Molecular diagnostics

The second generation TMA. The Gen-Probe APTIMA-Combo 2.

Principles of the test.

Fig 1. Second generation TMA nucleic acid amplification test in pictures (double click on each slide; approx 100 kb)


apt.GIF (96016 bytes)


tcdiag.GIF (112004 bytes)


tcpict.GIF (124254 bytes)

Fig 1a. The second generation test combines the technologies of target capture, TMA and Dual Kinetic Assay (DKA).

All these figures Gen-Probe Incorporated from their CD-ROM "Advances in molecular diagnostics"

Fig 1b. Diagram of target capture. Specific capture oligonucleotides, in solution, contain sequences complementary to the target rRNA and hybridise at 62C. When the temperature is decreased to room temperature the capture complex with its poly dA tail binds to poly dT covalently attached to the magnetic particles. Fig 1c. Illustration of the magnetic particles with captured target. Simple magnetic separation removes the potential amplification inhibitors from the reaction. After washing, the target is ready for amplification via TMA.


tcadv.GIF (94890 bytes)


tmaall.GIF (92901 bytes)


dk.GIF (97378 bytes)

Fig 1 d. Summary of the advantages of target capture. Fig 1 e. Transcription mediated amplification occurs as described before, but the subsequent detection step is different, employing a dual kinetic assay. Fig 1 f. In the DK assay, dual probes are used with slightly different kinetics of light emission as shown here. The fast 'flasher' probe is specific for CT, the slower 'glower' for GC. The luminometer plus software calculate the relative contribution of each..

The detection system is the Dual Kinetic Assay.

Click here for a Gen-Probe audio video presentation on target capture.

One advantage of the test is that the specimens, once in the transport media, are stable at environmental temperatures. Recommended storage for swab specimens are 2 to 30C for up to 60 days before testing, and for urine specimens 2 to 30C for up to 30 days [Manufacturer's package insert].

APTIMA-Combo 2: Clinical trial results & comparison with other tests

In their package insert [dated June 6th., 2001 p15] the manufacturers give the following information:

Performance characteristics were established in a multicenter study at seven geographical locations and they included, for C. trachomatis, PCR and LCx nucleic acid amplification tests. Swab and urine specimens were evaluated from 1,363 male and 1, 569 subjects attending STD, family planning or gynecology clinics. As many as three urethral swabs and a urine specimen were collected from males and up to four endocervical swabs and a urine specimen from females. Some pregnant women were also evaluated. For C. trachomatis, any two positive comparator assay results by any combination of swab and urine designated the subject as infected. If only one result was positive, the infection status was deemed inconclusive. Overall sensitivity and specificity for C. trachomatis in all specimens was approximately 95.8 and 98.2% respectively; for N. gonorrhoeae the corresponding results were 97.8% and 98.9%.

Martin et al., 2001 compared the sensitivity and specificity of the Gen-Probe APTIMA Combo 2 test, the Abbott LCx and the Roche AMPLICOR CT on 2,932 patients with suspected urogenital infection collected from seven clinical sites in the United States, using both first catch urine and urethral or endocervical swabs as appropriate. Sensitivities and specificities for C. trachomatis were as follows:

Sensitivity and Specificity of 3 Nucleic acid amplified tests for C. trachomatis infection

Swab

Urine

Women Combo 2 LCx AMPLICOR Combo 2 LCx AMPLICOR
Sensitivity 94.2 85.6 86.4 95.3 84.1 81.7
Specificity 97.6 99.4 99.1 98.9 99.4 99.4
Men
Sensitivity 96.9 91.1 nd 97.9 89.1 nd
Specificity 97.5 99.2 nd 98.5 99.9 nd

Combo 2 was more sensitive than LCx or AMPLICOR for C. trachomatis in men and women but the specificities of LCx and AMPLICOR were slightly higher than the APTIMA 2 unless the latter, being more sensitive, identified more truly infected patients.

Moncada et al., 2001 looked at the effect of urine testing in the evaluation of the sensitivity of the APTIMA Combo 2 assay for C. trachomatis on cervical swabs. 1411 patients with complete specimen and test results were enrolled in the study. The performance on cervical swabs is shown in the following table:

CT Sensitivity

CT Specificity

Test Specimen Infected Patient Specimen Infected Patient
APTIMA Combo 2 99.4%
(182/183)
92.1%
(186/202)
97.4%
(1196/1228)
97.7%
(1181/1209)
Abbott LCx 95.6%
175/183
86.6%
(175/202)
99.4%
(1221/1228)
99.4%
(1202/1209)
Roche AMPLICOR 95.6%
(175/183)
87.1%
(176/202)
99.3%
(1219/1228)
99.3%
(1201/1209)

There were 183 positive cervix specimens. APTIMA COMBO 2, LCx and PCR had 32, 7 and 9 cervical positives unique to each test. Since true positives were defined as specimens being positive by two different tests, these were categorized as false positives. However 5 of these specimens were also confirmed positive by urine testing. An additional 14 specimens positive only on urine were also identified, making a total of 19 positives. These reduced the sensitivity of cervical testing by 7-9% per test. By infected patient status there were thus 183 + 19 = 202 positives of whom 88.6% were positive on both cervix and urine, 4.5% on cervix only and 6.9% on urine only. Overall the sensitivity of the APTIMA Combo 2 was higher than that of the other tests, but alternate amplification tests would be required to determine the true specificity status of the test. In this respect the findings were similar to those of Martin _ et al _ 2001.

In a multi-centre study, Ferrero et al., 2001 collected endocervical swabs and matched urine from 1391 women patients plus urethral swabs and urine from 1095 male patients. Comparator tests were the Abbott LCx and the Roche COBAS AMPLICOR. A patient was defined as infected when two or more comparator assays were positive. For the diagnosis of C. trachomatis on female urine, the sensitivity and specificity of the APTIMA Combo 2 assay was 94.7% and 98.9% respectively, the negative predictive value was 99.1% and the positive predictive value was 93.8% (15% prevalence). For male urine the comparable results were sensitivity and specificity 97.9% and 98.9% respectively, the negative predictive value was 99.3% and the positive predictive value was 95.8% (25.8% prevalence). It was concluded that the new test had comparable performance to current FDA cleared NAATs. Combo 2 was particularly good for urine testing. The test was seen as potentially useful for urine screening programmes on asymptomatically infected patients.

A small study by Turner et al., 2001 was also reported at Berlin on 275 symptomatic and asymptomatic males and females attending either an STD or family planning clinic in Texas. Test results on swabs and urine were compared with chlamydial culture, LCx or COBAS AMPLICOR PCR. Twenty four of the 275 patients were excluded for various reasons. Patients were considered as infected if either culture or two amplification assays were positive. On this basis 38 of 251 patients were positive in the study, an overall prevalence of 15%. 26 of these 38 patients (68.4%) were positive by culture. The results were as follows:

Sensitivity, specificity, positive and negative predictive values and efficiency of three nucleic acid amplification tests for C. trachomatis. Combined results for swabs and urine.
Test

Sensitivity
%
swab / urine

Specificity
%
swab / urine
PPV
%
swab / urine
NPV
%
swab / urine
Efficiency
%
swab / urine
APTIMA Combo 2
COBAS PCR
LCx

100 / 92.1
92.1 / 92.1
94.7 / 81.5

99.1 / 98.6
96.2 / 97.7
100 / 99.5

95.0 / 92.1
81.4 / 87.5
100 / 96.9
100 / 98.6
98.6 / 98.6
99.1 / 96.8
99.2 / 97.6
95.6 / 96.8
99.2 / 96.8

It was concluded that the APTIMA Combo 2 assay for C. trachomatis performed at least as well as the two comparator assays, with excellent accuracy on both swabs and urines.

Bott et al., 2001 (Gen-Probe, San Diego) reported the analytical sensitivity of the APTIMA Combo 2 to be of the order of 0.01 IFU (inclusion forming units) / assay for C. trachomatis and 50 gonococcal cells / assay for N. gonorrhoeae. There was no impact of high levels of one target pathogen on the detection of low levels of the other target. Multiple strains of 17 Neisseria species were tested, including those known to cause cross reactions with other commercial nucleic acid amplification tests, and there were no non specific reactions. Anti-fungal agent, lubricants, anti itch cream, feminine spray, blood or white blood cells did not interfere with specific detection. No amplification inhibitors were detected among 409 swabs and 482 urines from known infected patients.

Fuller et al., 2002 (Indiana, USA) compared the APTIMA Combo 2 and BDProbetec™ assays for their ability to detect C. trachomatis and N. gonorrhoeae in urine and endocervical swabs from 460 women. A patient was considered positive if any combination of at least two of the four specimens (2 endocervical and 2 urines) was positive. The resolved combined CT/GC sensitivity for the APTIMA Combo 2 assay was 98% (61/62) for both swab and urine specimens, with a specificity of 99.8% for swabs and 100% for urine. The resolved combined CT/GC sensitivity for the BDProbeTec™ET was 96.8% (61/63) for swab and specimens and 89.7% (61/68) for urine specimens. Specificity of the BDProbetec was 99.8% for swabs and 100% for urine. The initial observed rate of amplification inhibition was 7.2% for the BDProbetec. For the Combo 2 assay 1.1% of the initial test results were equivocal, all of which were resolved as negative on repeat testing. It was concluded that for female urine specimens, the APTIMA Combo 2 test had better performance than the BDProbetec for the detection of C. trachomatis and N. gonorrhoeae and equivalent performance on endocervical swabs.

A small study by Martin et al., 2002 (New Orleans, USA) compared the APTIMA Combo 2 with LCx and BDProbeTec on urine and swab specimens from an initial 290 female patients. They used a rotating assay standard, whereby the swab and urine CT and GC results of each of three NAATs were compared to an infected patient standard based on the other two NAATs for each organism in separate analyses. The infected patient standard was defined as any two positive specimens by either of the two comparator NAATs. If only one of the comparator assays was positive the results were deemed inconclusive and were excluded. Single assay standards were also employed in which the performance of the APTIMA Combo 2 test was compared with either the LCx or BDProbetec. The results are summarized in the tables:

Table 1. Results of all three NAATs based on the rotating standard.

Organism

and test

Sensitvity %

Specificity %

Swab Urine Swab Urine
C. trachomatis
Combo 2
ProbeTec
LCx

91.5
81.5
80.3

94.9
78.5
74.2

94.6
100
100

99.0
99.5
100
N. gonorrhoeae
Combo 2
ProbeTec
LCx

100
96.4
92.7

100
94.5
89.1

99.0
99.5
98.6

100
100
100
Table 2. APTIMA Combo 2 compared to ProbeTec based on an LCx single test standard.
Organism

and test

Sensitvity %

Specificity %

Swab Urine Swab Urine
C. trachomatis
Combo 2
ProbeTec

91.8
85.2

93.4
82.0

93.8
99.5

97.1
98.1
N. gonorrhoeae
Combo 2
ProbeTec

94.7
91.2

94.7
89.5

98.6
99.1

99.5
99.5

Table 3. APTIMA Combo 2 compared to LCx based on a ProbeTec single test standard.

Organism

and test

Sensitvity %

Specificity %

Swab Urine Swab Urine
C. trachomatis
Combo 2
LCx

87.5
81.3

93.8
76.6

93.7
99.0

98.6
100
N. gonorrhoeae
Combo 2
LCx

98.2
91.1

98.2
87.5

99.1
98.6

100
100

The authors concluded:

  • Combo 2 was consistently more sensitive than LCx and BDProbeTec for both CT & GC, with the difference greatest for CT.
  • Specificity of Combo 2 was lower for CT than the other two assays for endocervical swabs. Whether this was due to carry over contamination or to an ability of Combo 2 to detect further infected patients was not resolved.
  • The Combo 2 assay, unlike LCx or BDProbeTec, performed consistently better on urine specimens than on endocervical swabs.

Ferrero et al., 2002 (San Joaquin, California) compared the performance of the APTIMA Combo 2 with LCx, BDProbeTec and the AMPLICOR PCR on a total of 503 urine specimens collected from 237 female and 266 male patients attending STD or juvenile detention center clinics. Patients were deemed infected if two or more assay results were positive. The overall prevalence of C. trachomatis infection was 7.8% (39/503). When compared to infected patient status, Combo 2 sensitivity and specificity was 100%. On the same basis, the specificity of all the other manufacturer's tests was also 100%. However, the sensitivities for female urine samples varied from 76% (16/21) for LCx to 86% for the BDProbeTec and AMPLICOR. For male urine, the sensitivity was 83% for LCx and AMPLICOR (15/18) to 89% (16/18) for BDProbeTec. There were 90 inhibited specimens detected; 89 for the BDProbeTec assay and 1 for the AMPLICOR assay. Two specimens that were positive by APTIMA Combo 2 but negative by all the other assays were retested by a TMA alternate amplification system and found to be positive. The authors noted that in the Combo 2 FDA clinical trial data the alternate amplification TMA confirmatory CT assay gave positive results for 97% of concordant positive specimens. It was therefore considered that this alternate TMA assay was reliable for resolving discrepant results. Thus the APTIMA Combo 2 was similar to the three other NAATs in specificity, but outperformed them on sensitivity. The high sensitivity of the Combo 2 assay on urine samples versus the other NAATs was particularly useful for screening programs.

Effect of urine inhibitors on the APTIMA Combo 2 assay.

The increased sensitivity of the APTIMA Combo 2 on urine specimens is attributable, at least in part, to the relative lack of female urinary inhibitors for this assay. Chong et al., 2003 collected first void urine specimens from 225 non-pregnant women (submitted for routine analysis to a clinical chemistry laboratory) and from 190 pregnant women attending an obstetrics clinic. The effect of inhibitors was assessed by adding to the urine samples approximately 12 elementary bodies of C. trachomatis serovar L2. In these circumstances the APTIMA Combo 2 test and the Abbott Chlamydia LCx test produced false-negative rates of 0.48% (2 of 415 specimens) and 13% (44 of 338 specimens), respectively. Reducing the amount of C. trachomatis added to one EB, a concentration closer to the APTIMA Combo 2 test cut-off, for a subset of 244 urine specimens increased the number of specimens with false-negative results by the APTIMA Combo 2 assay to 7 (2.9%), suggesting, [not surprisingly], that when the amount of C. trachomatis per specimen approaches the threshold detection level, the proportion of false-negative results increases. However, after overnight storage and retesting of the urine specimens either undiluted or diluted 1:4 [procedures known to reduce inhibition by urine inhibitors] there were no APTIMA Combo 2 false-negative results with the stronger inoculum and just 0.8% (2 of 244 specimens) with the weaker inoculum; the false-negative rate of the LCx assay was reduced to 5.4% (18 of 334 specimens). For LCx, nine of the 21 specimens to which EBs were added after processing and all of the 34 urine specimens to which the target was added before processing remained falsely negative on repeat testing at a 1:2 dilution, suggesting that input C. trachomatis DNA was lost during processing by the LCx assay. In contrast, the APTIMA Combo 2 assay had a higher sensitivity and either lost little nucleic acid during processing or demonstrated few problems with inhibitors of transcription-mediated amplification.

To summarise:

  • The APTIMA Combo 2 test was at least 100 fold more sensitive than the LCx test on serial dilutions of EBs;
  • The Combo 2 assay was subject to a much lower prevalence of urinary inhibitors than the LCx. Moreover these inhibitors were easily removed by storage overnight at 4C
  • The APTIMA Combo 2 assay with its greater sensitivity and lower susceptibility to false negatives has the potential to detect more cases of C. trachomatis infection.
  • Gaydos et al., 2003 evaluated the performance of the APTIMA Combo 2 for the simultaneous detection of gonococci and chlamydiae in endocervical swabs and urine from women. The results were compared with the patient infected status derived from other commercial amplification-based tests. Sensitivity and specificity for C. trachomatis in endocervical swabs were 94.2 and 97.6% respectively. Corresponding figures in first catch urine were comparable at 94.7 and 98.9%, respectively. For gonococci the relevant figures were sensitivity and specificity in swabs 99.2 and 98.7%, and in urine 91.3 and 99.3%. It was considered that the APTIMA Combo2 reliably detected both infections in co-infected patients.

    Recent advances

    [PICK The following information was kindly made available by Gen-Probe Inc. and the respective authors].

    Automation

    The FDA have approved a fully automated TIGRIS version of the APTIMA Combo 2 test . Details of the successful automation of the test using the DTS 1600 system, based on a Tecan Genesis 150 pipetting platform have also been presented by Bixby et al., 2003 (poster available Aptimadts.pdf as a pdf).

    Confirmation tests: Scicchitano et al., 2003 have described the described the development of APTIMA Combo 2 confirmatory tests for C. trachomatis and N. gonorrhoeae, using different primers. A high level of agreement with the original test result was obtained (poster available Aptimaconfirm.pdf as a pdf)

    Use with cytology screening.

    Accurate chlamydial and gonococcal diagnosis can be paired with Pap smear testing from a single sample. A poster by Fuller et al., 2003 describes the successful use of the APTIMA Combo 2 assay with the PreservCyt component of the CyTyc Corporation ThinPrep 2000 system for papilloma virus screening (poster available Aptimacytoprep.pdf as a pdf).

    Retesting samples collected for LCx or Probe Tec: In an important study, Cherneskey et al., 2003 and Jang et al., 2003 investigated whether a laboratory wishing to change to the APTIMA Combo 2 test could test specimens which had been originally collected in the LCx or Probe Tec collection systems. Both of the latter transport systems were found to be compatible with the APTIMA Combo 2 test (posters available retesting.pdf and Jang_ASM.pdf as pdf files).

    Stand-alone APTIMA CT assay.

    A non-combo assay, for the detection of C. trachomatis alone, has been described by Shih et al., 2003 of Gen-Probe (poster available aptimact.pdf as pdf). This new assay could be used either for stand-alone C. trachomatis diagnosis or as a confirmatory assay. The test showed no cross reaction with any of 154 organisms tested and all C. trachomatis serovars were detected to the level of 0.1 IFU per assay. This assay is likely to be of particular interest in countries where N. gonorrhoeae infection is insufficient to justify the routine use of a combination assay.

    [MEW] February 2004

    NEXT: NASBA

    Reference

    Bixby, T., Light, J., Turner, S., Shaw, J. (2003). Poster C-031: Aptimadts.pdf. [Acrobat]

    Bott, M., Bixby, T., Castillo, M., Light, J., Smith, M., Spencer-Covill, S., Turner, S., Watson, M., Williams, R. & Shaw, J. (2001). APTIMA Combo 2 analytical performance with C. trachomatis (CT) and N. gonorrhoeae (GC). Presented at ISSTDR meeting Berlin, Germany, June 24 - 27.

    Chernesky, M., Jang, D., Patel, J., Castriciano, S., Chong, S. & Kapala, J. (2003). Simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae in the APTIMA Combo 2 assay by testing cervical swabs previously tested by LCx or Probe Tec assays. Poster P1174, ECCMID. retesting.pdf. [Acrobat]

    Chong S, Jang D, Song X, Mahony J, Petrich A, Barriga P, Chernesky M. (2003). Specimen processing and concentration of Chlamydia trachomatis added can influence false-negative rates in the LCx assay but not in the APTIMA Combo 2 assay when testing for inhibitors. Journal of Clinical Microbiology 41, 778 - 782.

    Ferrero, D. V., Buck, L., Burgess, N. A., Schultz, D. E., Stewart, S. & Traudt, L. (2002). Head-to-head study comparing performance of the Gen-Probe APTIMA Combo 2, Abbott LCx Probe, Becton Dickinson BDProbeTec ET and Roche AMPLICOR nucleic acid amplification assays in detecting Chlamydia trachomatis from first catch urine. Poster C-175. American Society of Microbiology Meeting, Utah, 2002.

    Ferrero, D. V., Gaydos, C. A., Quinn, T., Hook, E. W., Martin, D. H., Schachter, J., Weissfeld, A. & Willis, D. (2001). Multicenter clinical trial performance of the new Gen-Probe APTIMA Combo 2 assay for detecting Chlamydia trachomatis and Neisseria gonorrhoeae in female and male urine specimens. Presented at ISSTDR meeting Berlin, Germany, June 24 - 27.

    Fuller, D. D., Jasper, L., Milish, M. & Davis, T. (2002). Comparison of Gen-Probe APTIMA Combo 2 assay to BDProbeTec ET assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and endocervical swab specimens from females. Poster C-164. American Society of Microbiology Meeting, Utah, 2002.

    Fuller, D. D., Jasper, L., Milish, M., Brunnemer, C., Short, M. & Fomena, C. (2003). Clinical evaluation of Gen-Probe APTIMA Combo 2 assay for the direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae from CyTyc PreservCyt ThinPrep Pap smear collection. Poster C-024 American Society of Microbiology. Aptimacytoprep.pdf. [Acrobat]

    Gaydos, C. A., Quinn, T. C., Willis, D., Weissfeld, A., Hook, E. W., Martin, D. H., Ferrero, D. V. & Schachter, J. (2003). Performance of the APTIMA Combo 2 assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in female urine and endocervical swab specimens. Journal of Clinical Microbiology 41, 304 - 309. Full article [Acrobat]

    Hill, C. S. (2001) Molecular diagnostic testing for infectious diseases using TMA technology. Expert Reviews of Molecular Diagnostics 1, 445 - 455.

    Jang, D., Castriciano, S., Patel, J., Chong, S., Kapala, J. & Cherneskey, M. (2003). Identification of Chlamydia trachomatis infected women by APTIMA Combo 2 testing of cervical swab residual material previously tested by Prebe Tec or LCx. Poster C-028. American Society of Microbiology. Jang_ASM.pdf [Acrobat]

    Martin, D. H., Cammarata, C. L. & Smith, B. (2002). Comparison of Gen-Probe APTIMA Combo 2 (AC2) assay for C. trachomatis (CT) and N. gonorrhoeae (GC) with ProbeTec and LCx on urine and swab specimens from women using a rotating standard. Poster C-174. American Society of Microbiology Meeting, Utah, 2002.

    Martin, D., Hook, E. III., Ferrero, D., Willis, D., Schachter, J., Weissfeld, A., Gaydos, C. & Quinn, T. (2001). Comparison of three nucleic acid amplification tests (NAATS) and N. gonorrhoeae (GC) culture for the detection of C. trachomatis (CT) and GC using a rotating standard. Presented at ISSTDR meeting Berlin, Germany, June 24 - 27.

    Moncada, J., Schachter, J., Hook, E. W. III., Ferrero, D., Gaydos, C., Quinn, T., Willis, D., Weissfeld, C. & Martin, D. H. (2001). The effect of urine testing in evaluations of the sensitivity of Gen-Probe APTIMA Combo 2 assay on cervical swabs for Chlamydia trachomatis. The infected patient standard reduces sensitivity of single site evaluation. Presented at ISSTDR meeting Berlin, Germany, June 24 - 27.

    Scicchitano, L., Shetterly, B., Wood, C. & Bourbeau, P. (2003). Use of APTIMA Neisseria gonorrhoeae and APTIMA Chlamydia trachomatis analyste specific reagents for confirmation of Gen-Probe APTIMA Combo 2 assay results. Aptimaconfirm.pdf. [Acrobat]

    Shih, M., Bott, M., Mauricio, M., Spidle, J., Eng, C. & Johnson, R. (2003). Performance of the APTIMA CT assay for the detection of Chlamydia trachomatis. Gen-Probe Inc. aptimact.pdf[Acrobat]

    Turner, B., Weissfeld, A., Vance, P., Trevino, E., Simmons, D & Hightower, A. (2001). Comparison of the Gen-Probe APTIMA Combo 2 assay with chlamydial culture, and the pcr and lcr diagnostic tests. Presented at ISSTDR meeting Berlin, Germany, June 24 - 27.

    NEXT: NASBA.

    Registered users: Please add any comments below.

     

    Topic attachments
    I Attachment Action Size Date Who Comment
    PDFpdf Aptimacytoprep.pdf manage 7321.0 K 2011-03-12 - 18:55 MeWard  
    PDFpdf Aptimadts.pdf manage 599.1 K 2011-03-12 - 18:56 MeWard  
    PDFpdf Jang_ASM.pdf manage 827.2 K 2011-03-12 - 18:57 MeWard  
    PDFpdf aptimaconfirm.pdf manage 6350.6 K 2011-03-12 - 18:48 MeWard  
    PDFpdf aptimact.pdf manage 3726.3 K 2011-03-12 - 18:51 MeWard  
    PDFpdf retesting.pdf manage 4911.1 K 2011-03-12 - 18:59 MeWard  
    Unknown file formatasx tma.asx manage 403.6 K 2011-03-12 - 19:34 MeWard  
    Topic revision: r3 - 2011-03-12 - MeWard
     
    This site is powered by the TWiki collaboration platformCopyright © 2008-2014 by the contributing authors. All material on this collaboration platform is the property of the contributing authors.
    Ideas, requests, problems regarding TWiki? Send feedback