SCI 8008SEF Medical Microbiology & Virology II

Lecture 1
Advanced laboratory technologies
in Clinical Microbiology – Multiplex PCR

By Dr. Andy YY CHEUNG

[email protected]
The BioFire® Respiratory 2.1
(RP2.1) Panel (FDA Cleared)-22 targets
• VIRUSES:
Adenovirus Influenza A virus Respiratory syncytial virus
Coronavirus 229E Influenza A virus A/H1 Human Metapneumovirus
Coronavirus HKU1 Influenza A virus A/H3 Human
Coronavirus NL63 Influenza A virus A/H1-2009 Rhinovirus/Enterovirus
Coronavirus OC43 Influenza B virus
Severe Acute Respiratory Parainfluenza virus 1
Syndrome Coronavirus 2 Parainfluenza virus 2
(SARS-CoV-2)
Parainfluenza virus 3
Parainfluenza virus 4
Questions
1. Why are these viral pathogen targets included?

2. Why are Influenza A virus A/H1 and Influenza A virus A/H3 subtyped?

3. Why are Parainfluenza viruses 1 / 2 / 3 / 4 differentiated?

4. Why is Human Rhinovirus/Enterovirus not differentiated?

The BioFire® Respiratory 2.1
(RP2.1) Panel (FDA Cleared)-22 targets
• BACTERIA:
• Bordetella pertussis
• Bordetella parapertussis
• Chlamydophila pneumoniae (Chlamydia pneumoniae)
• Mycoplasma pneumoniae
Questions
1. Why are these bacterial pathogen targets included?

2. What are atypical pathogens?

3. Why are S. pneumoniae and H. influenzae not detected?

4. What is the specimen type for BioFire® Respiratory 2.1

(RP2.1) Panel?
BIOFIRE Respiratory
® 2.1 plus Panel
• RP2.1 Panel +MERS

BioFire® Respiratory 2.1-EZ (RP2.1- EZ) Panel

(EUA)*
• Product not available outside U.S.A.
• Four types of Parainfluenza Virus (PIV1, PIV2, PIV3 and PIV4) can be
detected and will be reported as Parainfluenza Virus Detected (type
information is not reported).
The BioFire® FilmArray® System - How It
Works

https://www.youtube.com/watch?v=V5fUwjodmz4
BioFire® FilmArray® Blood Culture
Identification Panels - 43 targets
• GRAM-NEGATIVE BACTERIA: • GRAM-POSITIVE BACTERIA:
• Acinetobacter calcoaceticus-baumannii complex • Enterococcus faecalis
• Bacteroides fragilis • Enterococcus faecium
• Enterobacterales • Listeria monocytogenes
• Enterobacter cloacae complex • Staphylococcus spp.
• Escherichia coli • Staphylococcus aureus
• Klebsiella aerogenes • Staphylococcus epidermidis
• Klebsiella oxytoca • Staphylococcus lugdunensis
• Klebsiella pneumoniae group
• Streptococcus spp.
• Proteus spp.
• Streptococcus agalactiae
• Salmonella spp.
• Streptococcus pneumoniae
• Serratia marcescens
• Streptococcus pyogenes
• Haemophilus influenzae
• Neisseria meningitidis
• Pseudomonas aeruginosa
• Stenotrophomonas maltophilia
BioFire® FilmArray® Blood Culture
Identification Panels - 43 targets
• ANTIMICROBIAL RESISTANCE GENES: • YEAST:
• Carbapenemases • Candida albicans
• IMP • Candida auris
• KPC
• Candida glabrata
• OXA-48-like
• NDM • Candida krusei
• VIM • Candida parapsilosis
• Colistin Resistance • Candida tropicalis
• mcr-1 • Cryptococcus (C. neoformans/C. gattii)
• ESBL
• CTX-M
• Methicillin Resistance
• mecA/C
• mecA/C and MREJ (MRSA)
• Vancomycin Resistance
• vanA/B
Questions
1. Why are these pathogen targets included?

2. What is the specimen type for BioFire® FilmArray® Blood Culture

Identification Panels?

3. Why are antimicrobial resistance genes detected?

4. Why are yeasts detected?

The BioFire® FilmArray®
Gastrointestinal (GI) Panel – 22 targets
• BACTERIA: • DIARRHEAGENIC ESCHERICHIA
• Campylobacter COLI/SHIGELLA:
(C. jejuni / C. coli / C. upsaliensis) • Enteroaggregative E. coli (EAEC)
• Clostridioides (Clostridium) • Enteropathogenic E. coli (EPEC)
difficile (toxin A/B) • Enterotoxigenic E. coli (ETEC) lt/st
• Plesiomonas shigelloides • Shiga-like toxin-producing E.
• Salmonella coli (STEC) stx1/stx2
• E. coli O157
• Yersinia enterocolitica
• Shigella/Enteroinvasive E. coli (EIEC)
• Vibrio (V. parahaemolyticus / V.
vulnificus / V. cholerae)
• Vibrio cholerae
Questions
1. Why are these pathogen targets included?

2. Why are Vibrio cholerae and E. coli O157 detected specifically?

3. Why and how is Clostridioides (Clostridium) difficile toxin A/B

detected?

4. Why and how are EAEC / EPEC / ETEC / STEC / EIEC detected?
The BioFire® FilmArray®
Gastrointestinal (GI) Panel – 22 targets
• PARASITES: • VIRUSES:
• Cryptosporidium • Adenovirus F40/41
• Cyclospora cayetanensis • Astrovirus
• Entamoeba histolytica • Norovirus GI/GII
• Giardia lamblia • Rotavirus A
• Sapovirus (I, II, IV, and V)
Questions
1. Why are these pathogen targets included?

2. Why are only Adenovirus types 40 and 41 detected?

3. Why are only Norovirus GI/GII detected?

4. Which are the most common viruses that cause gastroenteritis in HK?

5. What is the specimen type for BioFire® FilmArray® Gastrointestinal (GI)

Panel?
The BioFire® FilmArray®
Meningitis/Encephalitis (ME) Panel – 14 targets
• BACTERIA: • VIRUSES:
• Escherichia coli K1 • Cytomegalovirus (CMV)
• Haemophilus influenzae • Enterovirus (EV)
• Listeria monocytogenes • Herpes simplex virus 1 (HSV-1)
• Neisseria meningitidis • Herpes simplex virus 2 (HSV-2)
• Streptococcus agalactiae • Human herpesvirus 6 (HHV-6)
• Streptococcus pneumoniae • Human parechovirus (HPeV)
• Varicella zoster virus (VZV)
• YEAST:
• Cryptococcus
(C. neoformans/C. gattii)
Questions
1. Why are these pathogen targets included?

2. Why is Escherichia coli K1 specifically detected?

3. What is the specimen type for BioFire® FilmArray®

Meningitis/Encephalitis (ME) Panel?
The BioFire® FilmArray®
Pneumonia (PN) Panel – 33 targets
• BACTERIA: (Semi-Quantitative)
• [Bacteria reported with bins of 104, 105, 106, or ≥107 copies/mL
• Acinetobacter calcoaceticus- • Klebsiella aerogenes • Staphylococcus aureus
baumannii complex
• Klebsiella oxytoca • Streptococcus agalactiae
• Enterobacter cloacae complex
• Klebsiella pneumoniae group • Streptococcus pneumoniae
• Escherichia coli
• Moraxella catarrhalis • Streptococcus pyogenes
• Haemophilus influenzae
• Proteus spp.
• Pseudomonas aeruginosa
• Serratia marcescens
The BioFire® FilmArray®
Pneumonia (PN) Panel – 33 targets
• ATYPICAL BACTERIA: • VIRUSES:
(Qualitative) • Adenovirus
• Chlamydia pneumoniae • Coronavirus
• Legionella pneumophila • Human metapneumovirus
• Mycoplasma pneumoniae • Human rhinovirus/enterovirus
• Influenza A virus
• Influenza B virus
• Parainfluenza virus
• Respiratory syncytial virus
Questions
1. Why are some bacterial pathogen targets reported semi-
quantitively?

2. Why are some bacterial pathogen targets reported qualitatively?

3. Why are all viral pathogen targets reported qualitatively?

4. What is the specimen type for BioFire® FilmArray®

Pneumonia (PN) Panel?
The BioFire® FilmArray®
Pneumonia (PN) Panel – 33 targets
• ANTIMICROBIAL RESISTANCE GENES:
• ESBL: • Methicillin resistance:
• CTX-M • mecA/C and MREJ (MRSA)

• Carbapenemases:
• IMP
• KPC
• NDM
• OXA-48-like
• VIM
The BioFire®
Joint Infection (JI) Panel – 39 targets
• GRAM-POSITIVE BACTERIA: • GRAM-NEGATIVE BACTERIA:
• Anaerococcus prevotii/vaginalis • Bacteroides fragilis
• Clostridium perfringens • Citrobacter
• Cutibacterium avidum/granulosum • Enterobacter cloacae complex
• Enterococcus faecalis • Escherichia coli
• Enterococcus faecium • Haemophilus influenzae
• Finegoldia magna • Kingella kingae
• Parvimonas micra • Klebsiella aerogenes
• Peptoniphilus • Klebsiella pneumoniae group
• Peptostreptococcus anaerobius • Morganella morganii
• Staphylococcus aureus • Neisseria gonorrhoeae
• Staphylococcus lugdunensis • Proteus spp.
• Streptococcus spp. • Pseudomonas aeruginosa
• Streptococcus agalactiae • Salmonella spp.
• Streptococcus pneumoniae
• Streptococcus pyogenes • Serratia marcescens
The BioFire®
Joint Infection (JI) Panel – 39 targets
• ANTIMICROBIAL RESISTANCE GENES:
• Carbapenemases • Methicillin Resistance
• IMP • mecA/C and MREJ (MRSA)
• KPC • Vancomycin Resistance
• NDM • vanA/B
• Oxa-48-like
• VIM
• ESBL • YEAST:
• CTX-M • Candida spp.
• Candida albicans
Questions
1. Why are those highlighted pathogen targets included?

2. What is the specimen type for BioFire® Joint Infection (JI) Panel?
FDA-cleared examples
The BioFire® Respiratory 2.1 (RP2.1) Panel (FDA Cleared)
• https://www.fda.gov/media/137583/download
• https://www.accessdata.fda.gov/cdrh_docs/pdf20/DEN200031.pdf
• https://www.biomerieux.com/content/dam/biomerieux-com/investor/regulated-
informations/2021/releases/pr_biomerieux_-_de_novo_biofire_rp2.1_-
_20210318_final.pdf.coredownload.pdf
The BioFire® Joint Infection (JI) Panel
• https://www.biofiredx.com/wp-content/uploads/BFR0002-0332-01-Lab-CLSI-Document-for-
BIOFIRE-JI-Panel.pdf
• https://www.biofiredx.com/wp-content/uploads/BFR0000-9503-02-BioFire-JI-Verification-Tech-
Note.pdf
• https://www.accessdata.fda.gov/cdrh_docs/pdf20/DEN200066.pdf
Roche cobas ® 6800/8800 System
• cobas® Babesia • cobas® MTB • cobas® SARS-CoV-2
• cobas® CHIKV/DENV • cobas® MTB-RIF/INH Qualitative
• cobas® CMV • cobas® MAI
• cobas® DPX • cobas® EBV
• cobas® HEV Test • cobas® Influenza A/B • cobas® HBV
• cobas® MPX & RSV UC • cobas® HCV
• cobas® WNV Test • cobas® SARS-COV-2 • cobas® HIV-1/HIV-2
• cobas® Zika Test • cobas® SARS-CoV-2 & Qualitative
Influenza A/B

MPX: HIV, HCV and HBV; DPX: parvovirus B19 (B19V) and hepatitis A virus (HAV)
cobas ® DPX test
• The cobas® DPX test is an in vitro test for the direct quantitation of
parvovirus B19 genotypes 1, 2, and 3 DNA and the direct qualitative
detection of Hepatitis A virus (HAV) genotypes I, II, and III RNA in
human plasma.
• This test is intended for use as an in-process test to quantify
parvovirus B19 DNA alone or to simultaneously quantify parvovirus
B19 DNA and detect HAV RNA in plasma intended for further
manufacture collected from donors of whole blood, blood
components, or plasma. Plasma from all donors or manufacturing
pools may be tested as individual samples or in pools comprised of
aliquots of individual samples.
cobas ® MPX
• The cobas® MPX test, for use on cobas® 5800/6800/8800 Systems is a qualitative in vitro test for
the direct detection of Human Immunodeficiency Virus Type 1 (HIV1) Group M RNA, HIV-1 Group
O RNA, Human Immunodeficiency Virus Type 2 (HIV-2) RNA, Hepatitis C Virus (HCV) RNA, and
Hepatitis B Virus (HBV) DNA in human plasma and serum.
• This test is intended for use to screen donor samples for HIV-1 Group M RNA, HIV-1 Group O RNA,
HIV-2 RNA, HCV RNA, and HBV DNA in plasma and serum samples from individual human donors,
including donors of whole blood, blood components, and other living donors.
• This test is also intended for use to screen organ and tissue donors when donor samples are
obtained while the donor's heart is still beating and in testing of cadaveric (non-heart beating)
donors. Plasma and serum from all donors may be screened as individual samples.
• For donations of whole blood and blood components, plasma and serum samples may be tested
individually or plasma may be tested in pools comprised of aliquots of individual samples. For
donations from cadaveric (non-heart beating) organ and tissue donors, samples may only be
screened as individual sample.
• This test is not intended for use as an aid in diagnosis of infection with HIV, HCV, or HBV.
Panther Fusion® Respiratory Assays
Aptima® STIs
• Aptima Combo 2® (for chlamydia / gonorrhea)
• Aptima Trichomonas vaginalis assay
• Aptima Mycoplasma genitalium assay
• Aptima Herpes Simplex Viruses 1 & 2 assay
• Aptima Zika virus assay
Hologic Transplant Assays
• Aptima® CMV Quant Assay

• Panther Fusion® BKV Quant Assay

• Panther Fusion® EBV Quant Assay

Aptima® Virology Assays
Design your own multiplex
QuantStudio™ 3 Real-Time PCR System,
96-well
ViiA™ 7 Real-Time PCR System with 96-Well
Block
• Detection Method
• SYBR, Primer-Probe Detection
• Optics
• 6 Emission Filters (500-720 nm),
• 6 Excitation Filters (450-670 nm),
• OptiFlex™ System (enhanced
fluorescence detection)
LightCycler® 480 System
LightCycler® 480 System
Filter Set of the LightCycler® 480 Instrument II

Try this-> https://www.biosearchtech.com/qpcr-multiplex-spectral-overlay-tool

 LightMix Universal Color Compensation
Hexaplex Plus

https://pim-eservices.roche.com/eLD/api/downloads/8b966057-cd2d-e911-a49d-00215a9b3428?countryIsoCode=pi
What shall you do to develop a multiplex PCR?
1. Check primers and probes interactions in silico
https://www.thermofisher.com/hk/en/home/brands/thermo-
scientific/molecular-biology/molecular-biology-learning-center/molecular-
biology-resource-library/thermo-scientific-web-tools/multiple-primer-
analyzer.html
2. Redesign if interaction is expected
3. Choose correct reporter dyes according to PCR machine
4. Evaluate LoD in a monoplex manner
5. Reject the primers/probe pair if LoD > 10 copies of template per
reaction
What shall you do to develop a multiplex PCR?
6. Perform color compensation in a multiplex manner
7. Perform no template control test and negative control (H2O) test in
duplicate or triplicate
8. Redesign primers and probe design if there is cross-reactivity
i.e. any fluorescence signal is detected with NTC or NC

?Why?
What shall you do to develop a multiplex PCR?
9. Perform multiplex with different target template individually
i.e. multiple primers & probe pairs + 1 target template only (high conc. -> low
conc. by 3 or 10-fold serial dilution)
10. Compare the LoD of multiplex with that of monoplex
11. Redesign primers and probe design if detection sensitivity
decreases > 1-log
12. You may optimize primers / probes concentration(s) / PCR
condition(s) instead, but it usually does not work
13. Accept the multiplex design if LoD is comparable (i.e. LoD increase
within 1-log)
What shall you do to develop a multiplex PCR?
• Do NOT forget to test template concentration effect if more than 1 PCR targets are
expected to be positive. Be aware of PCR reagents competition!

Tips:
• Use FAM channel for important target
• Use weak channel/reporter dye for internal gene
• Minimize primers and probe concentration for internal gene
• Choose reporter dyes with far-apart spectrum
• Crosstalk is fine if both channels shall be positive for identical target
• Multiplex > 4 PCR targets usually failed
• Carefully choose the annealing temperature
! Why ?
References
• https://www.biofiredx.com/products/the-filmarray-panels/
• https://diagnostics.roche.com/
• https://www.hologic.com/hologic-products/diagnostic-
solutions/panther-system
• https://www.thermofisher.com/hk/en/home.html
Supplementary information
• https://www.clinicalmicrobiologyandinfection.com/article/S1198-
743X(14)64501-8/fulltext
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324208/#:~:text=Cl
ostridium%20difficile%20PCR%20is%20designed,for%20a%20toxin%2
0regulatory%20pathway.
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548099/
• https://cdis.chp.gov.hk/CDIS_CENO_ONLINE/disease.html
• https://www.chp.gov.hk/en/statistics/data/10/641/717/6992.html