Dr Bhavini Sandip Shah

Director : Microbiology
Department of Microbiology
Neuberg Supratech labs
Ahmedabad.
Biofire : Filmarray
 Introduction
• The FilmArray is a multiplex PCR system that integrates

(a) sample preparation,

(b) amplification,

(c) detection and analysis.

• It is designed to be used with comprehensive panels that offers

testing for sets of pathogens associated with some of today's
MOST pressing healthcare challenges
One system. Fully integrated
The Skeleton of Filmarray
 15 pathogens Meningitis / Encephalitis
Bacteria: Viruses:
E. coli Cytomegalovirus (CMV)
H. influenzae Enterovirus
L. monocytogenes Epstein-Barr virus (EBV)
N. meningitidis Herpes simplex type 1 (HSV-1)
S. agalactiae Herpes simplex type 2 (HSV-2)
S. pneumoniae Human herpesvirus 6 (HHV-6)
Parechovirus
Fungi: Varicella zoster virus (VZV)
Cryptococcus
neoformans / gattii

Sample : Cerebral Spinal Fluid

 Lets discuss....
Viruses Bacteria

CMV Foscarnet
Gancyc E. coli
We Know therapy
H. influenzae
varies in all of
Foscarnet L. monocytogenes
these bacteria.
Gancyc N. meningitidis
(More in compromised)
HHV 6 S. agalactiae
Acyclovir S. pneumoniae
HSV 1 higher dose
( 14-21 days)
chronic if half
heartedly treated)
 
VZV Acyclovir
(along with Cryptococcus fungal
steroids) Neoformans
EBV
MENINGITIS PANEL
 Respiratory Panel
22 pathogens
Viral Bacterial
Adenovirus Bordetella pertussis
Coronavirus 229E Chlamydophila pneumoniae
Coronavirus HKU1 Mycoplasma pneumoniae
Coronavirus OC43
Coronavirus NL63
Human Metapneumovirus
Human Rhinovirus/
Enterovirus
Influenza A
Influenza A/H1
Influenza A/H1-2009
Influenza A/H3
Influenza B
Parainfluenza 1
Parainfluenza 2
Parainfluenza 3
Parainfluenza 4
Sample : Nasopharyngeal Swabs
RSV
RESPIRATORY PANEL
 Blood Culture ID Panel
27 pathogens

Gram + Bacteria: Gram - Bacteria : Fungi:

Enterococcus spp. A. baumannii C. albicans
L. monocytogenes Enterobacteriaceae C. glabrata
Staphylococcus Enterobacter cloacae C. krusei
S. aureus Complex C. parapsiolosis
Streptococcus spp. E. coli C. tropicalis
S. agalactiae (Group B) H. influenzae
S. pyogenes (Group A) K. oxytoca
S. pneumoniae K. pneumoniae
N. meningitidis
Antibiotic Resistance: P. aeruginosa
mecA Proteus
Van A/B S. marcescens
KPC
Sample : Positive Blood culture
 22 pathogens GI Panel

Bacteria: Protozoa:
Aeromonas
Cryptosporidium
Campylobacter
Cyclospora cayetanensis
Clostridium difficile (Toxin A/B)
Entamoeba histolytica
Plesiomonas shigelloides
Giardia lamblia
Salmonella
Vibrio
Viruses:
Vibrio cholerae
Adenovirus F 40/41
Yersinia enterocolitica
Astrovirus
Norovirus GI/GII
Diarrheagenic E. coli / Shigella Rotavirus A
E. coli O157 Sapovirus
Enteroaggregative E. coli (EAEC)
Enteropathogenic E. coli (EPEC)
Enterotoxigenic E. coli (ETEC)
Shiga-like toxin-producing E. coli (STEC)
Shigella/Enteroinvasive E. coli (EIEC)

Sample : Stool resuspended in Cary Blair

 Parameters Meningitis Respiratory

Conventiona Filmarray Conventiona Filmarray -

l methods - Biofire l methods Biofire

Reduction in time from 132 hrs 6 hrs 120 hrs 4 hrs

admission to diagnosis

Diagnostic yield 6% 16.9 % 12 % 49.6 %

Patient management cost X ↓ by 80% X ↓ by 40%

Hospital stay 7 days 3.8 days 5.8 days 3 days

Mortality rate 8% 2% 4% 1%
 Introduction
• A mass spectrometer is usually defined as any device that operates
by a process used to produce a mass spectrum.

• Among these designs is the innovation known as time-of-flight

mass spectrometry (April 1946, TOFMS), a technique that
determines the molecular weight of a substance by accelerating
ions toward a detector.

• The time it takes to travel from the ion source to the detector is
measured, then converted to mass with high accuracy.

• The greater the ratio of mass to charge, the slower the ion speeds
toward the detector as it is accelerated.
• https://masspec.scripps.edu/mshistory/perspectives/rkiser.php
 The MALDI-TOF
Matrix-Assisted Laser Desorption / Ionization
Spectroscopy, Time Of Flight.
 Applications of MALDI
• 1. Pharmaceutical Industry

• 2. Chemical and Petrochemical industry.

• 3. Metal industry

• 4. Forensic Sciences

• 5. Food and Agriculture

• 6. Clinical Diagnostics...........we talk on microbial identification

• 7. Material sciences and many

• www.Brukerindia.com
 Microbial identification :
A clinical tool in Diagnostics

• From Growth

• Bacterial Identification

• Fungal (Candida spp

and filamentous fungii)

• Mycobacterial species
identification.
 Bacterial ID on MALDI
• Misidentification of bacteria have played its role in AMR

• Mortality rate in infected patients have increased.

• Huge financial losses have been documented retrospectively

in addition to loss of life.

• LOS has increased significantly due to delays in

identifications and misidentifications.
• https://www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC

• J Clin Microbiol. 2015 Aug; 53(8): 2793–2794.Published online 2015 Jul 20. Prepublished online 2015 Jun 10. 
 Case 1:Bacterial ID
(2017)

• A 48 year old male patient, known complaints of

Hypertension and Hypothyroidism for 3 to 4 years.

• Admitted with history of RTA on 26/07/2018 at Bhuj. He was

primarily admitted in a local hospital, where he was intubated
and kept on the ventilator. Central line also inserted there.

• On July 28th , patient was shifted to Ahmedabad . He was

admitted with multiple fracture.

• Patient was intubated and had a central line and foley’s at the
time of admission.
• CONTD. Case 1

• Patient was admitted on the evening of 28th July with high Total
counts and fever but stable.

• He was shifted from a primary health centre to this tertiary care unit.
Expected sepsis due to RTA and injury. Antibiotics were changed to
colistin and merupenum.

• Started detiorating on 05th August .

• Surgeon upset. (What more than colistin !!!!!)

• Hospital personal data, 2017
 Date Total Count Date : Temperature
07/28/2017 17960 07/30/2017 99.4'F
18530 (Patient on Colistin and
07/30/2017 merupenum) 08/01/2017 100.3'F
08/02/2017 16550 08/02/2017 100.1'F (single blood cs : Sterile)
08/04/2017 20590 08/03/2017 100.4'F
08/06/2017 21170 08/04/2017 100.2'F
08/08/2017 13610 08/05/2017 100.1'F
08/09/2017 17620 08/06/2017 99.2'F
08/11/2017 10680 08/07/2017 100.6'F (2 sets of Blood cs sent)
08/14/2017 9570 08/08/2017 100.8'F : (4/4 Positive for GNB)
08/09/2017 101'F : Identified on Maldi TOF

08/10/2017 101'F Antibiotic ??????

08/11/2017 98.6'F

08/12/2017 98.6'F

08/13/2017 98.6'F

IMG 08/14/2017 98.6'F

PMG 08/15/2017 98.6'F

GMG
 Lets discuss....
• Blood culture Positive for Gram negative bacilli, Oxidase
Positive bacteria.

• Would you

• 1. Change the antibiotic

• 2. Wait for the culture sensitivity reports

• 3. Interact with the Microbiologist !

 Case 1 contd..

• Organism identified as Chrysobacterium gleum on MALDI-

TOF with a score of 2.3.

• No Interpretation criteria in CLSI for antibiotics against this

bug.

• Patient was deescalated to pip/Taz. Afebrile in 48 hours.

• Research papers available in abundance.

 Fungal Identification

• The application of MALDI TOF is useful in both Candida

spp, and Filamentous fungi.

• Excellent database for candida spp, and definitely saves

lives. Filamentous fungii remains a challenge.

• Can be processed from growth and Positive blood culture

bottles.(Sepsityper)

• Why do we need an early identification !!(Fungus gives us

very little time ….)
 Candida speciation

• Not speciating Candida spp. isolated from a critical

site/specimen is ethically a crime.
 Present scenerio…
• Blood culture/Critical sample Positive for Candida spp.

• Informed Consultant, with request for speciation.

• Request for ID, after repeated reminders request sent.

• Allow candida spp to grow on sabaurauds agar and mature

for 36 hours before you push it into Vitek-2.

• 18 hours for identification protocol, which means a total of

54 hours post positivity.

• Phoneix ??. Chrome , Would the patient hold on with

fungemia for 58 hours with an incorrect drug ????
 Case Study 2
(2016)

• 36 yr old male patient, post renal transplant .

• Hospitalization of 11 days

• 103 degree C fever – 2 sets of blood cultures drawn ( Bactec 9050).

• All 4/4 cultures positive for candida sp. between 22-23 hours.

• Germ tube : Negative,

• Patient put on Echinocandin.

• Request for speciation.(at 18 hours of growth)

• Maldi was expensive for the clinician so Speciation on Vitek-2

 Case 2 Contd.

• After 18 hours low discrimination

• Low discrimination between Candida parapsilosis and
Candida famata (time to ID imp)
• Patient on Echinocandin. : not responding
• Repeat ID on Vitek-2 (Culture was 36 hours old)
• Identified as Candida parapsilosis, good Identification.
• Patient deescalated to fluconazole.
• We lost the patient on the same day after one dose.
 Triazoles—Spectrum of Activity
Fluconazole Itraconazole Voriconazole Posaconazole

C. albicans +++ ++ +++ +++

C. glabrata + + ++ ++
C. krusei -- + +++ ++
C. tropicalis +++ ++ +++ +++
C. parapsilosis +++ ++ +++ +++
C. lusitanae ++ ++ +++ +++
Aspergillus -- ++ +++ +++
Cryptococcus +++ +++ +++ +++
Coccidioides +++ +++ +++ +++
Blastomyces ++ +++ ++ +++
Histoplasma + +++ ++ +++
Fusarium -- -- ++ ++
Scedosporium -- +/- + +/-
Zygomycetes - - - ++
 Zygomycetes -
Scedosporidium -
Echinocandins—Spectrum of Activity

Fusarium -
Histoplasma --
Blastomyces ++
Coccidioides ++
Cryptococcus --
Aspergillus +++
guilliermondii +
lusitanae +++
parapsilosis +
Candida tropicalis +++
krusei +++
glabrata +++
albicans +++
 Let’s do a diagnostic mortality morbidity meet

• Would MALDI have helped save that life ??

• We would have identified it at 24 hours of drawn
cultures.
• 23 hours to positivity and one hour of identification
directly from blood (sepsytitre).
• We reported Candida parapsilosis at almost 80 hours.
Vitek run takes 18 hours for Identification of candida
spp.
• We were so foolish ……..
 Advantages
• Saves time, Saves Life.

• Gives us confidence to report as scoring probability is reported.

• Space occupancy is reasonably compact with routine equipment requirements.

• Sturdy Equipment and software., each run economical.

• Remote access by manufacturer so troubleshooting is easy.

• Using different culture media and culture time, does not influence microbial identification by
MALDI-TOF MS.

• Valentine et al. (2005) , Carbonnelle et al 2007

•
 Disadvantages
• Very Expensive
• Very Expensive
• Very Expensive
• Needs pure culture only (in liquid media).
• There is an inherent limitation- "The database".
• Misidentifications do occur. But not to a great extent (E coli and shigella
spp, Strep Pneum and mitis)
Really nano...
The membrane...
Nanopore Seq. Technology
This is how it shows..
Sr.no Type of Sequencer Sequencing chemistry Generati
. on
1 Genetic Analyzer 3500xL dideoxy chain termination First
(Thermofisher inc.) sequencing know as sanger
sequencing
2 454 GS FLX (Roche inc.) Pyro Sequencing(luciferase Second
enzyme + PPI)
3 SOLiD Sequencing (Thermofisher inc.) Supported Oligo Ligation Detection Second
(SOLiD)
4 Solexa (Illumina inc.) sequencing Sequencing by synthesis using Second
reversible terminator technology

5 Ion torrent: Ion S5/S5xl sequencing Sequencing by synthesis using Second

(Thermofisher inc.) Ionic strength fluctuation on
semiconductor chip (pH)

6 PacBio RS II (Pacific Biosciences inc.) Single molecule real Third

time (SMRT)sequencing using
 zero-mode waveguide (ZMW)

7 MinIon/GrodION xs/PromethION Nanopore sequencing relies on the Third

(Oxford Nanopore technologies) use of transmembrane proteins,
that are embedded in lipid
membranes
 2nd 3rd Generation
1st
Generation Sequencing
Generation
Sequencers
Sequencer
 Generation Of Advantage
Sequencing technology
First Generation • Robust Sequencing
Sequencing technology consider till date.
• Can able to sequence long
DNA read with good quality
score.
Second Generation • Cost and time effective high
Sequencing throughput mass parallel
sequencing against 1st
generation sequencing
technology.
Third Generation • Cost effective against 2nd
Sequencing Generation of sequencing.
• Less complex wet lab
protocol and generate long
read sequencing.
Disadvantage
• Expensive and tedious and
time consuming process.
• Having PCR bias in Complex
and multi steps wet lab and
dry lab processes may
compromise quality of
sequencing data.
• Generate short read of
sequencing will difficult in
assembly of non modal
organisms.
• Required improve the dry lab
tools and protocols to
overcome quality data issues.
• Time consuming sequencing
process.
Ion Torrent Sequencing
technology
 THIS IS TOMORROW
That was then………
 Lets take home

• Stay Current, Interact with your Microbiologist / lab for

current diagnostics !

• Filmarray / MALDI-TOF/ Nanopore is a vision to the future

of Diagnostics.

• Lets also learn to understand, accept and convince our

colleagues regarding advantages and limitations in new
Technologies.

• These Magic boxes save life... Lets make the most of it.
 This method of data acquisition ensures all compounds eluting have molecular ion data and fragmentation spectra acquired all of the time – hence no
compounds are missed! This is achieved by continuously cycling between TOF-MS and bbCID MS/MS resulting in the seamless collection…  r Brochure:
TargetScreener, 08-2017 (1854882)  0.64 MB | 29.09.2017