Specimen pooling for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urogenital specimens

Abstract number: 1733_417

Altwegg M., Süess O., Jaton K., Greub G., Egli K.

Objectives: To test whether pooling of 3 clinical specimens in combination with automated DNA extraction and a home-brew duplex test for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) performs similarly as a well established commercial system with individual specimens and thus might help reducing costs for analysis.

Methods: 231 clinical specimens (swabs in Roche transport medium, native urines) were analyzed for CT and NG using the CobasAmplicor system (Roche). Inhibited specimens were retested after DNA extraction and NG+ specimens were confirmed using an independent test. The same specimens were further analyzed individually (N = 99) and pooled (N = 231; 3 consecutive specimens per pool). This included DNA extraction using the easyMAG system (bioMérieux) and analysis with a home-brew duplex test for CT and NG (5'exonuclease format) on the LightCycler (LC; Roche). In case of a positive result for a given pool, the 3 specimens were analyzed individually using the same system. The volumes of clinical specimens analyzed per PCR on the Cobas were 50ul and 25ul for urines and swabs, respectively, 29ul for individual and 18ul for pooled specimens in our own assay. Inhibition of PCR was determined using the internal amplification control on the Cobas and an external control on the LC.

Results: Of the 231 specimens, 4 were CT+, 1 NG+ and 2 CT+/NG+. An additional 2 specimens were false positive for NG on the Cobas. All pools gave the expected result when tested by home-brew PCR. Analysis of individual specimens from positive pools correctly identified the positive specimens (which were all in separate pools). The 2 false positive NG specimens did not result in a positive pool signal. Inhibition occurred in 19/231 (8.2%) of the individual specimens tested on the Cobas, in 1/99 (1.0%) individual specimens and in 2/73 (2.7%) pools tested on the LC. No inhibition was detected when the members of inhibitory pools were individually extracted and tested on the LC.

Conclusions: Pooling of urogenital specimens is a promising strategy to reduce costs for CT/NG testing. Because of the small number of positive results the sensitivity of this procedure requires confirmation. Optimal pool sizes depend on the number of specimens to be analyzed and on the prevalence of the two organisms. Amplification controls are recommended for pools, however, this may not be necessary for individual specimens because for those the use of automated DNA extraction results in an inhibition rate of only 1%.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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