MycAssay™ Aspergillus
Contents
1. Sample ASP IAC Interpretation Further MycAssay Ct MycAssay Ct Action Negative 37 0 or Within 27 7 31 9 Negative Control Patient results Control Undetected acceptable are valid Negative 37 0 or lt 27 7 or gt 31 9 Failure in Repeat entire Control Undetected Negative Control run Negative lt 37 0 Within 27 7 31 9 Contamination Repeat entire Control run Positive Within 15 0 N A Positive Control Patient results Control 20 0 acceptable are valid Positive lt 15 0 or gt 20 0 N A Failure in Repeat entire Control Positive Control run Patient gt 33 5 Within 27 7 31 9 Negative for Report result Aspergillus at Outcome 1 CCO Patient 33 5 N A Positive for Report result Aspergillus at Outcome 2 CCO Patient 37 0 or lt 27 7 or gt 31 9 IAC failure in Repeat Undetected sample sample Outcome 3 CCO clinical cut off All results at or below this level are considered clinically positive Other samples may report Ct values gt 33 5 but these are reflective of normal or background levels of Aspergillus load in the respiratory sample See Clinical Reporting Outcome 1 2 or 3 English 9 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 4 Troubleshooting 4 1 The Negative Control has generated a positive signal in the FAM channel gt Contamination occurred during the set up Results from the entire run cannot be relied2. Aspergillus spp not detected Outcome No 2 Aspergillus spp detected Positive result This assay also detects Penicillium spp Outcome No 3 Test failed inhibitors or other unknown substance present English 13 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 Limitations of Procedure The principal limitation of this procedure relates to the quality of the primary sample Ifthe sample is very small or not collected from the affected area of lung the test will be less sensitive and may be falsely negative BAL samples should be centrifuged prior to DNA extraction from the pellet Data also demonstrated that a reduction in the volume of supernatant used in the extraction process achieved by the centrifugation step decreases the proportion of inhibitors entering the system False positive results are possible if the infecting agent is Penicillium spp which cannot be differentiated from Aspergillus spp using this kit While the MycxXtra Fungal DNA extraction procedure is designed to remove PCR inhibitors not all drugs or patient populations have been evaluated The procedure has not been fully assessed with sputa nor has it been assessed with induced saline samples or on samples from children False positive results may arise from external contamination of the original sample or test Such contamination could arise
3. Proceed to Section 2 promptly MycAssay Aspergillus reactions are stable on the bench for up to 60 minutes Following the PCR set up ensure the work area is thoroughly cleaned using DNA decontaminating reagents Performing the run Open up the AB 7500 SDS software version 1 4 and enter your username and password Insert the MycAssay Aspergillus Myconostica Protocol CD ROM In the Quick Startup menu select the first option Create New Document Choose the settings as shown below Select the template MycAssay Aspergillus v1_2 sdt from the CD ROM via Browse Give the run an appropriate Plate Name An example is shown below New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Standard Curve Absolute Quantitation Container 96 Well Clear Z Browse Template MycAssay Aspergillus v1_2 sdt Run Mode Operator your namel Comments Plate Name Aspergillus_O4JAN10_name Finish Cancel English 6 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 2 6 Click Finish A new document will open containing the PCR parameters and detectors automatically set for this assay In the Plate view of the Setup tab use Well Inspector select a well and press Ctrl 1 or right click with the mouse to name the wel
4. 11 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 Performance Characteristics and Limitations AB7500 Analytical Performance Data The kit was initially validated using the Cepheid SmartCycler Certain of the assay performance claims were re validated on the AB7500 platform using 0 2 mL 96 well semi skirted PCR plates with optical adhesive film Star Lab Cat E1403 8200 and Applied Biosystems Cat 4311971 and are reported below Where the differences between platforms were not expected to affect the performance of the assay and therefore the claim the study was not repeated These results obtained using the SmartCycler are considered transferable to the AB7500 platform Analytical Sensitivity Using the AB7500 protocol described above and PCR templates generated at Myconostica the LoB for the MycAssay Aspergillus was determined to be a Ct of 37 0 while the LoD was determined to be lt 25 copies of target DNA using the AF293 strain of A fumigatus Analytical Selectivity Genomic DNA extracted from Penicillium spp generates 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 Analytical selectivity was tested using D
5. The results were analysed against the Limit of Blank LoB and the clinical cut off CCO At a concentration of 3 times the Limit of Detection LoD the results from 100 of all samples tested were in agreement positive for LoB and 83 of the samples were in agreement at the CCO At concentrations higher than 3 x LoD results from 100 of all samples were in agreement at both the CCO and LoB For negative templates all samples tested were negative at the CCO Transfer of the Clinical Cut Off The clinical cut off of 36 0 had been established on the SmartCycler This cut off was transferred analytically to the AB7500 platform using a template with an Aspergillus concentration that had been shown to yield 295 positive results at the CCO on the SmartCycler A Ct value of 33 5 was determined which was then confirmed using templates of 3 different concentrations The following Performance Claims were established using the Cepheid SmartCycler 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 English 12 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use Respiratory Samples chloride sodium cromoglicate terbutaline Tobramycin Performance Evaluation Respiratory samples BAL that had been c
6. Clin Microbiol Infect 4 710 716 Perfect JR Cox GM Lee JY Kauffman CA de Repentigny L Chapman SW Morrison VA Pappas P Hiemenz JW Stevens DA and the Mycoses Study Group 2001 The impact of culture isolation of Aspergillus species A hospital based survey of Aspergillosis Clinical Infectious Diseases 33 1824 33 Maertens J Van Eldere J Verhaegen J Verbeken E Verschakelen J Boogaerts M 2002 Use of Circulating Galactomannan Screening for Early Diagnosis of Invasive Aspergillosis in Allogeneic Stem Cell Transplant Recipients The Journal of Infectious Diseases 186 1297 306 8 Tillie Leblond Tonnel AB 2005 Allergic bronchopulmonary aspergillosis Allergy 60 1004 13 Greene R 2005 The radiological spectrum of pulmonary aspergillosis Med Mycol 43 Suppl 1 8147 54 10 Denning DW Riniotis K Dobrashian R Sambatakou H 2003 Chronic cavitary and fibrosing pulmonary and pleural aspergillosis Case series proposed nomenclature and review Clin Infect Dis 37 Suppl 3 S265 80 Camuset J Lavol A Wislez M Khalil A Bellocq A Bazelly B Mayaud C Cadranel J 2007 Bronchopulmonary aspergillosis infections in the non immunocompromised patient Rev Pneumol Clin 63 155 166 English 1 030 150 Version 1 1 O9NOV10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 the presence of Aspergillus spp in respiratory samples is important to prevent delay in the re
7. fibrosing aspergillosis Invasive aspergillosis IA occurs in at risk patient groups including those treated with corticosteroids and those with neutropenia or phagocyte dysfunction i e chronic granulomatous disease and HIV infection About 80 of cases of invasive aspergillosis involve the lungs Routine diagnosis of invasive pulmonary aspergillosis includes Computed Tomography CT scanning bronchoscopy and bronchoalveolar lavage microscopy and culture Aspergillus antigen testing of blood or histological examination of surgical biopsy specimens In this scenario cultures are frequently falsely negative Indeed bronchoalveolar lavage is only positive by culture in approximately 40 of cases even when the diagnosis is proven by other means gt showing the lack of sensitivity of culture in detecting invasive aspergillosis and chronic pulmonary aspergillosis Cultures are however important if positive because many diagnostic tests do not indicate either the genus or species of fungus causing the disease or the susceptibility profile of the isolate causing infection Allergic bronchopulmonary aspergillosis complicates asthma and cystic fibrosis and may rarely present with no underlying disease Most cases are associated with A fumigatus with other fungi occasionally implicated Episodic airway obstruction with thick sputum plugs full of Aspergillus hyphae serum total IgE gt 1 000 IU mL and detectable A fumigatus specific IgE and
8. 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 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 Part of this product is covered by an exclusive license to a patent application held by the Fred Hutchinson Cancer Centre Seattle USA Applied BioSystems is a registered trademark of Applera Corporation or its subsidiaries in the US and or certain other countries Myconostica Limited South Court Sharston Road m Sharston Manchester M22 4SN United Kingdom Telephone 44 0 161 998 7239 Facsimile 44 0 161 902 2496 Email mycotech myconostica co uk English 14 030 150 Version 1 1 O9NOV 10
9. For in vitro Diagnostic Use MycAssay Aspergillus Applied Biosystems 7500 myconos Ica Respiratory Samples MycAssay Aspergillus Applied Biosystems 7500 Respiratory Samples 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 respiratory specimens from the lower respiratory tract e g bronchial samples as an aid to diagnosis in adult patients suspected of having Aspergillus infection or allergy MycAssay Aspergillus has been validated for use with the Applied Biosystems 7500 using SDS software version 1 4 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 infections 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 infections and allergies caused by Aspergillus spp affect the respiratory tract Allergic syndromes include allergic bronchopulmonary aspergillosis ABPA and allergic Aspergillus sinusitis and are usually caused by A fumigatus Chronic pulmonary aspergillosis includes aspergilloma chronic cavitary pulmonary aspergillosis and chronic
10. IgG antibodies or a positive Aspergillus skin prick test are the characteristics of the disease Sputum cultures may be positive for A fumigatus and bronchiectasis may be seen on a CT scan of the chest Current methods of diagnosing chronic pulmonary aspergillosis are a mixture of radiology which is not specific and serology Aspergillus gG antibodies or precipitins which is positive in most forms of aspergillosis and thus not specific for any particular manifestation of aspergillosis Cultures are positive in only 40 65 of cases proven by radiology and serology As the differential diagnosis is wide including tuberculosis atypical mycobacteriosis sarcoidosis histoplasmosis coccidioidomycosis pneumoconiosis rheumatoid lung ankylosing spondylitis and others documenting 3 Species Database in www aspergillus org uk 2 Hope WW Walsh TJ Denning DW 2005 The invasive and saprophytic syndromes due to Aspergillus spp Medical Mycology 43 suppl 1 8207 38 Hope WW Walsh TJ Denning DW 2005 Laboratory diagnosis of invasive aspergillosis Lancet Infectious Diseases 9 609 22 4 Levy H Horak DA Tegtmeier BR Yokota SB Forman SJ 1992 The value of bronchoalveolar lavage and bronchial washings in the diagnosis of invasive pulmonary aspergillosis Respir Med 86 243 8 5 Greub G and Bille J 1998 Aspergillus species isolated from clinical specimens suggested clinical and microbiological criteria to deteremine significance
11. NA extracted from a variety of different fungal and non fungal species The following species did not report out a positive result Blastomyces capitatus Candida albicans C glabrata C parapsilosis C tropicalis Cladosporium spp Cryptococcus neoformans Doratomyces microsporus Fusarium solani 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 Corynebacterium 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 Human genomic DNA does not report a positive result with this assay The following 3 fungal species were not tested on the AB7500 system but had been tested on the SmartCycler Pneumocystis jirovecii Histoplasma capsulatum and Alternaria alternata All 3 gave negative results with the assay Reproducibility and Repeatability Repeatability and reproducibility were determined by 4 different operators testing a panel of 7 different templates in triplicate for a total of 168 assays Experiments were performed using 1 manufactured batch of MycAssay Aspergillus kit on 2 different instruments situated at 2 different locations in the UK
12. arget sequence Tris HCl Buffer The kit also contains MycAssay Aspergillus 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 English 3 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 Equipment Materials required but not provided A Equipment required Applied Biosystems Real Time PCR System including user manual attached computer and SDS software version 1 4 96 well skirted raised rim plates and optical grade sealing film Star Lab Cat E1403 8200 and Applied Biosystems Cat 4311971 Support rack for 96 well plate Centrifuge fitted with buckets which hold 96 well plates B Common equipment 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 Specimen The specimen for the MycAssay Aspergillus assay is total genomic DNA extracted from clinical BAL and other lower respiratory tract samples The following isolatio
13. bration Data Print Setup Print Preview Print CtrleP Spectra Component Delta Rn Ct Dissociation Results ee cic 3 6 To avoid confusion save the file with the same name as used for the run file itself Remember to save the file to an appropriate location 3 7 When prompted activate Export only selected wells and click OK 3 8 Open the saved csv file with Excel or similar spreadsheet software 3 9 Analyse each sample starting with the controls as shown in the flowchart below details can also be found in the table shown beneath the flowchart English 8 030 150 Version 1 1 O9NOV 10 MycAssay Aspergillus Applied Biosystems 7500 For in vitro Diagnostic Use Respiratory Samples Check the Negative Control Is the ASP MycAssay Ct 37 0 or Undetected Check the Negative Control Is the IAC MycAssay Ct 27 7 31 9 Run is contaminated ACTION Repeat the run Run failure ACTION Repeat the run Positive for Aspergillus DNA ACTION Report Outcome 2 Negative for Aspergillus DNA ACTION Report Outcome 1 Run failure ACTION Repeat the run Check the Positive Control Is the ASP MycAssay Ct 15 0 20 0 Check the Patient Sample Is the ASP MycAssay Ct 33 5 Check the Patient Sample Is the IAC MycAssay Ct 27 7 31 9 IAC failure ACTION Repeat sample If the same result again Report Outcome 3
14. ce 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 fluorescence 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 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 This test is only for use with the Applied Biosystems 7500 with SDS software version 1 4 Do not use reagents or controls if the protective pouches are op
15. cognition of pulmonary aspergillosis and mistreatment 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 within 4 hours compared to fungal culture 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 sequen
16. ded tubes or plates were used Thresholds are only valid when using the recommended 0 2ml 96 well semi skirted PCR plates with optical adhesive film Star Lab Cat E1403 8200 and Applied Biosystems Cat 43119771 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 well compared to others The Positive Control was incorrectly positioned in the instrument Take care that the wells are annotated correctly within the software Non recommended tubes or plates were used Thresholds are only valid when using the recommended 0 2ml 96 well semi skirted PCR plates with optical adhesive film Star Lab Cat E1403 8200 and Applied Biosystems Cat 4311971 4 4 Pat
17. ed PCR plates with optical adhesive film Star Lab Cat E1403 8200 and Applied Biosystems Cat 4311971 Use of other sources types of plastic could invalidate the thresholds and therefore the claims made in the IFU It is recommended that should an alternative source be used that local validation should be conducted with the positive and negative control reactions 1 Tyagi S Kramer FR 1996 Molecular beacons Probes that fluoresce upon hybridization Nature Biotechnology 14 303 308 English 2 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use Respiratory Samples MycAssay Aspergillus Applied Biosystems 7500 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 Tube 1 Orange Cap Tube 2 Green Cap Tube 3 Clear Cap Tube 4 Black Cap Volume dNTPs 66 uL MgCl Buffered solution of DNA Polymerase complex lt 0 01 Primers 66 uL 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 Negative Control 25 uL Water Positive Control 25 uL lt 0 0001 Positive Control DNA The Positive Control molecule is a recombinant plasmid containing the Aspergillus t
18. egative Patient Positive control sample control Tube 1 Orange 7 5 uL 7 5 uL 7 5 uL cap Tube 2 Green 7 5 uL 7 5 uL cap 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 immediately 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 Make a note of the positions of each sample in the plate on a plate plan Carefully and thoroughly seal the plate with the optical grade sealing film taking care to ensure the edges are firmly stuck down Spin down the plate in a centrifuge at 900 g for 1 minute Check that there are no bubbles present in the bottom of the wells If there are repeat spin
19. en 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 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 anew 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 Do not eat drink or smoke in areas where specimens or kit reagents are being handled 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 This kit was validated using 0 2ml 96 well semi skirt
20. ient sample s give Outcome 3 Invalid gt It is likely that the extracted clinical sample s contain PCR inhibitors We recommend that DNA from clinical samples is extracted using the MycXtra Fungal DNA Extraction kit 4 5 There are no results for any channel with any samples or controls English 10 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 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 see the kit box pouch label and repeat with an unexpired kit if necessary 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 file If you have further questions or you experience any problems please contact Technical Support mycotech myconostica co uk English
21. ls according to the positions of the samples in 1 12 For example 2 7 When all the wells are named appropriately save the run keeping the Plate Name as the file name 2 8 Start the run in the Instrument tab by clicking on the Start button 2 9 To determine how long the run will take to complete a countdown is shown next to the Start button English 7 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 3 Data Analysis and Interpretation 3 1 Once the run has finished click on the green arrow on the top menu bar to update 3 2 Open the Amplification Plot view of the Result tab On the right hand side set the thresholds for each channel as follows Asp MycAssay 12000 IAC MycAssay 4000 The Manual Baseline should remain at 3 15 for both detectors 3 3 Click the Analyze button to activate these changes For example Sta kycet E Stat eyelet 5 End leveled 15 End feyetet 15 Anayee Bryce Hep Heb 3 4 Save the changes 3 5 Select the wells containing samples and export the Report file File gt Export gt Results as shown below 4 7500 System SDS Software RandRPro_031209_ADS sds Standard Curve B GGF view Tools Instrument Analysis Window Help New Ctrl N Open Ctrl O Close Save As Import Sample Setup Ctrl I Export g Sample Setup View Exported Results z Cali
22. n kit and equipment is recommended for this purpose and was used during validation MycXtra Fungal DNA Extraction kit REF 080 005 available from Myconostica Vortex Genie 2 Scientific Industries Inc New York USA Vortex Adaptor Plate REF 080 015 available from Myconostica English 4 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 Procedural Notes Read the entire protocol before commencing The entire MycAssay Aspergillus process excluding DNA extraction takes approximately 2 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 However 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 posi
23. ollected from 2 hospitals extracted using the MycXtra kit and stored were used to evaluate the performance of the MycAssay Aspergillus kit with clinical samples Comparisons were made to both clinical diagnosis and culture The cut off value of a Ct of 36 0 was established following review of a dataset of samples sourced from different sites and different patient populations Different cut offs were evaluated for the probability of differentiating between disease state and non disease state PCR v Clinical Diagnosis MycAssay Aspergillus Applied Biosystems 7500 formoterol fumarate dehydrate ipratropium bromide lidocaine mannitol salbutamol sulphate salmerterol sodium Clinical Clinical positive negative PCR 31 1 0 97 PPV positive PCR 2 10 0 83 NPV negative 0 94 0 91 Sensitivity Specificity PCR v Aspergillus Culture Culture Culture positive negative PCR 29 3 0 91 PPV positive PCR 2 10 0 83 NPV negative 0 94 0 77 Sensitivity Specificity Of the samples tested 0 8 contained PCR inhibitors as reported by the IAC following extraction using the MycXtra kit Clinical Reporting 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
24. so 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 1 7 Allow the tubes contents to thaw by placing on the laboratory bench for 5 10 minutes ensuring that the contents of each tube are completely thawed 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 1 8 Place the 96 well plate in a support rack Take care to only touch the rim of the plate with your hands 1 9 Always set up the negative control first followed by the patient samples The positive control should always be set up last 13 For example see Mifflin T E 2003 Setting up a PCR Laboratory In PCR Primer 2nd Ed eds Dieffenbach and Dveksler Cold Spring Harbour Laboratory Press Cold Spring Harbour NY USA English 5 030 150 Version 1 1 O9NOV 10 For in vitro Diagnostic Use MycAssay Aspergillus Respiratory Samples Applied Biosystems 7500 1 10 1 15 1 16 2 1 2 2 2 3 2 4 2 5 Reagent and DNA volumes are shown in the table below Reaction Reagent N
25. tive test results Wear gloves at all times All reagent tubes must be capped following use and prior to disposal Take care to accurately note the positions of samples within the 96 well plate on a plate plan Procedure for Use 1 1 1 1 2 1 3 1 4 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 dry 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 Maximum number of patient Pouches samples 6 1 5 Remove the appropriate number of pouches from the freezer Do not use any pouch that is no longer sealed If 1 6 the patient samples were frozen after extraction al
26. upon as accurate 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 Make sure that the work area and instruments are properly decontaminated before and after use The Negative Control was incorrectly positioned in the instrument Take care that the wells are annotated correctly within the software Non recommended tubes or plates were used Thresholds are only valid when using the recommended 0 2ml 96 well semi skirted PCR plates with optical adhesive film Star Lab Cat E1403 8200 and Applied Biosystems Cat 4311971 4 2 The Negative Control IAC Ct value is not within the acceptable range gt The PCR has been inhibited 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 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 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 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 well compared to others Non recommen
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