LIGHTS ON/OFF: CLINICAL EVALUATION IN SOUTH AFRICA
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1 LIGHTS ON/OFF: CLINICAL EVALUATION IN SOUTH AFRICA ROB WARREN LARRY WANGH, BARRY KREISWIRT, JOHN RICE, MARGARETHA DE VOS, MICHAL WHITFIELD, SALVATOR MARRAS
2 OUTLINE Background LATE-PCR description LATE-PCR M(X)DR-TB assay LATE-PCR PZA assay
3 SIMPLIFIED DIAGNOSTIC ALGORITHM IN SOUTH AFRICA Sputum 1 Xpert Rif resistant HIGH TECH Sputum 2 Decontamination Smear Smear - Smear + LPA INH and RIF Culture RIF resistant cases Culture LPA INH and RIF DST amikacin and ofloxacin (isoniazid) LOW TECH
4 SOUTH AFRICAN TB TREATMENT GUIDELINES Time to initiation of treatment All patients diagnosed with drug susceptible TB must be started on TB treatment within 2 days and those with drug resistant TB within 5 days of suspicion. Laboratory confirmed TB patients who are never started on treatment are referred to as initial loss to follow up (Initial defaulters). These patients could have died (but confirmation not available) before initiation of treatment or could not be found on tracing. This measures the efficiency of the TB programme in ensuring that all bacteriologically confirmed patients are started on treatment.
5 PROPORTION OF MDR-TB CASES RECEIVING > 3 EFFECTIVE 2 ND LINE DRUGS 3 drugs 47% > 3 drugs 53% Assuming 100% susceptibility to Terizidone
6 LATE - PCR - PHASE 1: Exponential Amplification of DOUBLE - STRANDED DNA n Strand Denaturation Limiting Primer Tm Excess Primer Tm Primer Extension Limited Number of ds-dna Molecules Temperature Primer Annealing
7 LATE - PCR - PHASE 2: Exponential Amplification of SINGLE - STRANDED DNA Temperature n Strand Denaturation Primer Extension Excess Primer Tm Primer Annealing nmoles Single-stranded DNA
8 LIGHTS-ON/LIGHTS-OFF PROBES On probe Single stranded Target Fluorophore Quencher Off probe Quencher Temperature
9 On probe X Single stranded Target Fluorophore Quencher Off probe Quencher Temperature
10 STUDY DESIGN DNA from cultured isolates Drug susceptible and resistant Clinical Specimens Drug susceptible and resistant Reference samples (n=95) Blinded samples (n=1000) Set A (n=3268) Set B (n=3145) Input: 0.25ng/µl copies Input: ng/µl 2000 copies Residual sputum specimen Genolyse extraction Culture isolate Residual sputum specimen Culture isolate 95 signatures 852 signatures Banked + routine diagnostic data Randomly select a 1000 for evaluation
11 LATE-PCR LIGHTS-ON/LIGHTS-OFF M(X)DR ASSAY Rifampicin: rpob Isoniazid: katg and inha promoter Aminoglycosides: rrs and eis promoter Fluoroquinolones: gyra and gyrb
12 Detecting Rifampicin and Isoniazid resistance Detecting Aminoglycoside resistance inha promoter rpob IC katg EC eis promoter rrs Detecting Fluoroquinolone resistance gyrb gyra
13 Detecting rifampicin and isoniazid resistance inha promoter katg rpob IC
14 Detecting Rifampicin and Isoniazid resistance n=50 inha promoter katg rpob IC
15 -df/dt, QSR670 Detecting different rpob 526 mutations inha promoter katg rpob IC CTC 526TAC 526GAC 526AAC Temperature C
16 rpob codon rpob base change No of isolates RIF MIC range bp deletion 2 < T>C 14 < GA>TG 1 < A>C 2 < C>A 3 <1 Rifampicin critical concentration = 1ug/ml in MGIT media
17 Detecting rifampicin and isoniazid resistance n=10 (in total 89/852) inha promoter katg rpob IC rpob 531TTG inha promoter -15C/T katg wildtype
18 Detecting rifampicin and isoniazid resistance n=10 (in total 45/852) inha promoter katg rpob IC rpob 516GTC inha promoter -17G/T katg 315ACC
19 Detecting aminoglycoside resistance n=10 (in total 68/852) External control eis promoter rrs wildtype 1401G wildtype
20 -df/dt, CR610 External control Detecting aminoglycoside resistance eis promoter rrs -10C/T: 1/852-12G/A: 6/852 wildtype G/A wildtype -10 C/T -14 G/A Temperature C
21 -df/dt, CO560 Detecting fluoroquinolone resistance resistance 150 gyrb 125 wildtype Temperature C
22 -df/dt, FAM Detecting fluoroquinolone resistance resistance TGC 94GCC wildtype 94AAC 94GCC Temperature C
23 rpob 510 CCG 511 CCG 512 AAA 513 AAA 516 GCC 516 GTC 516 TAC 526 AAC 526 CTC = 526 CGC 526 GAC = 526 AGC 526 TAC = 526 GGC 531 TTG 533 CCG Del ( ) Del ( ) Del (518) Del ( ) 516 GGC CCG 526 TAC +533 CCG inha promoter gyra rrs eis promoter -15 C/T -17 G/T -8 T/A 88 TGC = 94 GGC 89 GGC = 94 GCC 90 GTG 91 CCG 94 AAC 94 GAC 94 TAC 94 TGC 1401 A/G -10 G/C -12G/A -14G/A katg 315 ACC 315 ACA
24 LATE-PCR PZA ASSAY Association between PZA resistance and level of resistance
25 LATE-PCR PZA ASSAY Quasar Cal Orange or Tamra Cal Red
26 Association between PZA resistance and level of resistance M Whitfield (submitted)
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36 LATE-PCR PZA ASSAY Total isolates tested = 654 Sanger sequencing of pnca: pnca WT = 240 pnca polymorphism = 414 Results of LATE-PCR PZA assay Sanger WT + LATE WT = 234 Sanger WT + LATE SNP = 6 Sanger SNP + LATE SNP = 391 Sanger SNP + LATE WT = different pnca polymorphisms assessed (SNPs, insertions, deletions, double SNPs)
37 LATE-PCR PZA ASSAY Sensitivity = 94.4% (95% CI: 92.2% %) Specificity = 97.5% (95% CI: 95.6% %)
38 Stellenbosch University MRC Centre for Tuberculosis Research DST/NRF Centre of Excellence for Biomedical Tuberculosis Research Prof R Warren Dr M de Vos Dr E Streicher Dr K Jacobson Dr RG van der Merwe Brandeis University, Waltham, MA Prof L Wangh Dr J Rice NHLS Prof N Ismail Mrs T Dolby PHRI, Newark, NJ Dr B Kreiswirth Dr N Kurepina Dr S Marras NIH Funding award R01AI Hain LifeSciences D Hain Dr V Allerheiligen Dr M Eckhard
39 LATE-PCR PZA assay Discordant isolates Sanger WT + LATE SNP = 6 (Sanger no underlying peaks) Sanger SNP + LATE WT = 23 2 isolates with insertion (codon 35 and 152) 2 isolates with deletion (codon 70 and 82) 19 isolates with SNP (all different SNPs spanning entire pnca gene)
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