TABLE 2.
Results of method validation using 35 well-characterized N. gonorrhoeae isolatese
| Target | Sequence type result(s) for the 35 control isolates (no. of isolates with result) determined by: |
Tm (°C) |
Mean ΔTm ± SD (°C) | Sensitivityc (%) (95% CI) | Specificityc (%) (95% CI) | ||
|---|---|---|---|---|---|---|---|
| DNA sequencing | Real-time PCR/HRM analysis | Range | Mean ± SD | ||||
| opa | Positive (35) | Positive (35) | 76.63–77.22 | 76.98 ± 0.13 | NA | 100 (90–100) | NAd |
| Negative (0) | Negative (0) | NA | NA | ||||
| porA | Positive (35) | Positive (35) | 73.79–74.88 | 74.36 ± 0.20 | NA | 100 (90–100) | NAd |
| Negative (0) | Negative (0) | NA | NA | ||||
| penA Gly545Ser | Nonmosaic (23) | Nonmosaic (23) | NAa | NAa | 100 (66–100) | 100 (87–100) | |
| Nonmosaic Gly545 (GGC) (3) | Nonmosaic Gly545 (GGC) (3) | 85.05–85.23a | 85.14 ± 0.08a | 0.46 ± 0.05 | |||
| Mosaic Gly545Ser (AGC) (9) | Mosaic Gly545Ser (AGC) (9) | 84.09–84.72 | 84.47 ± 0.20 | ||||
| penA Ala501 | Nonmosaic (26) | Nonmosaic (26) | NAb | NAb | 100 (66–100) | 100 (87–100) | |
| Mosaic (9) | Mosaic (9) | 83.59–84.35 | 84.17 ± 0.19 | NI | |||
| gyrA Ser91Phe | GyrA Ser91 (TCC), Ala92 (GCA) (11) | GyrA Ser91 (TCC), Ala92 (GCA) (11) | 77.97–78.16 | 78.08 ± 0.05 | 100 (86–100) | 100 (72–100) | |
| GyrA Ser91Phe (TTC), Ala92 (GCA) (23) | GyrA Ser91Phe (TTC), Ala92 (GCA) (23) | 77.29–77.59 | 77.47 ± 0.07 | 0.61 ± 0.06 | |||
| GyrA Ser91Phe (TTC), Ala92Ser (TCA) (1) | GyrA Ser91Phe (TCC), Ala92Ser (TCA) (1) | 76.15–76.17 | 76.16 ± 0.02 | 1.25 ± 0.01 | |||
| 23S rRNA A2059G | A2059 (34) | A2059 (34) | 81.33–81.52 | 81.44 ± 0.03 | 0.22 ± 0.02 | 100 (3–100) | 100 (90–100) |
| A2059G (1) | A2059G (1) | 81.61–81.70 | 81.67 ± 0.03 | ||||
| 23S rRNA C2611T | C2611 (34) | C2611 (34) | 75.69–76.33 | 76.12 ± 0.16 | 0.75 ± 0.05 | 100 (3–100) | 100 (90–100) |
| C2611T (1) | C2611T (1) | 75.08–75.55 | 75.30 ± 0.20 | ||||
| rpsE Thr24Pro | Thr24 (ACC) (34) | Thr24 (ACC) (34) | 73.87–74.34 | 74.08 ± 0.07 | 0.68 ± 0.01 | 100 (3–100) | 100 (90–100) |
| Thr24Pro (CCC) (1) | Thr24Pro (CCC) (1) | 74.66–74.94 | 74.76 ± 0.09 | ||||
| 16S rRNA C1192T | C1192 (34) | C1192 (34) | 81.38–81.72 | 81.56 ± 0.08 | 0.69 ± 0.01 | 100 (3–100) | 100 (90–100) |
| C1192T (1) | C1192T (1) | 80.74–80.94 | 80.82 ± 0.09 | ||||
Only nonmosaic pattern XIX (with penA Gly545) showed cross-amplification.
No amplification was observed for all other nonmosaic penA patterns tested.
Sensitivity is the probability that an isolate was correctly identified as being positive by HRM analysis for the target sequence (species identification, mosaic, or mutation); specificity is the probability that an isolate was correctly identified as being negative by HRM analysis for the target sequence (species identification, mosaic, or mutation).
Specificity was 100% considering that all 19 nongonococcal control strains were correctly characterized as non-N. gonorrhoeae isolates (see Table S1 in the supplemental material).
NA, not applicable; NI, not interpretable.