1. A New Age in Microbiology: New Molecular Diagnostics Oct 13, 2015 Jutta K Preiksaitis, University of Alberta, Edmonton, Alberta

2. Progress in Molecular Detection over the last 15 years

3. Potential uses of quantitative viral load assays As triggers for pre-emptive therapy for disease prevention (CMV, EBV, BKV) For disease diagnosis (CMV, EBV, BKV) For treatment monitoring (CMV, EBV, BKV) As surrogate markers for antiviral resistance (CMV) For safety monitoring in clinical trials [new immunosuppressive agents] (CMV, EBV, BKV) For tailoring immunosuppression in individual patients (EBV)

4. Pang et al, Am J Transplant. 2009;9:258-68 Log 10 variation in reported results (relative to expected) Variability in the result reporting of CMV viral load assays: only 58% results + 0.5 l og 10 expected results variation observed 2.0 l og 10 (minimum), 4.3 l og 10 (minimum) Plasma spiked with HCMV Merlin Clinical samples

5. High accuracy, but low precision High precision, but low accuracy International Reference Standards defines the bull's-eye at a global level; standards for CMV approved in October 2010, EBV in Oct 2011 and BKV standards are in development In most laboratories with extensive molecular diagnostic experience working within a quality systems framework, results have good precision and are linear over the clinically relevant dynamic range

6. I st International Standard for HCMV HCMV Merlin 5x10 6 IU/ml Using laboratory/kit calibrators Using WHO international standard as calibrators 2 log 10 range 0.5 log 10 range

7. Difference from mean of reported results Log 10 IU/ml 52bp 64/76bp 86bp <100bp 95/105bp 105bp 254bp 340bp #1 #2 CMV DNA (IU/ml) Log 10 variation of all clinical samples in each assay relative to geometric mean (GM) for each sample result range minimum :1.02 log 10,maximum: 2.74 log 10 Result harmonization has improved since IS implementation Clinically relevant variability persists!

8. Reported results CMV DNA IU/ml(Log10 ) 6A 6B Individual CMV DNA positive samples sorted by increasing mean of log10 transformed results for all assays identical commercial analyte specific reagents different nucleic acid extraction techniques, plasma input volume, detection system identical :All components and processes different: input volume

9. What are the implications from the clinician’s perspective (CMV, EBV, BK QNAT) Iindividual patients should be monitored in a single laboratory using the same assay ; result comparison among assays /institutions requires prior formal cross-referencing using clinical samples Exception: CMV DNA measured in plasma Results among laboratories using identical assays ( all reagents and procedures) can be compared The precision of quantitative NAT assays, even using state of the art technology, is such that changes in values should differ by at least 3 fold or (0.5 log10) [ 5 fold (0.7 log 10) near the assay’s limit of detection] to be considered biologically important changes

10. More implications… Clinical practice guidelines that rely on “trigger points”, negativity endpoints, or quantitative dynamic changes in viral load are difficult to develop and implement globally Multi-center clinical trials must use central reference laboratories for NAT testing results to reliably interpret integrated data. Do we need a standard free measurement tool- digital PCR?

11. Case 1 35 yr old M, 17 years post sibling allo-HSCT for AML, fully engrafted, no GVHD Presented to ER with 3-5 day history of fever, coryza, non-productive cough, progressive SOB CXR/ CT chest - diffuse interstitial infiltrates Hypoxic, intubated, BAL performed PJP stains neg, galactomannan neg, no significant bacterial or fungal pathogens isolated Metapneumovirus detected by RVP ( Luminex) Despite oral ribavirin, IVIG and ECMO support, died 7 days later

12. Examples of highly multiplexed respiratory PCR assays PathogenxTAG RVP Luminex Molecular Diagnostics FilmArray Bonfire Diagnostics FluA/B, Flu A H1,H3X H5 classic versionX also H1 2009 PIV 1-4xx RSV A/Bxx hMPVxx CoV includes HKU1, OC43,NL63, 229E, X SARS classic version x Adenovirusxx RhV /EnVxx B pertussisx M pneumoniaex C pneumoniaex hBoVx (FAST version) Gray Epidemiol Infect 2014;142:1, Zumla Lancet Infect Dis 2014;14:1123,Babady Exp Rev Mol Diag2015;13:779

13. Case 2 63 yr old female 4 years after double lung Tx (emphysema), on sirolimus/ azathioprine Admitted for investigation of two month history of persistent and severe diarrhea after episode of acute gastroenteritis Creatinine 220 μMol/L ( baseline) to 404 μMol/L Stools for C&S, O&P, C diff toxin neg, CMV and EBV VL undetectable, colonoscopy unremarkable Stool positive for sapovirus detected using in house developed multiplex enteric virus panel (detects rotavirus, norovirus, adenovirus 40/41, astrovirus, sapovirus)

14. Target (Analytes) SensitivitySpecificity Bacteria & Bacterial Toxins Shigella97.7%97.8% Campylobacter97.5%97.8% Yersinia enterocoliticaN/A*100.0% Enterotoxigenic E. coli (ETEC) LT/STN/A*97.3% E. coli O15788.2%98.8% Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2100.0%99% Salmonella82.1%99.1% Clostridium difficileToxin A/B97.7%94.9% Vibrio choleraeN/A*100.0% Viruses Adenovirus 40/41100.0% Rotavirus A94.7%99.8% Norovirus GI/GII93.5%98% Parasites Giardia lamblia100.0%98.2% Cryptosporidium87.5%100% Entamoeba histolytica100.0%99.4% *Due to low sample size, clinical sensitivity was not assessed for ETEC, Yersinia enterocolitica and Vibrio cholerae. However, analytical accuracy for these analytes was demonstrated in the limit of detection and reactivity studies with cultural isolates or plasmids. Examples of highly multiplexed gastroenteritis PCR assays Luminex xTAG GGP

15. Examples of highly multiplexed PCR assays F ilmArray GE Panel (BioFire Diagnostics) Bacteria Diarrheagenic E. coli ETEC EPEC STEC/EHEC -O157:H7 EIEC EAEC Aeromonas spp. Salmonella spp. Vibrio spp. -V. cholorae Shigella spp. -S. dysenteriae Campylobacter group Yersinia enterocolitica Clostridium difficile -C.difficile Nap1* Plesiomonas shigelloides* Viruses Norovirus (GI, GII, and GIV) Adenovirus F (40/41) Rotavirus (A, B, and C) Human Astrovirus* Sapovirus* Protozoa Cryptosporidium group Giardia lamblia Entamoeba histolytica Cyclospora cayetanensis* *Assays added to panel subsequent to presented data set. http://filmarray.com/the-evidence Multiplexed gastroenteritis PCR assays reviewed by Zhang Clin Lab Med 2015;35:461

17. Case 3 49 year old female with alpha 1- antitrypsin deficiency rapid deterioration with exertional hypoxia and recurrent “infections” over past year, worked up for lung Tx purulent sputum – multiple courses of abx – responds best to moxifloxacin, sputum cx grow only mixed oral flora CT chest – chronic collapse RLL with some bronchiectasis Mycobacterial cx ( all neg on direct smear) –Nov 14 2014 BAL – mycobacterium lentiflavum ( 2 sites) –May 22, 2014 sputum –mycobacterium xenopi –May 27, 2014 sputum -mycobacterium xenopi –July20, 2014 sputum – mycobacterium lentiflavum –July 20, 2014 sputum – mycobacterium avium –July21, 2014 sputum – mycobacterium lentiflavum August 2014- started on Rx ( azithromycin, ethambutol, rifampin)

18. Xpert MTB/RIF (Cepheid) Endorsed by WHO 2011 <2 hours to result, minimal hands-on technical time can be used for near patient testing Replacing smear microscopy sens 89%, spec 99% (23% ) Add on test ( neg smear microscopy) sens 67% spec 99% Smear pos, cx pos TB sens 98% Among 180 NTM; pos in only one sample that grew NTM Rifampin resistance (mutations rpoB gene) surrogate MDR-TB Rifampin resistance :sens 95%, specificity 98% Steingart KR Cochrane Database of Systematic Reviews 2014 DOI:10.1002/14651858.CD009593.pub3.

19. Three generations of sequencing technologies First: single chain sequencing (ie Sanger dideoxy chain termination sequencing) Second: sequencing analyzes an ensemble of DNA molecules simultaneously by “wash and scan” techniques Third: sequencing by synthesis, degradation or direct inspection of the DNA moleucle ( single molecule resolultion ie SMRT system from Oxford Nanopore) Bahassi Mutagenesis 2014;29:303 Dunne Eur J Clin Microbiol Infect Dis 2012; DOI 10.1007/s10096-012-1641-7

20. Identification of NTM Ingen Clin Chest Med 2015;36:43 Morozova O Ann Rev Genomics HumGenet 2009:10: 135

21. Into the future… Whole genome sequencing directly from clinical samples (pathogen identification, virulence factors, antimicrobial sensitivity, molecular epidemiology ) Urine - Hasman J Clin Microbiol 2014;32:139 TB - Brown AC J Clin Microbiol 2015;53:2230 Respiratory viruses – Zoll J Clin Microbiol 2015;66:6 Exploring the microbiome /virome –In mice, abx Rx after exogenously administered VRE, allows VRE to nearly completely displace the normal microbiota of both small and large intestine; inn HSCT, dominance of VRE in faceal flora precedes blood stream infection Ubeda J Clin Invest 2010;120:4332 –The lower respiratory tract in lung Tx recipients has a higher bacterial burden, frequent dominant organisms and decreased microbial richness and diversity when compared to controls Charlson Am J Crit Care Med 2012;186:536 –Could anellovirus load in plasma be used as a marker of “ global immunosuppression”? De Vlaminck Cell 2013;155:1178 the

22. The challenge Do “better tests” result in –Any significant change in patient management –Improved patient outcomes –Better antimicrobial stewardship –Reduction in infection transmission –Improved disease surveillance Evidence to date limited, more work required Gray Epidemiol Infect 2014:142:1