Literature review > Issue_2 > Review Gomes et al. 

Since nucleic acid amplification tests (NAATs) are expensive, it would be desirable to be able to decrease the cost of such assays by testing more than one specimen at a time in one "unit test dose". Indeed it has been shown that it is possible to "pool" more than one sample together for testing without marked loss of sensitivity and that this approach can save costs and be cost-effective [1]. The accuracy of pooling has been demonstrated for LCR, PCR, and for strand displacement amplification (SDA), but has not been reported for transcription mediated amplification (TMA) [1-9]. Gomes et al. report for the first time a sensitivity evaluation of pooling urine samples for the Gen-Probe AMP-CT assay in this paper [10].

The objective of determining the sensitivity of two pool sizes versus testing urine samples individually in the AMP-CT assay is clearly stated. The authors reported that the sensitivity of individual testing was 91.9%, while for a 4X pool it was 94.3% and for an 8X pool, it was 86.5%. The methodology was different from other pooling studies in that previously positive urine samples (33) tested by the Amplicor-PCR assay were used in pools with "fresh" previously untested urine samples. Each pool started with one of the previously positive urines and the remaining additions to the pool were new untested urines. Since both "old" and "fresh" urines were included, the study design was somewhat artificial and the accuracy will need to be evaluated in a more prospective manner also.

Each pool of 4 or 8 was made using 40 ul of aliquots of solubilized urine pellets, prepared by manufacturer's instructions, and then 50 ul from the mixture was tested by AMP-CT. Four additional positives were found from the "fresh urines" and these were also positive when tested by Roche PCR, confirming their positivity for a total of 37 positive samples. Because 2 of the newly positive samples were not contained in the 4X pools, they could not be included in the denominator used for the 8X pools. The strategy that was used makes it difficult to compare exactly the 2 sensitivities of 4X and 8X pools. This is one limitation of this study design. However, the strategy of pooling does appear to be highly sensitive for pools of 4 and the authors conclude accurately that the 8X pool may dilute low positives too much to be highly sensitive and conclude that the 4X strategy is the most useful for future pooling strategies using the AMP-CT assay.

Additionally, since the new APTIMA Combo 2 assay for Chlamydia has now been FDA cleared and has a different nucleic acid extraction method, which uses hybrid-capture, this new format will require evaluation in future pooling studies [11].

References:

1.  Kacena KA, Quinn SB, Howell MR, Madico GE, Quinn TC, Gaydos CA. Pooling urine samples for ligase chain reaction screening for genital Chlamydia trachomatis infections in asymptomatic women. J Clin Micro 36, 481-485. 1998.

2. Peeling RW, Toye B, Jessamine P, Gemmill I. Pooling of urine specimens for PCR testing: a cost saving strategy for Chlamydia trachomatis control programmes. Sex Trans.Inf. 74, 66-70. 1998.

3.  Clark AM, Steece R, Crouse K, Campbell J, Zanto S, Kartchner D et al. Multisite pooling study using ligase chain reaction in screening for genital Chlamydia trachomatis infections. Sex Trans Dis 2001; 28:565-568.

4.  Kapala J, Copes D, Sproston A, Patel J, Jang D, Petrich A et al. Pooling cervical swabs and testing by ligase chain reaction are accurate and cost-saving strategies for diagnosis of Chlamydia trachomatis. J Clin Microbiol 2000; 38:2480-2483.

5. Morre SA, Meijier CJLM, Munk C, Kruger-Kjaer S, Winther JF, Jorgensens HO et al. Pooling of urine specimens for detection of asymptomatic Chlamydia trachomatis infections by PCR in a low-prevalence population: cost saving strategy for epidemiological studies and screening programs. J Clin Microbiol 2000; 38:1679-1680.

6 Morre SA, van Dijk R, Meijer CJ, Van den Brule AJ. Pooling of cervical swabs for detection of Chlamydia trachomatis infections by PCR: sensitivity, dilution, inhibition, and cost-saving aspects. J Clin Microbiol 2000; 39:2375-2376.

7. Krepel J, Patel J, Sproston A, Hopkins F, Jang D, Mahoney J et al. The impact on accuracy and cost of ligase chain reaction testing by pooling urine specimens for the diagnosis of Chlamydia trachomatis infections. Sex Trans Dis 1999; 26:504-507.

8. Wood BJ, Theodore M, Quinn TC, Gaydos CA. Pooling of urine specimens for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by the Becton Dickinson (BD) ProbeTec ET Assay. J STD AIDS 2001; 12 Suppl2:115.

9. Gomes J, Ferreira M, De Sa A, Catry M. Pooling urine samples for PCR screening of C. trachomatis urogenital infections in women. Sex Transm Infect 2001; 77:76-77.

10. Gomes JP, Viegas S, Paulino A, Catry MA. Sensitivity evaluation of the Gen-Probe AMT-CT assay by pooling urine samples for screening of Chlamydia trachomatis urogenital infection. Int J STD AIDS 2002; 13:540-542.

11. Gaydos CA, Quinn TC, Willis D, Weissfeld A, Hook EW, Martin DH et al. Performance of the APTIMA Combo 2 assay for the multiplex detection of Chlamydia trachomatis and Neisseria gonorrheae in female urine and endocervical swab specimens. J Clin Microbiol 2003; 41:304-309

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