Standard Operating Procedures for Quantitative HIV-1 viral load testing using GeneXpert technology. June 2015

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1 Standard Operating Prcedures fr Quantitative HIV-1 viral lad testing using GeneXpert technlgy June Versin 0 Effective date: 10 June 2014

2 Table f Cntents I. Title f Prcedure... 3 II. Test Summary... 3 III. Principle... 3 IV. Specimen Handling and Preparatin... 4 V. Quality Cntrl... 4 VI. Reagents, Materials, & Equipment... 6 VII. Prcedure... 7 VIII. Interpretatin f Results IX. Pssible reasns fr repeating the assay X. Limitatins XI. Perfrmance Characteristics XII. References Figure 1: Overview f the GeneXpert test cartridge... 4 Figure 2: GeneXpert quality cntrl result scenaris... 6 Figure 3: GeneXpert transfer pipette and aerial view f the reactin cartridge... 8 Figure 4: Summary f sample lading in GeneXpert HIV-1 quant... 9 Figure 5: GeneXpert HIV-1 quantitative detectable VL Figure 6: GeneXpert HIV-1 quantitative nt detected VL Table 1: GeneXpert HIV-1Viral lad result interpretatin Versin 0 Effective date: 10 June 2014

3 I. Title f Prcedure Quantitative HIV-1 viral lad testing using GeneXpert technlgy II. Test Summary State the physilgic and diagnstic reasns fr perfrming the test. Human Immundeficiency Virus (HIV) is the etilgic agent f Acquired Immundeficiency Syndrme (AIDS). It can be transmitted thrugh sexual cntact, expsure t infected bld r bld prducts, prenatal infectin f a fetus, r perinatal r pstnatal infectin f a newbrn. HIV diagnstics have evlved significantly in the past tw decades and cntinue t be significantly imprtant in saving lives f millins f HIV infected patients. Tday, measurement f bld plasma HIV-1 RNA cncentratin knwn as viral lad using nucleic acid-based mlecular diagnstic assays has been established as standard f care in assessing HIV-psitive patient prgnsis and respnse t antiretrviral therapy. Untreated HIV-1 infectin is characterized by high-level viral prductin and CD4 T-cell destructin, despite an ften lengthy clinical latency, t significant net lss f CD4 T cells and AIDS. Assessment f viral lad levels is a strng predictr f the rate f disease prgressin and, by itself r in cmbinatin with CD4Tcell cunts, has great prgnstic value. The HIV-1 Quant Assay, perfrmed n GeneXpert Instrument Systems, is designed fr the rapid quantitatin f Human Immundeficiency Virus type 1 (HIV-1) in human plasma frm HIV-1 infected individuals ver the range f 40 t 10,000,000 cpies/ml. HIV-1 Grup M subtypes A, B, C, D, AE, F, G, H, AB, AG, J, K and Grups N and O are detected and quantified by the HIV-1 Quant Assay. The HIV-1 VL Assay uses reverse transcriptase plymerase chain reactin (RT-PCR) technlgy t achieve high sensitivity fr the quantitative detectin f HIV-1 RNA in human plasma frm HIV-1 infected individuals. III. Principle GeneXpert Instrument Systems autmate and integrate sample preparatin, nucleic acid extractin and amplificatin, and detectin f the target sequence in simple r cmplex samples using real-time reverse transcriptase PCR (RT-PCR). The systems cnsist f an instrument, persnal cmputer, and preladed sftware fr running tests and viewing the results. The systems require the use f single-use dispsable GeneXpert cartridges that hld the RT-PCR reagents and hst the RT-PCR prcesses. Because the cartridges are self-cntained, crsscntaminatin between samples is minimized. The HIV-1 VL Assay includes reagents fr the detectin f HIV-1 RNA in specimens and tw internal cntrls used fr quantitatin f HIV-1 RNA. The internal cntrls are als used t mnitr the presence f inhibitr(s) in the RT and PCR reactins. The Prbe Check Cntrl (PCC) verifies reagent rehydratin, PCR tube filling in the cartridge, prbe integrity, and dye stability. 3 Versin 0 Effective date: 10 June 2014

4 Figure 1: Overview f the GeneXpert test cartridge IV. Specimen Handling and Preparatin Whle bld shuld be cllected in EDTA, EDTA-PPT, r ACD cllectin tubes and centrifuged t separate the plasma and red bld cells per the manufacturer's instructins. A minimum f 1 ml plasma is required fr the HIV-1 VL Assay. If using the transfer pipette included in the kit, a minimum f 1.2 ml plasma is required. Alternatively, if using a precisin pipette, a minimum f 1 ml plasma is required. Whle bld may be held at C fr up t 8 hurs r at 2 8 C fr up t 72 hurs, prir t preparing and testing the specimen. After centrifugatin, plasma may be held at C fr up t 24 hurs r at 2 8 C fr up t 6 days, prir t testing. Plasma specimens are stable frzen ( -18 C and -70 C) fr six weeks. Plasma specimens are stable up t three freeze/thaw cycles. Plasma specimens must be thawed and equilibrated t rm temperature prir t transfer t cartridge. V. Quality Cntrl Each GeneXpert test cartridge is a self-cntained test device with an in-built cntrl fr each sample. Nrmally, n external cntrls are required. The internal cntrls enable the system t detect specific failure mdes within each fr each sample Instrument system cntrl: Check status-it checks the ptics, temperature f the mdule and the mechanical integrity f each cartridge. If the system cntrls fail, an ERROR test result will be reprted. 4 Versin 0 Effective date: 10 June 2014

5 Prbe Check cntrl (PCC): after sample preparatin, bead recnstitutin and tube filling (prir t thermal cycling), multiple flurescent readings are taken at different temperatures and cmpared t default setting. PCC cntrls fr: Missing target specific reagent (TSR) and r enzyme reagent beads which cntain all primers, prbes and internal cntrl template. Incmplete reagent recnstitutin Incmplete reactin tube filling Prbe degradatin If the PCC fails, an ERROR test result will be reprted. Sample prcessing cntrl (SPC) assesses the effectiveness f the sample prcessing steps, including and up-t reactin tube filling. SPC ensures that the sample was crrectly added t the cartridge and detects degradatin f the enzyme(s) r ther cmpnents f the system. SPC des nt cmpete with target DNA. SPC must be Psitive when target is Negative SPC can be Psitive r Negative when the target is Psitive SPC passes if it meets the validated acceptance criteria. E2097: t less sample vlume added (<1mL), E2096: n sample added Internal Quantitative Standard High and Lw (IQS-H and IQS-L): IQS-H and IQS-L are tw dry bead armred RNAs nnspecific t HIV in the frm f a dry bead that ges thrugh the whle GX prcess. The IQS-H and IQS-L are standards calibrated against the WHO 3rd Internatinal Standard. They are used fr quantificatin by using lt specific parameters fr the calculatin f HIV-1 RNA cncentratin in the sample. The IQS-H and IQS-L pass if they meet the validated acceptance criteria. They run internally with every cartridge and they cntrl fr reagent perfrmance due t imprper strage and they cnfirm that reactin cmpnents are set up crrectly. External Cntrls: nt available in the kit, but they can be used (psitive and negative cntrls). 5 Versin 0 Effective date: 10 June 2014

Figure 2: GeneXpert quality cntrl result scenaris VI. Reagents, Materials, & Equipment The HIV-1 VL Assay kit cntains sufficient reagents t prcess 10 specimens r quality cntrl samples.

6 Figure 2: GeneXpert quality cntrl result scenaris VI. Reagents, Materials, & Equipment The HIV-1 VL Assay kit cntains sufficient reagents t prcess 10 specimens r quality cntrl samples. The kit cntains the fllwing: HIV-1 VL Assay Cartridges with Integrated Reactin Tubes. Each cartridge cntains: Bead 1, Bead 2, and Bead 3 (freeze-dried) Lysis Reagent (Guanidinium Thicyanate) 2.0 ml Rinse Reagent 0.5 ml Elutin Reagent 1.5 ml Binding Reagent 2.4 ml Prteinase K Reagent 0.48 ml 2. Dispsable 1 ml Transfer Pipettes 10 per kit 3. CD 1 per kit Assay Definitin File (ADF) Instructins t imprt ADF int GeneXpert sftware Package Insert Strage and Handling Stre the HIV-1 VL Assay cartridges at 2 8 C. D nt pen the cartridge lid until yu are ready t perfrm the test. Use cartridge within fur hurs after pening the cartridge lid. D nt use a cartridge that has leaked. Materials Required but Nt Prvided 6 Versin 0 Effective date: 10 June 2014

7 Printer Bleach Warnings and Precautins Treat all bilgical specimens, including used cartridges, as if capable f transmitting infectius agents. Because it is ften impssible t knw which might be infectius, all bilgical specimens shuld be treated with standard precautins. D nt pen the HIV-1 VL Assay cartridge lid until yu are ready t add the plasma specimen D nt use a cartridge that has been drpped after remving it frm the packaging. D nt shake the cartridge. Shaking r drpping the cartridge after pening the lid may yield invalid results. D nt place the sample ID label n the cartridge lid r n the barcde label. Each single-use HIV-1 VL Assay cartridge is used t prcess ne specimen. D nt reuse spent cartridges. D nt use a cartridge that has a damaged reactin tube. Single-use dispsable pipette is used t transfer ne specimen. D nt reuse dispsable pipettes. Wear clean lab cats and glves. Change glves between prcessing each sample. In the event f cntaminatin f the wrk area r equipment with samples r cntrls, thrughly clean the cntaminated area with a slutin f 1:10 dilutin f husehld chlrine bleach and then 70% ethanl. Wipe wrk surfaces dry cmpletely befre prceeding. VII. Prcedure 1. Preparing the Specimen If using frzen specimens, place the specimens at rm temperature C until cmpletely thawed and equibrate t rm temperature befre use. Plasma samples stred in 2 8 C shuld be remved frm the refrigeratr and equilibrated t rm temperature befre use. Vrtex plasma fr 15 secnds befre use. If the specimen is cludy, clarify by a quick spin. 2. Preparing the Cartridge Wear prtective dispsable glves. Inspect the test cartridge fr damage. If damaged, d nt use it. Open the lid f the test cartridge. Allw samples t cme t rm temperature prir t lading plasma int the cartridge Optin 1: If using the transfer pipette included in the kit (Figure 1), fill t just belw the bulb but abve the line t transfer at least 1 ml plasma frm the cllectin tube int the sample chamber f the test cartridge (Figure 2). D NOT pur the specimen int the chamber! 7 Versin 0 Effective date: 10 June 2014

Figure 3: GeneXpert transfer pipette and aerial view f the reactin cartridge Clse the cartridge lid. Lad the cartridge int the GeneXpert instrument r Infinity system.

8 Figure 3: GeneXpert transfer pipette and aerial view f the reactin cartridge Clse the cartridge lid. Lad the cartridge int the GeneXpert instrument r Infinity system. Nte There is a thin plastic film that cvers the inner ring f 13 prts f the test cartridge. This film shuld nt be remved. Optin 2: If using an autmatic pipette, transfer at least 1 ml f plasma int the sample chamber f the test cartridge (Figure xx). D NOT pur the specimen int the chamber! Imprtant Start the test within fur hurs f adding the sample t the cartridge Lading less than 1 ml plasma int the cartridge will trigger an insufficient vlume errr (ERROR 2097), preventing the instrument frm running the sample Starting the Test This sectin lists the basic steps fr running the test. Fr detailed instructins, see the GeneXpert System Operatr Manual n the cmputer desktp. a. Turn n the GeneXpert instrument. First turn n the instrument and then turn n the cmputer. The GeneXpert sftware will launch autmatically. If it desn't, duble-click the GeneXpert b. Sftware shrtcut icn n the Windws desktp. 8 Versin 0 Effective date: 10 June 2014

9 c. Lg n t the GeneXpert Instrument System sftware using yur user name and passwrd. Click Create Test d. Scan in the Patient ID (ptinal). If typing the Patient ID, make sure the Patient ID is typed crrectly. The Patient ID is assciated with the test results and is shwn in the View Results windw. e. Scan r type in the Sample ID. If typing the Sample ID, make sure the Sample ID is typed crrectly. The Sample ID is assciated with the test results and is shwn in the View Results windw and all reprts. The Scan Cartridge dialg bx appears. f. Scan the barcde n the Xpert HIV-1 Viral Lad cartridge. The Create Test windw appears. Using the barcde infrmatin, the sftware autmatically fills the bxes fr the fllwing fields: Select Assay, Reagent Lt ID, Cartridge SN, and Expiratin Date. g. Click Start Test. Enter yur passwrd, if requested. h. Open the instrument mdule dr with the blinking green light and lad the cartridge. Clse the dr. The test starts and the green light stps blinking. When the test is finished, the light turns ff. Wait until the system releases the dr lck befre pening the mdule dr and remving the cartridge. The used cartridges shuld be dispsed in the apprpriate specimen waste cntainers accrding t yur institutin s standard practices. Figure 4: Summary f sample lading in GeneXpert HIV-1 quant 9 Versin 0 Effective date: 10 June 2014

10 WARNING: GeneXpert HIV Quant cartridges cntain a highly txic chemical (t human and envirnment), Guanidinium Thicyanate, and shuld be dispsed f after use, in an incineratr capable f reaching 1200 C. VIII. Interpretatin f Results Click the View Results icn t view results. Upn cmpletin f the test, click the Reprt buttn f the View Results windw t view and/r generate a PDF reprt file. The test results take apprximately 90minutes t be prduced The results are interpreted autmatically by the GeneXpert Instrument System frm measured flurescent signals and embedded calculatin algrithms and are clearly shwn in the View Results windw (Figure 5 and Figure 6). Pssible results are shwn in Table 1. Table 1: GeneXpert HIV-1Viral lad result interpretatin 10 Versin 0 Effective date: 10 June 2014

11 11 Versin 0 Effective date: 10 June 2014

12 Figure 5: GeneXpert HIV-1 quantitative detectable VL Figure 6: GeneXpert HIV-1 quantitative nt detected VL 12 Versin 0 Effective date: 10 June 2014

13 IX. Pssible reasns fr repeating the assay An INVALID result indicates that the sample was nt prperly prcessed, PCR was inhibited, r the sample was inadequate. An invalid result als indicates that the IQS-H and/r IQS-L Cts are nt within valid range. An ERROR result indicates that the Prbe Check Cntrl failed r maximum pressure limits were exceeded. An errr result indicates that the assay was abrted. Pssible causes include: insufficient vlume f sample was added, the reactin tube was filled imprperly, a reagent prbe integrity prblem was detected, r the maximum pressure limit was exceeded. A NO RESULT indicates that insufficient data were cllected. Fr example, the peratr stpped a test that was in prgress, a lad errr ccurred, r the sftware was clsed prematurely. Retest Prcedure Fr retest f an INVALID, ERROR, r NO RESULT, use a new cartridge (d nt re-use the cartridge). 1. Remve a new cartridge frm the kit. 2. Sectin VII n preparing cartridge and starting the test n page 7. X. Limitatins Gd labratry practices and changing glves between handling specimens are recmmended t avid cntaminatin f specimens r reagents. Factrs that negatively affect results Interfering substance False negative test results r invalid results may be bserved in the presence f interfering substance like; Albumin (90g/L), bilirubin (20mg/dL), haemglbin (500mg/dL), human DNA (0,4mg/dL) and triglycerides (3000mg/dL). Bilgical specimens, including used cartridges, shuld be treated as capable f transmitting infectius agents and shuld be discarded in hazardus clinical waste bins. 13 Versin 0 Effective date: 10 June 2014

14 XI. Perfrmance Characteristics Limit f Detectin (LD) The LOD fr the HIV-1 VL Assay is 40 cp/ml fr HIV-1 subtype B in EDTA plamsa. Limit f Quantitatin The LQ is defined as the lwest cncentratin f HIV-1 RNA that is determined with acceptable precisin and trueness. The LOQ f the HIV-1 VL Assay is 40 cp/ml (1.60 lg10). Linear Range The HIV-1 VL Assay is linear within a range 30 t 1x10 7 cp/ml Specificity The specificity f the HIV-1 VL Assay was evaluated using EDTA plasma specimens frm HIV-1 negative bld dnrs. The HIV-1 VL Assay specificity was demnstrated at 100% (95% CI = ). TECHNICAL SUPPORT Raynlds Mangena Pinte care diagnstics Harare Chepeid Suth Africa MOBILE FAX dipti.lallubhai@cepheid.cm 14 Versin 0 Effective date: 10 June 2014

15 XII. References 1. Barre-Sinussi F, Chermann JC, Rey F, et al. Islatin f a T-lymphtrpic retrvirus frm a patient at risk fr acquired immune deficiency syndrme (AIDS). Science 1983;220: Ppvic M, Sarngadharan MG, Read E, et al. Detectin, islatin and cntinuus prductin f cytpathic retrviruses (HTLV-I) frm patients with AIDS and pre-aids. Science 1984;224: Gall RC, Salahuddin SZ, Ppvic M, et al. Frequent detectin and islatin f cytpathic retrviruses (HTLV-I) frm patients with AIDS and at risk fr AIDS. Science 1984;224: Curran JW, Jaffe HW, Hardy AM, et al. Epidemilgy f HIV infectin and AIDS in the United States. Science 1988;239: Schchetman G, Gerge JR, editrs. AIDS testing: a cmprehensive guide t technical, medical, scial, legal, andmanagement issues. 2nd ed. New Yrk: NY Springer-Verlag; Nduati R, Jhn G, Mbri-Ngacha D, et al. Effect f breastfeeding and frmula feeding n transmissin f HIV-1: arandmized clinical trial. Jurnal f the American Medical Assciatin 2000;283: Perelsn AS, Neumann AU, Markwitz M, Lenard JM, H DD. HIV-1 dynamics in viv: virin clearance rate, infected cell life-span, and viral generatin time. Science 1996; 271: Wei X, Ghsh SK, Taylr ME, Jhnsn VA, Emini EA, Deutsch P, Lifsn JD, Bnheffer S, Nwak MA, Hahn BH, et al. Viral dynamics in human immundeficiency virus type 1 infectin. Nature 1995; 373: H DD, Neumann AU, Perelsn AS, Chen W, Lenard JM, Markwitz M. Rapid turnver f plasma virins and CD4 lymphcytes in HIV-1 infectin. Nature 1995; 373: Katzenstein DA, Hammer SM, Hughes MD, Gundacker H, Jacksn JB, Fiscus S, Rasheed S, Elbeik T, Reichman R, Japur A, Merigan TC, Hirsch MS. The relatin f virlgic and immunlgic markers t clinical utcmes after nucleside therapy in HIV-infected adults with 200 t 500 CD4 cells per cubic millimeter. AIDS Clinical Trials Grup Study 175 Virlgy Study Team. N Engl J Med 1996; 335: Mellrs JW, Munz A, Girgi JV, Marglick JB, Tassni CJ, Gupta P, Kingsley LA, Tdd JA, Saah AJ, Detels R, Phair JP, Rinald CR, Jr. Plasma viral lad and CD4+ lymphcytes as prgnstic markers f HIV-1 infectin. Ann Intern Med 1997; 126: Mellrs JW, Rinald CR, Jr., Gupta P, White RM, Tdd JA, Kingsley LA. Prgnsis in HIV-1 infectin predicted by the quantity f virus in plasma. Science 1996; 272: O'Brien WA, Hartigan PM, Martin D, Esinhart J, Hill A, Benit S, Rubin M, Simberkff MS, Hamiltn JD. Changes in plasma HIV-1 RNA and CD4+ lymphcyte cunts and the risk f prgressin t AIDS. Veterans Affairs Cperative Study Grup n AIDS. N Engl J Med 1996; 334: Ruiz L, Rmeu J, Cltet B, Balague M, Cabrera C, Sirera G, Ibanez A, Martinez-Picad J, Ravents A, Tural C, Segura A, Fz M. Quantitative HIV-1 RNA as a marker f clinical stability and survival in a chrt f 302 patients with a mean CD4 cell cunt f 300 x 10(6)/l. Aids 1996; 10:F Saag MS, Hldniy M, Kuritzkes DR, O'Brien WA, Cmbs R, Pscher ME, Jacbsen DM, Shaw GM, Richman DD, Vlberding PA. HIV viral lad markers in clinical practice. Nat Med 1996; 2: Versin 0 Effective date: 10 June 2014

16 16. Centers fr Disease Cntrl and Preventin. Bisafety in Micrbilgical and Bimedical Labratries. Richmnd JY and McKinney RW (eds) (1993). HHS Publicatin number (CDC) Clinical and Labratry Standards Institute. Prtectin f Labratry Wrkers frm Occupatinally Acquired Infectins; Apprved Guideline. Dcument M29 (refer t latest editin). 18. Clinical and Labratry Standards Institute. Evaluatin f Detectin Capability fr Clinical Labratry Measurement Prcedures; Apprved Guideline. Dcument EP17-A2 (Secnd Editin). Wayne, PA: Clinical Labratry Standards Institute; Clinical and Labratry Standards Institute. Evaluatin f the Linearity f Quantitative Measurement Prcedures: AStatistical Apprach; Apprved Guideline. NCCLS dcument EP06-A [ISBN ]. NCCLS, 940 West Valley Rad, Suite 1400, Wayne, PA USA, Clinical and Labratry Standards Institute. Evaluatin f Precisin Perfrmance f Clinical Chemistry Devices; Apprved Guideline. Dcument EP5-A2. 16 Versin 0 Effective date: 10 June 2014

ABIOpure TM Total RNA (version 2.0)

ABIOpure TM Total RNA (version 2.0) ABIOpure TM Ttal RNA (versin 2.0) Bld Extractin Handbk Cat N: M541RP50-B FOR RESEARCH USE ONLY Table f Cntents Cntents Page Kit Cmpnents 3 Precautins 3 Stability & Strage 4 General Descriptin 4 Limitatins