Multiplexing; what is it good for? Dr. S.M. Jazayeri MD, PhD, Virologist. Tehran University of Medical Sciences

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1 Multiplexing; what is it good for? Dr. S.M. Jazayeri MD, PhD, Virologist Tehran University of Medical Sciences

2 In a clinical laboratory setting

3 In a clinical laboratory setting 1.Speed is utmost

4 In a clinical laboratory setting 1.Speed is utmost 2.Sensitive methods

5 In a clinical laboratory setting 1.Speed is utmost 2.Sensitive methods 3.Cost

6 Introduction Traditional tests typically becomes positive at least hours after sampling. However, early detection and adequate treatment of causative pathogens is critical for a favorable outcome in critically ill patients. To date, several attempts have been made to translate the more universal diagnostic approach of these tests including blood-cultures into molecular-based assays. Development of a new sensitive and specific multiplex-based assay (simultaneous detection of multiple pathogens in a single reaction).

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8 Multiplexing APPLICATIONS In Microbiology Simultaneous diagnosis and studying agents of infectious diseases, Clarifying infectious diseases process, Epidemiological studies, Clarifying of emerging (and re-emerging) agents, Genotyping of multiple genotypes

9 What is the value of multiplexing? 1. It is unlikely that clinicians are correct about the exact pathogen 2. Rapid negative leads to a focus on other pathogens 3. Positive is believable and can lead to a reduction in other tests and drugs 4. Better value for money per pathogen. 5. Saves time, once established 6. Great for limited and valuable samples

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11 LightCycler SeptiFast The LightCycler SeptiFast (Roche) Can identify 19 pathogens (8 Gramnegative bacteria including 6 Grampositive bacteria and 5 fungi) from 1.5 ml of whole blood in 3.5 to 5 hours.

12 MagicPlex Sepsis Test (Seegene, Seoul, Korea) Associates conventional PCR and real-time PCR. This system allows the detection of more than 90 pathogens at the genus level, including 25 pathogens at the species level (19 bacteria and 6 fungi) and the resistance genes meca, vana and vanb.

13 PCR and sequencing: SepsiTest The SepsiTest (Molzym, Germany) The amplicons are sequenced and analysed by BLAST. This approach allows the identification of 345 pathogens (bacteria and fungi). The time to result is ranging from 8 to 12 hours.

14 PCR/ESI-MS analysis The PLEX-ID (Abbott Molecular) The PLEX-ID can detect and identify up to 800 pathogens. The PLEX-ID associates microbe detection by PCR and amplicon analysis by ESI-MS.

15 DNA GeneChip technology DNA microarray (or chip ) technology is the ideal tool for the rapid simultaneous analysis of thousands of genes (at least about ) in a single experiment. The total time needed to complete the assay is 8 h from specimen extraction to microarray detection, in a single working day.

16 PCR- MS PlexID: 6 h. PCR Multiplex : 6 h. MagicPlex: 6 h. SeptiFast : 6 h. SeptiTest : 6 h. Multiplexing, Nucleic Acid Testing for Infectious Diseases Diagnostics Microarray (2.5-5 h) Verigene PCR + Sequencing (12 h) MALDI- TOFF - MS PlexID: <1 h.

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18 Negative vs Positive Results 5% 34% Negative 61% Positive with same request Positive with different request

19 Negative vs Positive Results Using the algorithms properly? 5% 34% Negative 61% Positive with same request Positive with different request

20 Flu A Flu B Flu C PF1 PF2 PF3 PF4 Rhino CoV Adenov HmPV RSV Myco Ent Mixed Negative?clinicians misdiagnosing other pathogens as H1N1? Using the algorithms properly N=1820 samples Collected between May and July 2009 Sent to the virus lab for H1N1 testing

21 Single/Multiple Pathogen Report 13% 87% Single pathogen Multiple pathogen

22 Transplant Panels Adenovirus, BKV, CMV, EBV NEGATIVE 55% POSITIVE 45%

23 BCE Urine and Plasma Pathogen Results Comparison 43% 57% Different pathogen Same pathogen

24 Multiplex Pathogen Report in Kidney Transplant Recipients (Urine) 26% 7% 36% BKV CMV,BKV CMV,EBV CMV,BKV,EBV 5% 7% 19% CMV EBV

25 Multiplex Pathogen Report in Kidney Transplant Recipients (Plasma) 1% 3% 22% 5% 22% 47% CMV CMV,BKV CMV,EBV BKV EBV Adenovirus

26 Influenza Multiplex Test Report 8% 6% 2% 2% 82% Negative FLU B FLU A-H1N1 FLU A Rhino virus

27 Influenza Multiplex Test Report 8% 2% 6% 2% 82% Never Used Other Respiratory Multiplex Tests Negative FLU B FLU A-H1N1 FLU A Rhino virus

28 EYE Infection Multiplex Test Report 34% Negative Positive 66%

29 EYE Multiplex Test Specimen Type 12% 13% Aqueous fluid Eye swab Vitrous fluid 75%

30 EYE Positive Reports Based on Specimen Type %39 %25 %14 %61 %75 %86 AQUEOUS FLUID EYE SWAB VITROUS FLUID Negative Positive

31 EYE Multiplex Test Pathogen Type Results HSV-1 14% 5% 29% VZV 14% CMV VZV,Adenovirus 14% 24% VZV,EBV Adenovirus

32 Viral Encephalitis Multiplex Panel Details HSV-2 1% Entero 1% HHV-7 2% HHV-6 1% HSV-1 13% Adeno 15% VZV 15% EBV 13% CMV 39%

33 Sexually Transmitted Diseases Panel Ureaplasma. parvum 11% 6% 5% 5% 17% 56% Chlamydia ureaplasma. urealyticum Neisseria. gonorrhoeae HSV1 Mycoplasma. hominis

34 HPV Result Positive 45% Negative 55%

35 Multiple HPV Type Report 22% Single type 78% Multiple type

36 HPV Result (High& Low risk) 20% High Risk 33% Low Risk 47%

37 HPV Multiplex Genotype Report 45 (2%) 51 (7%) 52 (1%) 56 (3%) 59 (1%) 58 (3%) 39 (11%) 6 (42%) 35 (3%) 33 (1%) 31 (5%) 16 (12%) 18 (3%) 11 (6%)

38 Conclusion Multiplex assays have been introduced to the clinic allows the detection of numbers of pathogens at the same time. In the future, more developed molecular tests incorporate wider test panels of pathogens expected to provide greater diagnostic sensitivity, giving clinicians greater confidence in a rapid test result. The current issue is results interpretation about clinically important pathogens.

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