ESCMID Online Lecture Library. by author

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1 . Diagnostic molecular microbiology: first and next 25 years Mario Poljak Institute of Microbiology and Immunology Faculty of Medicine, University of Ljubljana, Slovenia

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3 Molecular methods methods based on the detection and partial or complete characterisation of microbial DNA or RNA in the clinical sample - diagnostic tests - epidemiological tools - research tools

4 Molecular methods dramatically changed clinical microbiology allowed discovery of several clinically important and previously unrecognized or uncultivable pathogens reduced the dependency of laboratory on culture-based methods became gold diagnostic standards for several microorganisms (C. trachomatis, HSV encephalitis, enteroviral meningitis, CMV reactivation, hepatitis C, )

5 . Diagnostic molecular microbiology: first and next 25 years Mario Poljak Institute of Microbiology and Immunology Faculty of Medicine, University of Ljubljana, Slovenia

6 David H. Persing American Society for Microbiology, Jan 1993

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8 standardisation automation miniaturisation

9 standardisation automation miniaturisation

10 - Sensitive and specific detection of PCR products - Control of PCR-amplicon carryover contamination

11 standardisation automation miniaturisation

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13 Molecular methods Commercially interesting microorganisms vs. Commercially less interesting microorganisms

14 Chlamydia trachomatis HIV HBV HCV HPV (CMV, EBV)

15 Roche Abbott Digene (Qiagen) Bayer Organon Teknika/bioMerieux Gen-Probe Becton-Dickinson PCR LCR, PCR Hybrid Capture b-dna, TMA NASBA TMA SDA

16 Cobas Ampliprep/TaqMan system (Roche)

17 Molecular diagnostic systems fully automated sample-to-result fashion - multiple tests performed concordantly - sample number flexibility - STAT test prioritization - random access

18 Panther System (Hologic-Gen-Probe)

19 Cobas 6800/8800 (Roche)

20 Molecular methods Commercially interesting microorganisms vs. Commercially less interesting microorganisms

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22 automated isolation of DNA or RNA + real-time PCR

23 automated isolation of DNA or RNA + real-time PCR

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26 automated isolation of DNA or RNA + real-time PCR

27 Real-time PCR PCR instrumentation and chemistry which allow the simultaneous amplification and quantification of specific nucleic acid sequences a combination of rapid thermal cycling and cycle-by-cycle basis detection of the reaction kinetics

28 Integrated systems automated isolation of DNA or RNA + real-time PCR

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30 BD MAX System (Becton Dickinson) Jaguar system (HandyLab)

31 GeneXpert (Cepheid) single-use disposable cartridges

32 3M Integrated Cycler (Focus)

33 FilmArray (Idaho Technology; BioFire Diagnostics; BioMerieux)

34 FDA approved: October 12, 2015

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36 Evaluating Multiplex Syndromic Panels? Improved Test Performance Improved Patient Care Improved Clinical and Cost Outcomes

37 Clin Infect Dis 2016;62:817-23

38 FDA approved: October 12, 2015

39 J Clin Microbiol 2016;54:785-7

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41 - specificity 1544/1556 = 99.2 % - 12 positives = 5 true positives and 7 false positives

42 standardisation automation miniaturisation

43 Lab-on-a-Chip microfluidic device that has closed channels, wells, pumps, virtual or micromechanical valves and other structures in which the sample is manipulated chemical reactions at a scale that is 100 to 10,000-fold smaller than traditional assays

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45 Pal R, Yang M, Lin R, et al. An integrated microfluidic device for influenza and other genetic analyses. Lab Chip 2005; 5:

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47 deep sequencing next generation sequencing metagenomics microbiome

48 J Clin Microbiol 2016; 54:

49 J Clin Microbiol 2016; 54:

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51 mbio 2016;7:3 - whole-genome sequencing of 308 invasive S. aureus isolates corresponding to a pan- European population snapshot - identification of high-risk clones on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content - in silico predictions of antibiotic resistance profiles at least as reliable as phenotypic testing

52 J Clin Microbiol 2015;53: rapid identification of the bacterial species and simultaneous determination of their antibiotic susceptibility profiles initial short cultivation step in the absence and presence of different antibiotics combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e. resistance) and no growth (i.e. susceptibility) a proof-of-concept study: - urinary tract infections - antibiotic susceptibility profiles of E. coli for ciprofloxacin and trimethoprim - 100% accuracy in 3.5 h

53 . Diagnostic molecular microbiology: first and next 25 years Mario Poljak Institute of Microbiology and Immunology Faculty of Medicine, University of Ljubljana, Slovenia

54 3R rule Rapid Relevant Right (in clinically relevant time frames) (clinically relevant) (specific and sensitive, analytical category) Right > Relevant > Rapid Relevant = Rapid > Right

55 faster cheaper 24/7

56 Two testing places evolving concept H H H H POC POC POC POC clin micro lab POC = point-of-care

57 POC = point-of-care N Engl J Med 2013;368:

58 World's smallest PCR-based molecular diagnostics platform? Liat - Roche Cube - Spartan Bioscience Omni Cepheid Alere i - Alere/Abbott FlashDirect - Thermal Gradient Palm - Mesa Biotech Palm PCR - Ahram Biosystems

59 cobas Liat strep A assay vs. S. pyogenes LightCycler PCR assay sensitivity = 100% specificity = 98.3 % positive predictive value = 97.7% negative predictive value = 100.0% J Clin Microbiol 2016;54:815

60 Where is my instrument???

61 An ipad-like, sample-to-answer prototype Abou Tayoun AN et al. Am J Clin Pathol 2014;141:17-24.

62 A smartphone dongle as point of care device Sci Trans Med 2015; 7: 273re1

63 Lab-on-a-USB key microfluidic devices integrated with USB key data storage devices a device could be attached to other computational devices such as a cell phone or laptop computer to control molecular assays being done on the microfluidic biochip analysis transmitted to central databases for shared use and metaprocessing

64 J Clin Virol 2015;69:16-21

65 No electricity??

66 Solar thermal PCR system Sci Rep 2014;4:4137.

67 PATH NINA heater low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material thermal standard deviation <0.5 C at operating temperature a cost of approximately 0.06 USD per test for heater reaction materials HIV LAMP amplicon detection via Milenia test strips PLoS One 2014;9:e

68 rapid (<30 min) and sensitive (<10 copies) visual detection of amplified products using ph-sensitive dyes with minimal buffering capacity achieved with loop-mediated isothermal amplification (LAMP) specificity sensitivity

69 J Virol Methods 2016;234:90-5 gold nanoparticles (AuNP) attached to a single-stranded DNA probe for HPV16 and HPV18. LAMP incubation time of 20 min and a temperature of 65 C detection of the LAMP product by AuNP color change after LAMP amplification its products were hybridized with the AuNP probe for 5 min and then detected by the addition of magnesium salt

70 No electricity?? No instrument???

71 Ustar Biotechnologies (Hangzhou, China) Cross Priming Amplification technology developed by Qimin You, while conducting research in Canada & US - instrument free specimen processing - isothermal nucleic acid amplification - visual read-out detection and easy data interpretation - cross contamination prevention - glassified reagents for ambient temperature transport and storage

72 Non-microorganism detection based molecular diagnostic approach? (i)

73 direct detection of the presence of microorganism(s) in clinical specimens determination of gene expression patterns in patient s blood mononuclear cells specific for particular microorganism(s)

74 Sci Transl Med 2013;5:203ra126 Sci Transl Med 2016;8:322ra11 J Infect Dis 2015;212: adults vs. 41 healthy volunteers sensitivity 89% specificity 94% overall accuracy 87% - 238/273 concordant with clinical adjudication 118 patients sensitivity 95% specificity 92%

75 Transcriptional profile discrimination between bacterial and viral lower respiratory tract infection (LRTI) Suarez et al., J Infect Dis 2015;212:

76 culture-confirmed tuberculosis vs. culture-negative tuberculosis, diseases other than tuberculosis, latent tuberculosis 51-transcript signature identified that distinguishing tuberculosis from other diseases in the South African and Malawian children a risk score based on the signature for tuberculosis and for diseases other than tuberculosis showed a sensitivity of 82.9% (68.6 to 94.3) and a specificity of 83.6% (74.6 to 92.7) for the diagnosis of culture-confirmed tuberculosis the sensitivity of the Xpert MTB/RIF assay for molecular detection of M. tuberculosis DNA in cases of culture-confirmed tuberculosis was 54.3% (37.1 to 68.6), specificity 100% RNA expression signatures provided data that helped distinguish tuberculosis from other diseases in African children with and those without HIV infection

77 - prospective cohort study - Healthy South African adolescents; years; infected with M. tuberculosis for 2 years - a prospective signature of risk derived from whole blood RNA sequencing data by comparing participants who developed active tuberculosis disease (progressors) with those who remained healthy (matched controls) - 46 progressors and 107 matched controls - 16 gene signature of risk identified Lancet 2016;387: the signature predicted tuberculosis progression with a sensitivity of 66.1% (95% CI ) and a specificity of 80.6% (95 CI ) in the 12 months preceding tuberculosis diagnosis

78 Non-microorganism detection based molecular diagnostic approach? (ii)

79 Categorising tumours by the genetic and epigenetic changes in their cells, rather than by anatomy and histology

80 mbio 2014; 5:e

81 J Virol 2014; 88: modulating the host response is a promising approach to treating influenza host transcriptomic profiling used to computationally predict drugs that reverse the host response to H7N9 infection six FDA-approved drugs identified that could potentially be repurposed to treat H7N9 and other pathogenic influenza viruses

82 Molecular real-time in vivo tests?

83 rapid detection and localization of bacterial infections in living animals Nat Med 2014;20:301-6 molecular imaging approach for the specific, noninvasive detection of S. aureus based on the activity of the S. aureus secreted nuclease, micrococcal nuclease several short synthetic oligonucleotides, rendered resistant to mammalian nucleases by various chemical modifications and flanked with a fluorophore and quencher

84 Surprise with revolutionary non-molecular technology?

85 16S RNA sequencing for bacterial identification

86 intrinsic fluorescence spectroscopy of whole cells mbio 2013;4:e multistage algorithm correctly classified 99.6% of unknown samples to the Gram level, 99.3% to the family level, and 96.5% to the species level

87 Emerg Infect Dis 2015;21: second noninvasive diagnosis of Plasmodium falciparum infection without drawing blood or using any reagent

88 Clin Infect Dis 2014;59: thermal desorption-gas chromatography/mass spectrometry - prospectively collected breath samples - patients with proven or probable invasive aspergillosis vs. patients without aspergillosis detection of α-trans-bergamotene, β-trans-bergamotene, a β-vatirenene like sesquiterpene, or trans-geranylacetone identified patients with invasive aspergillosis with 94% sensitivity (95% CI, 81% 98%) and 93% specificity (95% CI, 79% 98%)

89 Relative abundance of Aspergillus terpene metabolites in the breath Clin Infect Dis 2014;59:

90 Journal of Clinical Microbiology 2012; 50:

91 Current tuberculosis diagnostics pipeline Pai M, Schito M. J Infect Dis 2015;211:S21-8.

92 Intelligent chips?

93 remote controller Ehrlich GD et al. Intelligent hip implants to battle biofilms. ASM News 2004; 70: remote controller

94 JAMA Published online June 23, 2016 glucose levels in sweat; sodium and chloride levels in sweat

95 Molecular methods - summary dramatically changed clinical microbiology have become more standardized, accurate, reliable, automated, rapid, costeffective, and their clinical value for the diagnosis and management of a several infectious diseases has been proven when applied wisely and selectively, molecular testing represents indispensable part of the routine laboratory practice of clinical microbiologist today future is bright with many promising technologies in pipeline

96 Supplementary slides

97 Molecular testing in clinical microbiology (i) Detection of microorganisms: - that grow slowly in culture (mycobacteria, chlamydia, Bordetella pertussis), - for which reliable cultivation methods are not widely available or not existing (Tropheryma whippelii, human papillomaviruses, novel parvoviruses and novel polyomaviruses), - for minimizing the spread of antimicrobial-resistant bacteria in health care institutions (methicillin resistant S. aureus), - when high possibility of false-negative culture result exists due to current antibiotic therapy (Neisseria meningitidis, Streptococcus pneumoniae, endocarditis), - when cultivation methods are too slow and insensitive for clinically relevant decisions and/or are not cost-effective (HSV encephalitis, enteroviral and parechoviral meningitis, CMV and EBV infection in immunocompromised individuals). - in archival clinical specimens (paraffin-embedded tissue, Pap smears, Giemsa-stained slides)

98 Molecular testing in clinical microbiology (ii) Diagnosis of selected viral infections: - in early phase of acute infection e.g. before seroconversion or in individuals with indeterminate results of confirmatory tests (HIV, HBV, HCV), - in infants born to infected mothers (HIV, HCV, CMV), - for screening of blood, organ and tissue donors in addition to conventional serological markers (HIV, HCV, HBV, West Nile virus), - for resolving diagnostic uncertainties following serological testing (HIV, HCV, HBV).

99 Molecular testing in clinical microbiology (iii) Molecular identification: - slow growing bacteria (mycobacteria) and fungi, - when common bacteria exhibits an unusual phenotypic pattern (optochin-resistant S. pneumoniae, Pseudomonas aeruginosa in cystic fibrosis patients), - when conventional phenotypic methods are not reliable (nonfermenting Gram negative rods), - simultaneous detection and identification of several microorganism in a single test (respiratory viruses, central nervous system infections, diarrheal diseases), - identification of pathogens for which non-molecular tests are not available (novel parvoviruses, novel polyomaviruses).

100 Molecular testing in clinical microbiology (iv) Molecular detection of antimicrobial resistance: - in bacteria with well defined genetic background of resistance (methicillin resistance in S. aureus, glycopeptide resistance in enterococci, rifampin resistance in M. tuberculosis) - in viruses where phenotypic susceptibility testing is too slow and/or too expensive (HIV, CMV) or not existing (HBV, HCV). Prognosis: - disease progression (HIV-1 viral load, HBV viral load, HPV partial genotyping), - progression to death (HIV-1 viral load), - for predicting risk and distinguishing active disease from asymptomatic infection in transplant recipients (CMV viral load, EBV viral load, BK polyomavirus viral load).

101 Molecular testing in clinical microbiology (v) Prediction and monitoring: - patient response to antiviral therapy (HIV viral load and drug resistance testing in HIV-infected individuals; viral genotype, viral load and IL-28 polymorphism in HCV-infected patients, HBV viral load in HBVinfected patients; drug resistance testing in CMV-infected transplant recipients), - determination of duration of antiviral therapy (viral genotype, viral load kinetics and IL-28 polymorphism in HCV-infected patients), - guiding clinicians in initiating (determination of HIV viral tropism) and changing antiviral therapy (HIV, HBV and CMV drug resistance testing), - deciding when to initiate preemptive therapy in transplant recipients (CMV viral load).