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VOL.
36, 1998 INTERNAL CONTROL FOR ROUTINE DIAGNOSTIC PCR 195
TABLE 2. Enhancement of test sensitivity using IC

COBAS Sensitivity (%) Specificity (%)
AMPLICOR test and
specimen Without IC With IC Without IC With IC
C. trachomatis
Swab 94.8 (110/116) 97.4 (111/114) 99.0 (832/840) 99.0 (771/779)
Urine 92.0 (104/113) 94.0 (110/117) 99.1 (844/852) 99.0 (828/836)
N. gonorrhoeae
Swab 93.9 (108/115) 94.8 (109/115) 98.0 (814/831) 97.9 (775/792)
Urine 83.8 (98/117) 89.7 (104/116) 98.9 (828/837) 98.8 (818/828)
M. tuberculosis
Smear positive 98.7 (151/153) 98.7 (152/154) 90.0 (27/30) 89.7 (26/29)
Smear negative 73.6 (92/125) 75.6 (93/123) 99.0 (4027/4068) 99.0 (3906/3947)
HCV 72.6 (752/1036) 78.2 (802/1025) 98.2 (912/929) 98.2 (903/920)
fore, excluded from the sensitivity calculations. When the IC the IC result has no effect on the interpretation of positive
results were ignored, these specimens were classified as false specimens.
negative. Included in this category were 3 C. trachomatis swab An IC can also be used selectively when past experience has
specimens, 1 C. trachomatis urine specimen, 2 N. gonorrhoeae demonstrated very low rates of inhibition. Use of an IC can be
urine specimens, 2 M. tuberculosis smear-negative specimens, reserved for specific specimen or patient conditions where
and 11 HCV specimens (Table 1). inhibition is more likely to occur; examples include specimens
contaminated with blood or other interfering substances and
DISCUSSION urogenital specimens from pregnant women (11). An IC
should also be selectively employed when the probability of
The results of this study demonstrate that incorporating an infection is high and the consequence of a false-negative result
IC into PCR-based tests increases sensitivity by enabling the is severe. An example is testing acid-fast bacillus smear-posi-
user to identify and retest samples inhibitory to PCR. Further- tive patients for M. tuberculosis, where a false-negative test
more, a positive IC result indicates that amplification has oc- result would lead the clinician to conclude that the patient has
curred and thus provides assurance that negative test results a nontuberculosis mycobacterial infection.
are truly negative. The COBAS AMPLICOR tests evaluated in Even when the IC was used, test sensitivity was not 100%.
this study exhibited low rates of inhibition. Because inhibition Thus, factors other than inhibition must have caused false-
was infrequent, use of the IC resulted in only a relatively negative results. Sample-to-sample variation is one source of
modest improvement in test sensitivity. false-negative results. Localization of infection may also be a
The IC can also be used to monitor competition between
factor when testing female urine samples for C. trachomatis
multiple targets in multiplex PCR tests. A negative IC result in
and N. gonorrhoeae because PCR and culture are performed
specimens that are positive for one target indicates that com-
on specimens collected from different anatomical sites. Low
petition and/or inhibition reduced amplification efficiency be-
target concentration represents a second potential source of
low the threshold required to generate a positive result from a
false-negative results, especially when testing for M. tuberculo-
low-level target. In such specimens, negative results for the
other targets are considered invalid. The data presented here sis where the volume of sample used for PCR testing is 5% of
demonstrate that competition will not cause a false-negative that used for culture. Finally, the reference test result could
result unless the concentration of one target is 104-fold greater actually be false positive. This is almost certainly a factor in
than the concentration of the second target. HCV testing where a positive EIA result for anti-HCV anti-
The IC is used at a concentration of 20 copies per test body indicates prior exposure but does not serve as a marker
sample to monitor amplification at the limit of test sensitivity. for current infection.
PCR inhibitors decrease amplification efficiency, thereby re- Inhibition has been shown to affect the sensitivity of LCR-
ducing the amount of PCR product generated from each target based amplification tests (2, 4–6, 11, 22, 25), TMA tests (17,
molecule. A high target load can compensate for reduced am- 21), and a nucleic acid sequence-based amplification test (18).
plification efficiency, yielding enough product to generate a It is, however, difficult to determine the frequency of inhibition
positive signal. If used at a higher concentration, the IC might because the commercially available LCR- and TMA-based
not detect weak inhibition that could cause false-negative re- tests lack an IC. Consequently, inhibition can be evaluated only
sults at extremely low target loads. in reference test-positive specimens that are expected to give
For routine clinical applications, the laboratory can max- positive amplification test results. Inhibition cannot be as-
imize test sensitivity by using an IC to monitor amplifica- sessed in reference test-negative specimens since they are ex-
tion in every specimen. Nevertheless, it may be possible to pected to give negative test results. The frequency of inhibi-
use an IC selectively to increase efficiency without sacrific- tory, positive specimens can be estimated from the frequency
ing performance. For example, the fully automated COBAS reference test-positive, amplification test-negative specimens.
AMPLICOR system can be programmed to perform addi- This provides an upper limit on the inhibition rate in reference
tional tests based on the outcome of an initial test. Thus, a test-positive specimens; the actual inhibition rate may be lower
COBAS AMPLICOR user can easily detect the IC only in because false-negative results may be caused by factors other
specimens that test negative for the primary target(s). Using than inhibition. The frequency of LCR false-negative results in
this algorithm will not compromise test performance because urogenital specimens were 2% (7 of 345) (6), 7% (3 of 44) (22),

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VOL.

36, 1998 INTERNAL CONTROL FOR ROUTINE DIAGNOSTIC PCR 195

TABLE 2. Enhancement of test sensitivity using IC

HCV 72.6 (752/1036) 78.2 (802/1025) 98.2 (912/929) 98.2 (903/920)

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