Comparison of LightCycler PCR, rapid antigen immunoassay, and culture for detection of group A streptococci from throat swabs

Abstract

We compared the performance characteristics of a real-time PCR method, the LightCycler Strep-A assay (Roche Applied Science, Indianapolis, Ind.), to those of a rapid antigen immunoassay, the Directigen 1-2-3 Group A Strep Test kit (BD Diagnostic Systems, Sparks, Md.), and a standard culture method for detection of group A streptococci (GAS) from 384 throat swabs. The LightCycler PCR produced more positive results (n = 58) than either culture (n = 55) or the Directigen immunoassay (n = 31). The results of the LightCycler PCR and the Directigen method were independently compared to the results of the accepted "gold standard," bacterial culture. The sensitivities, specificities, and positive and negative predictive values for this comparison were as follows: for the Directigen method, 55, 99, 97, and 93%, respectively; for the LightCycler PCR, 93, 98, 88, and 99%, respectively. In no case was a throat swab positive by both the LightCycler PCR and the Directigen method but negative by culture. The medical histories of patients whose throat swabs were negative by culture but positive by either the LightCycler PCR (n = 7) or the Directigen method (n = 1) were reviewed. All of these patients had signs or symptoms compatible with GAS disease, and therefore, all of these discordant positive results (along with positive results by either the Directigen method or the LightCycler PCR that agreed with the culture results) were counted as true positives for statistical analysis. For this analysis, the LightCycler PCR detected more true-positive results than the culture method (58 versus 55 swabs); however, this difference was not statistically significant (P = 0.5465). In contrast, statistically significantly more true-positive results occurred by culture than by the Directigen method (55 versus 31 swabs; P < 0.0001) and by the LightCycler PCR than by the Directigen method (58 versus 31 swabs; P < 0.0001). The LightCycler PCR is a suitable stand-alone method for the detection of GAS from throat swabs. Additionally, this method requires less than half the personnel time and the procedure can be completed in considerably less time ( approximately 1 h) than our standard approach (up to 2 days) for detection of GAS in throat swabs (i.e., testing by the Directigen method with negative results verified by culture).

FIG. 1.

Detection of GAS DNA with the LightCycler instrument and LightCycler Strep-A Primer/Hybridization Probes. In this quantitative analysis, positive results are indicated by an upwardly deflecting curve (cycle number, ∼31) for both the positive control and a positive sample. F2 refers to the fluorescence emission for the LC-Red640 fluorophore, and F1 is fluorescence emission for the fluorescein fluorophore.

FIG. 2.

Quantitative representation or cycling curve analysis of recovery template (internal control) for the LightCycler PCR assay. The recovery template FRET probe has a different reporter dye (LC-Red705) than the FRET probe used to detect target DNA in the sample (LC-Red640 dye) and is detected in channel F3 of the LightCycler instrument (see Fig. 4). F3 is the fluorescence emission for the LC-Red705 fluorophore, and F1 is the fluorescence emission for the fluorescein fluorophore. This quality control step indicates whether inhibition of the PCR occurred in any of the patient samples or the positive or negative controls. Amplification of the recovery template should occur with each of these analyses except when the amount of target DNA in the patient's sample significantly exceeds that of the recovery template DNA. In this case, the target DNA competes with the recovery template DNA, but because the sample is positive, assessment of PCR inhibition by means of recovery template DNA amplification is unnecessary.

FIG. 3.

Melting curve analysis for LightCycler PCR assays. The designation −d[F2/F1]/dT on the y axis refers to the negative derivative of fluorescence. F2 is the fluorescence emission for the LC-Red640 fluorophore, and F1 is the fluorescence emission for the fluorescein fluorophore. The T_m of GAS by melting curve analysis is 57.3 ± 2.5°C. Large-colony-forming group C and group G streptococci of human origin, which have recently been classified as the same species (S. dysgalactiae subsp. equisimilis), produce identical melting curves, with a T_m of 51.5 ± 2.5°C, but are not detected during the real-time quantification portion of the PCR.

FIG. 4.

Recovery template. The recovery template (internal control) has the same sequence as the PCR product, except that the probe region has been replaced with a sequence complementary to recovery template probes. FRET detection of the target DNA is with a probe labeled with the LC-Red640 dye in channel 2 of the LightCycler instrument, while the recovery template is detected with a probe labeled with the LC-Red705 dye in channel 3. A small amount of the recovery template is added to the PCR mixture and is amplified along with the target DNA by the same primers. Thus, the two reactions compete for the primers. Normally, the recovery template is amplified from all samples, including the negative control. If neither the recovery template nor the target DNA is amplified, it is assumed that inhibition of the PCR has occurred and the test for that sample is not valid. However, if the target DNA is amplified but the recovery DNA template is not, it is assumed that the target DNA is present in a proportionally greater amount. In this situation, partial inhibition of the PCR may be present but the target DNA is successfully amplified or the recovery template may not be able to compete for primers and the recovery template signal may be weak or not present. When this occurs, the positive result is valid because the recovery template amplification is unnecessary.

FIG. 5.

Distribution of GAS-positive results for each of the assays tested. The positivity rate (any assay positive) was 63 of 384 samples (16.4%). For four samples (1.0%) inhibition of amplification of the recovery template (internal control) was detected by the LightCycler PCR assay. Of the 321 negative samples, 10 (3.1%) were found to contain DNA from group G or group C streptococci by the Light- Cycler PCR assay.

FIG. 6.

Comparison of LightCycler PCR crossing points with culture plate colony scores. ○, culture and LightCycler PCR positive; □, LightCycler PCR positive only; ▵, culture positive only. Refer to Materials and Methods for a description of culture quantitation.