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Laboratory diagnosis: Pooling and replication

Pooling clinical specimens

The screening of women at risk for C. trachomatis is potentially an effective way of preventing the high cost of pelvic inflammatory disease due to untreated chlamydial genital tract infections. However in  populations with a low prevalence of infection, particularly once control measures begin to impact, the relatively high cost of diagnosing each infection using modern molecular methods is a major disadvantage. Pooling samples offers a method of saving costs, as only those pools containing a positive specimen will require component individuals to be screened separately. Such a strategy has been used to reduce the costs of screening for HIV antibody positive individuals.

Kacena and colleagues elaborated a model to determine the number of samples of first catch urine for C. trachomatis LCR testing to be pooled for optimal cost savings at various population prevalences. This model was applied in practice on a study of 1,088 urine specimens. After individual urine specimens were processed, 568 specimens were pooled by 4 into 142 pools and another 520 specimens were pooled by 10 into 52 pools. All 1,088 urine specimens were also tested individually. In a population with a prevalence of 8% genital C. trachomatis infection, pooling by four would reduce costs by 39%. At  a lower prevalence of infection of 2%, pooling eight samples would reduce costs by 59%.  Thus, unlike when samples are tested individually, the cost for finding one case does not increase dramatically as prevalence decreases [Kacena et al., 1998]. This essential result was confirmed by a number of other practical studies [Clark et al., 2000; Kapala et al., 2000; Krepel et al., 1999; Morre et al., 2000; Peeling et al., 1998]. This applied to both LCD and PCR testing. In one study, the pooling of urine samples increased apparent sensitivity by diluting out LCR inhibitors [Kapala et al., 2000], although this may have been fortuitous. The pooling of urine specimens for chlamydial nucleic acid testing is now generally considered a cost effective screening strategy for chlamydial infection [Morre et al., 2000] in both men and women which results in little loss of sensitivity.

Repeat sampling and replicate testing.

Where clinical specimens have a low number of organisms present, even the sensitivity of nucleic acid amplification based methods may be inadequate. One way of increasing the possibility of detecting low level infection might be by repeated sampling, or by replicated testing. Nucleic acid amplification of clinical specimens with low target concentration has variable sensitivity. Smieja et al., 2001 examined whether testing multiple aliquots of extracted DNA increased the sensitivity and reproducibility of Chlamydia pneumoniae detection by PCR. Nested and non-nested C. pneumoniae PCR assays were compared using 10 replicates of 16 serial dilutions of C. pneumoniae. The proportion positive versus the C. pneumoniae dilution was modelled by probit regression analysis. Additionally, ten replicates of twenty six previously positive specimens of peripheral blood mononuclear cells, sputum, or nasopharyngeal swabs  were tested. Not surprisingly, the proportion of replicates that were positive varied with the concentration of C. pneumoniae in the sample. At concentrations above 5 infection-forming units  per ml, PCR assay sensitivities were greater or equal to 90% or greater.  In clinical specimens in circulating mononuclear cells corresponding to about 0.07 inclusion forming units per ml, testing 1, 3, or 5 replicates detected respectively 3, 5, or 9 out of 10 positive specimens. Thus performing 5 or 10 replicates considerably increased the sensitivity and reproducibility of C. pneumoniae PCR. The corollary is that, due to sampling variability at low amounts of organism, PCR tests done without replication may miss a large proportion of positive specimens [Smieja et al., 2001].

[MEW] April 2002

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References

Clark, A. M., Steece, R., Crouse, K., Campbell, J., Zanto, S., Kartchner, D., Mottice, S. & Pettit, D. (2001). Multisite Pooling Study Using Ligase Chain Reaction in Screening for Genital Chlamydia trachomatis Infections. Sexually Transmitted Disease 28, 565 - 568.

Kacena, K.A., Quinn, S. B., Howell, M. R., Madico G. E., Quinn, T. C. & Gaydos, C. A. (1998). Pooling urine samples for ligase chain reaction screening for genital Chlamydia trachomatis infection in asymptomatic women. Journal of Clinical Microbiology 36, 481 - 485. Full article

Kapala, J., Copes, D., Sproston, A., Patel, J., Jang, D., Petrich, A., Mahony, J., Biers, K. & Chernesky, M. (2000). Pooling cervical swabs and testing by ligase chain reaction are accurate and cost-saving strategies for diagnosis of Chlamydia trachomatis. Journal of Clinical Microbiology 38, 2480 - 2483. Full article  

Krepel, J., Patel, J., Sproston, A., Hopkins, F., Jang, D., Mahony, J. & Chernesky, M. (1999). The impact on accuracy and cost of ligase chain reaction testing by pooling urine specimens for the diagnosis of Chlamydia trachomatis infections. Sexually Transmitted Diseases 26, 504 - 507.

Morre, S. A., Meijer, C. J., Munk, C., Kruger-Kjaer, S., Winther, J. F., Jorgensens, H. O. & van den Brule, A. J. (2000). 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. Journal of Clinical Microbiology 38, 1679 - 1680. Full article  

Peeling, R. W., Toye, B., Jessamine, P. & Gemmill, I.  (1998). Pooling of urine specimens for PCR testing: a cost saving strategy for Chlamydia trachomatis control programmes. Sexually Transmitted Infections 74, 66 - 70.

Smieja, M., Mahony, J. B., Goldsmith, C. H., Chong, S., Petrich, A. & Chernesky, M. (2001). Replicate PCR Testing and Probit Analysis for Detection and Quantitation of Chlamydia pneumoniae in Clinical Specimens. Journal of Clinical Microbiology 39, 1796 - 1801. Full article