples was due to the recently described new Swedish variant of C. trachomatis,18 and (ii) include a reference method using a different target than Cobas Amplicor PCR. Both the reference methods used their specific inhibition controls according to the instructions of the manufacturer.

Interpretation of results

All positive samples in each NAAT were confirmed by repeated testing. If discrepancies between initial and repeated testing would have been identified, the sample should have been analysed once more and if two of the three replicates would have been positive the sample should have been regarded as positive. A sample was regarded as true positive if the positive result with any of the Russian assays was confirmed using both reference methods (i.e. Cobas Amplicor PCR and LightMix 480HT PCR), only Cobas Amplicor PCR, and also if only LightMix 480HT PCR was positive and C. trachomatis was detected by all the Russian assays. However, this latter scenario was not observed in the present study.

Determination of the detection limits of the PCRs developed in Russia

The detection limits of the Russian PCRs were determined using Chlamydia trachomatis DNA control (VIRCELL, Santa Fe, Spain), in triplicates and 10- and 2-fold dilution series.

Results

In total, according to both the reference methods (i.e. Cobas Amplicor PCR and LightMix 480HT), 40 females (12.5%) and 16 males (12.6%) were C. trachomatis positive. Furthermore, based on the results from the LightMix 480HT, none of the Cobas Amplicor–negative samples was due to the recently described new Swedish variant of C. trachomatis.

Concordance of the six Russian NAATs and the reference methods

In general, all the Russian NAATs and the reference methods displayed a high level of concordance. Thus, the concordance for cervical, vaginal, urethral and FVU was 98.1%, 97.9%, 99.2% and 98.4%, respectively. Consequently, only six cervical samples, six vaginal samples, one urethral sample and two FVUs displayed any discrepant results. According to the reference methods, 3 false-positive and 12 false-negative samples were identified in the Russian NAATs (Table 2).

Table 2.  Discordant results using five PCR assays and one real-time NASBA assay developed and presently widely used in Russia in the diagnosis of C. trachomatis. Roche Cobas Amplicor PCR and LightMix 480HT (TIB MOLBIOL Gmbh) were used as reference methods
  cPCR-DTartPCR-DTacPCR-LyacPCR-EpartPCR-EpaNASBAaCobas Amplicor and LightMix 480HTNumber of samples
  • a

    cPCR-DT and rtPCR-DT, conventional and real-time PCR manufactured by DNA Technology, Moscow, Russia; cPCR-Ly, conventional PCR manufactured by Lytech, Moscow, Russia; cPCR-Ep and rtPCR-Ep, conventional and real-time PCR manufactured by the Research Institute of Epidemiology, Moscow, Russia; NASBA assay developed by the Research Institute of Epidemiology, Moscow, Russia.

  • + positive; – negative.

WomenCervix++++1
 +++1
 +++1
 +3
Vagina++++2
 +++1
 +3
MenUrethra+++++1
Urine++1
 ++1

Sensitivity and negative predictive value of the Russian NAATs

The sensitivities of the Russian tests in different specimens ranged from 86.1% to 100% (Table 3). Briefly, in females the highest sensitivities were exhibited by DNA Technology PCRs (92.3% and 91.7% in cervical and vaginal samples, respectively) and NASBA (89.7% in the cervix and 91.7% in the vagina). The sensitivities in males were slightly higher, ranging from 92.3% to 100%. NASBA was the most sensitive test for both urethral and FVU samples, but the PCRs manufactured by Lytech and the Central Research Institute of Epidemiology also displayed 100% sensitivity for urethral specimens. The negative predictive values ranged from 98.2% to 100%.

Table 3.  Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of five PCR assays and one real-time NASBA assay developed and presently widely used in Russia in the diagnosis of C. trachomatis [reference methods were Roche Cobas Amplicor and LightMix 480HT (TIB MOLBIOL, GmbH)]
SpecimenAssayaSensitivity (%)Specificity (%)PPV (%)NPV (%)
  • a

    cPCR-DT and rtPCR-DT, conventional and real-time PCR manufactured by DNA Technology, Moscow, Russia; cPCR-Ly, conventional PCR manufactured by Lytech, Moscow, Russia; cPCR-Ep and rtPCR-Ep, conventional and real-time PCR manufactured by the Central Research Institute of Epidemiology, Moscow, Russia; NASBA assay developed by the Research Institute of Epidemiology, Moscow, Russia.

Women
 Cervix (n = 319)cPCR-DT92.399.697.398.9
rtPCR-DT92.399.697.398.9
cPCR-Ly87.299.697.198.2
cPCR-Ep87.210010098.2
rtPCR-Ep87.210010098.2
NASBA89.710010098.6
 Vagina (n = 298)cPCR-DT91.799.697.198.9
rtPCR-DT91.799.697.198.9
cPCR-Ly86.199.696.998.1
cPCR-Ep86.110010098.1
rtPCR-Ep86.110010098.1
NASBA91.710010098.9
Men
 Urethra (n = 127)cPCR-DT92.910010099.1
rtPCR-DT92.910010099.1
cPCR-Ly100100100100
cPCR-Ep100100100100
rtPCR-Ep100100100100
NASBA100100100100
 Urine (n = 127)cPCR-DT92.399.192.399.1
rtPCR-DT92.399.192.399.1
cPCR-Ly92.310010099.1
cPCR-Ep92.310010099.1
rtPCR-Ep92.310010099.1
NASBA100100100100

Sensitivity of the Russian NAATs using different specimens

To calculate the sensitivities in females when testing cervical and vaginal samples separately, only the subset of females with both specimens were included (n = 298). In one female, C. trachomatis was only found in the vagina by all the Russian tests. Consequently, testing only cervical and only vaginal samples resulted in a detection rate of 97.2% (35 of 36) and 100% (36 of 36), respectively. In two males, C. trachomatis was detected in the urine only, and in three males, only the urethral sample was C. trachomatis positive. Accordingly, when only urethral swabs and only FVUs in males were analysed separately, the rate of detection of true C. trachomatis infection was 87.5% (14 of 16) and 81.3% (13 of 16), respectively.

Specificity and positive predictive value of the six Russian NAATs

The specificities of the Russian tests ranged from 99.1% to 100% and the positive predictive values ranged from 92.3% to 100% (Table 3).

Inhibition of the Russian NAATs

In total, specimens (n = 25) from eight females and seven males were found to comprise amplification inhibitory substances (Table 4). Briefly, the highest inhibition rates (25 of 871 samples, or 2.9%), which were most pronounced for urethral samples (5.5%), were displayed by the conventional PCRs that use DNA extracts obtained by the DNA-EXPRESS technology (i.e. cPCR-DT and cPCR-Ly). However, inhibition was rare in the rtPCR-DT, also using DNA-EXPRESS technology, and especially using silica-based nucleic acid isolation techniques (i.e. DNA-sorbA and NucliSens Isolation Reagents; Table 4). All inhibited samples were resolved by retesting after reprocessing or dilution (i.e. confirmed as true negatives). For comparison, using MagNA Pure LC DNA isolation and subsequently Cobas Amplicor PCR or LightMix 480HT PCR, none of the examined samples was inhibited.

Table 4.  Frequency of inhibition in five PCR assays and one real-time NASBA assay developed and presently widely used in Russia in the diagnosis of C. trachomatis. The inhibition was identified using the internal controls supplied by the manufacturer of each assay
Nucleic acid extraction methodAssayaNumber of invalid results (%)
WomenMen
Cervix (n = 319)Vagina (n = 298)Urethra (n = 127)Urine (n = 127)
  • a

    cPCR-DT and rtPCR-DT, conventional and real-time PCR manufactured by DNA Technology, Moscow, Russia; cPCR-Ly, conventional PCR manufactured by Lytech, Moscow, Russia; cPCR-Ep and rtPCR-Ep, conventional and real-time PCR manufactured by the Central Research Institute of Epidemiology, Moscow, Russia; NASBA assay developed by the Central Research Institute of Epidemiology, Moscow, Russia.

DNA-EXPRESScPCR-DT8 (2.5%)8 (2.7%)7 (5.5%)2 (1.6%)
rtPCR-DT1 (0.3%)2 (0.7%)2 (1.6%)0
cPCR-Ly8 (2.5%)8 (2.7%)7 (5.5%)2 (1.6%)
DNA-sorbAcPCR-Ep02 (0.7%)00
rtPCR-Ep02 (0.7%)00
NucliSens isolation reagentsNASBA1 (0.3%)1 (0.3%)00

Detection limits of the Russian PCRs

The detection limits of cPCR-DT, rtPCR-DT, cPCR-Ly, cPCR-Ep and rtPCR-Ep were, in the order given, 2–4, 4–6, 4–6, 2–4 and 2–4 genome copies per reaction (Table 1).

Discussion

The present study is the first ever comprehensive evaluation of the performance characteristics of any Russian C. trachomatis NAATs to internationally well-recognized and validated C. trachomatis NAATs [i.e. the FDA-approved Cobas Amplicor PCR (Roche Diagnostics) and LightMix 480HT PCR (TIB MOLBIOL GmbH)]. Notably, the reference methods displayed identical sensitivities, which was somewhat surprising due to the fact that the Cobas Amplicor PCR uses a multicopy target (the cryptic plasmid) and LightMix 480HT only a single-copy target (the omp1 gene), which theoretically should result in a lower sensitivity. The six evaluated Russian NAATs proved to be relatively sensitive and specific in the detection of C. trachomatis in both invasive and non-invasive genital samples. In particular, the NASBA appeared exceedingly promising, i.e. combined a high sensitivity (ranged from 89.7% to 100%) with a high specificity (100%) for all specimens. However, larger studies, including a larger sample of symptomatic and asymptomatic patients as well as both invasive and non-invasive genital and extra-genital samples, are crucial. Furthermore, ideally, these studies should be evaluated in relation to several of the international well-recognized, validated and FDA-approved C. trachomatis NAATs, performed in strict accordance with the instructions of the manufacturer.

Presently, nationally developed NAATs are the primary means used in the detection of C. trachomatis in routine clinical practice in Russia. This is because of the substantially higher costs of the international commercially available C. trachomatis NAATs. In a recent study,10 a cell culture method, two divergent DIFs and three in-house PCRs used in Russia in the diagnosis of C. trachomatis were compared. The sensitivities of the cell culture method and the DIFs were highly suboptimal, and hence, these methods should not be recommended for the purpose of diagnosis. The sensitivities of the PCRs varied significantly (i.e. from 79% to 100%) among the different assays, gender of the patients and types of sample. However, the specificities of all the PCRs were high, varying from 97% to 100%. It was concluded that, at least from a Russian perspective, adequate in-house PCR methods may be used quite effectively in the detection of C. trachomatis.

The results of the present study indicate that the NAATs used in Russia for C. trachomatis detection have relatively good performance characteristics (i.e. reasonable sensitivity and specificity for both invasive and non-invasive samples). The sensitivities of the Russian NAATs were somewhat higher in males (92.3–100%) than in females (86.1–92.3%). Furthermore, in agreement with previous findings,19–23 the vaginal sample was at least as good as the cervical swab for detecting chlamydial infection by NAATs. Similarly, in males, the results using urine samples were nearly identical to those obtained using urethral swabs, which is also consistent with many studies.24–30 In comparison with a recent systematic review assessing the sensitivity and specificity of PCR, TMA and SDA assays in the diagnosis of C. trachomatis in females and males,3 the sensitivities and specificities of the Russian PCRs and NASBA, a transcription-based amplification technique, were comparable with international commercially available PCRs and the transcription-based method TMA.

The overall prevalence of genital chlamydial infection in the investigated population (i.e. symptomatic attendees of youth centres) was 12.6% (56 of 446). When the results of testing separately cervical and vaginal samples in females and urethral and FVU samples in males were compared with patients defined as infected, the test sensitivities for the individual specimen types ranged from 81.3% (FVUs) to 100% (vaginal samples). It has previously been confirmed that testing multiple specimens tends to detect a higher number of infections.10,20,27,30,31 Simultaneous analysis of two sample in the present study resulted in a slight increase (2.8%) in the detection rate of C. trachomatis infection in females and an 18.7% increase in males.

To ensure that amplification can be performed in each specimen all the evaluated assays in the present study incorporated ICs. The frequency of inhibition varied over a substantial range though this variation depended on the specimen type, the DNA/RNA extraction technique and the NAAT used. The inhibition frequency was slightly higher for urethral (0–5.5%) and vaginal (0.3–2.7%) samples than for cervical swabs (0–2.5%) and FVU samples (0–1.6%). These findings are, in general, consistent with previous reports.32–35 Nevertheless, using the reference methods, none of the samples was inhibited. This is probably due to the fact that MagNA Pure LC was used for automated DNA isolation, which has previously been shown to increase the sensitivity and significantly lower the rate of inhibition.13,14,16,17 However, in the present study, the Russian NAATs that used silica-based nucleic acid isolation techniques had significantly lower inhibition rates. Moreover, in the NAATs developed by the Central Research Institute of Epidemiology (conventional and real-time PCR, as well as real-time NASBA), the ICs are added before the nucleic acid isolation that is highly recommended, in order to examine both the inhibition of amplification and the efficiency of DNA/RNA extraction.

Numerous studies have shown that, because of their high sensitivity and specificity, NAATs can be highly suitable in the screening for C. trachomatis even when only non-invasive specimens are used.3,36–39 Accordingly, after comprehensive assessment and validation of the Russian NAATs used in the diagnosis of C. trachomatis, finally, it will become possible to provide reliable figures on the incidence and prevalence of chlamydial infection in Russia and perhaps even in some of the other East European countries, which are utilizing those Russian NAATs. In addition, these NAATs may provide the necessary prerequisites for performance of epidemiological surveillance, using cost-effective pooling of samples in many of these geographical areas.11,40

Conclusions

All six evaluated Russian NAATs proved to be relatively sensitive and specific in the detection of C. trachomatis in both invasive and non-invasive samples. However, improvements of at least a few of them, especially in terms of sensitivity, may still be crucial. Further evaluation is also needed to thoroughly assess the performance of the NAATs in larger populations. This evaluation should be done in comparison with also other international well-recognized, validated, and ideally FDA-approved C. trachomatis NAATs, performed in strict accordance with the instructions of the manufacturer. It is hoped that the present and future studies can form the basis for recommendations regarding accurate, effective and quality assured diagnosis of C. trachomatis infection in Russia and as a basis for adequate evaluation of many diagnostic assays used in East European countries.

Acknowledgements

We thank the personnel at the youth centres for collecting samples and relevant patient data. We are especially grateful to Marina Ippolitova (the youth centre of Petrogradsky District), Tatyana Trubetskaya (the youth centre of Krasnogvargeysky District) and Olga Landina (the central youth clinic ‘Juventa’) for organizing the collection and transportation of the samples. The present study was performed as one of several projects with the aims of optimizing, harmonizing and quality assuring the STI diagnostics in Eastern Europe and was organized by the Eastern European Network for Sexual and Reproductive Health (EE SRH Network), which is supported by grants from the East Europe Committee of the Swedish Health Community, Stockholm, Sweden.

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