GeneXpert (Xpert MTB/RIF): what is the future?
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1 GeneXpert (Xpert MTB/RIF): what is the future? Professor Lesley Scott Department of Molecular Medicine and Haematology, University of the Witwatersrand & National Priority Program of the National Health Laboratory Service Johannesburg, South Africa
2 2015: South Africa in the global context HIV MDR-TB WHO Global Tuberculosis Report Geneva South Africa: 1. Second highest incidence rate TB, 2. Largest number HIV-TB co-infected 3. Second highest diagnosed MDR-TB, 4. High rates of XDR-TB XDR-TB
3 HIV & TB in South Africa in 2015 ~55 Million total Population ~6.3 million HIV infected, ~0.29 million TB infected (Xpert only) Second highest incidence rate TB, Largest number HIV-TB co-infected ~16% extrapulmonary TB WHO Global Tuberculosis Report Geneva Laboratory Context (National Health Laboratory Service, 265 laboratories) servicing ~80% public sector TB: Xpert MTB/RIF in 207 smear microscopy laboratories ~10.7% MTBC detected, ~6% Rif resistant (proxy for MDR) Liquid culture (16 labs): 0.56million tests for diagnosis in HIV+/Xpert- and DST/LPA for MDR/XDR confirmation Smear microscopy (1.0million tests) for treatment monitoring: 78% reduction in test volumes since 4 years Xpert implementation. HIV: ~ 3.9 million CD4 tests in 60 laboratories, 4 million viral loads and EID tests in 16 PCR laboratories Test Method Volumes #GXP #AFB #Cult #GXP
4 South Africa s TB and HIV control program goals Xpert MTB/Rif performed on a single sputum (in 8 of 9 provinces) to replace smear microscopy. TB screening program (household contacts, community mobilization, door-todoor) focused high burden regions. IPT uptake increased dramatically (still low in PLHIV, children and pregnant women). Infection control program with re-engineered primary health and key populations (PLHIV, children, HCW, correctional facilities, mines, schools, taxi s) Children: treatment outcomes good, diagnosis still challenging. Extra-pulmonary TB: diagnosis difficult and too late ART program aimed at national coverage and with TB integration of services MDR-TB: confirmation and loss to follow up challenges, now decentralised and de-institutionalised treatment and new drugs (Bedaquiline access to MDR and XDR). Surveillance: challenges with data collection and linkage (unique identifier!), DR survey complete
5 Research and Development focus Diagnostic services Need increased sensitivity to treat more (Xpert 60-80% in smear negative TB), need good NPV to eliminate empiric treatment..xpert Ultra or fast followers. Simplification and cost reduction of the algorithm. Children stool Xpert testing at point of care. EPTB with alternative Xpert protocols. Integration with HIV using polyvalent platform approach. Confirmation of MDR and XDR with molecular platforms. Expand services: mining community, correctional facilities, increase access with mobile Xpert.Omni. Novel rapid surveillance with molecular indicators. RealTime monitoring to improve services with Remote Xpert.
6 What about TB: one of our biggest challenges Are molecular TB tests good enough?... Will they work in patients with HIV? Smear microscopy = 59% sensitive HIV+: 54% sensitive, 100% specific
7 Xpert distribution in public sector by district (March March 2016) 314 GeneXpert instruments: 4180 modules GX4: Upon 115; GX16: Honourable 190; GX48: Minister 1; GX80: of Health s 8 in 207 sites endorsement, In March 2011 South Africa both developed urban and and rural initiated settings- a across National 9 provinces Plan for phased implementation of the GeneXpert technology, as a replacement for smear microscopy; phased approach 314 GeneXpert instruments of varying sizes (GX4: 115; GX16:190; GX48: 1; GX80:8) have been placed in 211 sites both urban and rural settings- across 9 provinces A total of 1,943 laboratory and 9762 clinical staff have been trained since December 2011 Performed over tests to date (31 October 2015) MTB positivity rate in GXP assays decline from 16% to 9% Stable Rif resistance rate of 6-7% 207 sites servicing Significant support from CDC, Global fund, BMGF, UNITAID, amongst others ~4710 facilities from Resulted in faster time to treatment, increased laboratory confirmed previous microscopy diagnosis, significantly reduced time to treatment for Rif Res cases centres Further expanded access to Correctional Services inmates and Placement miners based on estimated number of smear tests/patient and per site
8 Test Method Volumes #GXP #GXP #AFB #Cult Phase 1 (Pilot) High Burden Districts: 25 sites in 9 provinces Phase 2 (Expand) Capacitate High Burden Districts Phase 3 (Embed) Clinical Impact studies Phased implementation starting in high burden districts Phase 4 (National coverage 2013) 207 sites Paediatric EPTB Phase 5 (Parallel) Correctional Facilities Peri-Mining (Mobile)
9 Impact of Xpert MTB/RIF on patients with RR-TB: (Example of Therapeutic Impact) Median time to treatment (days) by year and diagnostic method (Courtesy Mark Nicol; 2014)
10 National Task Team established by NPP with all stakeholders: NDoH, DCS, Clinicians, NHLS, human rights and advocacy groups, facility security, M&E group, research group, donors; infection control Laboratory Backbone facilitated expansion to Correctional services Vulnerable populations: Correctional Services Total DCS facilities- red Total NHLS GXP labs (207)- green 242 facilities in 48 clusters GIS mapped to NHLS lab offenders ( in remand) 7- mini labs on-site at Max Security (sample volume/security issues) assays conducted since (Oct 2014-March 2016) Average MTB positivity of 4.5% RIF resistance positivity of 4.3% ( n =420 new MDR cases) Analyzers linked to central lab. Infection Control: Major issue for inmates and healthcare workers.
11 Mines and Peri-mining Communities >1600 mines, different sizes, ~23 minerals TB Incidence in miners: 3000/ (Churchyard 2014) Global Fund- to provide services aimed at improving TB and HIV/AIDS management for peri-mining communities (~ people in six main mining districts). NHLS tests people, but low yield (2-3%). Contact tracing may be more successful. Use of vehicles for hotspots. Districts in red
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15 Why the GeneXpert? First in class (TB lab in a cartridge) Improved sensitivity Simultaneous Rifampicin susceptibility Modular format addresses volume needs Automation Increased TAT Simplicity and safety No need for cold chain Connectivity Still of concern: HIV+/Xpert- TB disease patients, currently followed up with culture. Test utility Xpert as initial diagnostic test to replace smear Add-on test to negative smear Smear-positive, culturepositive Among HIV+ Among HIV- Performance 88% sensitive, 99% specific 68% sensitive, 99% specific 98% sensitive, 79% sensitive, 86% sensitive Detect Rif resistance 95% sensitive, 98% specific
16 Xpert: single to high throughput Closed platform for extraction, amplification and detection of Mycobacterium tuberculosis (Mtb) complex and Rif resistance Direct from unprocessed sputum (0.5 4ml) Time to result: 2 hours 2. Shake then stand 10 minutes 4. Transfer 2ml to cartridge 1. Add 2:1 Sample Buffer to sample 3. Shake then stand tests per 8 hour shift (GX4) (possible to do 64/shift/GX16 Begin Test or 192/shift/GX48 further 5 minutes
17 86% sensitive, 97% specific HIV+: 84%, 96% specific
18 Xpert MTB/RIF for the diagnosis of TB Lawn, Nicol. Future Microbiol 2001; 6(9):
19 Internal control Probe B not hybridized
20 MTBC negative MTBC detected with RIF resistance MTBC detected but no RIF resistance detected
21 Presumptive TB clients who are Xpert positive are classified as TB cases
22 Xpert MTB/RIF vs Xpert Ultra Xpert MTB/RIF Specimen volume 1ml sputum, 500ul sediment 1ml sputum, 500ul sediment Ultra PCR reaction volume 25ul 50ul Limit of detection in spike sputum cfu/ml 5-25cfu/ml Target rpob with 5 probes IS16110, IS1081 and rpob 4 probes Quantification Semi-quantification with Cycle Threshold (Ct) Semi-quantification with Cycle Threshold (Ct) Mutation coverage rpob Delayed and drop out hybridization Melting temperature (may identify codons) Sensitivity smear negative TB 60-80% 94% Test time ~2hrs ~1hr 15min South Africa s strategy for initial diagnosis Smear replacement Smear and Culture replacement Alland.D et al, CROI 2015, abstract 91
23 Xpert Ultra: exciting preliminary results Collaboration with FIND and clinical partners in Johannesburg (10 country study). July 2016: n=160 patients n=91 complete (Ultra is non-inferior to Xpert) Considerations for implementation: Manage swap over (experience with G3/G4): National and rapid/local and focussed, stock management, FAQ/hotline, national clinical and laboratory training. Instrument software upgrade and interface to LIS, gate keeping. Result reporting now to include Trace Clinical/laboratory algorithm changes (only targeted culture), treatment monitoring. A 0 Cycle Threshold (Ct) can still produce a melt curve with no mutation. 100 HIV+ TB suspects 80 TB- 20 TB+ 16 Gx+ Test 84 with liquid culture 20 Gx+ All 20 detected with Ultra and only require culture for resistance testing (~6% MGIT tubes)
24 TB cases (millions) The Need for New Diagnostics: Mind the Gap in TB case detection Estimated incidence 5.8 Cases detected and notified Courtesy, David Alland
25 4 probes identify rifampin-r mutations in rpob by shifting their Tm away from a wild type reference value. Four probes Courtesy, David Alland A clear change in Tm distinguishes wild type from resistant mutant rpob core region. Any mutation = Rifampin resistance Probes over lap rpob sequence Rif -S Rif R
26 Ultra: easily wins the mutation panel challenge codo n Courtesy, David Alland rpo1 rpo2 rpo3 rpo4 Wild type L511P (-6.1) Q513K (-3.8) D516V (-3.7) DEL (-6.4) DEL (-7.1) S522L (-3) H526Y (-2.5) 67.8 H526D (-3.4) 67.5 H526L (-3.4) 66.5 R529K (-2.9) S531Q (-4.3) 66.4 S531L (-2.3) 73.6 (+6.8) S531W (-2.5) 70.5 (+3.7) L533P (-5.4) L533R (-5.4) All mutations supplied by FIND detected with a wide Tm shift.
27 Research considerations for evaluating new molecular tests Components: Biosafety, technical, diagnostic, therapeutic, patient impact. Stabilised and appropriate safe material (quality panels) Limited volumes and quality of sputum (especially HIV positive individuals) Imperfect gold standard of culture and not tested on the same specimen as molecular Inactivation limits culture growth, requires several specimen. Standard of care algorithms rely on a single sputum Sputum not easy to split Decontaminated sediment (pellet) requires splitting (smear, liquid and solid culture, DST molecular and phenotypic) or frozen storage. Patient reimbursement to reduce loss to follow up Unravelling discordant results (PCR detects dead organisms) Response to treatment currently monitored with much reduced sensitive test High throughput molecular platforms will require high throughput routine testing beyond the slower limit of strict informed consent driven trials. Future evaluations: Abbott MTB and RIF/INH, NGS, Fluorotype (Hain LifeSciencies), Xpert XDR (Cepheid).. Schumacher.S et al. Impact of Molecular TB diagnostics for important outcomes: review study methodologies. Plos One March 2016
28 TB diagnosis in children: no universal diagnostic algorithm - combination of clinical symptoms and non-specific tests WHO: Xpert sensitivity is 66%, specificity 98% Sputum quality and quantity poor. Studies limited and most focus on induced sputa and required laboratory decontamination and concentration prior to Xpert. Our experience: Johannesburg study, (June 2011 May 2012) 484 patients, median age 24 months, single sputum no induction. 63% below testing volume (<0.5ml) of Xpert MTB/RIF More Xpert positive in older children (>132 months). Xpert MTB/RIF: Sensitivity 62.5% (25, 91), specificity 99.1% (98, 99.9) Smear microscopy: sensitivity 33% (8, 70), specificity 99.5% (98, 99.9) Xpert is better than smear but not good enough using sputum! Gous N, et al. Diagnosing childhood pulmonary tuberculosis using a single sputum specimen on Xpert MTB/RIF at point of care. South African Medical Journal 105:12 (2015) DOI: /SAMJ.2015.v105i
29 Alternative specimen processing using stool Developed by Priya Banada at Alland laboratory (Rutgers University). Preliminary data Sample processing device easy to use at POC and feasible from nappies (0.6g, 1.2g, swab) Stool (0.6g) as good as respiratory and good option for non-invasive specimen in young children.
30 Xpert MTB/RIF pulmonary and Extrapulmonary (EPTB) national data (12m, 2014/2015) FINE NEEDLE ASPIRATE BIOPSY 0% TISSUE 1% LAVAGE GASTRIC BRONCHIALVEOLAR ASPIRATE FNA 1% (DAY 1) 1% 1% FLUID / ASPIRATE 7% ABSCESS (SUPERFICIAL) ASPIRATE 1% NASOPHARYNGEAL ASPIRATE 1% ASPIRATE GASTR 1% TRACHEAL ASPIRATE 4% CSF 14% FLUID 0% BRONCHIAL WASHING 0% BRONCHIAL ALVEOLAR LAVAGE BLC,BLE 0% CSF 43% 0% LAVAGE BRONCHIALVEOLAR URINE 1% 1% WASHINGS FLUID BRONCHIAL C,CF PLEURAL 1% 1% 1% FLUID / ASPIRATE 26% PUS 1% SWAB (SUPERFICIAL) 1% DRAIN FLUID (STERILE CAVITY) PLEURAL FLUID 1% TISSUE 1% 2% FINE NEEDLE ASPIRATE FNA 2% TRACHEAL ASPIRATE 7% BIOPSY 2% ABSCESS (SUPERFICIAL) ASPIRATE 3% GASTRIC WASH 4% 12 m specimen type>100 records from >13yrs BLOOD 2% GASTRIC WASH 68% 12m specimen type >100 records, <13yrs Total number MTB positive % Sputum <13yrs 294, % EPTB<13yrs CSF Fluid/Aspriate Gastric wash 20,745 2,612 1,363 12, % 1.88% 9.54% 2.25% Sputum>13yrs 2,069, % EPTB>13yrs CSF Gastric Wash Trachael asprirate Fluid/Aspirate 36,803 12,192 1,073 1,932 7, % 3.1% 13.6% 8.85% 13.01%
31 EPTB Xpert testing: Our experience (Johannesburg study, n=1175) Xpert detection (260/1175) equivalent to MGIT (277/1175) Overall Xpert sensitivity is 59% (CI 53, 65) and specificity is 92% (CI 90, 94). Highest sensitivity in pus, aspirates, then fluids (ascitic then pleural). Highest sensitivity is amongst thick specimens 87% (CI 76, 94), and significantly different to clear specimens 48% (CI 36, 61). This was unchanged with traces of blood 52% (CI 44, 60) or pre-centrifugation 57% (CI 28, 82) among clear specimens. Xpert (124 additional results) is less affected by contaminating bacteria than MGIT (10.5% contamination) with earlier RIF diagnosis. The specificity of the Xpert was lower among pus and aspirates (76% and 78% respectively) but likely true-positive cases. Specimen type Lymph node tissue and aspirate WHO Sensiti vity 85% CSF 80% Pleural fluid 44% Gastric lavage and aspirate 84% Other tissues 81%
32 Additional applications for Xpert (South African studies) Fine needle aspirates from lymph nodes: In HIV clinic (n=152 adults) Xpert sensitivity 94.2% (85, 98) Median time to diagnosis and treatment 1 day compared to 5 days for smear and up to 47 days for culture DST In Flow Cytometry laboratory to exclude lymphoma: Xpert sensitivity, 50% (CI:26 75%), specificity 99% (CI:91 100%) respectively. Xpert detected 8.5% not sent for culture. Additional interest: Xpert in urine (TB cases, n = 139) Not part of national implementation Feasible in hospitalized patients unable to produce sputum Requires 20ml with centrifugation Detection within first 24 hrs hospital admission: Xpert testing sputum, 39/139 (28.1 %), Xpert testing urine 89/139 (64.0 %) Xpert testing both sputum and urine combined 108/139 (77.7 %)
33 EPTB Volume (ml) Processing procedure Fluids: thick/purulent (pus, aspirates, purulent pleural) >1.0 Split sample: Xpert and then culture 1.0 Xpert only Fluids: clear (pleural) 1.0 Centrifuge, culture only (if enough residual perform Xpert) 3.0 Centrifuge, split deposit, Xpert and then culture CSF 2.0 Centrifuge, Xpert deposit 1.0 Xpert only FNA 1.0 Xpert >1.0 Centrifuge (if clear), Xpert and culture Lymph nodes and other tissue biopsies Considerations: Remote labs not testing EPTB specimens, still referral. Anticipate good performance from Ultra ( same specimen just better assay sensitivity ) Quick Guide to NHLS EPTB Algorithm Add 2ml PBS Cut/grind, homogenise, split sample, Xpert and culture
34 EPTB GXP Result Interpretation GXP (+) and RIF (R) Or GXP (-) GXP (+) RIF (I) or GXP unsuccessful GXP (+) RIF (S) Culture and LPA/DST from split specimen/request 2 nd specimen Request 2 nd sample No further testing
35 HIV/TB integration of services South Africa has a well established laboratory footprint: ~ 3.9 million CD4 tests 4.0 million viral loads and EID test. Acceleration of VL requirements exceeds expectations. Polyvalent platform: Xpert HIV-1 VL VL labs Red POC VL labs yellow Maximizing platform integration Small circles (current GeneXpert TB) = 207 blue = 54Gx4, green = 144Gx16, red = 9 (n=2 Gx48, n=7 Gx80)
36 GeneXpert Xpert HIV-1 Viral Load Xpert HIV-1 VL assay Fully automated Real-time RT-PCR in a cartridge Two internal quantification standards Requires 1 ml plasma LODetection: 18.3 cp/ml LOQuantification: 40 cp/ml Linear range: million cp/ml Targets: 5 end LTR Detects: HIV Group M, N, O and recombinants. TAT: 90 minutes Performance on plasma specimens: Good accuracy (0.3copies/ml) compared to reference. Low (3%) misclassification at 1000c/ml (93% sensitivity, 97% specificity compared to Roche CAP/CTM) Dried blood spot and whole blood testing under development
37 Polyvalent platform Workflow POC birth testing, HIV viral load and TB sputum and stool tested at POC on same modules. Xpert MTB/RIF =120minutes/module, therefore 4 TB specimens/module/8hr Xpert VL =90minutes/module, therefore 5 HIV specimens/module/8hr
38 Connectivity for monitoring and surveillance Laboratory information system (NHLS central data warehouse) Result reporting, Billing and Programme M&E Track patients across provinces Push information (sms printer) Operational Dashboards (Remote Xpert, Roche, Abbott): Near real time data monitoring Web based and user friendly Improve technical support
39 Laboratory Information System Instant data stream to central powerhouse data repository National data Result reporting Billing Central data warehouse Program M&E
40 Remote Xpert Dashboard real-time monitoring through cloud-based connectivity A Web portal that is a device relationship management platform
41 How are we using the LIS and Remote Xpert Laboratory audit indicators Changes in MTBC positive and Rif rates by: laboratory, user, module, cartridge lot, utility, module failure, Xpert Check, unsuccessful test (errors, invalids), training needs. Increases in Rif resistant mutations (using Xpert Ct and probes) not linked to clinical outcome November laboratory X High Medium Low Very Low
42 big data 7,188,164 GeneXpert results [as of 12/02/2016] Hot spots of TB/MDRTB transmission Test and Collect and Molecular Epidemiolog y Frequency of Rif probe hybridization: E:531bp(55.7%), D:526bp(18.6%), :516bp(18.2%), A (6.0%), C(1.5%).
43 Molecular surveillance: a new tool Cycle threshold (Ct) =sputum mycobacterial load and may indicate hot spots of transmission and response to interventions The frequency of mutation probes may indicate trends in transmission and circulation of new strains. National data may indicate population trends over time and is available in near real time Frequency of Rif probe hybridization: E:531bp(55.7%), D:526bp(18.6%), :516bp(18.2%), A (6.0%), C(1.5%).
44 Crude Real time molecular surveillance
45 GeneXpert the game changer (accurate, easy, safe, fast). lets implement! What about QUALITY? How to check the instrument is fit for purpose before testing patient specimens? Verification material needs whole Mycobacterium tuberculosis. SAFETY
46 Developing the Dried Culture Spot for instrument verification whole inactivated, quantified M.tb Endorsed by WHO The testing process Global external quality assessment program US Patent Number 8,709,712 Scott LE, et al. Dried Culture Spots for Xpert MTB/RIF External Quality Assessment: Results of phase 1 pilot study from South Africa. J Clin Microbiol Dec;49(12):
47 Result reporting through TBgx dashboard 1. DCS panel 2. Information 3. SOP 4. Barcodes Inactivated dried TB culture spots tested on a GeneXpert instrument for verification and EQA. Automated result reporting part of requirement for laboratory auditing
48 Added advantage to a standard material tested globally: Gx performance through global EQA Since 2011, n=295 Gx testing sites have participated in the DCS quality program across 22 countries: 97.4% modules functioning correctly on installation 98.6% correct EQA >12 months stability, testing done anywhere, by anyone, and on any molecular platform Program now commercialised: Spin- off from WITS University: SmartSpot Quality Pty.Ltd
49 Added advantage to a standard material tested globally: Gx performance through global EQA Number of Instruments Since 2011, n=295 Gx testing sites have participated in the DCS quality program across 22 countries: 97.4% of the modules functioning correctly on installation 98.6% correct results for external quality assessment GX1 0% Verification EQA Number of Modules GX 48 1% GX80 16% GX4 17% GX1 Program now commercialised: Spin- off from WITS University: SmartSpot Quality Pty.Ltd GX16 66% GX4 GX16 GX 48 GX80
50 NPP TB innovation supports implementation 207 centres service >3900 facilities Mobile expansion, mines and correctional facilities Linkage to care Quality Connectivity LIS capability Dashboard SMS: Rapid result
51 EXIT-RIF study 542 individuals enrolled (52% female, 76% HIV co-infected). Van Rie. A, Vossdelima.Y, Stevens.W, Scott. LIUTLD, October Barcelona 2014 Patient outcomes stratified by mode of Rif resistance diagnosis DST/LPA N=264 Xpert MTB/RIF N=278 P value Time to diagnosis (days) 43 0 < % Initiate treatment Time to treatment (days) < Mortality all HIV negative HIV positive Overall >1:3 patients died, Xpert only reduced mortality from 38% to 34% (unfamiliarity with technology may contribute), but did significantly reduced mortality of HIV negative individuals (2.4%). On Gx tested patients (n=278) 40% had no confirmatory test for Rif (46% within 14 days) Only 20% assessed XDR-TB 16% discordance between Gx and LPA/DST
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54 Linkage to care
55 Linkage to care & adherence M-Health Hub (smart phones) ensuring linkage to care.
56 Outside the lab: Diagnostics response required in HBC Screening assay required: innovation in diagnostics (Triage test?) Active case finding & prompt treatment Treatment almost immediate reduction in infectiousness Gaps in diagnosis; HIV positive Xpert ve, Better drug resistance assays (second-line in particular), EPTB, infants Linkage to care: mhealth strategies, sms printers Targeted Case finding: screening individuals at greater risk of disease peri-mining communities & general population screening: lower yield in SA Correctional services Optimised case finding will differ within SA geographically, risk factor, hotspots Household Contacts: Is this the place for POCT? geographical program data
57 Single module 2017 Release Battery life: 4 hours
58 Lessons learnt and ways forward Engagement of public, private and civil society sectors, NGO s, donors, treasury, all tiers of government Multi-disciplinary teams required One national plan; initial vertical implementation worked best with standardization Costing, modeling, forecasting, procurement models led to a full investment case for HIV and TB together Clinical algorithm required simplification Ability to validate new technologies and allow flexibility (geography, prevleance, testing volumes) Gaps in appropriate diagnosis need to be fixed Sustainability through National funding Needs based innovation Significant investment in Health Care Worker s training and safety
59 Acknowledgements South African Ministry of Health National Health Laboratory Service, National Priority Program, Prof Wendy Stevens and team Department of Molecular Medicine and Haematology research and development team Centre Excellence of TB Research Funders, Laboratory and Clinical Clinical partners, (WRHI/RTC/PHRU/FIND) Participants Commercial collaborators
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