-
Phenotypic DST was performed on 100 Mtb strains using the CLSI-recommended broth microdilution MIC method. The phenotypic DST results of the 100 Mtb isolates selected in this study are listed in Supplementary Table S1, available in www.besjournal.com. Of the 100 Mtb isolates, 65 (65%) were MDR strains, 22 (22%) were mono-INH-resistant strains, 4 (4%) were mono-RIF-resistant strains, and 9 (9%) were INH- and rifampin-sensitive strains. The results for each probe detected by MDR-LAMP are shown in Figure 1. The performance of MDR-LAMP is summarized in Table 1. The sensitivity and specificity of MDR-LAMP reagents for detecting RIF resistance were 85.5% and 93.5%, respectively. The PPV and NPV were 96.7% and 74.4%, respectively. The sensitivity and specificity of detecting INH resistance were 80.5% and 92.3%, respectively, and the PPV and NPV were 98.6% and 41.4%, respectively. Considering that the P values are both lesser than 0.01, we investigated the reason why the coverage of the resistant genes tested or the accuracy of the MDR-LAMP reagents affected the consistency between MDR-LAMP and phenotypic DST results. As shown in Table 2, the sensitivity of the MDR-LAMP detection of INH (katG and inhA) resistance gene mutations was 93.2%, and the specificity was 92.3%. The PPV and NPV were 97.2% and 82.8% compared with whole-genome sequencing (WGS). The sensitivity and specificity of RIF (rpoB) resistance gene mutations were 89.1% and 88.9%, respectively, and the PPV and NPV were 93.4% and 82.1%, respectively. The results showed that the accuracy of the MDR-LAMP reagents to detect rpoB gene mutations must improve. We calculated the detection sensitivity, specificity, PPV, and NPV of each probe compared with the mutation results obtained by WGS analysis to evaluate the efficiency of each probe for detecting rpoB. The consistency rate of each probe for detecting rpoB S531L/L533P and rpoB H526Y/H526D mutations presented a significant difference to WGS (P < 0.05). The specificity of the probe for detecting rpoB H526Y/H526D mutation was 93.7%, whereas that of the other probes was 100% (Table 3). As shown in Table 4, all the 11 rpoB S531L mutations detected by the FAM filter in rpoB S531L mutations were detected by the Cy5 filter, whereas the sensitivity of the probe rpoB S531L/L533P was 29.7%. In conclusion, the number of resistance-related mutation sites should be increased in the MDR-LAMP assay to cover the phenotypic resistance of INH and RIF. In addition, the probes for detecting gene mutations should be optimized to improve accuracy, especially for the rpoB gene.
Table S1. The phenotypic DST results of the 100 Mtb isolates
LAMP MDR assay Strain No. MIC Cutoff-value DST (S:Sensitivity, R:Resistance) WGS Gene: (W:Wild, M:Mutant) LAMP MDR (+:Detect, −:Not Detect) INH:
> 0.2RIF:
> 1INH RIF rpoB site katG site inhA-
Promotor-
15Reagent
AT Reagent
BT Reagent
CT 031024 2 > 16 R R M S531L M S315T W + 55 °C + Cy5:73 °C − 033059 4 > 16 R R W − W − M − − + 64 °C 033061 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033062 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033063 2 > 16 R R M H526Y M S315T W + 56 °C − + 65 °C 033064 2 > 16 R R M H526Y M S315T W + 56 °C − + 65 °C 033065 2 > 16 R R M H526Y M S315T W + 54 °C − + 62 °C 033066 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033067 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033068 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033070 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033071 2 > 16 R R M H526Y M S315T W + 54 °C − + 64 °C 033102 1 > 16 R R M S531L W − W − + Cy5:71 °C − 033106 1 > 16 R R M S531L W − W − + Cy5:72 °C − 033109 1 > 16 R R M S531L W − W − + Cy5:72 °C − 033127 2 > 16 R R M S531L M S315T W + 55 °C − + 62 °C 033129 1 > 16 R R M H526Y M S315T W + 55 °C − + 66 °C 033159 4 > 16 R R M S531L M S315T W + 55 °C + Cy5:72 °C − 033214 4 > 16 R R M S531L M S315T W + 55 °C + Cy5:72 °C − 061021 0.5 > 16 R R M S531L W − W − + Cy5:71 °C − 061026 4 > 16 R R W − M S315T W + 54 °C − + 61 °C 061045 4 ≤ 0.12 R S W − M S315T W + 54 °C − − 061046 4 > 16 R R W − M S315T W + 54 °C − − 061102 0.06 > 16 S R M H526Y W − W − − + 62 °C 061105 4 ≤ 0.12 R S W − M S315T W + 55 °C − − 061260 0.06 > 16 S R M S531L W − W − − + 68 °C 061300 4 ≤ 0.12 R S W − M S315T W + 55 °C − − 061301 4 ≤ 0.12 R S W − M S315T W + 55 °C − − 061305 4 > 16 R R W − M S315T W + 55 °C − − 061315 > 4 1 R S W − W − M − − − 071014 4 16 R R M S531L M S315T W − − − 071023 4 0.12 R S W − M S315T W + 55 °C − − 071028 4 0.5 R S W − M S315T W + 55 °C − − 071031 4 0.25 R S W − M S315T W + 55 °C − − 071040 2 16 R R M H526Y M S315T W + 54 °C − + 62 °C 071061 4 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C+ 64 °C 071070 2 16 R R M H526D W − W − − + 67 °C 071075 4 0.12 R S W − M S315T W + 55 °C − − 071097 4 16 R R W − M S315T W + 54 °C − − 071107 4 16 R R W − M S315T W + 55 °C − − 071121 4 0.12 R S W − M S315T W + 55 °C − − 071126 4 0.12 R S W − M S315T W + 55 °C − − 071127 4 16 R R W − M S315T W + 55 °C − − 071140 4 1 R S W − M S315T W + 55 °C − − 081005 2 > 16 R R M H526D M S315T W + 54 °C − + 68 °C 081044 2 > 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C− 081051 2 > 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C− 081056 2 > 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C− 081067 4 > 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C− 081161 0.06 > 16 S R M S531L W − W − + Cy5:72 °C − 081217 0.12 > 16 S R M S531L W − W − + Cy5:71 °C − 082248 4 16 R R M S531L M S315T W + 54 °C + Cy5:71 °C − 082308 0.25 > 16 R R M S531L W − W − + Cy5:72 °C − 082312 4 > 16 R R M L533P W − W + 48 °C − − 091079 4 0.25 R S W − M S315N W + 54 °C − − 091104 4 0.25 R S W − M S315T W + 55 °C − − 091119 4 0.25 R S W − M S315T W + 55 °C − − 102238 4 16 R R M S531L M S315T W + 54 °C + FAM:62 °C,
Cy5:72 °C− 104186 0.03 0.12 S S W − W − W − − − 104191 0.03 0.25 S S W − W − W − − − 104200 0.06 0.5 S S W − W − W − − − 104218 0.03 0.12 S S W − W − W − − − 104229 0.03 0.12 S S W − W − W − − − 151022 2 16 R R M H526D M S315T W + 54 °C − + 67 °C 153058 4 16 R R M H526Y M S315T W + 55 °C − − 154089 2 16 R R M H526Y M S315T W + 54 °C − + 66 °C 154214 4 0.25 R S W − M S315T W + 55 °C − − 154285 4 0.12 R S W − M S315T W + 55 °C − − 154286 2 16 R R M H526Y M S315T W + 55 °C − + 66 °C 171019 2 16 R R M D516V W − W − + Cy5:60 °C − 171026 0.5 > 16 R R M S531L W − W − + FAM:62 °C,
Cy5:72 °C− 171052 > 4 > 16 R R M S531L W − W − + FAM:62 °C,
Cy5:72 °C− 171064 > 4 > 16 R R M H526D M S315T W + 54 °C − + 67 °C 171078 > 4 16 R R M D516V M S315T W + 54 °C − − 171079 > 4 16 R R M D516V W − W − + Cy5:60 °C − 171292 2 > 16 R R M H526Y M S315T W + 56 °C − + 64 °C 171607 4 > 16 R R M S531L M S315T W + 56 °C + Cy5:72 °C − 172096 0.06 ≤ 0.12 S S M S531L M S315T W + 56 °C + Cy5:72 °C − 172300 4 > 16 R R M D516V M S315T W + 56 °C + Cy5:60 °C − 172314 > 4 > 16 R R M S531L M S315T W + 56 °C + Cy5:72 °C − 172573 2 > 16 R R M S531L M S315N W + 54 °C + Cy5:72 °C − 173001 0.25 > 16 R R M S531L W − W + 46 °C + FAM:62 °C,
Cy5:71 °C− 173093 > 4 > 16 R R M S531L W − W − + FAM:62 °C,
Cy5:72 °C− 173422 0.5 ≤ 0.12 R S W − W − W − − − 174067 0.06 0.25 S S W − M S315T W − − − 184403 4 16 R R M S531L M S315T W + 55 °C + Cy5:71 °C − 185209 4 0.5 R S W − M S315T W + 55 °C − − 185544 4 16 R R M S531L M S315T W + 56 °C + Cy5:67 °C − 185551 4 0.25 R S W − M S315T W + 55 °C − − 19200003 4 0.25 R S W − M S315T W + 55 °C − − 19200399 4 16 R R M S531L M S315T W + 55 °C + Cy5:72 °C − 221211 2 1 R S M D516V M S315N W − + Cy5:60 °C − 221227 0.25 16 R R M S531L W − W − + FAM:62 °C,
Cy5:71 °C− 221437 2 16 R R M S531L M S315T W + 56 °C + Cy5:72 °C − 221518 4 16 R R M S531L W − M + 48 °C + Cy5:72 °C − 221571 4 16 R R M S531L M S315T W + 55 °C + Cy5:72 °C − 222280 4 16 R R M D516V M S315T W + 55 °C + Cy5:60 °C − 233077 0.06 0.25 S S W − W − W − − − 235254 0.06 0.12 S S W − W − W − − − 320005 2 2 R R M S531L M S315T W + 55 °C − − Table 1. Efficacy of MDR-LAMP test compared with the broth microdilution MIC method
Drugs MIC MDR-LAMP Sub-total Sensitivity
(95% CI)aSpecificity
(95% CI)aPPV
(95% CI)aNPV
(95% CI)aχ2 b Pc Resistance Sensitive RIF Resistance 59 10 69 85.5%
(74.5–92.5)93.6%
(77.2–98.9)96.7%
(87.6–99.4)74.4%
(57.6–86.4)10.73d 0.039 Sensitive 2 29 31 Sub-total 61 39 100 INH Resistance 70 17 87 80.5%
(70.3–87.9)92.3%
(62.1–99.6)98.6%
(91.4–99.9)41.4%
(24.1–60.9)17.64e < 0.01 Sensitive 1 12 13 Sub-total 71 29 100 Note. aWilson 95% confidence interval: including continuity correction; bν = 1 the chi-square value reported is the Yates chi-square, corrected for continuity (dν = 1, P = 0.0011, eν = 1, P < 0.0001); cMcNemar’s test, two-tail. Table 2. Comparison of gene mutation identified by WGS and MDR-LAMP assay
Drugs (genes) WGS MDR-LAMP Sub-total Sensitivity
(95% CI)aSpecificity
(95% CI)aPPV
(95% CI)aNPV
(95% CI)aχ2 b Pc Mutation Wild INH (katG, inhA) Mutation 69 5 74 93.2%
(84.3–97.5)92.3%
(73.4–98.7)97.2%
(89.3–99.5)82.8%
(63.5–93.5)5.33d 0.453 Wild 2 24 26 Sub-total 71 29 100 RIF (rpoB) Mutation 57 7 64 89.1%
(78.2–95.1)88.9%
(73.0–96.4)93.4%
(83.3–97.9)82.1%
(65.9–91.9)9.62e 0.549 Wild 4 32 36 Sub-total 61 39 100 Note. aWilson 95% confidence interval: including continuity correction; bν = 1 the chi-square value reported is the Yates chi-square, corrected for continuity (dν = 1, P = 0.021, eν = 1, P = 0.0019); cMcNemar’s test, two-tail. Table 3. Evolution efficiency of each probe of MDR-LAMP
Reagent Probes WGS MDR-LAMP Sub-total Sensitivity
(95% CI)aSpecificity
(95% CI)aPPV
(95% CI)aNPV
(95% CI)aχ2 b Pc Mutation Wild Reagent B FAM: rpoB
S531L/L533PMutation 11 26 37 29.7%
(16.4–47.2)100%
(92.8–100)100%
(67.9–100)70.8%
(60.1–79.7)27.6d < 0.01 Wild 0 63 63 Sub-total 11 89 100 Reagent B Cy5: rpoB
D516V/S531LMutation 37 5 42 88.1%
(73.6–95.5)100%
(92.3–100)100%
(88.3–100)92.1%
(81.7–97.0)3.28e 0.063 Wild 0 58 58 Sub-total 37 63 100 Reagent C FAM: rpoB
H526Y/ H526DMutation 20 1 21 95.2%
(74.1–99.8)93.7%
(85.2–97.7)80%
(58.7–92.4)98.7%
(91.8–99.9)4.3f 0.219 Wild 5 74 79 Sub-total 25 75 100 Reagent A FAM: katG
S315N/S315TMutation 68 3 71 95.8%
(87.3–98.9)100%
(85.4–100)100%
(93.3–100)90.6%
(73.8–97.6)1.35g 0.25 Wild 0 29 29 Sub-total 68 32 100 Reagent A FAM:
inhA Promotor-15Mutation 1 2 3 33.3%
(1.8–87.5)100%
(95.3–100)100%
(5.5–100)98%
(92.2–99.7)0.51h 0.5 Wild 0 97 97 Sub-total 1 99 100 Note. aWilson 95% confidence interval: including continuity correction; bν = 1 the chi-square value reported is the Yates chi-square, corrected for continuity (dν = 1, P < 0.0001, eν = 1, P = 0.0701, fν = 1, P = 0.0381, gν = 1, P = 0.2453, hν = 1, P = 0.4751); cMcNemar’s test, two-tail. Table 4. Degree Celsius distribution of each probe by MDR-LAMP reagents
Drugs (genes) Mutation site Number of strains Predict degree Celsius Actual degree Celsius (Number) INH (katG、inhA) katG S315T 68 FAM:56 °C 54 °C (25), 55 °C (32), 56 °C (9), -(2) katG S315N 3 FAM:54 °C 54 °C (2), -(1) inhA Promotor-15T 3 FAM:46 °C 46 °C (2), 48 °C (1) RIF (rpoB) rpoB S531L 36 FAM:63 °C and / or Cy5:73 °C FAM:62 °C and Cy5:71 °C (2), FAM:62 °C and
Cy5:72 °C (4), FAM:63 °C and Cy5:72 °C (5),
Cy5:67 °C (1), Cy5:71 °C (5), Cy5:72 °C (14),
Cy5:73 °C (1), -(4)rpoB D516V 6 Cy5:61 °C 60 °C (5), -(1) rpoB L533P 0 − − rpoB H526Y 17 FAM:67 °C 62 °C (3), 64 °C (2), 65 °C (8), 66 °C (3), -(1) rpoB H526D 4 FAM: 69 °C 67 °C (3), 68 °C (1) Note. “−” indicates “not detected.”
doi: 10.3967/bes2021.085
Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of Mycobacterium tuberculosis
-
Abstract:
Objective To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the rpoB, katG, and inhA genes at the Chinese Center for Disease Control and Prevention. Methods MDR-LAMP assay was evaluated using 100 Mycobacterium tuberculosis (Mtb) isolates obtained from the National Reference Laboratory for Tuberculosis in China. Phenotypic resistance to isoniazid and rifampicin and whole-genome sequencing served as reference standards. Results The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of Mtb cultured from smear-positive sputum samples, respectively. When DNA sequencing was used as the reference standard, the sensitivity, specificity, PPV, and NPV of MDR-LAMP were 93.1%, 92.3%, 97.2%, and 82.8% for the detection of katG and inhA gene mutations, respectively, and 89.1%, 88.9%, 93.4%, and 81.1% for the detection of rpoB gene mutation, respectively. Conclusion MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of Mtb isolates. -
Key words:
- MDR-LAMP /
- Mycobacterium tuberculosis /
- Diagnostic method
注释: -
S1. The phenotypic DST results of the 100 Mtb isolates
LAMP MDR assay Strain No. MIC Cutoff-value DST (S:Sensitivity, R:Resistance) WGS Gene: (W:Wild, M:Mutant) LAMP MDR (+:Detect, −:Not Detect) INH:
> 0.2RIF:
> 1INH RIF rpoB site katG site inhA-
Promotor-
15Reagent
AT Reagent
BT Reagent
CT 031024 2 > 16 R R M S531L M S315T W + 55 °C + Cy5:73 °C − 033059 4 > 16 R R W − W − M − − + 64 °C 033061 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033062 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033063 2 > 16 R R M H526Y M S315T W + 56 °C − + 65 °C 033064 2 > 16 R R M H526Y M S315T W + 56 °C − + 65 °C 033065 2 > 16 R R M H526Y M S315T W + 54 °C − + 62 °C 033066 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033067 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033068 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033070 2 > 16 R R M H526Y M S315T W + 54 °C − + 65 °C 033071 2 > 16 R R M H526Y M S315T W + 54 °C − + 64 °C 033102 1 > 16 R R M S531L W − W − + Cy5:71 °C − 033106 1 > 16 R R M S531L W − W − + Cy5:72 °C − 033109 1 > 16 R R M S531L W − W − + Cy5:72 °C − 033127 2 > 16 R R M S531L M S315T W + 55 °C − + 62 °C 033129 1 > 16 R R M H526Y M S315T W + 55 °C − + 66 °C 033159 4 > 16 R R M S531L M S315T W + 55 °C + Cy5:72 °C − 033214 4 > 16 R R M S531L M S315T W + 55 °C + Cy5:72 °C − 061021 0.5 > 16 R R M S531L W − W − + Cy5:71 °C − 061026 4 > 16 R R W − M S315T W + 54 °C − + 61 °C 061045 4 ≤ 0.12 R S W − M S315T W + 54 °C − − 061046 4 > 16 R R W − M S315T W + 54 °C − − 061102 0.06 > 16 S R M H526Y W − W − − + 62 °C 061105 4 ≤ 0.12 R S W − M S315T W + 55 °C − − 061260 0.06 > 16 S R M S531L W − W − − + 68 °C 061300 4 ≤ 0.12 R S W − M S315T W + 55 °C − − 061301 4 ≤ 0.12 R S W − M S315T W + 55 °C − − 061305 4 > 16 R R W − M S315T W + 55 °C − − 061315 > 4 1 R S W − W − M − − − 071014 4 16 R R M S531L M S315T W − − − 071023 4 0.12 R S W − M S315T W + 55 °C − − 071028 4 0.5 R S W − M S315T W + 55 °C − − 071031 4 0.25 R S W − M S315T W + 55 °C − − 071040 2 16 R R M H526Y M S315T W + 54 °C − + 62 °C 071061 4 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C+ 64 °C 071070 2 16 R R M H526D W − W − − + 67 °C 071075 4 0.12 R S W − M S315T W + 55 °C − − 071097 4 16 R R W − M S315T W + 54 °C − − 071107 4 16 R R W − M S315T W + 55 °C − − 071121 4 0.12 R S W − M S315T W + 55 °C − − 071126 4 0.12 R S W − M S315T W + 55 °C − − 071127 4 16 R R W − M S315T W + 55 °C − − 071140 4 1 R S W − M S315T W + 55 °C − − 081005 2 > 16 R R M H526D M S315T W + 54 °C − + 68 °C 081044 2 > 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C− 081051 2 > 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C− 081056 2 > 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C− 081067 4 > 16 R R M S531L M S315T W + 54 °C + FAM:63 °C,
Cy5:72 °C− 081161 0.06 > 16 S R M S531L W − W − + Cy5:72 °C − 081217 0.12 > 16 S R M S531L W − W − + Cy5:71 °C − 082248 4 16 R R M S531L M S315T W + 54 °C + Cy5:71 °C − 082308 0.25 > 16 R R M S531L W − W − + Cy5:72 °C − 082312 4 > 16 R R M L533P W − W + 48 °C − − 091079 4 0.25 R S W − M S315N W + 54 °C − − 091104 4 0.25 R S W − M S315T W + 55 °C − − 091119 4 0.25 R S W − M S315T W + 55 °C − − 102238 4 16 R R M S531L M S315T W + 54 °C + FAM:62 °C,
Cy5:72 °C− 104186 0.03 0.12 S S W − W − W − − − 104191 0.03 0.25 S S W − W − W − − − 104200 0.06 0.5 S S W − W − W − − − 104218 0.03 0.12 S S W − W − W − − − 104229 0.03 0.12 S S W − W − W − − − 151022 2 16 R R M H526D M S315T W + 54 °C − + 67 °C 153058 4 16 R R M H526Y M S315T W + 55 °C − − 154089 2 16 R R M H526Y M S315T W + 54 °C − + 66 °C 154214 4 0.25 R S W − M S315T W + 55 °C − − 154285 4 0.12 R S W − M S315T W + 55 °C − − 154286 2 16 R R M H526Y M S315T W + 55 °C − + 66 °C 171019 2 16 R R M D516V W − W − + Cy5:60 °C − 171026 0.5 > 16 R R M S531L W − W − + FAM:62 °C,
Cy5:72 °C− 171052 > 4 > 16 R R M S531L W − W − + FAM:62 °C,
Cy5:72 °C− 171064 > 4 > 16 R R M H526D M S315T W + 54 °C − + 67 °C 171078 > 4 16 R R M D516V M S315T W + 54 °C − − 171079 > 4 16 R R M D516V W − W − + Cy5:60 °C − 171292 2 > 16 R R M H526Y M S315T W + 56 °C − + 64 °C 171607 4 > 16 R R M S531L M S315T W + 56 °C + Cy5:72 °C − 172096 0.06 ≤ 0.12 S S M S531L M S315T W + 56 °C + Cy5:72 °C − 172300 4 > 16 R R M D516V M S315T W + 56 °C + Cy5:60 °C − 172314 > 4 > 16 R R M S531L M S315T W + 56 °C + Cy5:72 °C − 172573 2 > 16 R R M S531L M S315N W + 54 °C + Cy5:72 °C − 173001 0.25 > 16 R R M S531L W − W + 46 °C + FAM:62 °C,
Cy5:71 °C− 173093 > 4 > 16 R R M S531L W − W − + FAM:62 °C,
Cy5:72 °C− 173422 0.5 ≤ 0.12 R S W − W − W − − − 174067 0.06 0.25 S S W − M S315T W − − − 184403 4 16 R R M S531L M S315T W + 55 °C + Cy5:71 °C − 185209 4 0.5 R S W − M S315T W + 55 °C − − 185544 4 16 R R M S531L M S315T W + 56 °C + Cy5:67 °C − 185551 4 0.25 R S W − M S315T W + 55 °C − − 19200003 4 0.25 R S W − M S315T W + 55 °C − − 19200399 4 16 R R M S531L M S315T W + 55 °C + Cy5:72 °C − 221211 2 1 R S M D516V M S315N W − + Cy5:60 °C − 221227 0.25 16 R R M S531L W − W − + FAM:62 °C,
Cy5:71 °C− 221437 2 16 R R M S531L M S315T W + 56 °C + Cy5:72 °C − 221518 4 16 R R M S531L W − M + 48 °C + Cy5:72 °C − 221571 4 16 R R M S531L M S315T W + 55 °C + Cy5:72 °C − 222280 4 16 R R M D516V M S315T W + 55 °C + Cy5:60 °C − 233077 0.06 0.25 S S W − W − W − − − 235254 0.06 0.12 S S W − W − W − − − 320005 2 2 R R M S531L M S315T W + 55 °C − − Table 1. Efficacy of MDR-LAMP test compared with the broth microdilution MIC method
Drugs MIC MDR-LAMP Sub-total Sensitivity
(95% CI)aSpecificity
(95% CI)aPPV
(95% CI)aNPV
(95% CI)aχ2 b Pc Resistance Sensitive RIF Resistance 59 10 69 85.5%
(74.5–92.5)93.6%
(77.2–98.9)96.7%
(87.6–99.4)74.4%
(57.6–86.4)10.73d 0.039 Sensitive 2 29 31 Sub-total 61 39 100 INH Resistance 70 17 87 80.5%
(70.3–87.9)92.3%
(62.1–99.6)98.6%
(91.4–99.9)41.4%
(24.1–60.9)17.64e < 0.01 Sensitive 1 12 13 Sub-total 71 29 100 Note. aWilson 95% confidence interval: including continuity correction; bν = 1 the chi-square value reported is the Yates chi-square, corrected for continuity (dν = 1, P = 0.0011, eν = 1, P < 0.0001); cMcNemar’s test, two-tail. Table 2. Comparison of gene mutation identified by WGS and MDR-LAMP assay
Drugs (genes) WGS MDR-LAMP Sub-total Sensitivity
(95% CI)aSpecificity
(95% CI)aPPV
(95% CI)aNPV
(95% CI)aχ2 b Pc Mutation Wild INH (katG, inhA) Mutation 69 5 74 93.2%
(84.3–97.5)92.3%
(73.4–98.7)97.2%
(89.3–99.5)82.8%
(63.5–93.5)5.33d 0.453 Wild 2 24 26 Sub-total 71 29 100 RIF (rpoB) Mutation 57 7 64 89.1%
(78.2–95.1)88.9%
(73.0–96.4)93.4%
(83.3–97.9)82.1%
(65.9–91.9)9.62e 0.549 Wild 4 32 36 Sub-total 61 39 100 Note. aWilson 95% confidence interval: including continuity correction; bν = 1 the chi-square value reported is the Yates chi-square, corrected for continuity (dν = 1, P = 0.021, eν = 1, P = 0.0019); cMcNemar’s test, two-tail. Table 3. Evolution efficiency of each probe of MDR-LAMP
Reagent Probes WGS MDR-LAMP Sub-total Sensitivity
(95% CI)aSpecificity
(95% CI)aPPV
(95% CI)aNPV
(95% CI)aχ2 b Pc Mutation Wild Reagent B FAM: rpoB
S531L/L533PMutation 11 26 37 29.7%
(16.4–47.2)100%
(92.8–100)100%
(67.9–100)70.8%
(60.1–79.7)27.6d < 0.01 Wild 0 63 63 Sub-total 11 89 100 Reagent B Cy5: rpoB
D516V/S531LMutation 37 5 42 88.1%
(73.6–95.5)100%
(92.3–100)100%
(88.3–100)92.1%
(81.7–97.0)3.28e 0.063 Wild 0 58 58 Sub-total 37 63 100 Reagent C FAM: rpoB
H526Y/ H526DMutation 20 1 21 95.2%
(74.1–99.8)93.7%
(85.2–97.7)80%
(58.7–92.4)98.7%
(91.8–99.9)4.3f 0.219 Wild 5 74 79 Sub-total 25 75 100 Reagent A FAM: katG
S315N/S315TMutation 68 3 71 95.8%
(87.3–98.9)100%
(85.4–100)100%
(93.3–100)90.6%
(73.8–97.6)1.35g 0.25 Wild 0 29 29 Sub-total 68 32 100 Reagent A FAM:
inhA Promotor-15Mutation 1 2 3 33.3%
(1.8–87.5)100%
(95.3–100)100%
(5.5–100)98%
(92.2–99.7)0.51h 0.5 Wild 0 97 97 Sub-total 1 99 100 Note. aWilson 95% confidence interval: including continuity correction; bν = 1 the chi-square value reported is the Yates chi-square, corrected for continuity (dν = 1, P < 0.0001, eν = 1, P = 0.0701, fν = 1, P = 0.0381, gν = 1, P = 0.2453, hν = 1, P = 0.4751); cMcNemar’s test, two-tail. Table 4. Degree Celsius distribution of each probe by MDR-LAMP reagents
Drugs (genes) Mutation site Number of strains Predict degree Celsius Actual degree Celsius (Number) INH (katG、inhA) katG S315T 68 FAM:56 °C 54 °C (25), 55 °C (32), 56 °C (9), -(2) katG S315N 3 FAM:54 °C 54 °C (2), -(1) inhA Promotor-15T 3 FAM:46 °C 46 °C (2), 48 °C (1) RIF (rpoB) rpoB S531L 36 FAM:63 °C and / or Cy5:73 °C FAM:62 °C and Cy5:71 °C (2), FAM:62 °C and
Cy5:72 °C (4), FAM:63 °C and Cy5:72 °C (5),
Cy5:67 °C (1), Cy5:71 °C (5), Cy5:72 °C (14),
Cy5:73 °C (1), -(4)rpoB D516V 6 Cy5:61 °C 60 °C (5), -(1) rpoB L533P 0 − − rpoB H526Y 17 FAM:67 °C 62 °C (3), 64 °C (2), 65 °C (8), 66 °C (3), -(1) rpoB H526D 4 FAM: 69 °C 67 °C (3), 68 °C (1) Note. “−” indicates “not detected.” -
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