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Malaria Pf - Molbio Diagnostics

1. Malaria Pf 100 Parasite species Plasmodium falciparum Pf n 119 Microscopy 78 57 Truenat Malaria Pf reported more number of positives than the other methods INTERFERENCE Effect of interferences of elevated serum parameters such as lipid cholesterol and triglycerides were evaluated in this study Specimen total 10 samples showing higher level of serum parameters were spiked with known amount of P falciparum positive samples These spiked samples were tested using Truenat Malaria Pf The Ct values obtained were found not to be significantly impacted Ct average 27 93 standard deviation 1 7 variance 2 89 by the presence of elevated biochemical parameters as described above Carry Over E ffect To determine whether the Truenat Malaria Pf microchip PCR assay showed any signs of carryover of PCR products between runs alternating runs of positive Pf high titre samples and negatives samples were performed in triplicates 10 positives samples and 10 negatives samples were used for the study Results of carry over evaluation are below Sr No Samplel D CtPf1 CtPf2 CtPF3 Average STDEV 1 Positive 23 5 23 23 2 23 23 0 25 2 egative 3 Positive 21 8 22 22 3 22 03 0 25 4 egative 5 Positive 22 1 22 4 22 5 22 33 0 21 6 egative 7 Positive
2. 23 23 1 23 3 23 13 0 15 8 egative 9 Positive 23 1 23 4 23 5 23 33 0 21 10 egative 11 Positive 21 9 22 22 2 22 03 0 15 12 egative 13 Positive 21 6 21 8 22 21 80 0 80 14 egative 15 Positive 21 4 21 6 21 5 21 50 0 10 16 egative 17 Positive 23 2 23 4 23 6 23 40 0 20 18 egative 19 Positive 22 1 22 3 22 6 22 33 0 25 20 egative All the positive samples were accurately detected and all negative samples were undetected indicating absence of carryover of PCR products between runs using the Truenat Malaria Pf chip based RealTime PCR test 18 REFERENCES 1 Rich S M etal 2009 The origin of malignant malaria Proceedings of the National Academy of Sciences 106 35 14902 14907 2 Perlmann P et al 2000 Malaria blood stage infection and its control by the immune system Folia biologica 46 6 210 8 3 Marchand RP et al 2011 Co infections of Plasmodium knowlesi P falciparum and P vivax among Humans and Anopheles Mosquitoes Southern Vietnam Emerg Infect Dis J ul 2011 17 7 1232 9 4 MoodyA 2002 Rapid diagnostic tests for malaria parasites Clin Microbiol Rev 2002 15 66 78 5 Andrade et al 2010 Towards a precise test for malaria diagnosis in the Brazilian Amazon comparison among field microscopy a rapid diagnostic test nested PCR and a computational expertsystem based on artificial neural networks Malaria J ournal 2010 9 117 6 Khairnar K et al 2009 Multiplex real time quantitative
3. PCR microscopy and rapid diagnostic immuno chromatographic tests for the detection of Plasmodium spp performance limit of detection analysis and quality assurance Malar 2009 8 284 7 Shokoples S E et al 2009 Multiplexed real time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections Clin Microbiol 2009 47 975 980 8 WHO FIND CDC Malaria RDT ProductTesting Methods Manual Version 1 05 2008 9 Adams JH etal 1992 A family of erythrocyte binding proteins of malaria parasites Proc Natl Acad Sci USA 89 7085 7089 Pascal Michon et al 2002 Evolutionary Relationships of Conserved Cysteine Rich Motifs in Adhesive Molecules of Malaria Parasites SYMBOL KEYS Consult itro Di a c Ta ico amare ppd rores J Suet REF anc Date of h Numb ins suffici For singl M iae pag Bow ime NZ earar Q cea wl molbio Molbio Diagnostics Pvt Ltd M 46 47 Phase Ill B Verna Industrial Estate Verna Goa 403 722 India E mail sales tulipgroup com TNMAL 0413 VER OL
4. also display the C value and the parasites per uL P uL for positive specimen The result screen also displays the validity of the test run as VALID or INVALID Invalid samples have to be repeated with fres specimen from the sample preparation stage While IPC will co amplify in most positive cases also in some specimen having a high target load the IPC may not amplify however the test run is still considered valid QUALITY CONTROL PROCEDURES To ensure thatthe Truelab Uno Real Time micro PCR Analyzer is working accurately run positive and negative controls from time to time The Truenat Universal Control Kit containing Positive Control and Negative Control must be ordered separately It is advisable to run controls under the following circumstances Whenever a new shipment of test kits is received When opening a new testkitlot 2 Ifthe temperature of the storage area falls outside of 2 30 C By each new user prior to performing testing on clinical specimen DISPOSAL AND DESTRUCTION 1 Submerge the used Truenat Malaria Pf chip based Real Time PCR test in freshly prepared 1 sodium hypochlorite solution for 20 minutes before disposal as per the standard medical waste disposal guidelines Disinfect the solutions and or solid waste containing biological samples before discarding them according to local regulations Samples and reagents of human and animal origin as well as contaminated mate
5. labeled luorescent probe in the Truenat Malaria Pf chip based Real Time PCR test to release the luorophore in an exponential manner which is then captured by the built in opto electronic sensor and displayed as amplification curve on the analyzer screen on a real time basis during the test run The Cycle threshold Ct is defined as the number of amplification cycles required for the fluorescent signal o cross the threshold i e exceed the background signal Ct levels are inversely proportional to the amount of target nucleic acid in the sample i e the lower the Ct level the greater is the amount of arget nucleic acid in the sample Ct value is linearly correlated with the amountof target DNA present in the sample enabling quantitative estimation of the analyte Standard values for every batch are preset in the Truenat Malaria Pf chip based Real Time PCR test and the analyzer automatically compares these with the Ct value of the test sample to provide a quantitative result In the case of negative samples amplification does notoccur and a horizontal amplification curve is displayed on the screen during the test run Atthe end of the test run Falciparum DETECTED or NOT DETECTED resultis displayed and in positive cases Ctvalues and parasites per microlitre P u L is also displayed on the screen Based on the Ct of the Internal Positive Control IPC the validity of the testrun is also displayed The IPC is a full process control t
6. samples obtained from National Institute of Malaria Research NIMR Bangalore and 50 Pf negative samples The WHO Nested PCR protocol was run in parallel to confirm the status of the samples All the 50 Pf samples were detected as positive andthe 50 Pf negative samples were negative respectively by both Truenat Malaria Pfand the WHO nested PCR protocol REPRODUCIBILITY AND PRECISION Truenat Malaria Pf kit was tested for reproducibility and precision between users analyzers and reagent lots Pf DNA was extracted from a high titre clinical specimen and serially diluted for this study The same sample panel was provided to 3 users who ran it on 3 differentTruelab micro PCR analyzers with 3 different lots of Tuenat Malaria Pf The tests were runin triplicates High inter user inter analyzer and inter lot reproducibility was observed average standard deviation 0 26 Ct and no significant difference was noted in the Ct values obtained from different users or analyzers or reagentlots METHOD CORRELATION Validation at National Institute of Malarial Research NIMR Bangalore Results of Truenat Malaria Pf chip based RealTime PCR test from 119 archived clinical specimens were correlated with microscopy and a commercially available rapid diagnostic test RDT Relative clinica sensitivity normalized with the Truelab results as 100 is tabulated below Summary of validation study using NIMR samples RDT 77 14 Truenat
7. c Truenat Malaria Pf Chip based Real Time PCR test for Plasmodium falciparum INTENDED USE Truenat Malaria Pf REF 601120005 601120020 is a chip based Real Time Polymerase Chain Reaction qPCR test for the quantitative detection and diagnosis of Plasmodium falciparum Pf in human blood specimen and aids in the diagnosis of infection with Malaria caused by Plasmodium falciparum Pf Truenat Malaria Pf runs on the Truelab Uno Real Time micro PCR Analyzer INTRODUCTION Falciparum malaria is a life threatening disease caused by the protozoan parasite Plasmodium falciparum which is transmitted via the bite of an infected female Anopheles mosquito In the human body the parasites multiply in the liver and then infect red blood cells Plasmodium falciparum i known to cause the most dangerous malignant malaria which has the highest rate of complication and mortality Early and accurate diagnosis of Falciparum malaria is necessary to initiate appropriate treatment check transmission of the disease and prevent death Microscopic examination of stained blood smears is still considered as the gold standard for detection of malaria parasitemia However in comparison to PCR methods these are reported to be only 80 90 sensitive Nested PCR and rea time PCR presenthigher sensitivity and specificity to malaria diagnosis compared to light microscopy HoweverPCR orRealTime PCR tests have so far been restricted to centralized reference labo
8. ctuser and enterpassword Selectthe test profile for MALARIA Pf on the Analyzerscreen Enter the patient details as prompted in the Truelab Uno Real Time micro PCR Analyzer screen Press Start Reaction Device will promptas Please load sample Press the ejectbutton to open the chip tray Open a pouch of Truenat Malaria Pfand retrieve the micro PCR chip Label the chip with the patient ID using a marker pen atthe space provided onthe back side of the chip 9 Place the Truenat Malaria Pf chip based RealTime PCR teston the chip tray withouttouching the white reaction well The reaction well should be facing up and away from the Analyzer Gently press the chip to ensure thatitis seated in the chip tray properly WNP po En 10 Using the filter barrier tip provided in the pouch pipette six 6 uL of the purified DNA from the Elute Collection Tube into the centre of the white reaction well Take care not to scratch the internal well surface and notto spill elute on the outside ofthe well 11 Slide the chip tray containing the Truenat Malaria Pf chip based Real Time PCR test loaded with the sample into the Truelab Uno RealTime micro PCR Analyzer 12 Press the powerbutton on the Analyzer to turn iton The green LED should glow 13 Press Doneon the Please Load Sample Alertmessage 14 Atthe end of the test observe the optical plot for any irregularities R efer to the Truelab Uno Real Time mic
9. hatundergoes all the processes the specimen undergoes from extraction to amplification thereby validating the test run from sample to result Absence of or shift of IPC Ct beyond a pre set range in case of negative samples invalidates the test run While IPC will co amplify in most positive cases also in some specimen having a high target load the IPC may not amplify however the testrun is still considered valid The results can be printed via Bluetooth using the Truelab micro PCR printer or transferred to the lab computer or any remote computer via Wifi network or GPRS network Upto 5000 test results can be stored on the analyzer for future recall and reference TARGET SELECTION The target sequence for this kithas been taken from the erythrocyte binding protein EBP gene which is expressed on the surface of the merozoites EBP is involved in the parasite s invasion of the red blood cells RBC by pathways that are specific to Plasmodium falciparum The region selected is specific for P falciparum CONTENTS OF THE Truenat Malaria Pf KIT A Individually sealed pouches each containing a 1 Truenat Malaria Pf micro PCR chip 2 DNase amp RNase free pipette tip 3 Desiccant pouch B Package Insert 601020005 VW 5T STORAGE AND STABILITY Truenat Malaria Pfis stable for one year from the date of manufacture if stored between 2 30 C Itis also stable for upto
10. nce ofthe concerned pathogen 3 The instruments and assay procedures are designed to minimize the risk of contamination by PCR amplification products However it is essential to follow good laboratory practices and ensure careful adherence to the procedures specified in this package insert for avoiding nucleic acid contamination from previous amplifications positive controls or specimens 4 A specimen for which the Truenat assay reports Not Detected cannot be concluded to be negative for the concerned pathogen As with any diagnostic test results from the Truenat assay should be interpreted in the context of otherclinical and laboratory findings CLEANING AND DECONTAMINATION 1 Spills of potentially infectious material should be cleaned up immediately with absorbent paper tissue and the contaminated area should be decontaminated with disinfectants such as 0 5 freshly prepared sodium hypochlorite 10 times dilution of 5 sodium hypochlorite household bleach before continuing work 2 Sodium hypochlorite should not be used on an acid containing spill unless the spill area is wiped dry first Materials used to clean spills including gloves should be disposed off as potentially bio hazardous waste e g ina biohazard waste container TEST PROCEDURE Please also refer Section 4 in the Truelab Uno Real Time micro PCR Analyzer user manual Switch on the Truelab Uno RealTime micro PCR Analyzer touch screen Sele
11. nd package inserts of all the components of the Truelab Real Time micro PCR System before use Allmaterials of human origin should be handled as though potentially infectious Do notpipette any material by mouth 7 Donoteat drink smoke apply cosmetics or handle contact lenses in the area where testing is done 8 Use protective clothing and wear disposable gloves whe performing sample extraction am handling samples and while PROCEDURAL PRECAUTIONS 1 Check all packages before using the kit Damage to the packaging does not prevent the contents of the kit from being used However if the outer packaging is damaged the user must confirm hatindividual components of the kit are intact before using them 2 Do not perform the test in the presence of reactive vapours e g from sodium hypochlorite acids alkalis or aldehydes or dust 3 While retrieving the Truenat Malaria Pf chip based Real Time PCR test and the DNase amp RNase free pipette tip from the pouch ensure that neither bare hands nor gloves that have been sed for previous tests run are used PROCEDURAL LIMITATIONS 1 Optimal performance of this test requires appropriate specimen collection handling storage and ransportto the testsite 2 Though very rare mutations within the highly conserved regions of the target genome where the Truenat assay primers and or probe bind may result in the under quantitation of or a failure to detectthe prese
12. ratories s they require skilled manpower and elaborate infrastructure Also the turnaround time for results could take a few days The Truelab Real Time micro PCR System enables decentralization and near patient diagnosis of alciparum malaria by making real time PCR technology rapid simple robust and user friendly and offering sample to result capability even at resource limited settings This is achieved through a combination of lightweight portable mains battery operated Truelab Uno Real Time micro PCR Analyzer and Trueprep MAG Sample Prep Device and room temperature stable Truenat chip based Real Time PCR test and Trueprep MAG Sample Prep kits so that even the peripheral laboratories with minimal infrastructure and minimally trained technician can easily perform these tests routinely in their facilities and report PCR results in less than an hour Moreover with these devices PCR testing can also be initiated in the field level onsite Truenat Malaria Pf is a disposable room temperature stable chip based Real Time PCR test with dried down PCR reagents for performing Real Time PCR test for detection and diagnosis of Plasmodium falciparum and runs on the Truelab Uno Real Time micro PCR Analyzer It requires only six 6 uL of purified DNA to be added to the reaction well for the analysis The intelligent chip also carries test and batch related information including standard values for quantitation The Truenat Mala
13. ria Pf chip based Real Time PCR test also stores information of used test to preventany accidental re use ofthe test NOTE Truelab Truelab Uno Trueprep MAG Truepet Truenat are all registered trademarks of Molbio Diagnostics P Limited The Truelab Real Time micro PCR Analyzer is protected by the following patents and patents pending IN 2313 CHE 2007 WO 2009 047804 and corresponding claims of any foreign counterpart s thereof The Truenat micro PCR chip is protected by the following patents and patents pending IN 2312 CHE 2007 WO 2009 047805 and corresponding claims of any foreign counterpart s thereof The Truenat Malaria Pf test is protected by the following patents and patents pending IN 421 CHE 2009 WO 2010 097803 and corresponding claims of any foreign counterpart s thereof S S w o PRINCIPLE OF THE TEST Truenat Malaria Pf works on the principle of R eal Time Polymerase Chain Reaction The DNA from the patient blood sample is first extracted using Trueprep MAG Sample Prep device and Trueprep MAG Blood Sample Prep Kit Six 6 uL of the purified DNA is then dispensed into the reaction well of the Truenat Malaria Pf chip based Real Time PCR test The Truenat Malaria Pf chip based Real Time PCR test is then inserted in the Truelab Uno Real Time micro PCR Analyzer where thermal cycling takes place A positive amplification causes the dual
14. rials disposables neutralized acids and other waste materials must be discarded according to local regulations after decontamination by immersion in a freshly prepared 0 5 of sodium hypochlorite for 30 minutes 1 volume of 5 sodium hypochlorite for 10 volumes of contaminated fluid or water Do notautoclave materials or solutions containing sodium hypochlorite Chemicals should be handled in accordance with Good Laboratory Practice and disposed off according to the local regulations N w ve SPECIFIC PERFORMANCE CHARACTERISTICS ASSAY RANGE Plasmids carrying cloned PCR amplicons were used as template DNA The samples were quantified by UV spectrophotometry to determine the copy number microlitre The plasmids were serially diluted and tested using Truenat Malaria chips to obtain Ct values which were plotted against the plasmid concentration per reaction to determine the range of the assay The assay was found to have a broad linear dynamic range over 7 orders of magnitude The Truenat Malaria Pf testwas able to detect as lowas 2 parasites per uL equivalent of blood CLINICAL SENSITIVITY CLINICAL SPECIFICITY Truenat Malaria Pf was tested against a nested PCR protocol according to SOP 5 8 of WHO Methods Manual for Product Testing of Malaria Rapid Diagnostic Tests Version 2 2009 and clinical sensitivity and specificity were both found to be 100 Truenat Malaria Pf was tested with 50 archived Pf positive
15. ro PCR Analyzer manual 15 Read the resultfrom the screen 16 Press the powerbutton on the Analyzer to turn itoff The green LED should stop glowing 17 Take outtheTruenat Malaria Pf chip based RealTime PCR testatend ofthe test and dispose itoffas perthe section on Disposal and Destruction Section 16 18 TumonTruelab micro PCR printer and select printon the screen for printing out hard copy of the results Test results are automatically stored and can be retrieved any time later Refer to the Truelab Uno Real Time micro PCR Analyzer manual 19 Switch off the Truelab Uno Real Time micro PCR Analyzer touch screen RESULTS amp INTERPRETATIONS Two amplification curves are displayed onthe Truelab Uno Real Time micro PCR Analyzerscreen to indicate the progress of the test Both the target and the internal positive control IPC curves wil take a steep exponential path when the fluorescence crosses the threshold value in case of positive samples The Ct will depend on the number of parasite genomes in the sample The target curve wil remain horizontal throughout the test duration and the IPC curve will take an exponential path in case of negative samples In case the IPC curve remains horizontal in a negative sample the test is considered as Invalid At the end of the test run the results screen will display DETECTED for Positive result or NOT DETECTED for Negative result The result screen would
16. three 3 months attemperatures upto 40 C Avoid exposure to light 601020020 20T MATERIALS REQUIRED BUT NOT PROVIDED WITH THE KIT Truelab RealTime micro PCR Workstation REF 603010001 consisting of 1 Trueprep MAG Sample Prep Device REF 603040001 2 Truelab Uno Real Time micro PCR Analyzer REF 603020001 3 Truelab micro PCR Printer REF 603050001 4 Truepet Precision Micropipettes 6uL 50uL 100uL 500uL 1000uL REF 604010006 604020050 604030100 604040500 604051000 Also required additionally are Trueprep MAG Blood Sample Prep Kit REF 609100050 Truenat Universal Control Kit REF 601100008 DNase and RNase free pipette tips 2 200uL 200 1000u L microtips with filter barrier which may also be procured from Molbio REF 604072200 REF 604062010 respectively Powderfree disposable gloves waste disposal containerwith lid SPECIMEN PREPARATION Truenat Malaria Pf requires purified nucleic acids from blood specimen that are extracted using the Trueprep MAG Sample Prep Device and Trueprep MAG Blood Sample Prep Kit Refer to the User Manual of Trueprep MAG Sample Prep Device and the package insert of Trueprep MAG Blood Sample Prep Kitfor details SAFETY PRECAUTIONS 1 Forinvitro diagnostic use only 2 Bringallreagents and specimen to room temperature 20 30 C before use 3 Donotuse kitbeyond expiry date 4 Carefully read the User Manuals a

Malaria Pf - Molbio Diagnostics

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