MycAssay™ Aspergillus
Contents
1. see the kit box pouch label and repeat with unexpired kit if necessary Either Tube 1 or 2 reagent was not added to the PCR reaction or double the amount of Tube 2 was added gt Repeat the run taking care in the set up stage Such errors can be detected by seeing higher or lower levels of liquid in one reaction tube compared to others 030 169 Version 1 1 O5MAR12 English 16 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler 4 3 The Positive Control is negative gt The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired Please check correct storage conditions of the kit have been followed Check the expiry date of the reagents see the kit box pouch label and repeat with an unexpired kit if necessary An error occurred during step 1 12 and the Positive Control template Tube 4 was placed in the wrong reaction tube Repeat the run taking great care during the set up stage Such errors can be detected by seeing a higher level of liquid in one reaction and a lower level in another compared to normal Either Tube 1 or 2 reagent was not added to the reaction Repeat the run taking care in the set up stage Such errors can be detected by seeing lower levels of liquid in this reaction compared to others The Positive Control was incorrectly positioned in the instrument Take care that the reaction tubes are placed2. 030 169 Version 1 1 O5MAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use Invasive fungal disease IFD rates are nearly seven times higher in allogeneic HSCT patients than in autologous transplant patients and invasive aspergillosis IA is responsible for approximately half of infections Aspergillosis is largely confined to the early post transplant neutropenic phase in autologous HSCT patients Allogeneic HSCT patients are at risk for much longer periods not only up to but also beyond 100 days owing to their more frequent GvHD and slow T cell recovery In patients receiving chemotherapy for acute leukaemia or salvage regimens for relapsed leukaemia or lymphoma IA is a leading cause of death Consensus definitions of Invasive Fungal Diseases have been revised and published by the European Organisation for Research EORTC and the Mycoses Study Group MSG including defined criteria for diagnosis of proven probable and possible IA in patients with hematologic malignancy or following HSCT Currently the criteria for probable IA are defined as one host factor plus one clinical criterion plus one microbiological test Diagnosis of possible IA does not require a microbiology criterion The microbiological tests accepted in the probable IA criteria include a serum based ELISA test that detects the presence of galactomannan Galactomannans are polysaccharides that are released as the Aspergillus fungus g
3. For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler Extraction Protocol shaded areas identify those steps that are modified from manufacturer s instructions Add 400 uL of Binding buffer and 80 uL of Proteinase K mix o5mLserum immediately incubate for 10 0 5 mL serum minutes at 70 C Add 200 uL of lsopropanol mix well and apply to High Pure filter tube by repeat addition centrifuge for 1 min a Add 500 uL Inhibi xm uL Inhibitor Discard flow through Centrifuge for 1 removal buffer and collection tube f minute at 8 000xg K Add 500 uL wash Discard flow through Centrifuge for 1 a buffer and collection tube minute at 8 000xq Add 500 uL wash Discard flow through seeti a buffer and collection tube ventri uge tor minute at 8 000xg a Pa Discard flow through retain collection tube Centrifuge for 1 minute at a Add new sterile 1 5 3 3 mL tube and 65 uL Discard collection Centrifuge for 1 Elution Buffer 70 C minute at 8 000xg Purified DNA 030 169 Version 1 1 05MAR12 English 9 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use 1 2 1 3 1 4 1 5 1 6 1 7 Real Time PCR Set Up To begin switch on the Real Time PCR System instrument and associated computer and launch the relevant software Enter usernames and passwords as required Ensure the work area has been cleaned using DNA decontaminating reagents and allowed to d
4. Laboratory Press Cold Spring Harbour NY USA English 7 030 169 Version 1 1 O5MAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use All reagent tubes must be capped following use and prior to disposal Take care to identify the SmartCycler reaction tubes appropriately when multiple patient samples are being processed Take care when selecting the protocol run file Select SERUM MycAssay Asp v1 Do NOT select MycAssay Aspergillus Dx1 7b v1 3 or MycAssay Aspergillus v1_3 Procedure for Use The procedure has 2 stages DNA extraction from serum followed by Real Time PCR quantification DNA extraction is achieved using the Roche High Pure PCR Template Preparation Kit The High Pure PCR Template Preparation Kit is designed to purify nucleic acids from a variety of sample types The extraction protocol detailed in this IFU has been optimised to isolate Aspergillus spp DNA from serum and is suitable for use with the MycAssay Aspergillus Serum kit IMPORTANT NOTE The manufacturer s instructions have been modified to improve the yield of DNA recovered from a serum sample and to improve the sensitivity of the test Certain reagents detailed in steps 1 and 2 of Section 2 3 in the High Pure PCR Template Preparation Kit IFU will be depleted before others and will need to be replaced During the validation process Proteinase K from Sigma Aldrich was used 030 169 Version 1 1 OS5MAR12 English 8
5. privileges are required in the software to Retrieve Run s or Import an assay These can only be assigned by the Administrator of the instrument 2 1 SmartCycler Dx Diagnostic software version 1 7b 2 1 1 Open up the SmartCycler Dx Diagnostic software version 1 7b and enter your username and password 2 1 2 Insert the MycAssay Aspergillus Myconostica Protocol CD ROM and click on the Define Assays tab 2 1 3 Got to Retrieve Run s via the Tools directory on the top menu bar and click Proceed 030 169 Version 1 1 O5MAR12 English 12 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler 2 1 8 2 1 9 2 1 10 2 1 11 Smart Cycler Dx User Logs Setup Tools Help Data Management F archive Run s Installation Qualification Complete Backup Control Trending Complete Restore System Monitoring Report EEEE ee Compact Database Retrieve Run s G This will retrieve the selected run s into the main database Would you like to continue Proceed Select the file SERUM MycAssay Asp v1 DXA from the CD ROM WARNING This file will be one of two recognised by the software ensure you pick the correct file On the next screen highlight the filename SERUM MycAssay Asp v1 and click OK followed by Proceed and OK Close the software When it is reopened the SERUM MycAssay Asp v1 assay will be available for use when creating a new run Click on the Create Run tab En
6. Assay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use prompting you to select the run you wish to retrieve Select the run to be retrieved and click OK A message screen will appear stating how many runs are to be retrieved if this number is correct click Proceed 3 8 If a hardcopy of the results is also required click on Report and Print 4 Troubleshooting 4 1 The Negative Control has generated a positive signal in the FAM channel Contamination occurred during the set up Results from the entire run cannot be relied upon as accurate gt Repeat the entire run taking great care when adding the templates in particular the Positive Control Tube 4 to ensure that cross contamination does not occur gt Make sure that the work area and instruments are properly decontaminated before and after use The Negative Control was incorrectly positioned in the instrument gt Take care that the reaction tubes are placed in their designated sites 4 2 The Negative Control IAC Ct value is not within the acceptable range The PCR has been inhibited gt Ensure that the work area and instruments are thoroughly dry after the use of decontaminating agents prior to PCR set up The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired gt Please check correct storage conditions of the kit have been followed Check the expiry date of the reagents
7. For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler my conosjica MycAssay Aspergillus Cepheid SmartCycler Serum REF 080 045 Intended Use MycAssay Aspergillus is indicated for use by qualified laboratory professionals for the qualitative detection of Aspergillus spp genomic DNA extracted from serum as an aid to diagnosis of pulmonary aspergillosis MycAssay Aspergillus SERUM has been validated for use with the Cepheid SmartCycler using Dx software versions 1 7b and 3 0 Summary and Explanation Aspergillus spp are ubiquitous opportunistic moulds which cause both allergic and invasive syndromes The genus is comprised of approximately 300 species of which 41 have been associated with human disease The majority of diseases are caused by A fumigatus A flavus A terreus and A niger less commonly A nidulans and other rarer species such as A sydowii A versicolor A lentulus and A pseudofischeri have been implicated Most diseases caused by Aspergillus spp affect the respiratory tract Invasive aspergillosis IA occurs in at risk patient groups including those having treatment for leukemia and lymphoma hematopoetic stem cell HSCT and solid organ transplant patients as well as patients treated with corticosteroids and those with neutropenia or phagocyte dysfunction i e chronic granulomatous disease and HIV infection Species Database in www aspergillus org uk English 1
8. acterium diphtheriae Escherichia coli Haemophilus influenza Lactobacillus plantarum Legionella pneumophila Moraxella catarrhalis Mycoplasma pneumonia Neisseria meningitides Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pneumonia S pyogenes S salivarius The following were specifically tested for potential presence in serum and did not report out a positive result Acinetobacter baumannii Aeromonas hydrophilia Burkholderia cepacia Citrobacter koseri Enterobacter cloacae Enterococcus faecium Klebsiella pneumoniae Morganella morganii Proteus mirabilis Salmonela enterica Serratia marcescens Stenotrophmonas maltophila Human genomic DNA does not report a positive result with this assay Interfering Substances contraindications for use The following compounds were tested at clinically relevant concentrations and found not to inhibit the assay acteylcysteine amphotericin beclometasone dipropionate budesonide colistimethate sodium fluticasone propionate formoterol fumarate dehydrate ipratropium bromide lidocaine mannitol salbutamol sulphate salmerterol sodium chloride sodium cromoglicate terbutaline tobramycin The following were tested for potential presence in serum Clinically relevant concentrations were tested and were found not to inhibit the PCR reaction Amoxicillin with clavulanic acid atovaquone azathioprine azotreonam ceftazidime ciproflaxicin chlorphenamine maleate clindamycin
9. ame format as the Patient Results but for each individual target 3 5 If a Patient sample reports an Invalid result this is due to a failed IAC result indicated by Unresolved in the Sample Results tab repeat the reaction plus Positive and Negative controls If the reaction continues to fail an inhibiting substance may be present in the template and a Negative result cannot be relied upon 3 6 To export run data to allow transfer to another computer go to the Tools directory at the top of the screen and select Data Management followed by Archive Run s from the drop down menu A message screen will appear click Proceed Select the run to be archived by ticking the box to the left and click OK A warning message will appear stating how many runs are to be archived if this number is correct click Proceed Select a destination to save the run file e g USB data stick Click Save and make a note of the file name A message screen will appear stating how many runs are to be archived if this number is correct click Proceed 3 7 To import run data go to the Tools directory at the top of the screen and select Data Management followed by Retrieve Run s from the drop down menu A message screen will appear click Proceed Go to Look In and select the storage device used to archive the run data see section 3 4 above Select the run file to be retrieved and click Open Another screen will appear English 15 030 169 Version 1 1 O5MAR12 Myc
10. e at 22 2 U 0 5mL serum may have caused Aspergillus DNA degradation prior to extraction During validation batches of Proteinase K were obtained and used that were subsequently found to be contaminated at source with Aspergillus Source all materials carefully and use recommended sources wherever possible False positive results may arise from external contamination of the original sample or test Such contamination could arise from Aspergillus contaminated air poor experimental technique with respect to the positive control or external especially pipettor contamination with Aspergillus DNA As a true positive result may be obtained from patients who are transiently or persistently colonised by Aspergillus spp clinical judgment is required in interpretation of the test results in the context of disease 030 169 Version 1 1 O5MAR12 English 22 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler LICENSING TopTaq Hot Start is provided by QIAGEN QIAGEN is a registered trade mark of Qiagen GmbH Hilden Germany This product is sold under license from the Public Health Research Institute Newark New Jersey USA and may be used under PHRI patent rights only for human in vitro diagnostics SmartCycler is a registered Trademark of Cepheid 904 Caribbean Drive Sunnyvale CA 94089 USA High Pure is a registered Trademark of Roche Diagnostics GmbH 68298 Mannheim Germany Part of this prod
11. e which can take several days to produce positive results This assay offers advantages over currently available diagnostic methods for acute invasive and chronic pulmonary aspergillosis These advantages include faster detection of Aspergillus spp and the potential for increased sensitivity for Aspergillus spp in highly immunocompromised patients suspected of having invasive pulmonary aspergillosis Principles of the Procedure Following mixing of the reagents in the MycAssay Aspergillus kit with a sample containing the Aspergillus target DNA sequence a section of the Aspergillus ribosomal 18S gene thermocycling will result in DNA amplification occurring The assay also contains an Internal Amplification Control IAC a DNA fragment not present in Aspergilli other fungal bacterial or human genomes to detect PCR inhibitory substances and confirm the functionality of the assay reagents The amplified DNA targets are detected using Molecular Beacon technology Molecular Beacons are single stranded oligonucleotide hybridisation probes that form a stem and loop structure The loop contains a probe sequence that is complementary to a target sequence and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence A fluorophore which fluoresces when excited by light of the appropriate wavelength is covalently linked to the end of one arm and a quencher which suppresses the fluoresc
12. e will be listed SmartCycler Dx Diagnostic software version 3 0 Open up the SmartCycler Dx Diagnostic software version 3 0 and enter your username and password Insert the MycAssay Aspergillus Myconostica Protocol CD ROM and click on the Define Assays tab and Import the SERUM MycAssay Asp v1 sca file from the CD ROM WARNING This file will be one of two recognised by the software ensure you pick the correct file Click on the Create Run tab Enter an appropriate Run Name it is recommended that this includes the date and operators initials as a minimum or leave blank if you wish the name to be created automatically by the software Select SERUM MycAssay Asp v1 as the assay Enter the Lot Number and Expiration Date of the kit as printed on the kit box and each pouch The lot number will be in the form of M XXXXXXXX Enter the Patient Sample ID and the Number of specimens replicates in the appropriate boxes and click Apply Do this for all patient samples being tested The software will automatically include a Negative and Positive control in the Real Time PCR run Carefully place the reaction tubes into the designated sites in the SmartCycler block and click Start Run N B Take care when placing the reaction tubes into the designated sites as they may not be in the same order as your set up Make a note of the run name and click OK The run will now start and red lights will appear above each site in use on the block To dete
13. ence of the fluorophore when in close physical proximity is covalently linked to the end of the other arm Molecular beacons do not fluoresce when they are free in solution However when they hybridise to a nucleic acid strand containing a target sequence they undergo a conformational change that physically separates the fluorophore and the quencher enabling them to fluoresce upon excitation The amount of fluorescence at any given cycle or following cycling depends on the amount of specific amplicons present at that time The Real Time PCR System simultaneously monitors the fluorescence emitted by each beacon English 3 030 169 Version 1 1 O5MAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use Precautions The kit is intended for use only by laboratory professionals Procedures are required for non aerosol manipulations of specimens Standard precautions and institutional guidelines should be followed in handling all samples A Material Safety Data Sheet is available from Myconostica Ltd This test is for in vitro diagnostic use only In analytical validation studies levels of transaminase of 22 2 U per 0 5 mL serum were shown to have a possible degradation effect on Aspergillus DNA This assay has been evaluated with serum collected in Greiner Red Top serum collection tubes Other serum blood collection tubes may contain inhibiting or competing substances that have not been tested This assay has been va
14. gillus Cepheid SmartCycler Do not eat drink or smoke in areas where specimens or kit reagents are being handled Serum may be stored up to 48h in a refrigerator 2 8 C or freezer 15 to 25 C Low concentrations of DNA can be unstable if not stored correctly It is recommended that DNA extractions from clinical samples are stored at 80 C to preserve their integrity Multiple rounds of thawing and refreezing should also be avoided whenever possible Kit Contents Description The kit consists of five 3 compartment sealed foil pouches each of which can be removed from the box and used separately Each pouch contains sufficient reagents for 8 reactions Volume Tube 1 dNTPs 66 uL Orange Cap MgCl Buffered solution of DNA Polymerase complex Tube 2 lt 0 01 Primers 66 uL Green Cap lt 0 01 Molecular Beacons lt 0 0001 Internal Amplification Control IAC The Internal Amplification Control is a recombinant DNA plasmid containing a non infective sequence unrelated to target Aspergillus sequence Tris HCl Buffer Tube 3 Negative Control 25 uL Clear Cap Water Tube 4 Positive Control 25 uL Black Cap lt 0 0001 Positive Control DNA The Positive Control molecule is a recombinant plasmid containing the Aspergillus target sequence Tris HCl Buffer English 5 030 169 Version 1 1 O5MAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use The kit also contains MycAssay Asper
15. gillus Myconostica Protocol CD ROM Instructions for Use Certificate of Analysis Storage The kit should be stored frozen 15 to 25 C until the expiry date indicated on the kit box label when it should be disposed of according to local regulations Once a pouch has been opened the contents must be used immediately not re frozen or re used at a later date Equipment Materials required but not provided A Equipment required SmartCycler Real Time PCR System including user manual attached computer and SmartCycler Dx software versions 3 0 or 1 7b SmartCycler reaction tubes Mini centrifuge adapted for SmartCycler reaction tubes Plastic support rack for SmartCycler reaction tubes B Common equipment materials required Micro centrifuge Vortex mixer Micropipettes volumes required 7 5 uL 20 uL Sterile low retention filtertips Disposable gloves powderless Proprietary DNA decontaminating solution Permanent marker pen DNA isolation kit see below 030 169 Version 1 1 O5MAR12 English 6 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler Specimen The specimen for the MycAssay Aspergillus assay is total genomic DNA extracted from serum samples The following DNA extraction kit and equipment used during validation is recommended for this purpose High Pure manual DNA extraction kit Roche Diagnostics Cat No 11 796 828 001 Proteinase K solutio
16. ile If you have further questions or you experience any problems please contact Technical Support productsupport lab21 com 030 169 Version 1 1 OS5MAR12 English 18 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler Performance Characteristics and Limitations Analytical Sensitivity Using the protocol described above and PCR templates generated at Myconostica the Limit of Blank LoB for the MycAssay Aspergillus was determined to be a Ct of 39 0 while the Limit of Detection LoD starting from a serum sample was determined to be lt 5 genome equivalents This was determined using genomic DNA from the AF293 strain of Aspergillus fumigates spiked into serum of non infected individuals The genome of the AF293 strain has been fully sequenced and it is known there are 37 copies of the target within the genome determined by optical mapping Analytical Specificity This was initially determined during the validation studies for use with respiratory samples and was not repeated Analytical specificity was tested using DNA extracted from 15 different Aspergilli species including several strains each of A fumigatus A niger A terreus and A nidulans Signals detected above the LoB were recorded as a positive result All of the 15 Aspergillus spp tested were positive with the assay In addition to those previously mentioned this includes A flavus A versicolor A glaucus A sclerotiorum A
17. in their designated sites 4 4 Patient sample s give Outcome 3 Invalid gt It is likely that the extracted clinical sample s contain PCR inhibitors Follow the Roche High Pure manual DNA modified extraction procedure and not manufacturer s instructions for optimal DNA extraction Some blood collection tubes for serum may contain PCR inhibitors that have not been tested 4 5 There are no results for any channel with any samples or controls gt The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired Please check correct storage conditions of the kit have been followed Check the expiry date of the reagents see the kit box pouch label and repeat with an unexpired kit if necessary English 17 030 169 Version 1 1 OSMAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use The equipment used is not functioning optimally gt Please check that your Real Time PCR instrument has an up to date service history and has been fully calibrated as described in its Installation and Maintenance Guide An incorrect protocol file was used during the software set up gt Please refer to Section 2 and choose the correct Protocol file as specified for each software type version from the Myconostica Protocol CD ROM Only the file appropriate to the software can be loaded Repeat the run using the correct protocol f
18. lidated for serum specimens Validation data are not available for plasma or whole blood This test is only for use with the Cepheid SmartCycler system with Dx diagnostic software versions 1 7b and 3 0 Do not use reagents or controls if the protective pouches are open or broken when received Reagents and controls are not interchangeable between kits with different lot numbers Never pool reagents or controls from different tubes even if they are from the same lot Never use the reagents or controls after their expiry date Ensure all reagents used are free from fungal contamination Reagents and controls should not be re frozen or re used after opening Wear protective clothing and disposable gloves while handling kit reagents Avoid microbial and deoxyribonuclease DNAse contamination of reagents when removing aliquots from tubes The use of sterile DNAse free low retention disposable filter tips or positive displacement pipette tips is recommended Use a new tip for each specimen or reagent Dispose of unused reagents and waste in accordance with country federal state and local regulations To avoid contamination with Aspergillus or IAC amplicons do not open the reaction tubes after amplification Additional controls may be tested according to guidelines or regulations of local state provincial federal or accrediting organisations 030 169 Version 1 1 O5MAR12 English 4 For in vitro Diagnostic Use MycAssay Asper
19. mmediately discard it and any remaining contents into a sealable clinical waste container Unused reagents cannot be saved for later use Take extra care when pipetting Tube 4 positive control DNA to ensure it does not contaminate any other reaction tube Closing the lids on the other reaction tubes before opening Tube 4 can reduce the risk of cross contamination English 11 030 169 Version 1 1 OSMAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use 1 14 Make sure all reaction tube lids are firmly closed and then label each lid using a permanent marker pen e g POS for positive control NEG for negative control and patient ID for patient samples Spin down the reaction tubes for 10 seconds using the specially adapted mini centrifuge Visually check that there are no bubbles present in the reaction mixtures 1 15 Proceed to Section 2 promptly MycAssay Aspergillus reactions are stable on the bench for up to 60 minutes 1 16 Following the PCR set up ensure the work area is thoroughly cleaned using DNA decontaminating reagents 2 Performing the run Before proceeding with the following section please check which version of the Dx software you have installed on your computer Open the software choose Help from the toolbar and click About For version 1 7b follow the instructions below in Section 2 1 For version 3 0 follow the instructions below in Section 2 2 Please also be aware that certain user
20. n Sigma Aldrich Chemicals Cat No P4850 5ML 2 Propanol Sigma Aldrich Chemicals Cat No 19516 25ML Vortex Genie 2 Scientific Industries Inc New York USA Vortex Adaptor Plate REF 080 015 available from Myconostica Procedural Notes Read the entire protocol before commencing The entire MycAssay Aspergillus process including DNA extraction takes approximately 212 hours dependent on the number of samples tested Setting up of the test should be performed in a PCR workstation or pre PCR laboratory If a PCR workstation is not available then the test should be set up in a dedicated area of the laboratory separated from areas used for DNA extractions that is regularly cleaned with DNA decontaminating reagents Avoid using DNA decontaminating reagents when performing the Real Time PCR set up as they can inhibit the assay Use micropipettes for the transfer of fluids Dedicated micropipettes should be used for the set up of these reactions and they should be regularly decontaminated Low retention filtertips are recommended for use to ensure that no DNA is lost during the set up procedure Exercise caution when handling Tube 4 This contains positive control DNA material and contamination could cause false positive test results Wear gloves at all times T For example see Mifflin T E 2003 Setting up a PCR Laboratory In PCR Primer 2nd Ed eds Dieffenbach and Dveksler Cold Spring Harbour
21. niveus A lentulus A unguis A candidus A wentii A tubingensis and A foetidus Genomic DNA extracted from Penicillium spp also generated positive results This is due to the fact that the sequences of the molecular targets are highly conserved between Aspergillus and Penicillium Therefore it must be noted that a positive result with this assay may be the result of infection by Penicillium rather than Aspergillus 8 Nierman WC Pain A Anderson Mu et al 2005 Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigates Nature 438 1151 6 English 19 030 169 Version 1 1 05MAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use Analytical Selectivity Analytical selectivity was tested using DNA extracted from a variety of different fungal and non fungal species The following species were tested during the initial validation for respiratory samples and did not report out a positive result Alternaria alternata Blastomyces capitatus Candida albicans C glabrata C parapsilosis C tropicalis Cladosporium spp Cryptococcus neoformans Doratomyces microsporus Fusarium solani Histoplasma capsulatum Pneumocystis jirovecii Rhizomucor pusillus Rhodotonila rubra Saccharomyces cerevisiae Scedosporium apiosperinu S prolificans Sporothrix schenkii Trichosporon capitatu The following bacterial species did not report a positive result Bordetella pertussi Coryneb
22. phosphate co trimoxazole creatinine dapsone dexamethasone sodium phosphate fluconazole meropenam 030 169 Version 1 1 05MAR12 English 20 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler metoclopramide hydrochloride paracetamol primaquine phosphate prednisone sodium phosphate prednisone prochlorperazine vancomycin and voriconazole The following were found to inhibit PCR reactions cefuroxime heparin methylpredisolone sodium succinate transaminase and urea When these inhibiting substances were added at clinically relevant levels to serum containing Aspergillus DNA and extracted with the modified High Pure DNA extraction kit no inhibition was observed However transaminase appeared to degrade the Aspergillus DNA prior to extraction as 25 of the replicates were negative for Aspergillus Clinical Reporting NOTE When inspecting the Results Report ensure that the correct protocol has been used For serum samples SERUM MycAssay Asp v1 must be used Use of the wrong protocol will give invalid results The MycAssay Aspergillus kit is intended as an aid to diagnosis The results need to be taken in context of the clinical condition of the patient and other diagnostic test results The following are recommended reports each depending on the assay result interpretation Outcome No 1 Aspergillus spp not detected Outcome No 2 Aspergillus spp detected Positive result This assay al
23. re completely thawed 030 169 Version 1 1 O5MAR12 English 10 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler 1 8 1 9 1 13 before proceeding Vortex mix the tubes contents and the patient samples follow by a short spin in a microcentrifuge to ensure collection of all the contents at the base of the tubes before use Place the required number of SmartCycler reaction tubes in their support rack s Take care to only touch the neck of the reaction tubes with your hands Always set up the negative control first followed by the patient samples The positive control should always be set up last Reagent and DNA volumes are shown in the table below Reaction Reagent Negative Patient Positive control sample control Tube 1 Orange cap 7 5 uL 7 5 uL 7 5 uL Tube 2 Green cap 7 5 uL 7 5 uL Tube 3 Clear cap 10 uL Patient Sample 10 uL Tube 4 Black cap Total volume 25 uL Add reagents in the order shown in the table above Tube 1 then Tube 2 followed by the template Negative control Patient sample or Positive control Take care when taking aliquots from Tube 1 the liquid is slightly viscous and can stick on the inner ridge of the tube If this happens re spin to collect the final contents in the base of the tube before attempting to remove the final aliquots Use a new pipette tip for every liquid transfer Re cap each reagent tube after use and i
24. rmine how long the run will take to complete click on the Check Status tab The run name and subsequent run time will be listed Data Analysis and Interpretation The results can be viewed in Dx software by selecting the View Results tab Click on the View Another Run button at the bottom of the page select the run you wish to view then click OK 030 169 Version 1 1 O5MAR12 English 14 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler 3 3 The Patient Results should already be selected in the Views list The patient sample ID and the subsequent assay result will be clearly listed The results can be interpreted using the table below Out Patient Colour Interpretation Further Action come Result 1 Negative Green Negative for Aspergillus Report result 2 Positive Red Positive for Aspergillus Report result 3 Invalid Light Grey IAC failure in sample Repeat sample 4 Invalid Light Grey Failure in Positive or Repeat entire run Negative Control If the result is reported as ND in light grey this corresponds to error code 3079 the result of high fluorescence in one or more channels If a Ct value of lt 39 0 is recorded in the Aspergillus channel report as positive 3 4 To view individual sample results for either Aspergillus or IAC separately select Sample Results from the Views list and click on the individual tabs for each target The results will be displayed in the s
25. rows In serum the test has a sensitivity in adult patients of 79 3 95 C I 61 6 90 2 and a specificity in adult patients of 88 8 95 C I 82 6 93 0 in patients not receiving prophylaxis with azoles active against moulds Two consecutive positive GM tests are recommended to improve diagnostic accuracy A meta analysis by Mengoli et al reported on gt 10 000 blood serum and plasma samples from 1618 patients at risk for IA They calculated the sensitivity and specificity of a single PCR positive blood sample to be 0 88 95 C I 0 75 0 94 and 0 75 95 C I 0 63 0 84 respectively and the diagnostic odds ratio for proven and probable cases to be 16 41 95 C I 6 43 41 88 MycAssay Aspergillus is a molecular diagnostic kit for the detection of Aspergillus spp genomic DNA using Molecular Beacon Real Time PCR technology The whole test procedure including extraction of DNA from the clinical sample can be completed a Kontoyiannis DP et al Clin Infect Dis 2010 50 8 1091 1100 Ascioglu S et al Clin Infect Dis 2002 34 7 14 4 BioRad Platelia Aspergillus EIA product information gt Mengoli C et al The Lancet ID 2009 9 86 96 Tyagi S Kramer FR 1996 Molecular beacons Probes that fluoresce upon hybridization Nature Biotechnology 14 303 308 030 169 Version 1 1 O5MAR12 English 2 For in vitro Diagnostic Use MycAssay Aspergillus Cepheid SmartCycler within 4 hours compared to fungal cultur
26. ry completely avoid use during assay set up as excess cleaning solution may inhibit the PCR reactions A pouch contains one each of Tube 1 Tube 2 Tube 3 and Tube 4 There are sufficient reagents in one pouch to run 8 reactions At least one positive control and one negative control reaction must be performed per run where the reagents are from a single kit lot One pouch therefore can analyse 6 patient samples If more than 6 samples need to be tested more than one pouch can be used if the pouches used are from the same kit lot A maximum of 38 patient samples may be tested using the 5 pouches in a kit Calculate the number of reactions required referring to the table below Number of Pouches Maximum number of patient samples 6 1 2 3 4 5 Remove the appropriate number of pouches from the freezer Do not use any pouch that is no longer sealed If the patient samples were frozen after extraction also remove these from the freezer Tear open the required number of pouches and remove the tubes If more than one pouch is being used but only one set of positive and negative controls are being run it is only necessary to remove Tubes 3 and 4 from one pouch Exercise caution when handling Tube 4 This contains positive control DNA material and contamination could cause false positive test results Allow the tubes contents to thaw by placing on the laboratory bench for 5 10 minutes ensuring that the contents of each tube a
27. so detects Penicillium spp Outcome No 3 Test failed inhibitors or other unknown substance present English 21 030 169 Version 1 1 05MAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use Limitations of Procedure The principal limitation of this procedure relates to the quality of the primary sample If the levels of Aspergillus DNA in the blood are low extraction efficiency may impact the result and the test may give a false negative outcome Preliminary data indicate that freezing and storage of serum samples may affect the quantity of viable DNA available for assaying No data are available on the stability of Aspergillus DNA in unprocessed whole blood or serum It is recommended therefore that samples are processed as quickly as possible after collection No data are available on the performance of serum collected in blood collection tubes other than the recommended Greiner Red Top serum collection tubes No data are available on the performance characteristics of the assay starting with Aspergillus DNA extracted from plasma or whole blood False positive results are possible if the infecting agent is Penicillium spp which cannot be differentiated from Aspergillus spp using this kit While the High Pure DNA extraction procedure may remove PCR inhibitors not all drugs or patient populations have been evaluated During analytical validation studies it was noted that transaminas
28. ter an appropriate Run Name it is recommended that this includes the date and operators initials as a minimum or leave blank if you wish the name to be created automatically by the software Select SERUM MycAssay Asp v1 as the assay Enter the Lot Number and Expiration Date of the kit as printed on the kit box and on each pouch The lot number will be in the form of M XXXXXXXX Enter the Number of specimens in the box and click Apply The Sample ID for each specimen will automatically be named SPEC by the software Therefore rename each site appropriately for identification purposes i e double click on SPEC to highlight it and then type in the sample ID The software will automatically include a Negative and Positive control in the Real Time PCR run Carefully place the reaction tubes into the designated sites in the SmartCycler block and click Start Run N B Take care when placing the reaction tubes into the designated sites as they may not be in the same order as your set up Make a note of the run name and click OK The run will now start and red lights will appear above each site in use on the block English 13 030 169 Version 1 1 O5MAR12 MycAssay Aspergillus Cepheid SmartCycler For in vitro Diagnostic Use 2 1 12 2 2 2 2 1 2 2 2 2 2 3 2 2 4 2 2 5 2 2 6 2 2 7 2 2 8 3 1 3 2 To determine how long the run will take to complete click on the Check Status tab The run name and subsequent run tim
29. uct is covered by an exclusive license to a patent application held by the Fred Hutchinson Cancer Centre Seattle USA Lab21 184 Cambridge Science Park il Cambridge CB4 0GA United Kingdom Telephone 44 0 1638 552 882 Facsimile 44 0 1638 552 375 Email productsupport lab21 com CE English 23 030 169 Version 1 1 05MAR12
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