Background
Methods
Study population
Acid-Fast Bacilli (AFB) smear and drug-susceptibility testing
DNA extraction, PCR and molecular targets
Genes | Target | Forward primer | Reverse primer | Sequencing primer | Detection range | Reference |
---|---|---|---|---|---|---|
Mtb Identification | IS6110 | Biotin-CCGCCAACTACGGTGTTTA | CAGGCCGAGTTTGGTCAT | GGCCACCTCGATGCC | Multiple | [9] |
Isoniazid-R |
katG
| Biotin-CGGAACCGGTAAGGACGC | CCATTTCGTCGGGGTGTTC | TCCATACGACCTCGAT | Codons 312 to 316 | [9] |
inhA
| Biotin-ACGCTCGTGGACATACCG | CAGTGGCTGTGGCAGTCA | TGTGGCAGTCACCCC | Position −4 to −20 | [9] | |
ahpC
| TCCTCATCATCAAAGCGGACAAT | Biotin-CGATGCCGATAAATATGGTGTGAT | CATTTGGTTGCGACAT | Position −4 to −23 | [9] | |
Rifampin-R |
rpoB1
| GGAGGCGATCACACCGCAGACGTT | Biotin-CCTCCAGCCCGGCACGCTCACGT | GCGATCAAGGAGTTCTTC | Codons 507 to 521 | |
rpoB2
| TTTCGATCACACCGCAGACGTT | Biotin-AAAGGCACGCTCACGTGACAGAC | CAGAACAACCCGCTG | Codons 522 to 533 | ||
Fluoroquinolone-R |
gyrA
| AATGTTCGATTCCGGCTTCC | Biotin-CGGGCTTCGGTGTACCTCAT | CAACTACCACCCGCAC | Codons 88 to 95 | [2] |
Injectable-R |
rrs
| TAAAGCCGGTCTCAGTTCGGAAaC | Biotin-CAGCTCCCTCCCGAGGGTTA | CTTGTACACACCGCC | Position 1397 to 1406 | [9] |
Kanamycin-R |
eis
| Biotin-GGCTACACAGGGTCACAGTC | GCCAGACACTGTCGTCGTAATATTC | CAGACACTGTCGTCG | Position −5 to −47 | This study |
Pyrosequencing (PSQ)
Pyrosequencing (PSQ) diagnostic performance
Analysis of pyrosequencing (PSQ) technical performance
Human research conduct
Results
Culture and Drug Susceptibility Testing (DST) results
Sensitivity and Specificity of Pyrosequencing (PSQ) as Compared to Phenotypic Testing
India (n = 492) | Moldova (n = 226) | South Africa (n = 196) | |||||||
---|---|---|---|---|---|---|---|---|---|
Sensitivity | Specificity | Agreement | Sensitivity | Specificity | Agreement | Sensitivity | Specificity | Agreement | |
INH | 98 % (0.96–0.99) | 97 % (0.90–1) | 98 % (0.96–0.99) | 94 % (0.88–0.97) | 96 % (0.87–0.99) | 95 % (0.90–0.97) | 71 % (0.53–0.85) | 94 % (0.87–0.98) | 88 % (0.81–0.93) |
RIF | 98 % (0.95–0.99) | 100 % (0.94–1) | 98 % (0.96–0.99) | 94 % (0.86–0.98) | 100 % (0.92–1) | 97 % (0.92–0.99) | 77 % 0.54–0.91) | 98 % (0.91–1) | 94 % (0.87–0.97) |
MOX | 96 % (0.92–0.98) | 96 % (0.92–0.98) | 96 % (0.94–0.98) | 67 % (0.39–0.87) | 100 % (0.97–1) | 97 % (0.92–0.99) | 82 % (0.48–0.97) | 99 % (0.96–1) | 98 % (0.94–1) |
OFX | 96 % (0.93–0.98) | 99 % (0.96–1) | 97 % (0.95–0.99) | 64 % (0.36–0.86) | 99 % (0.96–1) | 96 % (0.91–0.98) | 90 % (0.54–0.99) | 99 % (0.96–1) | 99 % (0.95–1) |
AMK | 94 % (0.82–0.98) | 100 % (0.98–1) | 99 % (0.98–1) | 33 % (0.11–0.65) | 99 % (0.96–1) | 95 % (0.91–0.98) | 92 % (0.60–1) | 98 % (0.94–0.99) | 98 % (0.93–0.99) |
KAN | 89 % (0.76–0.95) | 100 % (0.98–1) | 99 % (0.97–0.99) | 7 % (0.02–0.18) | 99 % (0.95–1) | 71 % (0.64–0.77) | 92 % (0.60–1) | 98 % (0.94–0.99) | 97 % (0.94–0.99) |
KAN (+eis) | 93 % (0.81–0.98) | 91 % (0.88–0.94) | 91 % (0.88–0.94) | 79 % (0.66–0.88) | 95 % (0.90–0.98) | 90 % (0.85–0.94) | 92 % (0.60–1) | 98 % (0.93–0.99) | 97 % (0.93–0.99) |
CAP | 94 % (0.81–0.98) | 99 % (0.98–1) | 99 % (0.97–0.99) | 40 % (0.14–0.73) | 99 % (0.96–1) | 96 % (0.92–0.98) | 85 % (0.54–0.97) | 98 % (0.94–0.99) | 97 % (0.92–0.99) |
KAN resistance upon eis promoter addition
Pyrosequencing (PSQ) success by smear- and culture-result
Pyrosequencing (PSQ) indeterminate analysis
Variable | Crude OR | 95 % CI | Adjusted OR | 95 % CI |
---|---|---|---|---|
AFB Smear | ||||
Negative |
5.30
| 4.76–5.90 |
2.19
| 1.91–2.51 |
Positive | 1.00 | 1.00 | ||
Culture | ||||
Negative |
8.44
| 7.51–9.49 |
7.74
| 6.67–8.99 |
Positive | 1.00 | 1.00 | ||
Site | ||||
Moldova |
2.07
| 1.84–2.34 |
2.86
| 2.47–3.31 |
South Africa |
1.86
| 1.64–2.10 |
1.50
| 1.29–1.74 |
India | 1.00 | 1.00 | ||
Target | ||||
katG |
1.52
| 1.19–1.92 |
1.69
| 1.29–2.21 |
inhA |
1.30
| 1.02–1.66 |
1.38
| 1.05–1.81 |
ahpC |
1.71
| 1.35–2.16 |
1.96
| 1.50–2.55 |
rpoB1 |
3.65
| 2.93–4.55 |
5.29
| 4.12–6.79 |
rpoB2 |
3.31
| 2.65–4.12 |
4.65
| 3.62–5.97 |
gyrA |
2.99
| 2.39–3.73 |
4.07
| 3.16–5.23 |
rrs |
1.90
| 1.50–2.39 |
2.24
| 1.72–2.91 |
IS6110 | 1.00 | 1.00 |
TARGET | No Read Error | Homopolymer Error | Instrument Error | Other Error | Mixed Population | New Mutation | TOTAL | Error Rate (/1128 reactions) |
---|---|---|---|---|---|---|---|---|
IS6110 | 112 (85 %) | 12 (9 %) | 4 (3 %) | 4 (3 %) | - | - | 132 | 11.7 % |
katG
| 91 (49 %) | 74 (40 %) | 1 (1 %) | 19 (10 %) | 1 (1 %) | - | 186 | 16.5 % |
inhA
| 121 (73 %) | 9 (5 %) | 3 (2 %) | 29 (17 %) | 2 (1 %) | 2 (1 %) | 166 | 14.7 % |
ahpC
| 182 (87 %) | 13 (6 %) | 6 (3 %) | 9 (4 %) | - | - | 210 | 18.6 % |
rpoB1
| 221 (61 %) | 76 (21 %) | 15 (4 %) | 45 (12 %) | 1 (0 %) | 3 (1 %) | 361 | 32.0 % |
rpoB2
| 269 (78 %) | 40 (12 %) | 7 (2 %) | 28 (8 %) | - | 1 (0 %) | 345 | 30.6 % |
gyrA
| 294 (91 %) | 2 (1 %) | 6 (2 %) | 18 (6 %) | 1 (0 %) | 1 (0 %) | 322 | 28.5 % |
rrs
| 149 (66 %) | 30 (13 %) | - | 48 (21 %) | - | - | 227 | 20.1 % |