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1. general purpose room as far as possible In order to limit the risk of contamination cultures where available should be processed in a different room Equipment and supplies 1 Equipment amp consumables supplied by FIND e Test kits including cartridges sample treatment reagent and sterile plastic Pasteur pipettes 3 5 ml with 0 5 ml graduation e GeneXpert device will remain on site after the study e Computer e Printer local purchase e 2D barcode scanner e UPS 1500 VA local purchase e Temperature logs 2 Equipment needed but not supplied by FIND e Adapters for European plug GeneXpert computer printer e Extension cables depending on set up of equipment e USB memory stick e 4 C refrigerator e Autoclave e 1 timer e Gloves N95 masks respirator recommended e Sterile plastic Pasteur pipettes 3 5 ml with 0 5 ml graduation if pouring of SR not desired e Bleach Reference laboratories need in addition e Facilities reagents consumables and equipment for solid or liquid culture incubator centrifuge e Freezer 20 C for pellets and culture isolates e Cryotubes for storage of pellet and isolates 1 8 ml cryorack cryoboxes e Disinfectants e Racks for cryotubes e Sterile plastic Pasteur pipettes 1 ml and 5 ml with graduation required to split pellet for culture etc 20 DATA ANALYSIS The sensitivity specificity predictive values and diagnostic accuracy for Xpert MTB Rif in comp
2. C Not at all Screening test for TB suspects replacing smear only L Screening test for TB suspects replacing culture only _ Screening test for TB suspects replacing both smear and culture _ Screening test for high risk groups e g MDR suspects HIV patients prisoners replacing smear only Screening test for high risk groups e g MDR suspects HIV patients prisoners replacing culture only Screening test for high risk groups e g MDR suspects HIV patients prisoners replacing both smear and culture L MTB confirmation of positive smear or of positive culture results Add on test to culture start Xpert culture in parallel to have a rapid result and a culture confirmation rif resistance screening with Xpert TB for culture positive patients Additional comments and or remarks Please specify in this section any additional issue you would like to point out 31 ANNEX 3 Process controls Why e To exclude rpoB amplicon contamination that affects the Xpert MTB Rif performance When e Prior to starting enrolment and monthly throughout the study What e Test 2 negative controls by Xpert MTB Rif using 2 ml of distilled water per test e Test 2 wet swabs by Xpert MTB Rif How e Take 4 cartridges and 4 bottles of sample reagent SR e Prepare 2 negative samples as follows 1 Label sterile tubes NC1 NC2 The tubes must have a capacity of at least 5 ml anda leak proof cap Add 1 ml of ste
3. MGIT or LJ for each specimen 41 ANNEX 8 Speciation with Tauns Capilia TB For study purposes all participating laboratories will follow the routine sputum processing procedures and species identification The Capilia TB procedure is described below Assay Principle This immunochromatographic assay is based on a double antibodies sandwich technique in which i antibody labeled by colloidal gold particles reacts with target antigens to form an antigen antibody complex ii this complex migrates across a chromatographic carrier such as a filter paper and iii the complex is captured by second antibody readily fixed in the middle of the chromatographic carrier If the target antigens are present in the test specimen a color reaction caused by the labeled colloidal particles is observed at the site on the chromatographic carrier where the second antibody is fixed and the specimen is interpreted as positive This kit employs colloidal gold labeled MPB64 monoclonal antibody mouse Capilia TB Test Procedure Capilia identification test should only be done on AFB positive cultures Preparation for LJ tubes Dispense 0 2 ml of Tauns extraction buffer into an Eppendorf tube Pick 1ul of bacteria equivalent to the amount of a 1mm diameter platinum micro loop from the bacterial colony that grew on the solid medium Suspend the collected bacteria in the buffer solution in the tube Close the tube with a lid and full
4. but autoclaving is recommended Laboratories currently doing NALC NaOH treatment of sputum for culture may use the pellet for testing in Xpert MTB Rif without the need to split the raw sputum prior to treatment RATIONALE In response to the growing problems of MDR and XDR TB as well as the continuing epidemic of HIV associated TB WHO and its partners have called for greatly enhanced global capacity for mycobacterial culture and DST as well as for implementation of rapid methods for screening for MDR TB i This massive scale up is thought to require that up to 2000 new culture capable TB laboratories be established and 10 000 new laboratory workers be trained by 2015 It is possible that much of this needed capacity could be addressed by molecular tests for case finding and MDR TB detection With the WHO endorsement in 2008 of line probe assays LPAs for MDR TB screening many countries are implementing this system However as noted above the test is sufficiently complex that decentralized testing is not feasible Therefore the Xpert MTB Rif assay is of great interest as it provides for highly accurate and simplified testing at a much lower level of the health system Although it provides information only on rifampicin resistance in addition to detection of M tuberculosis complex in nearly all settings rifampicin resistance is an accurate surrogate for MDR TB Although good performance of the Xpert MTB Rif assay has been demonstrat
5. DST preferable on liquid media e Measure increased yield in TB and MDR TB case detection rates compared to local routine diagnostic algorithm 2 Operational performance e Assess robustness of Xpert reagents and equipment temperature dust power irregularities contamination rates indeterminate results e Determine maintenance and customer support needs as well as e Determine minimal training needs through user proficiency testing tool e Confirm established biosafety needs and the ease of waste management e Assess user appraisal through user appraisal questionnaire e Develop recommendations on how to integrate automated molecular testing in resource poor countries using existing diagnostic algorithms for case detection and MDR TB screening 3 Impact e Time to detection of TB and MDR TB compared to local routine diagnostic algorithm e Time to reporting of Xpert MTB Rif results compared to conventional results e Time to initiation of appropriate treatment 4 Cost efficiency e Quantify per test and per patient costs for the health system compared to routine culture and DST Secondary endpoints optional 1 Impact e Patient dropout rate prior to diagnosis e Mortality for enrolled patients during follow up e Hospitalization frequency and duration e Time to return to work 2 Cost efficiency e Quantify costs to the health system and assess cost effectiveness of Xpert MTB RIF testing compared to baseline instead of and
6. Explanation At this point the testing of patients on TB treatment is not indicated according to package insert Reason DNA and AFB fragments might still be detected in culture converted patients If such patients were tested to determine the resistance status a What would be your treatment decision for a patient with Xp pos Rif resistance not detected at month 4 of 1 line TB treatment Culture result is neg L Continue current regimen as planned C IStop current regimen and switch to different regimen Depends on clinic If clinical suspicion of treatment failure switch if no suspicion stick to current therapy Don t know NA b What would be your treatment decision for a patient with Xp pos Rif resistance detected at month 4 of 1 line TB treatment Smear result is neg L Continue current regimen as planned C stop current regimen and switch to different regimen Depends on clinic If clinical suspicion of treatment failure switch if no suspicion stick to current therapy L Don t know NA c What would be your treatment decision for a patient with Xp neg at month 4 of 1 line TB treatment L Continue current regimen as planned C IStop current regimen and switch to different regimen Depends on clinic If clinical suspicion of treatment failure switch if no suspicion stick to current therapy Don t know NA 10 Assuming per test costs of 10 20 USD how would you use this test
7. Positive non contaminated MGIT and negative LJ gt Perform DST from MGIT Negative or contaminated MGIT and positive LJ gt Perform DST from LJ Positive contaminated MGIT and negative LJ gt Redecontaminate MGIT tube and perform DST from MGIT if successful Negative contaminated MGIT and negative LJ gt No DST Negative non contaminated MGIT and negative LJ gt NoDST Standard Drug Concentrations for Use in MGIT ug ml STR 1 0 INH 0 1 RIF 1 0 EMB 5 0 PZA 100 0 Perform all drug reconstitution addition steps in the class Il biosafety cabinet Label each MGIT tube with relevant drug concentration and patient study ID number Tubes and drug concentrations are listed below SIRE GC MGIT Tube SIRE growth control and will not contain any of the SIRE drugs STR 1 0 MGIT tube INH 0 1 MGIT tube RIF 1 0 MGIT tube EMB 5 0 MGIT tube Where done PZA GC PZA medium MGIT tube PZA medium growth control and will not contain PZA PZA 100 PZA medium MGIT tube Using a sterile pipette or transfer pipette reconstitute each of the SIRE drug vials with 4 ml of sterile distilled deionized water Use separate pipette for each drug Reconstitute PZA drug vial with 2 5 ml of sterile distilled deionized water Add 0 8 ml MGIT SIRE Supplement to each SIRE tube and the SIRE growth control tube Add 0 8 ml MGIT PZA Supplement to each PZA medium tube Add 0 1 ml 100 ul of the appropriate reconstituted drug solutions into e
8. Tauns Capilia test according to SOP provided below or analogous testing available on site Ziehl Neelsen Smear of Positive MGIT Cultures Mix the broth by vortexing and then remove a small aliquot using a sterile pipette Place 1 2 drops of this on the slide and spread it on a small area approximately 2 x 1 cm If the smear is negative for AFB and the tube does not appear to be contaminated broth is clear re enter the tube into the instrument for further monitoring See MGIT 960 System s User Manual Section 4 6 3 for returning positive tubes to instrument for further testing After 3 days visually inspect MGIT tube for growth and repeat AFB smear as above If AFB smear remains negative contamination is not found on the blood agar and the LJ slants are not positive by 8 weeks the culture is considered to be negative Work up of Negative MGIT Culture Open the desired drawer and press the negative key Remove the negative tube and scan 39 Visually inspect MGIT tube for potential mycobacterial growth If there is suspicion of mycobacterial growth in a negative tube proceed with AFB smear inoculation of blood agar plate and subculture on LJ Dealing with MGIT Contamination Isolation of Mycobacteria from Contaminated or Mixed Cultures whenever DST is indicated and no other Positive Non Contaminated Cultures are available for that Patient If contamination is confirmed with negative AFB smear from the broth dis
9. between chambers iv an area for capturing concentrating washing and lysing cells v lyophilized real time PCR reagents and wash buffers and vi an integrated PCR reaction tube that is automatically filled by the instrument 12 The assay is based on multiplex nested real time PCR A series of molecular beacons is used to simultaneously detect the presence of MTB and to diagnose rifampicin resistance as a surrogate marker for MDR disease Species specific primers allow amplification of the MTB rpoB core region Nested PCR is used in order to increase the sensitivity of the assay Up to six target sequences can be detected simultaneously with the six color assay one of the six molecular beacon probes was designed to detect DNA of the sample processing control Bacillus globigii the other five molecular beacons are designed to hybridize to overlapping segments of the rpoB core region the amplicon The analytic sensitivity of the real time PCR reaction was found to be 4 5 genomes per assay using genomic DNA Spiking M tuberculosis organisms into negative sputum in dilutions at 10 to 300 cfu per reaction in replicates of 20 found a limit of detection of 131 cfu per assay In wild type M tuberculosis the rpoB core region is highly conserved but is mutant in 95 of rifampicin resistant MTB The presence of all five fluorescence signals indicates that rifampicin susceptible MTB DNA has been detected At least two but less than five fluoresc
10. described below Principle The majority of clinical specimens sent to the mycobacteriology laboratory for cultural confirmation of suspected mycobacterial infection are contaminated by rapidly growing normal flora To maximize the mycobacterial yield contaminated specimens require treatment with a digestion and decontamination procedure NALC NaOH Citrate Solution NALC NaOH is a gentle but effective decontaminating agent NaOH Sodium Hydroxide can be used both as a digesting and decontaminating agent As mucolytic agent it is most effective at a final concentration of 2 However this concentration is not only toxic to contaminants but also to some mycobacteria NALC N Acetyl L cysteine is a mucolytic agent NALC loses its mucolytic activity on standing Therefore the reagents should be gently mixed before use and used within 24 h Sodium citrate Tri sodium citrate dihydrate is included in the NALC NaOH Citrate Solution to bind heavy metal ions which could inactivate NALC A phosphate buffer with a pH 6 8 decreases the activity of NALC NaOH Citrate Solution and lowers the specific gravity of the specimen before the mycobacteria are recovered in a concentrated form by centrifugation The resuspended pellets can be used for semi quantitative sputum smear and semi quantitative culture onto solid media and or liquid culture Safety e A safety cabinet biological class II is needed for the entire procedure e Working with Mycobacterium tuberc
11. for drugs H R E amp S one each for 107 and 10 suspensions total 12 LJ slopes Each LJ slope requires approximately 5 ml of LJ fluid The following table gives the number sets to be prepared is calculated in the following manner Number of sets Total volume of LJ solution required to be required for each set for prepared proportion method ml 10 600 20 1200 30 1800 40 2400 50 3000 60 3600 70 4200 80 4800 90 5400 100 6000 Drug containing LJ slopes are made by adding appropriate amounts of drugs asceptically to LJ fluid before inspissation A stock solution of the drugs is prepared based on the potency of the drug in sterile distilled water for streptomycin isoniazid and ethambutol and rifampicin is dissolved in absolute methanol The solutions are sterilized by membrane filtration Suitable working dilutions 44 are made in sterile distilled water and added to the LJ fluid dispensed in 5 ml amounts and inspissated once at 85 C for 50 minutes Drug stock solutions should be prepared fresh on the day of drug media preparation The medium can be stored in the cold for 3 4 weeks Isoniazid H Drug potency 1g to 1g substance Stock solution preparation Weigh out 20mg of isoniazid in 40ml of sterile distilled water 500ug ml Label with date of preparation Working solution Prepare the working solution on the day of drug media preparation Sterilize by filtering thro
12. in addition to scenarios e Establish innovative partnership models that increase cost efficiency impact and sustainability of Xpert MTB Rif testing integrating TB screening in HIV care in Uganda and South Africa Sample size The sample size for this study is largely a reflection of WHO s requirement to see a new test evaluated in a geographically and otherwise representative number of routine settings The sample size calculation was largely driven by the need to assess Xpert in a variety of different settings representative of the real world situation in high burden countries It will be important to determine how dependant Xpert performance is on population factors notably HIV prevalence daily workload user skills and laboratory infrastructure aspects We will have to assess whether the rate of DNA contamination goes up over time to follow the evolution of sensitivity and specificity and the impact of laboratory technician s fatigue and to monitor the robustness of the assay in the field error rate instrument robustness All these endpoints require longer study duration and a variety of sites Xpert sensitivity and specificity estimates were the primary endpoints for the minimum sample size calculation during phase 1 validation phase Based on discussions with the participating centers we assumed a minimum average daily enrolment number per site of 5 patients and an average TB prevalence of 15 The following minimum perform
13. potency is 0 731 the required amount of active drug is 20 0 731 27 35 mg 27 35 mg is dissolved in 50 ml of SDW to obtain 20 mg of active drug Always look for the potency mentioned on the drug bottle Do not store this solution Number of sets Number of bottles of S MI of stock Amount of Final required to be drug media required solution LJ fluid to concentration prepared each bottle with 5m 400ug ml be added of Sin LJ of LJ fluid ug ml 5 10 0 5 49 5 4 10 20 1 99 4 15 30 1 5 148 5 4 20 40 2 198 4 25 50 2 5 247 5 4 30 60 3 297 4 Rifampicin Preferred substance Sigma R3501 Correction for potency required Stock solution preparation Weigh out 40mg Potency of rifampicin and dissolve in 5 ml of absolute methanol followed by addition of 5 ml of 99 ethanol to get 4000ug ml of stock solution Do not store this solution Number of sets Number of bottles of MI of stock Amount of LJ Final required to be R drug media solution fluid to be concentration prepared required each bottle 4000ug ml added in ml of Rin LJ with 5m of LJ fluid ug ml 5 10 0 5 49 5 40 10 20 1 99 40 15 30 1 5 148 5 40 20 40 2 198 40 25 50 2 5 247 5 40 30 60 3 297 40 46 MGIT Drug Susceptibility Testing Perform DST according to the following scheme Positive non contaminated MGIT and positive LJ from this specimen gt Perform DST from MGIT
14. B Rif sensitivity and specificity for Rif and MDR detection 3 Xpert 3 smears 4 cultures sequencing data not taken into consideration preliminary study results In conclusion Xpert MTB Rif was not only suited for rapid MDR screening but also seemed very useful for early case detection in smear negative TB patients The high accuracy of a single Xpert MTB Rif result per patient and the time to diagnosis of below 2 hours should allow for on the spot diagnosis and TB treatment initiation based on one Xpert MTB Rif result only These findings need now to be confirmed in the intended setting of use We will inform you as soon as the submitted manuscript with evaluation study results has been accepted for publication OPERATIONAL PERFORMANCE OF Xpert MTB Rif TEST During the evaluation studies the indeterminate rate for Xpert MTB Rif was lt 5 at all sites This was equal or lower than the indeterminate rate for culture The overall indeterminate rate fell to 1 1 if successful repeats were counted The assay performed equally well on both NALC NaOH processed sputum and untreated sputum samples Xpert MTB Rif was designed as a closed system to reduce or eliminate the risk of amplicon contamination Once closed the cartridges are never reopened The Xpert MTB Rif cartridge works by trapping bacteria and in the first wash step free DNA is removed to the waste chamber Inoculation of small amounts of free M tuberculosis DNA
15. IRB approvals 6 months 12 months Xpert MTB Rif plus Auramine smear 1 smear plus culture Xpert MTB Rif Auramine smear 2 smears plus culture Standard diagnostic algorithm Auramine smear plus selected culture Smear culture Xpert MTB Rif DST for all cultures Smear culture Xpert MTB Rif Selected DST for Rif res As per routine and for Xpert MTB Rif or culture pos patients after 2 months As per routine and for Xpert MTB Rif pos patients after 2 months By May 09 Shipment of GeneXpert instruments amp cartridges May 09 Validation Phase enrolment Jun Aug 09 Implementation Phase enrolment Sep Nov 09 Continuation Phase 10 Dec 09 Jun 10 WHO expert committee meeting Apr 10 possibly Sep 10 DEMONSTRATION STUDY PROTOCOL BACKGROUND Despite the enormous global burden of TB approaches to TB diagnosis still rely on traditional methods that have major limitations The disproportionate amount of smear negative disease in sub Saharan Africa which shoulders two thirds of the global burden of HIV AIDS has greatly complicated TB case detection and disease control Multidrug resistant MDR TB defined as resistance to both isoniazid and rifampicin two of the most important first line anti TB drugs is a rapidly growing problem especially in Eastern Europe and countries of the former Soviet Union Even more alarming is the recently documented phenomen
16. J PACT slant Lay slants with medium face up for 30 min to allow the bacteria to adhere to the surface of the medium Incubate at 37 C for up to 8 weeks Examine for growth weekly for up to 8 weeks Continue examinations until the first of the following three events occurs a colonies are sufficiently large to count b contamination is apparent or c no growth is apparent after 8 weeks Any egg culture with growth believed to be Mycobacteria has to be confirmed by Ziehl Neelsen smear microscopy Cultures completely overgrown by bacterial or fungal contamination are discarded and the result recorded as contaminated if occurred within 3 weeks In case of mixed LJ culture pick isolated colonies and re culture for purity 40 e Species identification is to be carried out on the first positive tube MGIT or LJ using the Tauns Capilia test according to SOP provided below or analogous test available on site e Record the semiquantitative growth result according to table below Smear from LJ e Place 2 clean loops of sterile water on the slide Use of 10 mcl volume loops Pick up several pieces from several colonies on slant Put material onto the slide mix with water and spread it on a small area approximately 1 x 1 cm e If microscopy test reveals AFB with reliable color this may mean the culture belongs MTB complex e If the smear is negative for AFB or it appears blue or semi violet crimson color bacteria this may
17. TB Rif is easy and robust enough to be decentralized and allows to significantly increase case detection rates for TB and MDR TB as well as to shorten time to treatment initiation in comparison with the conventional diagnostic algorithm Patient management based on a single Xpert MTB Rif test carried out while the patient is waiting for his her result decreases the dropout rate prior to diagnosis and the costs and barriers for the patient The introduction of such testing is cost effective for the health system when compared to conventional culture and DST costs Setting of use The demonstration project will primarily assess the feasibility of using Xpert MTB Rif in sub district laboratories These are defined for study purposes as TB laboratories with a minimum workload of 600 sputum samples per month with continuous power supply a minimum of two rooms and at least two well trained TB staff microscopist or laboratory technician Quality assured routine use of ZN or FM microscopy and access to conventional culture and drug susceptibility testing DST are required Participating laboratories will not have any prior experience with molecular testing All satellite clinics sending diagnostic requests to participating laboratories will be integrated in the study and will be important for patient data collection Primary endpoints all sites 1 Clinical performance e Determine sensitivity and specificity compared to central culture and
18. ach of the corresponding labeled BACTEC MGIT 960 tubes Do not add drug solution to the GC tubes Preparation of Inoculum from MGIT Tube The day a MGIT tube is positive by the instrument is considered Day 0 The tube should be kept incubated for at least one more day Day 1 before drug susceptibility testing may be incubated in a separate incubator at 37 C 1 C A positive tube may be used up to and including the fifth day Day 5 after it becomes instrument positive 47 e A tube that has been positive for more than 5 days should be subcultured in a fresh MGIT tube supplemented with MGIT 960 growth supplement and should be tested in MGIT 960 instrument until it is positive Use this tube from one to five days of instrument positivity e f growth in a tube is of Day 1 or Day 2 mix well to break up clumps vortex Leave the tube undisturbed for about 5 10 minutes to allow large clumps to settle to the bottom Use the supernatant undiluted for inoculation of the drug set e The best way is to use the vortex or shaker and sterile 3 mm glass beads in the volume of 3 4 ml For this purpose do use it in 20 mm thick wall glass tube Perform vortex 15 20 min e f growth is on Day 3 4 or 5 mix well to break up the clumps Let the large clumps settle for 5 10 minutes and then dilute 1 0 ml of the positive broth in 4 0 ml of sterile saline This will be a 1 5 dilution Use this well mixed diluted culture for inoculation Inoculation f
19. al support Sites will contact Catharina or the designated study monitor by e mail or telephone if they have any difficulties or questions Catharina will be the interface with Cepheid for Xpert MTB Rif related issues Telephone conferences between FIND and study sites will be scheduled once a month Monitoring site visits and close out visits will be conducted by FIND Data from each site will be monitored at 2 week intervals for all sites checking for completion and consistency of all data as well as enrolment targets During each visit 10 of source data is to be reviewed for consistency with data on CRFs and entered into the database Additionally 10 of all completed CRFs are to be reviewed by FIND staff for consistency with 22 data entered into the database In both cases an error rate is to be recorded Error rates gt 3 of all questions require discussion and review Error rates of gt 5 of all questions require immediate CRF re training Any errors on primary endpoints are to be discussed with each site and reviewed individually ETHICAL CONSIDERATIONS During the implementation phase patient management and TB treatment decisions will be based on Xpert MTB Rif results for case detection and during continuation phase the Xpert MTB Rif results of Rifampicin resistance will also be used for patient managment and the routine diagnostic algorithm will be modified Ethical approval will therefore be required for all participating sit
20. ance criteria were set as go no go decision criteria for moving to the next project phase a Xpert MTB Rif sensitivity gt 80 of culture positive cases this should be the lower limit of the confidence interval b Xpert MTB Rif specificity gt 90 of culture negative cases this should be the lower limit of the confidence interval For each site enrolling 380 patients would provide 57 confirmed TB cases A sensitivity of 90 or above would produce a lower confident interval limit of at least 80 Of the 323 remaining 5 10 would be of indeterminate diagnosis However only 55 TB negative patients are sufficient to have a lower confidence interval limit of at least 90 with given the expected specificity of 98 8 This would leave us with an overall sample size need of 2660 TB suspects for phase 1 and an enrolment duration for this phase of 3 months The overall sample size for the study all phases was also determined by Rifampicin resistance endpoints While the expected sensitivity is 95 the lower limit of the confidence interval should be at least 92 requiring at least 315 Rif resistant cases among culture positives With an MDR prevalence rate of 5 across all sites this would require a total enrolment of 6300 TB suspects Equipment and reagent needs South Peru Philippines India Uganda Azerbaijan Africa GeneXpert 3 3 2 1 1 1 Xpert 2160 2160 720 1140 720 720 MTB Rif Total 9 instrumen
21. any technical difficulties during installation or use No lYes please describe Training 20 Did you feel the training you L Satisfied received on the Xpert MTB Rif L Unsatisfied please explain was Satisfactory L Don t know 21 How many days of training on LI 1 3 days the Xpert MTB Rif do you feel 4 6 days are necessary for some one O 7 10 days trained in smear microscopy C gt 10 days 22 How many days of training on L 1 3 days the Xpert MTB Rif do you feel 4 6 days are necessary for someone C 7 10 days trained in AFB culture _ gt 10 days 23 Are there any topics or specifics in using the Xpert MTB Rif assay which were not included in training that you _lYes please describe feel should be included Additional comments and or remarks Please specify in this section any additional issue you would like to point out 29 No Questionnaire Clinicians appraisal of Xpert MTB Rif assay Xpert MTB Rif Demonstration Project FIND foundation for innovative new diagnostics Pre su rvey Clinic Country Date of survey completion DD MM YYYY Study phase L Validation _ Implementation L Continuation Purpose To evaluate the potential utility of the Xpert MTG Rif assay in TB diagnosis and treatment Instructions To be completed by clinicians nurses responsible of TB treatment decisions Please answer all the questions by s
22. arison to culture and DST as the gold standard will be calculated Analysis will be done per patient applying the following diagnostic rules as outlined in the table below Additionally the secondary endpoints time to detection of TB and rifampicin resistance time to TB treatment and time to MDR treatment and time to reporting case detection and MDR will be calculated Time to detection will be calculated for all tests per sputum from the date of the sputum collected on which the test was performed to the date that the result is available Time to appropriate treatment and time to clinical reporting will be calculated per patient from the time of first sputum collection from the patient Indeterminate rates will be calculated out of all tests performed Diagnosis Smear positive Scanty positive smear Culture positive Contaminated culture Xpert MTB Rif positive Xpert MTB Rif resistant case Xpert MTB Rif invalid case Non TB case Indeterminate cases excluded from analysis Description 21 smear 210 100 fields or 2 scanty positive smears Smear positives with only negative or contaminated cultures will be excluded from analysis 1 9 AFB per 100 fields 2 2 scanty positive smears with 2 negative or contaminated cultures will be excluded from analysis 2 1 LJ and or MGIT culture growth confirmed MTB complex Cross Contamination A single LJ culture with20 colonies or a single MGIT culture with MTB growth 228
23. ated or standing Whenever possible subjects will be instructed to rinse the mouth twice with water Subjects will be instructed to inhale deeply cough vigorously and expectorate the material produced into collecting receptacle The subject should be told to cough the specimen from deep in the chest If the subject does not cough spontaneously instruct him her to take several deep breaths and then hold their breath momentarily repeating this step several times will often induce coughing 7 After coughing the subject will be instructed to hold the sterile specimen container to his her lower lip and gently release the specimen into the container Instruct the patient to avoid spills or soiling the outside of the container with the specimen The lid should be carefully placed on the container without touching the inside of the lid Lids should be firmly screwed back onto specimen containers to prevent leakage e Specimens visibly contaminated with oral material food particles or saliva will be discarded and the subject will receive a second sterile specimen container and be instructed to try again e Specimens to be taken to the laboratory for processing as soon as possible after collection or as per routine sputum collection practices 33 ANNEX 5 Sputum decontamination for culture amp microscopy Kubica For study purposes all participating laboratories will follow the routine sputum processing procedures A generic protocol is
24. ays During the on site training phase lab technicians from the sub district laboratory and the supervisor from the reference laboratory will receive training on the Xpert MTB Rif assay from the FIND study team Before the training begins a quality income check will be performed to ensure that cartridges and the GeneXpert are working properly The actual testing will only start once Cepheid has approved the QC income check At least 32 tests will be performed per site to gain practical experience and to determine the proficiency of the users This will be followed by proficiency testing with 4 well characterized samples per user The required frozen sputum samples will be collected and stored at the site before the training begins Training will also comprise other study related aspects such as study specific workflow and data management Oral and written feedback on overall impression of the assay will be provided by the training participants in the form of a user appraisal questionnaire 2 Validation Phase at least 3 months enrolment This study phase aims at the collection of baseline data for the routine diagnostic algorithm prior to intervention and at the validation of Xpert MTB Rif performance in this new environment The routine diagnostic algorithm at participating laboratories will be maintained except that one Xpert MTB Rif is done along with ZN In addition culture and DST for at least rifampicin and isoniazid will be pe
25. ble with No 1 lab room TB MDR ZN detection occasional short interruptions HXV Suburban Health Center Case 12 Stable with weekly No 1 lab room TB MDR ZN detection short interruptions Vellore India supervised by Christian Medical College Vellore CHAD Suburban Health Center Case 20 Stable with daily No 1 5 lab rooms TB ZN detection short MDR in interruptions Chennai Baku Azerbaijan STI Urban Referral Center MDR 60 Stable weekly BSC 1 4 lab rooms TB MDR ZN LJ MGIT detection short interruptions Cape Town South Africa supervised by NHLS laboratory Groote Schuur and University of Cape Town Paarl Urban Provincial Case 300 Stable with weekly BSC 1 3 lab rooms TB MDR Laboratory FM detection short interruptions MSF KH Urban Health Center Case 30 Stable with weekly BSC 1 2 lab rooms TB MDR FM detection short interruptions Kampala Uganda supervised by National Reference Laboratory and FIND Uganda Mulago ER Urban Referral Case 30 Stable with weekly No 1 room TB Hospital detection short Emergency unit interruptions ZN Manila Philippines supervised by Tropical Disease Foundation LCP Urban Referral Hospital MDR 10 Stable BSC 1 3 rooms TB MDR ZN Ogawa detection Table Sites description Study phases all sites except South Africa The project will be split in the following study phases 1 Training amp proficiency testing 1 3 d
26. card the specimen and report as contaminated If contamination is confirmed with a positive AFB smear from the broth but other specimens collected from a patient are not contaminated it is not necessary to attempt to salvage a contaminated culture If contamination is confirmed with a positive AFB smear from the broth and no other non contaminated cultures are available complete the following procedures Transfer the entire tube of MGIT broth into a 50 ml centrifuge tube Add an equal volume of sterile 4 NaOH solution Mix well and leave at room temperature 20 minutes mixing and inverting the tube periodically Add sterile phosphate buffer pH 6 8 up to 40 ml mark and mix well Centrifuge at 3500 x g for 20 minutes Carefully pour off the supernatant fluid into a suitable container with mycobactericidal disinfectant Re suspend the pellet in 1 0 ml of buffer and mix well Inoculate 0 5 ml into a fresh MGIT tube supplemented with MGIT Growth Supplement PANTA Additionally inoculate one LJ slant with 0 1 0 2 ml of reprocessed culture Wipe tubes and caps with a mycobactericidal disinfectant Leave inoculated MGIT tubes at room temperature for 30 min Load tube into MGIT 960 and observe for growth of mycobacteria as before LJ Primary Culture Inoculation Using the pipette used for adding phosphate buffer to the pellet or a new sterile pipette inoculate 0 2 ml of the resuspended processed sputum sample onto an egg slant 1 LJ or 1 L
27. ch experience do you have doing smear microscopy L LJ LJ O O 9 How many years of experience do you have doing AFB culture O EJ L O O 10 During the training study approximately lt 1 week 1 20 wk 20 100 wk gt 100 wk how many samples have you personally O O O O processed by Xpert MTB Rif 28 Ease of Use Question Response Comments 11 How difficult was the installation Difficult please explain of the Xpert MTB Rif assay Easy _ Don t know NA 12 Which would you use for sample _ Pouring reagent addition _ Pipette 13 How difficult is the sample _ Difficult please explain preparation addition of sample _ Easy reagent for Xpert MTB Rif L Don t know NA 14 In your opinion does Xp sample _ Yes please explain preparation require a biosafety C No please explain cabinet _ Don t know NA 15 How difficult was the first use of Difficult please explain the Xpert MTB Rif Easy L Don t know NA 16 How difficult is the Xpert _ Difficult please explain MTB Rif derived waste Easy management L Don t know NA 17 Is the display software for the _ Difficult please explain Xpert MTB Rif easy to use _ Easy Don t know NA 18 Is the automatically printed _ No would prefer a results report on completion of different report format the assay suitable to you explain _ Yes suitable L Don t know NA 19 Did you have
28. d outside indicator of the drawer Positive will be followed by continuously beeping sound signal Inside of the drawer after pushing the proper button Positive tubes will be displayed by the indicator light changing from red to green at the exact location of the tube in the instrument drawer Negative tubes will be displayed by a green indicator light at the exact location of the tube in the instrument drawer If capacity of the MGIT 960 becomes an issue remove negative tubes at 4 weeks and transfer to incubator checking manually with Wood s lamp or UV transilluminator for growth daily Work up of Positive MGIT Cultures Open the desired drawer and press the positive key This will be displayed by a green indicator light showing the exact location of the positive tube in the instrument drawer Remove the positive tube and scan Visually inspect MGIT tube for potential mycobacterial growth Mycobacterial growth typically appears granular with only slight turbidity M tuberculosis growth settles at the bottom of the tube Usually the growth time is more then 3 days Proceed with inoculation of a blood agar plate AFB smear and LJ subculture required for DST in case of MGIT contamination in that order and freeze aliquot of pos culture Incubate blood agar plate at 37 C for 48 hours checking for growth of contaminants at 48 hours Species identification is to be carried out on the first positive tube MGIT or LJ using the
29. days on MGIT culture positive at follow up only culture positive but missing speciation culture positive but NTM only and discrepant RIF in conventional DST 21 Case report forms CRFs will be provided by FIND The electronic data entry tool for the study will be connected to FIND s central database through secured VPN access which will have the advantage that study monitors have continuous access to the electronic data First data entry will have to be done as soon as results become available real time Second data entry will be done based on completed CRFs When final culture and DST results are available final data sets will be generated for analysis MINIMIZATION OF BIAS Avoiding sampling and selection bias Consecutive sampling method and community based study A consecutive series of patients with typical symptoms of TB will be included The study group will consist of all subjects who satisfy the criteria for inclusion and are not disqualified by one or more of the exclusion criteria The vast majority of samples will be collected from out patients and not from the more severely ill hospitalized patients Measurement bias Within subject comparison and blinded interpretation of results In order to increase the precision of the estimates for the accuracy of the Xpert MTB Rif the study will be based on a within subject and on a within sample comparison Due to the variability of sputum samples within individuals and within sa
30. days per patient will be excluded from analysis NTM Specimens with growth of mycobacteria other than MTB complex only will be excluded from analysis LJ Cultures completely overgrown by bacterial or fungal contaminations within 3 weeks discarded In case of mixed cultures isolated MTB colonies transferred to new LJ tube repeat culture e MGIT Instrument positivity w o detection of AFB e Contaminated culture results only among all available cultures will lead to exclusion from analysis Xpert MTB results TB positive Xpert MTB Rif resistance detected Persistent invalid or error or no result after repetition whenever possible e Smear negative culture negative patient without TB treatment on the basis of clinical criteria e For Xpert MTB Rif or Culture positive patients and random controls a follow up at 2 and 6 months with repeated clinical and bacteriological work up will be required to exclude TB with the highest possible likelihood Only if the bacteriological work up remains negative the patient is called Non TB Excluded from analysis as indeterminate cases will be patients not meeting inclusion criteria or with incomplete case report forms sputum 1 and 2 collected gt 7 days away no culture done no valid culture result due to contamination no valid Xpert MTB Rif result smear positive culture negative single positive culture with lt 20 colonies on LJ or gt 28
31. dizes to its target the stem helix is forced apart and the fluorophore is separated from the quencher permitting the fluorophore to fluoresce when excited by light of an appropriate wavelength 13 CLINICAL PERFORMANCE OF Xpert MTB Rif TEST After completion of development the assay has undergone clinical validation in a statistically powered number of patients in reference settings in South Africa India Peru Germany and Azerbaijan As summarized in the table below the Xpert MTB Rif assay was found to be highly sensitive and specific detecting M tuberculosis DNA in almost all smear positive sputum specimens and a very high percentage of smear negative culture positive specimens Rifampicin resistance and MDR was detected with high accuracy Assay performance even remained good when analyzing a single Xpert MTB Rif result in comparison to 4 culture results per patient The performance was very homogenous across countries Sensitivity Sensitivity Specificity in S C in S C in Non TB Total 99 5 90 0 97 9 98 4 99 8 84 6 93 7 96 4 98 8 Table Per patient analysis Xpert MTB Rif sensitivity and specificity 3 Xpert 3 smears 4 cultures preliminary study results Sensitivity in Rif Sensitivity in MDR Specificity in Rif resistant cases cases sensitive cases 96 1 96 5 98 6 93 0 98 3 97 2 99 3 92 4 98 0 Table Per patient analysis Xpert MT
32. e foundation for innovative new diagnostics STUDY PROTOCOL Xpert MTB Rif Demonstration Feasibility impact and cost efficiency of decentralizing molecular testing for detection of tuberculosis and rifampicin resistance using Xpert MTB Rif Version and date 1 0 04 April 2009 Project and study Cepheid 7210 3 1 Trial sites South Africa Peru Philippines Uganda India Azerbaijan Study Coordinator Dr Catharina Boehme email catharina boehme finddiagnostics org tel 41 22 710 93 16 Project Leader Dr Ranald Sutherland email ranald sutherland finddiagnostics org tel 41 22 710 09 53 _ SSS SSS ee Partnering for better diagnosis for all CONFIDENTIALITY STATEMENT The information contained in this document especially unpublished data is the property of FIND or under its control and may not be reproduced published or disclosed to others without written authorization TABLE OF CONTENTS TABLE OF CONTENTS PARTNER INSTITUTIONS STATEMENT OF INVESTIGATORS PROTOCOL SYNOPSIS DEMONSTRATION STUDY PROTOCOL BACKGROUND RATIONALE STUDY DESIGN Patient inclusion criteria Patient exclusion criteria Laboratory requirements MINIMIZATION OF BIAS STUDY COORDINATION TRAINING AND MONITORING ETHICAL CONSIDERATIONS ANNEX 1 Proficiency testing form ANNEX 2 User appraisal questionnaire ANNEX 3 Process controls ANNEX 4 Sputum collection ANNEX 5 Sputum decontam
33. e Add 50 ml 4 6 Sodium hydroxide solution NaOH and 50 ml 2 9 Tri sodium citrate dihydrate solution to 0 5 g N Acetyl L cysteine to arrive at a NaOH concentration of 2 3 e Add 2 3 NALC NaOH Citrate to the sputum container in a volume that is equal to the specimen volume final NaOH concentration after 1 1 dilution with specimen 1 1 5 e Vortex the alkaline suspension briefly and set at room temperature for 15 minutes Start timer when NaOH is added to the first specimen Place the specimens on a shaker to improve homogenization e When 15 minutes have passed add phosphate buffer to the 50 ml mark on the centrifuge tube Do not touch the centrifuge tube with the buffer container when dispensing buffer e Mix suspension by inverting the tubes several times or by vortexing e Place tubes in centrifuge in a balanced arrangement Subject tubes to centrifugation at 3500 times g 4000 rpm for 15 minutes If possible use a refrigerated centrifuge to maintain centrifugation temperature at 8 10 C e Remove tubes from centrifuge and decant supernatant into splash proof container in the biosafety cabinet leaving only sample pellet in the tube If necessary swab lip of tube with disinfectant soaked gauze e Add phosphate buffer to the centrifugation pellet to adjust volume to 1 5 ml For the pellets which will be used for an Xpert PCR comparison 2 0 ml will be required Recap and vortex briefly to resuspend the pellet e Spread a drop of the s
34. e SOP instructions The FIND monitor or study coordinator that supervises the assessment will complete the checklist For each correct item the operator will obtain 1 point Did the operator add the correct volume of SR compared to the reference material sputum cups Did the operator shake the sample mix 2 times before the end of incubation time Was the incubation time 15 minutes Was the input volume transferred to the cartridge correct Did the operator start each new test in the GX without any problem Did the operator clean the work bench with fresh bleach after performing the Xpert MTB Rif assay Are new unopened Xpert MTB Rif kits stored in a clean and dry place Are used Xpert MTB Rif reagents sealed in a plastic bag before discarding Are all infectious materials sputum cups pipettes properly disposed of as per local guidelines for hazardous materials Is the workflow in the lab correctly set up to avoid DNA cross contamination Unidirectional workflow from sample preparation area to DNA amplification area Score Number of correct items 25 OYES OYES OYES OYES OYES OYES OYES OYES OYES AYES LINO LINO LINO LINO LINO LINO LINO LINO LINO LINO B Questionnaire Instructions v The FIND monitors or study coordinator that supervises the assessment will ask the following questions to each operator in the context of the Xpert MTB Ri
35. e completed by each Lab technician performing Xpert MTB Rif at the end of each study phase Please answer all the questions by selecting the most suitable response Provide comments wherever necessary to aid in understanding or provide additional relevant feedback General Satisfaction Question Response Comments 1 Overall are you satisfied with the _ Satisfied performance of the Xpert _ Unsatisfied please explain MTB Rif assay L_ Don t know 2 Do you feel that the Xpert Yes MTB Rif is a practical means of C No please explain testing for MTB L Don t know 3 Do you feel that use of the _lYes Xpert MTB Rif meets your No please explain countries needs Don t know Xpert MTB Rif compared to Standard Testing 4 How difficult is use of the Xpert More difficult MTB Rif compared to smear L Same microscopy L Less difficult Don t know 5 How much hands on time is _ More hands on time required for Xpert MTB Rif L Same compared to smear microscopy C Less hands on time L Don t know 6 How difficult is use of the Xpert More difficult MTB Rif compared to AFB L Same culture L Less difficult _ Don t know 7 How much hands on time is _ More hands on time required for Xpert MTB Rif L Same compared to AFB culture L Less hands on time L Don t know Background None lt 6 months 6mos 2yrs 2 5yrs gt 5yrs 8 How mu
36. e growth into a sterile tube approximately16x128 mm containing 4 ml 0 85 saline or BBL Middlebrook 7H9 broth and 3 4 ml of 3 mm sterile glass beads e Tighten the cap and Vortex the tube for 2 3 minutes to break up any large clumps Repeat it as many as necessary The turbidity of the suspension should be greater than the McFarland number 1 standard e Let the suspension stand for 20 minutes undisturbed Using a sterile pipette carefully transfer the supernatant suspension into another sterile tube Avoid taking any growth that has settled on the bottom e Let this tube stand for another 15 minutes undisturbed Using a sterile pipette carefully transfer the supernatant suspension out with a pipette without disturbing the sediment and transfer into a third sterile tube e Again avoid taking any growth that has settled on the bottom The turbidity of this suspension should be greater than McFarland 0 5 standard 48 e Adjust the turbidity of this suspension to McFarland 0 5 standard by adding sterile saline and adjusting by visual comparison The turbidity should not be less than McFarland 0 5 Dilute 1 0 ml of this suspension in 4 0 ml of sterile saline and mix well This 1 5 dilution will be used as the inoculum for DST Inoculation from Solid Media Derived Specimen SIRE Growth Control Tube e Using sterile pipettes dilute 0 1 ml of MGIT specimen prepared in Section 8 4 into 10 ml sterile saline and mix well e This is the 1 500 Gro
37. e laboratories because of their great advantage of speed compared to culture However the complexity of existing commercial NAAT formats the labor demands and the need for a high degree of technical support and well trained staff make them unsuitable for decentralized settings FIND has signed a development agreement with Cepheid to deliver a fully automated molecular platform for TB case detection and rifampicin resistance testing for high endemic countries It is designed to purify concentrate amplify and identify targeted rpo nucleic acid sequences and provides results from unprocessed samples in less than 2 hours with minimal hands on technical time After completion of development the assay has undergone Clinical validation in a statistically powered number of patients in reference settings in South Africa India Peru Germany and Azerbaijan As outlined in more detail in the study protocol below Xpert MTB Rif performance characteristics were assessed in approximately 4500 sputum specimens from 1500 TB suspected patients The assay was found to be highly specific and sensitive detecting M tuberculosis MTB DNA in almost all smear positive sputum specimens and a high percentage of smear negative culture positive specimens Rifampicin resistance was detected with high accuracy In collaboration with National TB Programs and partner institutions FIND will now explore the feasibility of decentralized use of Xpert MTB Rif to impro
38. ed in carefully controlled laboratory settings data on testing in program conditions as well as information about cost and patient public health impact are needed to inform public policy on the use of the system These demonstration projects are intended to provide this information 15 STUDY DESIGN After having confirmed that assay performance targets are met in reference laboratories FIND will now explore the feasibility of decentralized use of Xpert MTB Rif to improve TB and MDR TB control In collaboration with National TB Programs and partner institutions large scale demonstration projects will be carried out to provide the evidence that this new generation of NAAT can be successfully implemented in regional and sub district laboratories and even microscopy centers and have significant medical and public health impacts in programmatic settings The demonstration project will primarily assess the feasibility of using Xpert MTB Rif in sub district laboratories Whereas the Xpert MTB Rif assay is implemented and used at the sub district laboratories the reference laboratory will be responsible for performing culture and phenotypic DST For study purposes a supervisory site will be assigned for each participating sub district laboratory demonstration site with its satellite clinics This supervisory site will ensure protocol and GCLP compliance and will be responsible for local project coordination and data ma
39. ediment 20 30ul onto a labeled glass microscope slide Place slides on slide rack to dry for about 20 30 min e Add 0 5 ml into the MGIT tube close the tube tightly and shake it twice upside down Leave the tube in the rack for 30 min and insert it then into the MGIT 960 e Add 0 2 ml 5 10 drops to LU medium Summary Receive and log in sputum sample Decant supernatant 4 4 Add equal volume NaOH NALC Citrate Resuspend pellet with PB to 1 5 ml to solution to sputum container final 2 0 ml for PCR comparison concentration 1 1 5 NaOH 4 Make smear for microscopy 1 drop Vortex for 30 s 4 Inoculate cultures and incubate 0 5 ml Shake at room temperature for 20 min for MGIT 0 2 ml for LJ v Buffer to 45ml with PB Sediment at 3500 x g 10 C for 15 min Vv 35 ANNEX 6 Semi quantitative mycobacterial smear For study purposes all participating laboratories will follow the routine sputum processing procedures A generic protocol is described below for Ziehl Neelsen smear microscopy Principle The goal of acid fast microscopy is to detect mycobacteria in clinical samples and to give a semi quantitative estimation of their number which serves as a rough guide to the bacterial burden of disease and the infectiousness of the diseased patient Mycobacteria and related organisms retain carbolfuchsin and other dyes despite washing with acid alcohol This feature is exploited to differentiate mycobacteria from the backgro
40. electing the most suitable response Provide comments wherever necessary to aid in understanding or provide additional relevant feedback Question Response Comments Are you confident in the TB _ Not confident detection results L Confident _ Don t know NA Are you confident in the Rifampicin _ Not confident resistance detection results L Confident L Don t know NA Would you use Xpert MTB Rif assay as a factor in starting appropriate TB treatment _ Never _ Hardly ever L Often L Don t know NA Would you use Xpert MTB Rif results alone to start TB treatment Never Hardly ever L Often _ Don t know NA Would you use Xpert MTB Rif results to change TB treatment regimen _ Never Hardly ever _ often L Don t know NA Would you use Xpert MTB Rif results in combination with most likely clinical diagnosis especially in smear negative cases _ Never Hardly ever L Often Don t know NA Would you use semi quantitative Xpert MTB Rif results in making clinical decisions _ Never Hardly ever L Often L Don t know NA Which clinical decision s would be L Isolation influenced by the semi L Follow up quantitative result schedule C Treatment regimen L Hospitalization L Other specify 30 9 Role of Xpert MTB Rif assay Xp for patients on TB treatment
41. emands including the need for a high degree of technical support and well trained staff make them suitable for central reference laboratories only FIND has signed a development agreement with Cepheid to deliver a fully automated molecular platform for TB case detection and drug resistance testing for high endemic countries It is designed to purify concentrate amplify and identify targeted rpoB nucleic acid sequences and delivers answers from unprocessed sputum samples in 120 minutes with minimal hands on technical time DESCRIPTION OF Xpert MTB Rif TEST The Xpert MTB Rif assay performed on the GeneXpert MTB Rif system is a rapid test for the detection of M tuberculosis MTB and rifampicin resistance The GeneXpert system consists of a GeneXpert instrument personal computer and disposable fluidic cartridges The system combines cartridge based sample preparation with amplification and detection in a fully integrated and automated nucleic acid analysis instrument 11 The manual sample pre treatment for the assay is simple Sample treatment buffer is added to sputum samples and a defined volume of this mixture is then transferred to the sample chamber of the cartridge The cartridge is then inserted in the GeneXpert From this point on all steps are automated GeneXpert first captures MTB organisms from the sputum sample on a filter membrane Inhibitors are then washed from the captured cells with buffer Finally the cap
42. ence signals indicate the presence of rifampicin resistant MTB The presence of a single fluorescence signal in addition to the signal for the sample processing control is interpreted as an invalid result since experience has shown that even the most mutant MTB isolates produce at least two positive signals No fluorescence signal from one or fewer of the rpoB probes indicates absence of MTB DNA the positive signal for the internal process control indicates that the assay worked Spiking M tuberculosis DNA containing clinically relevant mutations into the cartridges that were then run with M tuberculosis negative sputum demonstrated the ability to pick up all rpoB mutations reported to be present at a frequency at least 0 5 of all rifampicin resistant strains worldwide calculated by compiling sequence frequencies from 78 separate studies of over 4000 rifampicin resistant strains More detailed information can be shared as soon as the data have been published David Alland data submitted for publication 1 Molecular beacons are single stranded nucleic acid molecules that possess a stem and loop structure The loop portion of the molecule serves as a probe sequence that is complementary to a nucleic acid target sequence Short complementary arm sequences bind to each other to form the stem hybrid A fluorophore is covalently linked to the end of one arm and a nonfluorescent quencher to the end of the other arm When the molecular beacon hybri
43. es However no informed consent will be sought for this demonstration project and a waiver of informed consent is required based on the study benefits and risks Benefits amp Risks Numerous published studies and a large scale FIND demonstration project confirm the high accuracy of rooB based tests for rifampicin resistance detection In June 2008 the Strategic Technical Advisory Group STAG of WHO has approved line probe assays for MDR screening in developing countries However this use is restricted to central laboratories due to complexity and biosafety and equipment needs The Xpert MTB Rif has the additional advantages of simplicity reduced biosafety needs and much increased sensitivity which should allow using the assay for case detection in smear negative patients as well as for MDR screening in decentralized settings All results obtained so far have shown excellent assay performance Especially during later study phases patients will receive a better diagnostic work up than they would under routine conditions Thus participation in this project poses no foreseeable risk to patients Furthermore investigators will have no direct contact with patients All data will be obtained from records collected by the clinical and laboratory staff and will be stripped of patient identifiers prior to entry into project databases No sample bank will be created The stored pellets and culture isolates will only be needed to resolve discrepant cases F
44. f proficiency run v For each correct item the operator will obtain 1 point Answered Questions correctly 1 Which are the acceptable ranges for SR and input volume OYES ANO 2 For how long is the sample mix sputum SR stable and you can batch samples OYES UNO 3 What would you do if you have started a test and realize that you did not enter the Qyes ONO sample ID 4 What would you do if you drop sputum on the bench and other containers when Qyes ONO transferring it to the cartridge 5 What would you do if you drop SR and splashed on hands face and eyes OYES UNO 6 What would you do if you dropped a cartridge containing sample mix while Qyes ONO transporting it from the sample preparation area to the GeneXpert 7 What would you do if the Xpert result is error invalid or no result OYES ANO 8 Which are the biosafety requirements for the Xpert sample preparation step OYES UNO 9 What would you do if you receive an error message or if a run is aborted by YES UNO GeneXpert 10 How often do you have to calibrate the GeneXpert OYES UNO PART B Score Number of correct questions 26 C Assessment of the GX software Checklist Instructions v When the Xpert proficiency run is over the FIND monitor or study coordinator will ask the Xpert operator to perform the tasks in the below table Vv The supervisor will complete the checklist according to the operator s prof
45. iciency to perform each task v For each correct task the operator will obtain 1 point fash Saha 1 Create a test enter all the information requested by the software to start a new run OYES ONO 2 Enter information manually in case the barcode reader does not work OYES UNO 3 Edit the test information i e change the sample ID for a given sample result OYES ANO 4 Display test results for a given ID OYES UNO 5 Archive gxx files OYES UNO 6 Retrieve gxx files OYES UNO 7 Export test results into a csv file OYES ANO 8 Installa new ADF QYES UNO 9 Create reports assay definition report and installation qualification OYES UNO 10 Maintenance perform plunger rod disinfection valve maintenance and self test OYES UNO PART C Score Number of correct tasks z How would you evaluate the level of confidence shown by the operator while performing Xpert g 0 1 not confident u2 u3 u4 5 very confident lt x 2T ce FIA D ANNEX 2 User appraisal questionnaire Questionnaire User appraisal of Xpert MTB Rif assay foundation 5 for innovative new diagnostics Xpert MTB Rif Demonstration Project Pre survey Laboratory ID Country Date of survey completion DD MM YYYY Study phase L Training L Validation L Implementation L Continuation Purpose To evaluate the usability and of the Xpert MTG Rif assay and obtain feedback for improvement Instructions To b
46. inally it must be ensured by all sites that patients diagnosed by Xpert MTB Rif assay including MDR TB cases receive appropriate treatment according to program norms throughout the demonstration period Confidentiality There are no risks with respect to confidentiality Patient identifiers will be encrypted Only authorized medical personnel at the study sites will have access to patient names 23 CIN D ANNEX 1 Proficiency testing form ne Proficiency testing Xpert MTB Rif assay ounaation for innovative new diagnostics Xpert MTB Rif Demonstration Project This tool is intended to be used by FIND monitor and or study coordinator to assess the minimum training needs and the resulting proficiency of the laboratory technicians at different points during the study The assessment will be carried out after initial training and at defined time intervals as described in the training plan The operators will be asked to perform a complete Xpert run as per routine testing The supervisor will observe without intervening or correcting mistakes The testing will be comprised of the following parts A Observed hands on Xpert MTB Rif run Each operator will be provided with 4 sputa with known results and asked to perform a routine Xpert run The supervisor will observe the run and complete a proficiency checklist which includes the critical steps that should be performed according to the SOP to ensure reliable test results B Quest
47. ination for culture amp microscopy Kubica ANNEX 6 Semi quantitative mycobacterial smear ANNEX 7 Semi quantitative mycobacterial culture ANNEX 8 Speciation with Tauns Capilia TB ANNEX 9 Drug susceptibility testing a A O 11 15 16 18 19 20 22 22 23 24 28 32 33 34 36 38 42 44 PARTNER INSTITUTIONS 1 South Africa 2 sites Ministry of Health and National TB Programme NTP National Health Laboratory Services NHLS South African Medical Research Council SAMRC University of Cape Town UCT M decins Sans Fronti res 2 Peru 2 sites National TB Programme NTP amp Instituto Nacional de Salud INS Instituto de Medicina Tropical Alexander von Humboldt Universidad Peruana Cayetano Heredia UPCH 3 Philippines 1 site National TB Programme NTP Tropical Disease Foundation TDF Center for Disease Control Atlanta 4 India 1 site Central TB Division CTD CMC Vellore FIND India 5 Uganda 1 site National TB Programme NTP Infectious Diseases Institute IDI University of California UCSF FIND Uganda 6 Azerbaijan 1 site Main Medical Department MMD Ministry of Health MOH Special Treatment Institution for Detainees with Tuberculosis STI STATEMENT OF INVESTIGATORS In signing this page agree to conduct the study in accordance with the relevant current protocol applicable regulations and institutional policy will ensure that the requirements relating to obtai
48. into non infected sputum and loading in the Xpert MTB Rif does not result in positive assay results No false positive results have occurred in tests on healthy subjects During evaluation studies with sample workloads of up to 40 specimens per day the demonstrated specificity was high 14 The users regarded the minimal manual sample pretreatment as easy and suitable for the microscopy center level This 15 min sample pre treatment was found to be capable of inactivating up to 10 AFBs spiked In addition aerosol studies showed that significantly fewer aerosols are produced during sample pretreatment for Xpert MTB Rif than during preparation of direct smears No infectious aerosols were found to be released from cartridges even for input of up to 10 AFBs In conclusion the sample pre treatment steps for Xpert MTB Rif can be performed without biosafety cabinet on an open bench during demonstration studies Only laboratories which have biosafety cabinets BSC and are currently preparing microscopy smears in the BSC will carry out the sputum pre treatment steps for Xpert MTB Rif in the BSC The GeneXpert instrument will be placed in a general purpose laboratory without biosafety facility in all settings No live bacteria have been recovered from spent cartridges but it will be recommended to autoclave cartridges as well as sputum containers for the demonstration project No live bacteria have been recovered from spent cartridges
49. ionnaire These questions should be asked orally during after the observed hands on Xpert run to assess the level of understanding of the Xpert MTB Rif assay and the operator ability in coping with problems that might arise during the procedure C Assessment of the GX software Each operator will be asked to perform the most important tasks using the GX software The supervisor will complete a checklist D Appraisal At the end the FIND supervisor or study coordinator will estimate the level of confidence shown by the operator while performing Xpert and this will be criteria for proficiency Performance targets The operators will only start performing the Xpert MTB Rif assay if the following performance targets are met Individual scores for Part A Band C 2 8 AND an overall appraisal of the operator s confidence to perform Xpert of 2 4 If performance targets are not met after initial training the operator will undergo additional training on specific topics until targets are met Materials needed Abottles of SR Xpert MTB Rif cartridges and disposable pipettes 4viscous very viscous sputa 1 culture negative 3 culture positive and among them 2 DST Rif resistant Timer 24 A Observed hands on Xpert MTB Rif run Checklist Instructions SNL A SAMPLE PREPARATION XPERT TEST BLEACH STORAGE WASTE LAB SETUP PART A 1 10 Prepare the workspace Process 4 samples according to th
50. ll participating laboratories will follow the routine sputum processing procedures A generic protocol is described below for MGIT and Lowenstein Jensen culture Principle The goal of mycobacterial culture is to detect viable mycobacteria in clinical samples Mycobacteria grow on specific media after processing Factors such as the rate of growth the colony morphology and the microscopic appearance of culture isolates help distinguish mycobacteria from contaminating flora Liquid culture provides increased sensitivity and speed while solid culture yields information about morphology and purity of culture The higher the bacterial load in a sample the greater the number of colonies in solid culture and the faster growth detection in liquid culture Materials e Commercial LJ and MGIT media reagents e Sterile plastic pipettes 5 ml e Materials for microscopic culture confirmation and speciation of culture isolates Bactec MGIT 960 Please refer to the FIND MGIT Manual for detailed information the manual can be downloaded from the FIND Web site www finddiagnostics org Reconstitution of PANTA e Dissolve PANTA in 15 ml MGIT Growth Supplement e Add 0 8 ml of the resultant enrichment to each MGIT tube just prior to inoculation amount for manual MGIT is 0 1 ml of PANTA and 0 5 ml OADC e To maintain the CO concentration in the media open tubes one at a time and for as short a time as possible Do not leave multiple MGIT tubes uncapped a
51. m 43 ANNEX 9 Drug susceptibility testing For study purposes all participating laboratories will follow the routine sputum processing procedures A generic protocol is described below for LJ and MGIT drug susceptibility testing LJ proportion method Principle Enables precise estimation of the proportion of mutants resistant to a given drug several 10 fold dilutions of inoculum are inoculated in both control media and drug containing media at least one dilution should yield isolated countable 50 100 colonies When these numbers are corrected by multiplying by the dilution of inoculum used the total number of viable colonies observed on the control medium and the number of mutant colonies resistant to the drug concentrations tested can be determined The proportion of bacilli resistant to a given drug is then determined by expressing the resistant portion as a percentage of the total population tested Preparation of drug containing media Drug concentrations are as follows Rifampicin 40 ug ml Isoniazid 0 2 ug ml Ethambutol 2 ug ml Streptomycin 4 ug ml dihydro streptomycin sulfate concent corresponding to 4 mg ml base Preparation of drug stock solution is done along with preparation of drug free LJ media For each prepared batch quality must be assured by inoculation of a wild strain One set consist of one LJ slope each for two 10 drug free slopes two 10 drug free slopes eight LU drug containing slopes two each
52. mean the tube is contaminated Inoculation and reporting LJ Place 5 7 drops 30ul each onto the solid media 0 2 ml Leave plate face up until inoculum is absorbed Then incubate LJ slants at 37 C Solid media are examined weekly for 8 weeks Cultures completely overgrown by bacterial or fungal contamination are discarded and the result recorded as contaminated if occurred within 3 weeks In case of mixed LJ culture pick isolated colonies and re culture for purity Colonies on LJ suspected to be mycobacteria are examined by Ziehl Neelsen staining MGIT Inoculate MGIT tube with 0 8 ml of antibiotics PANTA and growth supplement and then with 0 5 ml of resuspended pellet Only insert into instrument after 30 min to avoid oxygen consumption by still living bacteria Incubate liquid media vials at 37 C for 6 weeks see above for details Positive liquid cultures are confirmed by Ziehl Neelsen staining and contamination excluded by inoculation of blood agar plates and LJ subculturing Results are reported semi quantitatively of AFB colonies Result 0 negative 1 19 record number 20 100 101 200 201 500 gt 500 The date of inoculation and the date of positivity are recorded and will provide the time to positivity in days Positive mycobacterial cultures must at least be speciated to the level of MTB complex vs NTM using Tauns Capilia test at least from the first positive tube
53. mples themselves a certain measurement bias cannot be avoided The Xpert MTB Rif data analysis is not done by the users but real time in the Xpert software based on predefined mathematical algorithms It will therefore not be necessary to blind the Xpert MTB Rif users against smear or culture results However it will be necessary to blind the lab technicians performing smear microscopy against Xpert MTB Rif results The lab technicians involved in smear microscopy will therefore not have access to the GeneXpert software in order to ensure blinding between the gold standard and the method under investigation The Xpert MTB Rif results will be documented in a separate result form as the standard methods Indeterminate or invalid GeneXpert results will be analysed as described above STUDY COORDINATION TRAINING AND MONITORING FIND is the study sponsor and is responsible for planning managing and monitoring the study Dr Catharina Boehme will be the FIND study coordinator The site study team will receive technical on site training by FIND during the study initiation visit Completion of case report forms CRF and data management will be part of the initial training Staff at clinical enrolment sites will be trained in checking study inclusion criteria applying questionnaire and will be familiarized with the SOP for sputum collection by the on site study team During the study Catharina will be responsible for troubleshooting and logistic
54. nagement The supervisory site can but does not have to be the reference laboratory Criteria for country selection e Agreement at National Level MOU with NTP NRL or MOH e High burden of TB e Low or middle income e Atleast 2 countries with high HIV prevalence and high prevalence of smear negative TB e Atleast 2 countries with significant MDR prevalence e Local presence of FIND or implementing partner e Representative of global TB situation Criteria for selection of microscopy centers National TB Programs will select the demonstration sites that shall participate in the project and will base their selection on the following criteria e Selected demonstration sites must be easily accessible by monitors from the respective supervisory site e Must have at least 10 15 smear positivity e Must have at least 2 rooms e Ideally should have a minimum of 150 sputum samples eligible for Xpert testing e 2 microscopists or laboratory technicians present with no prior experience with molecular biology 16 Country Location Level of the Primary Average Electricity Biosafety Infrastructure Access to Site health system Use smears day cabinet treatment Lima Peru supervised by Instituto Nacional de Salud and Universidad Peruana Cayetano Heredia HSJL Urban District Hospital Case 25 Stable with BSC 1 1 lab room 1 TB MDR ZN Ogawa detection occasional short storage room interruptions CSPL Urban Health Center Case 15 Sta
55. ne purposes e There are two basic types of sputum specimens 7 spot specimens collected at a single time in the health facility if the volume brought up from the lungs in a single cough is too small the patient may collect sputum produced over a period of 1 hour in the same container initial spot specimen usually taken when the patient first presents with symptoms and another spot specimen taken when the patient returns with the second i e the early morning specimen Depending o the sites specimens will be either spot and or morning morning specimens these are spot specimens collected in the early morning when respiratory secretions that have gathered in the lower airways are cleared If the volume brought up from the lungs in a single cough is too small the patient may collect sputum produced over a period of 1 hour in the same container e Patients will be told that nasal secretions and saliva are not sputum Subjects will be told that the desired sample is deep cough sputum consisting of the thick mucoid white yellow material from the lower airways and lung Subjects will be instructed not to touch the inside of the specimen containers or lids e Labeling of the container will be done before it is used Information written on the side not on the lid must comprise patient ID date of collection and sputum number 1 2 or 3 e Specimens will be collected in the following manner The subject will be preferably be se
56. ning the Institutional Review Board IRB review and approval are met will promptly report to the IRB all changes in research activity agree to notify FIND prior to making any changes in the protocol have understood that the Xpert MTB Rif Assay is a new technology with highly promising performance characteristics in reference settings The herein described demonstration study is designed to establish performance characteristics and effectiveness data in decentralized laboratories These demonstration study data will be compiled and reported by FIND to national TB programs and public health authorities as well as the WHO Stop TB Department in order to generate informed policy decisions As such use of the Xpert MTB Rif Assay outside of the frame ofthis controlled demonstration study in decentralized laboratories should only be implemented subsequent to the policy decision of the appropriate national health authorities and or WHO recommendations shall maintain confidentiality concerning the contents of the protocol and all other written materials relevant to the demonstration study agree to ensure that all colleagues and employees assisting in the conduct of the study are informed about their obligations in meeting all of the above requirements PROTOCOL SYNOPSIS Background Commercial nucleic acid amplification tests NAAT for the detection of tuberculosis TB and rifampicin resistance have come into routine use in referenc
57. on GU values as described for SIRE drugs If the GC tubes become positive in less than 4 days or remain negative up to 21 days or some other conditions occur which may affect the test results the instrument report will be as an Error X In such situations the test needs to be repeated World Health Organization The global MDR TB and XDR TB response plan WHO HTM TB 2007 387 49
58. on of XDR or extensively drug resistant TB defined not only as resistance to isoniazid and rifampicin but also to one second line injectable agent and a fluoroquinolone antibiotic XDR TB is often not curable with available drugs The low sensitivity of smear microscopy limits its impact on TB control Culture methods are complex slow and still only scarcely available in high endemic countries Drug susceptibility testing is even slower and more technically complex While patients await diagnosis their disease progresses with an increased chance of dying from tuberculosis and they continue to transmit drug resistant TB to others especially family members Early case detection is essential to interrupt transmission and to prevent the further spread of tuberculosis and multidrug resistant tuberculosis A new diagnostic approach is therefore urgently needed Only a small fraction of the estimated 489 000 MDR TB cases occurring each year have access to drug susceptibility testing DST Key obstacles to DST expansion over the past years were 1 the complexity of available tools and the laboratory infrastructure required for their implementation 2 the unaffordable price of better but equally complex technologies and 3 the unavailability of rapid and simple tools for identification of resistance Although nucleic acid amplification tests NAAT for the rapid detection of TB and rifampicin resistance exist their complexity along with the labor d
59. resistant cases where this is acceptable for the IRB Otherwise DST will have to be carried out as per routine diagnostic algorithm A follow up visit after 2 and 6 months for Xpert MTB Rif positive patients is desired Study phases South Africa only 1 Phase I at least 6 months enrolment Consecutive TB suspects are enrolled The diagnostic algorithm is changed on a weekly alternating schedule Auramine smear and MGIT culture vs Auramine smear MGIT culture and Xpert MTB Rif DST is done on all culture positive cases In all patients with a rifampicin resistant result the test will be confirmed using Hain MTBDRplus testing for independent confirmation of MDR TB Patients will be appropriately referred In addition Hain MTBDRplus testing will continue to be used independently of this study when indicated as per current clinical practice Auramine smear MGIT culture and Xpert MTB Rif are used for TB treatment decisions 2 Phase Il at least 12 months enrolment As for Phase I however during Xpert week only Xpert MTB Rif to be performed whilst in the routine week standard of care to be followed two sputum smears In both arms culture and DST will be performed when routinely requested according to standard diagnostic practice at the sites For details see country specific protocol 18 Patient inclusion criteria Case detection group e Clinical suspicion of pulmonary TB cough 2 2 weeks e Age2 18 years MDR ri
60. rformed for one untreated leftover sample per patient by the reference laboratory This will provide the comparative gold standard Patient 17 management however will only be based on routine results not on the Xpert MTB Rif or the gold standard result A patient follow up visit after 2 months and 6 months for culture or Xpert MTB Rif positive patients is required Participating demonstration sites will be able to move to the implementation phase if the following Xpert MTB Rif performance targets are met c Xpert MTB Rif indeterminate rate lt 5 d Xpert MTB Rif sensitivity gt 80 of culture positive cases e Xpert MTB Rif specificity gt 90 of culture negative cases Data will be reviewed after 4 5 months and review shall include a 3 months enrolment phase A report summarizing the local findings will be submitted to IRBs for review and approval for the respective sites to move to the implementation phase 3 Implementation Phase at least 3 months enrolment ZN and Xpert MTB Rif are used for TB treatment decisions but not for MDR management Culture and DST for at least rifampicin and isoniazid will be performed by the reference laboratory A patient follow up visit after 2 and 6 months for culture or Xpert MTB Rif positive patients is required 4 Continuation Phase at least 6 months enrolment ZN and Xpert MTB Rif are used for TB treatment decisions Conventional DST is done only for Xpert Rif
61. rile water to each tube Add 2 ml of SR to each tube Mix by shaking 20 times and repeat after 5 10 min Let stand for a total 15 minutes using a timer 6 Go to preparation of the cartridge for testing below e Prepare 2 swabs as follows 1 Label sterile tubes Swab1 and Swab2 2 Add 2 ml of SR directly to each sterile tube Use a separate bottle of SR for each sample 3 Dip the tip of 2 cotton swabs in sterile water Cotton swabs should only be moist not wet 4 Swab 1x the inner surface of the GeneXpert modules and 1x the bench where sample preparation is carried out 5 Dip the swabs in the SR containing labeled tubes and turn them several times while touching the tube all to wring out potential DNA 6 Securely close lids 7 Mix by shaking 20 times and repeat after 5 10 min 8 Let stand for a total of 15 minutes using a timer 9 Go to preparation of the cartridge for testing below e Preparation of the cartridge for testing 1 Label the cartridges NC1 NC2 Swab1 and Swab2 2 Open the cartridge lid of the first cartridge 3 Using a separate sterile transfer pipette for each sample transfer between 2 0 and 2 5 ml of sample to the specimen chamber of each of the cartridges Close each cartridge lid 5 And start a test for each one IAN Send gxx files for respective tests to catharina boehme finddiagnostics org 32 ANNEX 4 Sputum collection e Specimens will be collected in the same containers as done for routi
62. rom Positive MGIT Tube Specimen SIRE Growth Control Tube e Using sterile pipettes dilute 0 1 ml of MGIT specimen in 10 ml sterile saline and mix well e This is the 1 100 Growth Control Suspension for a 1 2 day culture and 1 500 for a 3 5 day culture e Inoculate 0 5 ml of this suspension into the GC labeled tube Immediately recap the tube tightly and mix by inverting the tube several times PZA Growth Control Tube e Dilute 0 5 ml of the MGIT specimen prepared in section 8 2 into 4 5 ml sterile saline and mix well e This is the 1 10 PZA Growth Control Suspension for a 1 2 day culture and 1 50 for a 3 5 day culture Inoculate 0 5 ml of this suspension into the PZA GC labeled tube e Immediately recap the tube tightly and mix by inverting the tube several times Drug Containing Tubes e Inoculate each labeled drug containing tube with 0 5 ml of the MGIT specimen prepared in section e Immediately recap the tube tightly and mix by inverting the tube several times e Wipe all tubes and caps with a mycobactericidal disinfectant Preparation of Inoculum from LJ Media e Colonies from solid media may be used if they are no more than 15 days from the first appearance of positive growth Do not use very young culture because the growth rate for sensitive and resistant colonies may be different e Using a sterile loop or proper applicator spatula scrape as many colonies as possible trying not to remove any of the solid medium e Transfer th
63. sk group Presenting to the clinic as a possible MDR suspect e Re treatment cases PTB within the last year or non converting PTB case e Symptomatic contacts of known MDR cases or specific MDR associated exposures Patients presenting specifically as MDR suspects will be analyzed only for the Rif detection endpoints and not for TB case detection endpoints Patient exclusion criteria e No provision of at least 2 soutum samples Routine symptom screening at participating clinics Inclusion criteria a Cough 2 2 weeks Age 218 years Collection of 2 3 sputa as per routine Parallel conduct of routine smear microscopy and Xpert Weekly alternation Routine 2 direct 3 direct 2 direct 2 direct 3 direct 2 FM on smear ZN Sp 1 pellet Sp 1 microscopy 2 3 2 Culture 1 MGIT Sp method 2 DST method MGIT MGIT Indirect LJ LJ Direct and SIRE SIRE LPA LJ proportion proportion indirect proportion LPA Figure 2 Study flow The first positive culture from each patient will undergo confirmation of M tuberculosis species by biochemical or molecular methods or by MPT64 antigen detection for study purposes if MTB confirmation is not done systematically 19 Laboratory requirements Space The sample processing for the Xpert MTB Rif assay will be done on the open bench in the smear microscopy room or under the same conditions as per preparation of soutum smears The GeneXpert will be set up in a 2
64. t the same time e Perform these steps in biosafety cabinet class 2 Inoculation of MGIT Medium e Perform all inoculation steps in the class Il biosafety cabinet e Mark each MGIT tube with patient study ID number label e Using the pipette used for adding the buffer or a fresh sterile pipette or transfer pipette for each specimen or adjustable pipettor with filter plugged tips add 0 5 ml of a well mixed processed concentrated specimen to the appropriately labeled MGIT tube e To reduce the risk of cross contamination and to maintain the CO concentration in the media open tubes one at a time and for as short a time as possible Do not leave multiple MGIT tubes uncapped at the same time e Tightly recap the tube and mix by inverting the tube several times e Wipe tubes and caps with a mycobactericidal disinfectant e Leave inoculated MGIT tubes at room temperature for 30 min Incubation e Open the desired MGIT 960 drawer and press the tube enter key The barcode scanner will light up 38 Scan the inoculated MGIT tube and load into the slot identified by the MGIT 960 Perform these steps in biosafety cabinet class 2 Be sure that the cap is tightly closed Do not to shake the tube during the incubation Check MGIT 960 daily for indicator lights flagging positive and negative cultures Incubate MGIT tubes until the instrument flags them as positive or negative Positive and negative tubes will be displayed on the screen an
65. ts and 7620 tests including already available instruments from previous study Study Phases y All sites except South Africa Study phase sputa patient Validation Implementation Continuation South Africa at 20 C Case Case Case Study phase sputa patient 3 5 days detection 2 3 MDR risk 2 3 detection 2 3 MDR risk 2 3 detection 1 MDR risk 1 2 if Rif res per Routine ZN Xpert MTB Rif Possible culture ZN Xpert MTB Rif ZN Xpert MTB Rif per Selected DST for Xpert Rif res cases Routin weeks e Treatment decision based on Routine smear culture MDR risk Routine DST Smear Xpert MTB Rif case detection only Selected DST for Rif res Smear Xpert MTB Rif Selected DST for Rif res Treatment decision based on FU at Clinic Requires at least 32 sputum samples with known smear culture and Rif result stored Culture amp DST Culture amp DST As per routine and for Xpert MTB Rif or culture pos patients after 2 and 6 months As per routine and for Xpert MTB Rif or culture pos patients after 2 and 6 months As per routine and for Xpert MTB Rif pos patients after 6 months FU at Clinic Requires 32 sputum samples with known smear culture and Rif result stored at 20 C Phase reported in this Lancet manuscript Phase Il ongoing Timelines Activity Event
66. tured washed cells are lysed with ultrasonic energy and the released DNA eluted through the filter membrane The DNA solution is finally mixed with dry PCR reagents and transferred into the PCR tube for real time PCR and detection To eliminate test to test contamination all fluids including amplicons are contained within the disposable cartridge Concentrates bacilli amp removes inhibitors Sample is Ultrasonic lysis of filter End of hands on work automatically captured organisms to filtered amp washed release DNA 4 DNA molecules are mixed with dry PCR reagents Semi nested real time amplification amp detection in integrated reaction tube Transfer of 2 ml after 15 min Sputum liquefaction amp inactivation with 2 1 SR Printable test result Figure 1 Xpert MTB Rif assay procedure Each instrument contains 4 individually accessible modules that are capable of performing separate real time polymerase chain reaction PCR protocols Other instrument sizes are available containing between 1 and 72 individually accessible modules Each module consists of a syringe drive for dispensing fluids an ultrasonic horn for lysing cells a thermocycler and optical signal detection components The single use cartridges contain i chambers for holding sample and reagents ii a valve body composed of a plunger and syringe barrel iii a rotary valve system for controlling the movements of fluids
67. ugh a membrane filter 2 ml of stock solution 500yg ml 48ml of sterile distilled water 50ml of 20ug ml Do not store this solution Number of sets Number of bottles of MI of working Amount of LJ Final required to be H drug media solution of H fluid to be concentration prepared required each bottle 20ug ml added ml of H in LJ with 5ml of LJ fluid ug ml 5 10 0 5 49 5 0 2 10 20 1 99 0 2 15 30 1 5 148 5 0 2 20 40 2 198 0 2 25 50 2 5 247 5 0 2 30 60 3 297 0 2 Ethambutol E Drug potency 1g to 1g substance Stock solution preparation Weigh out 20mg of ethambutol and dissolve in 100ml of sterile distilled water to get 200ug ml of stock solution Sterilize by filtering through a membrane filter Stock solution of E should not be stored Number of sets Number of bottles of E MI of stock Amount of LJ Final required to be drug media required solution fluid to be concentration prepared each bottle with 5m of 200ug ml added of E in LJ LJ fluid ug ml 5 10 0 5 49 5 2 10 20 1 99 2 15 30 1 5 148 5 2 20 40 2 198 2 25 50 2 5 247 5 2 30 60 3 297 2 45 Dihydro Streptomycin Sulphate S Preferred substance Sigma 7253 Correction for potency required Stock solution preparation Weigh out 20mg Potency of dihydro streptomycin sulphate and dissolve in 50 ml SDW to obtain 400ug ml of stock solution For example if the
68. ulosis grown in culture requires biosafety level 3 practices e NaOH is alkaline and cause burns In case of contamination take off contaminated clothes immediately Gloves must be worn In the event of eye or skin contact rinse for at least 15 min with tap water and seek medical advice If ingested drink milk and seek medical advice Materials e N Acetyl L cysteine 0 5 g measured in sterile 100 ml Erlenmeyer flask e 6 Sodium hydroxide solution NaOH autoclaved 50 ml e 2 9 Tri sodium citrate dihydrate solution autoclaved 50 ml e Phosphate buffer solution Potassium Di hydrogen phosphate pH 6 8 e MGIT vials 7 ml e BBLMGIT PANTA and BACTEC MGIT Growth Supplement e LJ medium L wenstein Jensen medium e Safety cabinet biological class Il vortexer shaker centrifuge with employments for 50 ml centrifuge tubes and aerosol protection hoods MGIT 960 Incubator 36 1 C refrigerator e Multipette timer racks e Graduated sterile cylinder 100 ml sterile centrifuge tubes 50 ml sterile transfer pipettes 5 ml sterile tips for multipette 10 ml e Camping gas or Bunsen burner microscopy slides pencil disinfectant autoclave bag 34 Processing e Sort the samples according to ascending sequence of numbers e Label all tubes cultural media MGIT LJ and slides with specimen numbers e Fill a report form for each investigation material arrived in the laboratory e Prepare NALC NaOH solution use reagent within 24 hours
69. und of cellular material and other organisms in clinical specimens Materials e Microscope slides w one frosted end pencil e Binocular microscope with 20 40 100x objectives e Ziehl Neelsen staining kit reagents e Camping gas or Bunsen burner and staining rack Procedure e Briefly flame fix after air dry e Stain for acid fast microscopy using Ziehl Neelsen technique Ziehl Neelsen Cover sample area with carbolfuchsin phenol Flame heat until steam rises do not boil Leave 2 min Rinse lightly with water Flood slide with acid alcohol leave to sit for 2 min Rinse lightly with water Flood slide with Methionine blue for 3 min Rinse lightly with water e Examine the slide using visual light Ziehl Neelsen ZN e Begin examination with low power screen of entire sample area to identify thick areas areas that have been lost during staining and washing process and other problem areas e Read several representative sections of the slide At least 100 fields should be read Slides should be read in a sweeping pattern avoiding repeated examination of the same area see figure below S 36 e Examine the slide using the 100x oil immersion objective Score the smear using the table below Number of acid fast Bacilli 0 1 9 100 fields 10 99 100 fields 1 10 field gt 10 field Result negative record number of AFB 37 ANNEX 7 Semi quantitative mycobacterial culture For study purposes a
70. ve TB and MDR TB control Large scale demonstration projects are required to provide the evidence that this new diagnostic test can successfully be implemented and have significant medical and public health impacts in programmatic settings Data from these projects will be shared with the WHO Stop TB Department that is expected to issue guidance on the use of this assay in 2010 Following established procedures WHO will convene an Xpert MTB Rif committee to review available data on the use and performance of the Xpert MTB Rif and draw up guidelines for the implementation of the assay in low and middle income countries This guidance will be reviewed by the WHO Strategic and Technical Advisory Group STAG for TB in June 2010 Subsequent to STAG approval WHO will issue formal guidance Project purpose The purpose of this demonstration project is to assess the implementation of Xpert MTB Rif in decentralized laboratories in low and moderate income settings The project aims at a confirming the Xpert MTB Rif performance in decentralized laboratories and b demonstrating the feasibility impact and cost efficiency of using Xpert MTB Rif at lower levels of the health system for enhanced detection of smear negative TB and MDR TB The data will be reported to national public health authorities and WHO in order to inform policy decision Study hypothesis Fully automated molecular testing with integrated specimen processing by Xpert M
71. wth Control Suspension e Inoculate 0 5 ml of this suspension to the GC labeled tube e Immediately recap the tube tightly and mix by inverting the tube several times PZA Growth Control Tube e Dilute 0 5 ml of the MGIT specimen prepared in section 8 4 into 4 5 ml sterile saline and mix well This is the 1 50 PZA Growth Control Suspension Inoculate 0 5 ml of this suspension into the PZA GC labeled tube Immediately recap the tube tightly and mix by inverting the tube several times SIRE and PZA Drug Containing Tubes e Inoculate each labeled drug containing tube with 0 5 ml of the MGIT specimen prepared e Immediately recap the tube tightly and mix by inverting the tube several times e Wipe all tubes and caps with a mycobactericidal disinfectant Incubation e Enter the inoculated set of DST specimens into the BACTEC 960 instrument using the AST set entry feature Be sure that the tubes are loaded according to the order specified for the AST set entry feature Be sure that the caps are tightly closed Do not to shake the tube throughout the incubation The BACTEC 960 instrument will monitor the inoculated media and will indicate once the test is complete within 4 21 days Growth Control reaches GU 400 or more At this point the susceptibility set can be taken out after scanning and a report can be printed out e The susceptibility report will indicate S susceptible or R resistant The instrument interpretation of results is based
72. y suspend with a vortex mixer For AFB positive MGIT tubes no preparation is required Capilia TB testing Apply 100ul of the AFB positive MGIT media or the LJ colony suspension to the sample well on the Capilia TB specimen placing area of the test plate directly without any manipulation Observe the reading area of the test plate for 5 minutes and for presence or absence of red to deep purple or faint bands both for the control and the test bands all results should be read within 1 hour after dispensing the sample into the sample well Interpret the results as explained below and report result For specimens with a negative or invalid test at 5 min read the test again at 1 hour Interpretation of Capilia TB test results C T C T Lp bo ooo T POSITIVE NEGATIVE INVALID Positive for Mycobacterium tuberculosis complex Red purple colour appears in both the T and C reading area The result is read as positive if area T shows red purple colour that is lighter than or the same as or darker than the colour in area C Negative for Mycobacterium tuberculosis complex A test is negative if red purple colour appears in area C but not in area T Invalid test A test is invalid if red purple colour does not appear in area C If a test is invalid repeat test using a new test plate preferably from a newly opened foil pouch If the repeated test result is valid enter this result on the case report for

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