The use of new diagnostic technologies in the clinical microbiology laboratory

1. The use of new diagnostic technologies in the clinical microbiology laboratory AW Dreyer

2. Introduction • Changes in Clinical Microbiology over the past 100 years have been prompted by changes in clinical needs (e.g. new treatment options associated with a need in more rapid diagnosis) • Virology, more advanced…difficulty in culture • Microbiologists struggle to abandon traditional culture techniques (comfort zone)

3. Function of the Clinical Microbiology Laboratory • Analyze samples from patients – direct link to patient management and outcome • Provide data…surveillance • Sentinel towards outbreaks (e.g. natural or bioterrorism) • Antimicrobial stewardship • Detecting emerging resistance

4. Evolution of clinical microbiology • Culture • Antigen detection • Serology • Nuceic acid amplification testing (NAAT) • High throughput extraction NAAT • Whole genome sequencing (WGS)

5. Why the need for change? • Rapid identification

6. Conventional culture Limitations Viable organisms Lengthy incubation TAT >24 hours Why rapid identification? Enables appropriate treatment/de-escalation Improve clinical outcome Infection control Various platforms available – molecular and proteomics

7. Molecular microbiology Organisms • Respiratory viruses • Enterovirus • HSV • B.pertussis • M.tuberculosis • N.gonorrhea • C.trachomatis • VRE • MRSA • C.difficile • Platforms • GeneXpert • Film array technology • PNA-FISH • BD Max • Septifast multiplex PCR • Nanosphere

8. Rapid diagnosis of M.tuberculosis • Xpert MTB/Rif assay has revolutionized detection • TAT 2hrs compared to conventional culture (2 -4 weeks +++) • MTBDRplus assay • Used for rapid confirmation of MDR (direct samples)

9. Bloodstream infections • Bacteraemia/Sepsis • Blood culture • Common (Both community and nosocomial) • Mortality 14-34% • Risk of death increase with 7% every hour until the start of appropriate therapy • High attributable cost • 2 bottle system • Prelim results in 1-3 days • Final results > 5 days • Difficult to modify therapy • Is there an alternative?

10. Peptide nucleic acid Fluorescent in situ Hybridization • (PNA-FISH) • Detects S. aureusspecific 16SrRNA directly from blood cultures • Sensitivity and specificity ~ 100% • Assays for Candida species, Enterococci and Gram negatives (E.coi vs. Pseudomonas) • Septifast (Roche) • SepsiTest (Molzym) • Whole blood • Multiplex realtime PCR • Targets 25 potential pathogens • TAT 3-30hrs • Whole blood • 16SrRNA • PCR combined with sequencing • Targets > 300 potential pathogens • TAT ~12hours • Broad based assays

11. MALDI-TOF MS • Separation of molecules based on the mass to charge ratio • Ionized, separated and detected • Comparedto mass spectra database M-Matrix A-Assisted L-Laser D-Desorption I-Ionization T-Time O-Of F-Flight M-Mass S-Spectrometry

12. Application in sepsis International journal of medical Microbiology 2013 • In-house protocol using Tween80 • Correct identification (GP 91.9%,GN 96.9%, anaerobes 100% to genus, yeast 48.2% to genus)

13. Rapid results for infection control Others: C. trachomatisand N. gonorrhea (CTNG) MRSA Clostridium difficile

14. Why the need for change? • Rapid identification • Accurate diagnosis

15. Conventional culture Limitations Viable organisms Lengthy incubation TAT >24 hours 5S, 16S, 23S rRNA Taxonomy

16. Accuracy - Sensitivity • Smear pos culture pos TB, pooled sensitivity was 98% (97% to 99%); 21 studies, 1936 participants • People with HIV infection, pooled sensitivity was 79% (70% to 86%); 7 studies, 1789 participants • People without HIV infection, it was 86% (76% to 92%); 7 studies, 1470 participants Steingart et al. The Cochrane Library 2014 Issue 1

17. Speciation Bezinniet al. ClinMicrobiol Infect 2010

18. Why the need for change? • Rapid identification • Accurate diagnosis • Clinically relevant

19. Pediatric tuberculosis Clinical diagnosis challenging Smear microscopy poor Xpert MTB/Rif better (still poor) Sampling difficult • Identified transcriptional signatures that can distinguish active TB from latent as well as other diseases in African children Anderson ST et al

20. Using PNA-FISH and Cons PNA-FISH – AMT • No active reporting from laboratory • No AMT guidelines/ support • No difference between LOS as well as Vancomycin usage between cases and controls • Holtzman et al. 2011. J ClinMicrobiol 49 (4): 1581-1582 • PNA-FISH + AMT • Lower hospital cost • Lower LOS • Lower Vancomycin usage • Forrest et al. 2006. J Antimicrob Chemother 58: 154-158

21. Why the need for change? • Rapid identification • Accurate diagnosis • Clinically relevant • Detect resistance mutations

22. Conventional culture Limitations Viable organisms Lengthy incubation TAT >24 hours Phenotypic detection of a resistance method ? Enzyme ? A mutation ? One of many

23. Next generation sequencing • Established in the infectious diseases domain: • Equipment feasible – minimal infrastructural requirements • Uncomplicated workflows • Reasonable cost DNA Preparation Sequencing Bioinformatics

24. Changing the Paradigm

25. Data generated

26. Applications of WGS • Correlate with phenotype • Compensatory mutations • Discovery of new mutations • Resolve discordance of routine molecular assays • Discover new targets for drug development

27. Why the need for change? • Rapid identification • Accurate diagnosis • Clinically relevant • Detect resistance mutations • Epidemiological tracking

28. Outbreaks • Epidemiologic curves • Strain relatedness • Clusters/hotspots • Map transmission patterns • Discover mutations (“drift” and “shift” phenomena) • Monitor changes over time

29. Why the need for change? • Rapid identification • Accurate diagnosis • Clinically relevant • Detect resistance mutations • Epidemiological tracking • Detect new and re-emerging diseases

30. NovalArenavirus

31. Why the need for change? • Rapid identification • Accurate diagnosis • Clinically relevant • Detect resistance mutations • Epidemiological tracking • Detect new and re-emerging diseases • Research – target product profiles, new drugs and vaccines

32. MALDI-TOF MS for beta-lactamase detection Hrabaket al. ClinMicrobiol Rev 2013 • Detects degradation products of the antibiotic • Validated for KPC, VIM, IMP, NDM, OXA etc • Good sensitivity • Also new methods to distinguish different types of beta-lactamases • Other mechanisms e.g. rRNAmethyltransferases looks promising but not for routine

33. Molecular determinants of virulence Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of resistance. ClinMicrobiol Rev 2003:16(3)

34. Resistance – “Superbug” mutations

35. Why the need for change? • Rapid identification • Accurate diagnosis • Clinically relevant • Detect resistance mutations • Epidemiological tracking • Detect new and re-emerging diseases • Research – target product profiles, new drugs and vaccines • Cost effective

36. The costs of false-positive diagnoses are poorly defined and often underestimated • Morbidity and mortality of undiagnosed conditions, increased cost associated with TB therapy, increase in acquired resistance • Diagnostic accuracy (i.e., sensitivity and specificity) is an inadequate proxy of outcomes important to patients and public health • Disease diagnosis and management is a dynamic process, assumption that all patients will be tested with a specific test is incorrect • Diagnostic testing often competes for resources with other TB-specific interventions • To role out Xpert MTB/Rif throughout India (at a very cost effective price) would consume the entire health budget vs. improving access to healthcare and effective microscopy

37. Costs • Whole genome sequencing • High set-up cost • 80 $ per sequence (50 x coverage) • Sequencing facilities is a potential option • MALDI-TOF MS • High set-up cost • Low-consumables, high throughput (0.50 $ per isolate)

38. Challenges toward implementation • Lack of expertise (leadership and bench level) to perform more complicated tests (molecular testing) • Lack resources for verification • Most assays await FDA approval • Interpretation issues e.g. MALDI-TOF MS new species (don’t now the outcome) limited clinical evidence • Lack of pK/pd (in vivo) • Point of care so far disappointing • Lack of coordination between clinical laboratories and public heath directives • Shortage of staff and low incentives will continue to get worse

39. Solutions/recommendations • Translating innovative research into actual use (timely, partnerships) • Competent staff (increased recruitment and incentives) • Optimization of point of care • Bi-directional communication between laboratories, clinicians and public health

40. Thank you