Pooling of processed urine specimens for detection of N. gonorrhoeae
by LCR produced accurate results compared to results of individual testing of specimens by LCR. The cutoff ratio of the LCR test for the pooled samples was not reduced from the cutoff for individual specimens, as was necessary in the pooled chlamydia testing described previously (6
). The high sensitivity and specificity of LCR were not affected by pooling of as many as 10 samples, whether they were stored frozen or were never frozen. Freezing of processed or unprocessed urine samples had no effect on the accuracy of testing of pooled samples. Consequently, the pooling algorithm could be used in laboratories with a high volume of samples or in laboratories conducting epidemiological research where specimens are stored frozen and tested at a later time.
It is unknown why one LCR pooling algorithm-positive specimen was unable to be adjudicated as a true positive. This specimen was pool positive and individual test positive but negative when tested later for adjudication by both the opa and the pilin gene LCR. N. gonorrhoeae from cervical or urethral swabs of patients with low levels of infection seems less likely to be cultured successfully. Similarly, low levels of target DNA in processed specimens could be less likely to be able to be amplified over time and after multiple cycles of freezing and thawing. Although it cannot be determined, it is possible that this specimen was from a patient with a low organism load. Because it was unable to be adjudicated, it was considered to be a false-positive result in this study according to our definition of a true positive.
A theoretical concern is that pooling would dilute the low-level positive sample below the limit of detection of the assay. However, review of the manufacturer’s data presented in the package insert from individually tested specimens (n = 3,362) indicated that “low-positive” samples (with S/CO of <2.0) constituted only 3.3% of positive specimens. In the FMR and MSS data presented in this paper, none of the specimens were low positives.
A potential limitation of the pooling algorithm is the chance for technician error in the pooling of processed samples in the LCR run. The use of tray maps simplifies this process. We have used the following process for eliminating technician error. Samples are be organized by skipping a space after each pool group in the specimen rack. Thus, pooling adds no significant complexity to the process of setting up individual unit dose assays. Additional technician error can be avoided when samples from presumptively positive pools (detected in the previous run) are retested individually at the beginning of the batch before the routine testing of the new pool groups. Therefore, each run has a combination of samples that are retested individually and new pooled samples from the next group of specimens.
Pooling is a technique which could be used in high-volume laboratories such as state public-health labs and reference labs for significant cost savings. Public-health screening programs which are currently using culture can benefit from the ease of specimen collection, higher sensitivity, and lower cost of pooled LCR. Specific populations or laboratories that might benefit from pooling include any laboratory where, as a minimum, both turnaround time and volume allow for a combination of 19 pools and retests per day. With 96 specimens at a population prevalence of about 4%, pooling by 6 would allow for the completion of one full run (38 test unit doses) per day. The run would theoretically include, on average, 16 pools of 6 and 22 individual retests.
Use of the pooling algorithm could benefit investigators and program planners in two ways: (i) money saved by using the pooling algorithm could be applied to other areas of disease prevention, and/or (ii) the amount of money allocated to screening would allow more specimens to be tested for the same total cost. Pooling of urine samples for the detection of genital N. gonorrhoeae
infection is a cost-saving strategy, simple to perform, and could be applicable in screening programs in the United States and in population-based research worldwide. In addition, a combined chlamydia and gonorrhea detection program which uses pooling of processed urine specimens for LCR testing could be used in populations at significant risk for both pathogens and would detect most infections for less cost, since the same processed urine specimen can be used for both the chlamydia and gonorrhea LCR tests. Although not considered here, technician cost can be estimated as previously described in detail for LCR pooling for the detection of chlamydia (12
). Running specimens pooled for both chlamydia and gonorrhea testing by LCR would most significantly reduce technician time, specimen processing costs, and LCR assay costs.
Laboratory managers should consider two points before using pooling. First, processed specimens from presumptive positively pools need to be amplified and detected individually. This additional step adds a minimum delay of 3 h to the laboratory turnaround time until individual test results on specimens in presumptively positive pools are known. Second, the estimated cost savings to be gained for a particular laboratory depend on a combination of the salaries of technicians and their benefits, institutional overhead, and the prevalence of gonorrhea in the populations the laboratory serves. Pooling of samples from patients in a population where the prevalence of gonorrhea may be 20% or greater is not advised and would be minimally cost saving. The pooling algorithm would be cost saving at lower prevalences of infection.
The study laboratory has met Clinical Laboratory Improvement Act requirements for the modification of a manufacturer’s package insert directions for performance of a test by a clinical laboratory using a diagnostic kit cleared by the Food and Drug Administration. The investigators considered the performance and documentation of the required study adequate for using the pooling algorithm protocol in testing of clinical specimens in the study laboratory. Each laboratory that wishes to introduce pooling must meet the requirements set forth to modify the package insert from a test cleared by the Food and Drug Administration. These requirements are explained more fully as regulations set forth in the Federal Register (4
Pooling of processed urine samples for LCR testing of N. gonorrhoeae
will decrease the cost of screening, providing more evidence to health planners that screening programs can and should be implemented. An additional application of pooling of urine specimens by LCR is the detection of genital C. trachomatis
). The cost savings of pooling of urine for both N. gonorrhoeae
and C. trachomatis
should also be considered. Although LCR failed to detect two cervical culture-positive specimens, this strategy of screening everyone in a population by testing urine specimens detected 11 more of the 39 true positives (28.2%) than the strategy of performing culture on specimens collected from the portion of females who received pelvic examinations where cervical swabs were taken or on specimens from males where urethral swabs were obtained due to their clinical presentation of signs and symptoms. Screening of urine from sexually active students by using the pooling algorithm was more sensitive (92 and 100%) than culture (79 and 88%) in women and men, respectively, and more cost saving than performing individual LCR assays for N. gonorrhoeae
In conclusion, the LCR urine pooling algorithm for the detection of N. gonorrhoeae was accurate compared to testing of specimens individually and selective culturing of specimens, and it could be used as a cost-saving public-health measure for screening of populations at risk for gonorrhea, especially when a cervical or urethral swab cannot be obtained.