Respiratory Samples
Contents
1. PCP relies on microscopic methods as P jirovecii cannot be cultured in routine microbiology laboratories Bronchoalveolar lavage BAL is the preferred means of sample collection Common methods for diagnosis include immunofluorescence IF or direct fluorescence and histological staining of samples MycAssay Pneumocystis is a molecular diagnostic kit for the detection of P jirovecii based on Molecular Beacon PCR technology The whole test procedure including extraction of DNA from the clinical sample can be completed within 4 hours or only 2 hours if extracted DNA is already available This assay brings the direct benefit of enhanced laboratory efficiency combined with a rapid test leading to likely clinical benefits The diagnostic accuracy of the test depends to a great extent on sample quality Principles of the Procedure Following mixing of the reagents in the MycAssay Pneumocystis kit with a sample containing Pneumocystis target DNA sequence a portion of the Pneumocystis mitochondrial ribosomal large sub unit thermocycling will result in DNA amplification occurring The assay also contains an Internal Amplification Control IAC sequence a DNA fragment not present in Pneumocystis 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 with Molecular Beacons single stranded oligonucleotide hybridization probes tha2. and are annotated correctly within the software 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 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 4 3 The Positive Control is negative 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 An error occurred during step 1 12 and the Positive Control template Tube 4 was placed in the wrong reaction tube gt Repeat the run taking great care during the set up stage Such errors can be detected by seeing a higher
3. drug treatment of Pneumocystis carinii pneumonia JAMA 286 2450 60 i Huang L Morris A Limper AH Beck JM ATS Pneumocystis Workshop Participants An Official ATS Workshop Summary Recent advances and future directions in pneumocystis pneumonia PCP Proc Am Thorac Soc 2006 3 655 64 Tyagi S Kramer FR 1996 Molecular beacons Probes that fluoresce upon hybridization Nature Biotechnology 14 303 308 English 1 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler 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 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 upon arrival Reagents and controls are not interchangeable between kits with differing 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 refrozen or reused after opening Wear protective clothing and disposable gloves whil
4. into the main database Would you like to continue Proceed 2 1 4 Select the file MycAssay Pneumocystis Dx1_7 v3_1 DXA from the CD ROM as shown below This file should be the only one recognised by the software an example is shown below English 5 Look in S MycAssayPne Protocol CD ROM E fat c B8 8 E MycAssay Pneumocystis Dx 1_7 v3_1 DXA File Name IMycAssay Pneumocystis Dx 1_7 v3_1 DXA Files of Type Smart Cycler Archive File dxa scr v Open Cancel 030 090 Version 3 2 09NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler 2 1 5 2 1 6 2 1 7 2 1 8 2 1 9 2 1 10 2 1 11 2 1 12 2 2 2 2 1 On the next screen highlight the filename Validation test 1 Myc Pne v3 1 and click OK followed by Proceed and OK Select Run s to Retrieve Status User one Name Date Validation test 1 Myc Pne v3 1 Unknown Jan 7 2010 02 09 PM Retrieve Run s A A total of 1 runis have been selected This process takes some time to finish Would you like to continue Cancel Proceed Close the software When it is reopened the MycAssay Pneumocystis Dx1 7b v3 1 assay will be available for use when creating a new run 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
5. DNA volumes are shown in the table below Reagent Negative Patient Positive control sample control 1 11 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 1 12 Use anew 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 English 4 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use Respiratory Samples MycAssay Pneumocystis Cepheid SmartCycler 1 13 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 1 14 Make sure all the SmartCycler 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 bu
6. For in vitro Diagnostic Use MycAssay Pneumocystis Cepheid SmartCycler mycor OS fi Ca Respiratory Samples MycAssay Pneumocystis Cepheid SmartCycler Respiratory Samples REF 080 035 Intended Use MycAssay Pneumocystis is indicated for use by qualified laboratory professionals for the qualitative detection of Pneumocystis jirovecii 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 P jirovecii pneumonia MycAssay Pneumocystis has been validated for use with the Cepheid SmartCycler using Dx software versions 1 7b and 3 0 Summary and Explanation Pneumocystis jirovecii formerly carinii pneumonia PCP is a common opportunistic pneumonia in immunocompromised patients especially those with advanced HIV infection and AIDS It is typically community acquired and sub acute in presentation leading to progressive respiratory failure and death if untreated Prophylaxis with trimethoprim sulphamethoxazole Bactrim or Septrin is routinely given to many at risk patients a practice which has substantially reduced the incidence of PCP but breakthrough occurs and those who do not know they are HIV positive may present with AIDS with PCP PCP also occurs in other immunocompromised patients including recipients of solid organ transplants hypogammaglobulinaemia and chronic leukaemia Currently the diagnosis of
7. bbles present in the reaction mixtures 1 15 Proceed to Section 2 promptly MycAssay Pneumocystis 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 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 Pneumocystis 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 User Logs Setup Tools Help Data Management gt Archive Run s Retrieve Run s Installation Qualification Complete Backup Control Trending Complete Restore System Monitoring Report ompact Database ra l Retrieve Run s fe This will retrieve the selected run s
8. blank if you wish the name to be created automatically by the software select MycAssay Pneumocystis Dx1 7 v3 1 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 To determine how long the run will take to complete click on the Check Status tab The run name and subsequent run time 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 English 6 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler 2 2 2 Insert the MycAssa
9. chenkii Trichosporon capitatum The following bacterial species did not report a positive result Bordetella pertussis Corynebacterium diphtheriae Escherichia coli Haemophilus influenzae Lactobacillus plantarum Legionella pneumophila Moraxella catarrhalis Mycoplasma pneumoniae Neisseria meningitidis Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pneumoniae S pyogenes S salivarius 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 septrin trimethoprim sulphamethoxazole sodium chloride sodium cromoglicate terbutaline and tobramycin Performance Evaluation The clinical cut off at a Ct of 39 0 was established following analysis of a set of clinical samples sourced from different patient populations Clinical samples collected by bronchoaleveolar lavage BAL that had been obtained at 2 hospitals extracted using the MycxXtra kit and stored were used to evaluate the performance of the MycAssay Pneumocystis kit Comparisons were made of the PCR results to immunofluorescent microscopy PCR v Microscopy Diagnosis M
10. d a Negative result cannot be relied upon 3 6 The lower the Ct value the higher the probability of disease Ct values close to the cut off of 39 0 are more likely to represent colonisation than infection but some patients may have disease with very little P jirovecii present representing a poor specimen prior treatment or the nature of fungal load in that particular patient English 7 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler 3 7 3 8 3 9 3 10 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 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 sec
11. e DNA material and contamination could result in false positive test results Wear gloves at all times All 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 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 3 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler Procedure for Use 1 Real Time PCR Set Up 1 1 To begin switch on the SmartCycler Real Time PCR System instrument and associated computer and launch the relevant software Enter usernames and passwords as required 1 2 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 Ae 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 sa
12. e 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 tipped 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 Pneumocystis or internal amplification control IAC amplicons do not open the reaction tubes post amplification Additional controls may be tested according to guidelines or requirements of local state provincial and or federal regulations 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 Kit Contents Description The kit consists of five 3 compartment sealed foil pouches each of which can be used separately Each pouch contains sufficient reagents for 8 reactions Volume Tube 1 dNTPs 66 uL Orange MgCl Cap Buffered solution of DNA Polymerase complex Tube 2 lt 0 01 Primers 66 uL Blue lt 0 01 Molecular Beacons Cap lt 0 0001 Internal Amplification Control IAC The I
13. econtaminating solution Permanent marker pen DNA isolation kit see below Specimen The specimen for the MycAssay Pneumocystis assay is total DNA extracted from clinical BAL samples The following DNA isolation kit and equipment supplied by Myconostica Ltd 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 Adapter Plate REF 080 015 available from Myconostica Procedural Notes Read the entire protocol before commencing The entire MycAssay Pneumocystis 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 which is regularly cleaned with DNA decontaminating reagents However avoid using DNA decontaminating reagents during 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 templat
14. ibitors entering the system While the MycXtra 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 from P jirovecii contaminated air poor experimental technique with respect to the positive control or external especially pipette contamination with P jirovecii DNA As a true positive result may be obtained from patients who are transiently or persistently colonized by P jirovecii clinical judgment is required in interpretation of the test result LICENSING TopTaq Hot Start 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 Myconostica Limited South Court Sharston Road Sharston Manchester M22 4SN United Kingdom Telephone 44 0 161 998 7239 Facsimile 44 0 161 902 2496 Email mycotech myconostica co uk English 12 030 090 Version 3 2 O9NOV10
15. icroscopy Microscopy pone meaane PCR 45 0 85 PPV pa ee PCR 2 33 0 94 NPV negative 0 96 0 80 Sensitivity Specificity Table 1 Diagnostic specificity and sensitivity of the MycAssay Pneumocystis kit compared to immunofluorescent microscopy Table 1 represents data obtained from patients with diagnosed HIV patients not infected with HIV and patients with undetermined HIV status Patients with Pneumocystis pneumonia have highly variable amounts of organism detectable the lower the Ct value the higher the likelihood of disease Patients with HIV and Pneumocystis pneumonia tend to have higher numbers of organisms detectable than patients who are not infected with the virus but the overlap is English 10 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler considerable The scatter plot in Figure 1 below demonstrates this overlap For completion as the data set in Table 1 included patients whose HIV status was unknown the scatter plot for this group is included in Figure 1 column 3 39 a 37 Ct Value Nm gt eecore 9 3 1 2 3 Category 1 HIV Microscopy 2 HIV Microscopy 3 HIV Unknown Microscopy 4 All Microscopy Figure 1 Scatter plot of Ct values obtained from DNA extracted from patient respiratory samples Four groups are described Clinical Reporting The MycAssay Pneumocystis kit is i
16. 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 gt 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 gt Take care that the reaction tubes are placed in their designated sites 4 4 Patient sample s give Outcome 3 Invalid It is likely that the extracted clinical sample s contain PCR inhibitors gt 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 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 soft
17. me kit lot A maximum of 38 patient samples may be tested using the 5 pouches in a kit 1 4 Calculate the number of reactions required referring to the table below Number of Maximum number of patient Pouches samples 1 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 1 6 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 required number of SmartCycler reaction tubes in their support rack s Never touch the diamond shaped reaction chamber of the reaction tubes 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 1 10 Reagent and
18. ntended as an aid to diagnosis of Pneumocystis pneumonia The results need to be taken in context of the clinical condition of the patient and other diagnostic tests results The following are recommended reports each depending on the assay result interpretation Outcome No 1 Pneumocystis jirovecii not detected Outcome No 2 Pneumocystis jirovecii detected Positive result State Ct value Outcome No 3 Test failed inhibitors or other unknown substance present The lower the Ct value the higher the probability of disease Ct values close to the cut off of 39 0 are more likely to represent colonisation than infection but some patients may have disease with very little P jirovecii present representing a poor specimen prior treatment or the nature of fungal load in that particular patient English 11 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler Limitations of Procedure The principal limitation of this procedure relates to the quality of the primary sample lf the 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 inh
19. nternal Amplification Control is a recombinant DNA plasmid harbouring a non infective sequence unrelated to either target Pneumocystis sequence Tris HCI Buffer Tube 3 Negative Control 25 uL Clear Water Cap Tube 4 Positive Control 25 uL Black lt 0 0001 Positive Control DNA Cap The Positive Control molecule is a recombinant plasmid harbouring the Pneumocystis target sequences Tris HCl Buffer The kit also contains MycAssay Pneumocystis 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 at which time 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 English 2 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler Equipment Materials required and not provided Cepheid SmartCycler Real Time PCR System including user manual attached desktop computer and Dx Diagnostic software version 1 7b or 3 0d Mini centrifuge adapted specifically for SmartCycler reaction tubes Micro centrifuge Vortex mixer SmartCycler reaction tubes Support rack for SmartCycler reaction tubes Micropipettes volumes required 7 5 uL 20 uL Sterile low retention filtertips Disposable gloves powderless Proprietary DNA d
20. rpretation 3 1 The results can be viewed in Dx software by selecting the View Results tab 3 2 Click on the View Another Run button at the bottom of the page select the run you wish to view then click OK 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 Patient Colour Interpretation Further Action Result Pneumocystis Pneumocystis Light Grey IAC failure in sample Repeat sample 4 a 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 39 0 is recorded in the Pneumocystis channel report as positive 3 4 To view individual sample results Ct values for either Pneumocystis or IAC separately select Sample Results from the Views list and click on the individual tabs for each target i e lt Pne gt or lt IAC gt The results will be displayed in the same format as the Patient Results but for each individual target 3 5 lf 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 an
21. t 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 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 enables them to fluoresce The amount of fluorescence at any given cycle or following cycling depends on the amount of specific amplicons present at that time The SmartCycler Real Time PCR System simultaneously monitors the fluorescence emitted by each beacon 1 Morris A Lundgren JD Masur H Walzer PD Hanson DL Frederick T Huang L Beard CB Kaplan JE 2004 Current epidemiology of Pneumocystis pneumonia Emerg Infect Dis 10 1713 20 Miller RF Allen E Copas A Singer M Edwards SG Improved survival for HIV infected patients with severe Pneumocystis jirovecii pneumonia is independent of highly active antiretroviral therapy Thorax 2006 61 716 21 Kovacs JA Gill VJ Meshnick S Masur H 2001 New insights into transmission diagnosis and
22. tion 3 4 above Select the run file to be retrieved and click Open Another screen will appear 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 When a run has completed its run data file will be automatically downloaded and can be located in the SmartCycler Dx Folder on the desktop This can be copied into any folder and stored electronically if required If a hardcopy of the results is also required click on Report and Print This will include both the overall Patient result Positive Negative or Invalid and the Ct value for each Sample Result English 8 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler 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
23. ware 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 9 030 090 Version 3 2 O9NOV10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Cepheid SmartCycler Performance Characteristics and Limitations Analytical Sensitivity Using the protocol described above and a recombinant Pneumocystis DNA molecule generated at Myconostica the Limit of Detection LoD for Pneumocystis was determined to be lt 35 copies This value was determined using a recombinant DNA plasmid harbouring the target sequence The Pneumocystis target sequence is mitochondrial therefore there will be numerous copies per cell but it is not known how many Analytical Selectivity Analytical selectivity was tested using DNA extracted from a variety of different fungal and non fungal species The following species did not report out a positive result Alternaria alternata Aspergillus flavus A fumigatus A niger A terreus 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 apiospermum S prolificans Sporothrix s
24. y Pneumocystis Myconostica Protocol CD ROM and click on the Define Assays tab and Import the MycAssay Pneumocystis v3_1 sca file from the CD ROM as shown below feed ieee imme E laim ar Leal Ls al LF i 2 2 3 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 2 2 4 Select MycAssay Pneumocystis v3 1 as the assay 2 2 5 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 2 2 6 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 2 2 7 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 determine how long the run will take to complete click on the Check Status tab The run name and subsequent run time will be listed 3 Data Analysis and Inte
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