The QIAsymphony RGQ as a platform for laboratory developed tests

Transcription

1 The QIAsymphony RGQ as a platform for laboratory developed tests James B. Mahony Ph.D., F.A.A.M., F.C.C.M. Director, Regional Virology Laboratory St. Joseph s Healthcare, Hamilton Assistant Dean Medical Sciences Graduate Program Professor, Department of Pathology & Molecular Medicine McMaster University, Hamilton, Ontario (AMP Nov )

2 Applications for QIAsymphony RGQ in Virology 1. Influenza virus subtyping 2. Respiratory virus M-PCR assays 3. Enteric virus M-PCR assay 4. Nucleic acid extraction removal of amplification inhibitors 5. Adaptable workflow in Virology lab

3 QIAsymphony - First application Influenza virus subtyping using matrix gene M-PCR instead of conventional HA and NA genes

4 Emerging Influenza A Strains Seasonal H1N1 and H3N2 Avian influenza H5N1 (1997, 1999) A/H7N2 (2003) A/H9N2 (2005) Swine 2009 Pandemic A/H1N1 Traditionally subtyped using serology or PCR targeting HA or NA gene

5 Influenza virus subtypes and resistance Strain identification assists WHO Reference labs with vaccine recommendatins Circulating strains determine makeup of annual vaccine (genetic drift) Influenza treatments are available (Amantadine and Oseltamivir) Resistance has developed to both antiviral drugs Treatment failures have led to development of antiviral susceptibility testing (H275Y allele)

6 Influenza virus subtyping HA and NA gene analysis has been used for both subtyping and anti-viral drug susceptibility testing (H275Y/H274Y genotypes) M-PCR has recently been used for combined subtyping and oseltamivir susceptibility testing (Mahony et al. J Clin Virol 2010) Influenza subtyping has been used as a surrogate test for antiviral susceptibility testing (not all clinical labs can sequence) The Matrix gene also contains subtype-specific sequences and can be used for subtyping

7 Influenza A Subtying Assay on RGQ Ojective was to develop a M-PCR subtyping assay on the QIAsymphony RGQ using stored flu A positive specimens from Assay used 1 Matrix gene primer pair and 3 probes (CAL Fluor Red 610, CAL Fluor Orange 560, FAM) one each for H1, H3 and 2009 H1 subtype 5 ul NA extracted on QIAsymphony SP was amplified in 20 ul reaction volume for 45 cycles and signals acquired in green (H1), orange (H3) and yellow (pandemic H1) channels

8 Subtying Assay Results 123 of 125 specimens gave clear cut typing result for either H1, H3 or 2009 A/H1 gene (2 specimens had no RNA) All 123 specimens gave signal in only 1 channel (no cross talk) with CTs between and The Matrix gene subtyping assay had equivalent sensitivity to CDC detection assay (LLOD of ~10 genome equivalents)

9 How good is QIAsymphony SP extraction? Selected 40 influenza positive NP specimens from 2011 and extracted 0.2 ml aliquots with biomerieux easymag and QIAsymphony SP and tested by Matrix gene M-PCR Assay on RGQ 39 out 40 specimens gave a positive result in both assays with the similar Mean CTs for EasyMAG and QIAsymphony (p<0.05) of and 21.05, respectively Extraction with QIAsymphony SP is as efficient as easymag extractor

10 QIAsymphony Second application Detection of respiratory viruses SARS Coronavirus

11 Identification of specific respiratory viruses 1. Some respiratory viruses are treatable (oseltamivir for influenza, Pleconaril for Rhinovirus, prophylactic Synagis for RSV) and new drugs on the way 2. Infection control practices differ for some viruses cohorting to prevent multiple infections 3. Some viruses are reportable to public health authorities 4. Some viruses such as SARS-CoV have huge public health and economic consequences 5. Monitoring newly emerging viruses during pandemic

12 Detection of 8 respiratory viruses on RGQ Multiplexed LDTs and commercial M-PCR assays have been developed for the detection of respiratory viruses (reviewed by Mahony et al. 2011) We developed two in-house M-PCR assays for the detection of 8 respiratory viruses using the RGQ First assay detects: Flu A, Flu B and RSV (uses 4 channels with IC) while the second assay detects ParaFlu 1, 2, 3, HMPV and Adenovirus (5 channels) Both assays in routine use since last winter Currently adding rhinovirus to Flu/RSV assay

13 QIAsymphony Third application Detection of enteric viruses using M-PCR Norovirus Rotavirus

14 Enteric virus M-PCR using RGQ Our objective was to develop a M-PCR assay for the detection of 4 Enteric viruses for the QIAsymphony RGQ Multiplex PCR was designed using 4 sets of primers and 4 probes to detect Rotavirus A, Norovirus GI and GII, and Adenovirus Stool suspensions (N=45) were extracted with QIAsymphony SP, NA (5 ul) was set up for M- PCR on RGQ using QIAsymphony AS Amplified products were identified by as follows: Norovirus GI (orange channel), Noro GII (yellow channel), Adenovirus (red channel) and Rotavirus A (green channel)

15 Enteric virus M-PCR results The number of positives was as follows: 21 Rotavirus, 6 Adenovirus, 18 Norovirus II with 3 dual positives and 2 negatives The sensitivities for Rotavirus, Adenovirus and Norovirus GII were 100% (22/22), 100% (6/6), and 100% (18/18) respectively The specificities for Rotavirus, Adenovirus and Norovirus GII were 100% (23/23), 100% (39/39), and 96.3%% (26/27) respectively The sensitivity and specificity of the M-PCR assay on the RGQ was equivalent to M-PCR assay run on a competitors platform

16 Development of a stool extraction protocol using the QIASymphony SP No existing protocol available!

17 Extraction of stool specimens PCR testing of stools for C. difficile indicated that ~15-20% of stools are inhibitory and the spiked MS2 internal control failed to amplify Some of these stools are very viscous due to the presence of mucous We evaluated a pre-treatment step that we have developed for NP/sputa specimens using SL- Solution (SLS) from Copan Italia to reduce the viscosity and inhibition rate (Smieja et al. 2011)

18 Stool extraction protocol for RGQ We selected 16 inhibitory stools that were extracted with biomerieux easymag and were inhibitory where the internal MS2 spike failed to amplify ( inhibitory specimens ) We developed an extraction protocol for the QIAsymphony SP that uses a 10% suspension of fecal material and SLS 15 stool specimens that were extracted by biomerieux easymag and were inhibitory were rendered non-inhibitory by the use of SLS and QIAsymphony SP

19 QIAsymphony SP - prospective study Prospectively processed 45 stools using QIAsymphony SP and 10% stool suspension in SLS then tested by M-PCR on the RGQ After resolution of discordants M-PCR detected 32 positives: 17 Rotavirus, 4 Adenovirus, 3 Norovirus GI, 9 Norovirus GII, 1 dual positive Rotavirus/Adenovirus and 11 negatives compared with only 18 using (CombiSTICK, SVC and EM) None of the 45 stools were inhibitory (MS2 control amplified for all stools) indicating that SLS plus QIAsymphony resulted in removed of all amplification inhibitors

20 Prospective evaluation II - QIAsymphony SP Evaluated the SLS/QIAsymphony SP extraction protocol for stool specimens collected from symptomatic children at Botswana University (Grand Challenges Canada/Gates Foundation Study) 147 prospectively collected stool specimens were shipped frozen to RVL in Hamilton and nucleic acid was extracted using SLS/QIAsymphony method 5 ul tested by xtag GPP Assay (LMD) for 9 bacterial, 3 viral and 3 parasitic pathogens

21 QIAsymphony extraction of African stools Only 1/147 (<1%) specimens were inhibitory (MS2 negative) - this compares to ~15% inhibition rates for domestic stool specimens extracted with water and biomerieux easymag We next compared SLS+easyMAG to SLS+ QIAsymphony SP on a subset of inhibitory stools QIAsymphony SP had a smaller number of inhibitory stools and appears to be superior to easymag when SLS was used with easymag and QIAsymphony SP

22 Workflow analysis in diagnostic and research laboratory

23 Scenario 1: Processing 4 different runs of 144 specimens 48 specimens for C. Difficile Testing 48 specimens for Respiratory VirusTesting 24 Specimens for CMV Testing 24 Specimens for C. Difficile Testing Specimen Set Up Purification PCR Assay Set Up PCR Cycling Time *Based on the availability of 2 Rotor-Gene Q instruments, Pathogen Extraction protocol for 200µl, PCR assay run times of 2 to 2.5 hrs. (Slides prepared by Sylvia Chong)

24 Scenario 2: Processing 6 different runs of 144 specimens on RGQ 24 specimens for C. Difficile Testing 24 specimens for C. Difficile Testing 24 specimens for Respiratory Virus Testing 24 specimens for Respiratory Virus Testing 24 specimens for CMV Testing 24 specimens for C. Difficile Testing Specimen Set Up Purification PCR Assay Set Up PCR Cycling Time *Based on the availability of 2 Rotor-Gene Q instruments, Pathogen Extraction protocol for 200µl, PCR assay run 2.5 hrs. Using 1QIAsymphony in place of 3 easymag extractors where tech needs to be present for lysis to add beads and end of run to remove eluates from cartridges.

25 Scenario 4: Batch Testing of Specimens in the Research Laboratory testing 120 specimens available at one time. Specimen Set Up Purification PCR Assay Set Up PCR Cycling Time *Based on the availability of 1 Rotor-Gene Q instrument, Pathogen Extraction protocol for 200µl, PCR assay run time of 2.5 hrs. Last PCR run left to run overnight. (Slides prepared by Sylvia Chong)

26 Summary 1. The QIAsymphony RGQ platform is readily amenable for running laboratory developed tests. 2. We have developed an influenza A M-PCR subtyping assay (H1, H3, pandemic H1) and an enteric virus M-PCR for detecting Rotavirus, Adenovirus and Norovirus GI and GII to run on the QIAsymphony RGQ. 3. We developed two M-PCR assays for 7 respiratory viruses on RGQ (adding rhinovirus 2011/2012). 4. All M-PCR assays run on the RGQ had excellent sensitivities and specificities.

27 Summary (cont d) 5. We have a developed an extraction protocol for stool specimens using SL-Solution (Copan Italia) and the QIAsymphony SP that removes amplification inhibitors (>99%). 6. SLS+QIAsymphony SP was better than easymag for removing inhibitors; there were fewer inhibitory stools and CTs were lower indicating more efficient extraction of nucleic acids. 7. The stool extraction protocol was validated in both retrospective and two prospective studies involving over 200 specimens.

28 Summary (cont d) 8. Workflow analysis indicated that results for 24 specimens could be generated in 4.75 hours using the QIAsymphony RGQ platform. Results 144 specimens involving 4-6 different PCR assays and 4-6 runs could be generated in ~11 hours.