Dr. Sanghamitra Datta

Consultant

Dept. of Clinical Microbiology & Immunology

Sir Ganga Ram Hospital

The Hard truth Despite dramatic advances in diagnostic

technologies, it’s optimal integration into clinical care

is still questionable:

Patients suspected of infections still receive

empiric anti-microbial therapy

Overuse of our small inventory of effective anti-

microbial

continue to dwindle due to increasing levels of

antimicrobial resistance.

Whether caring for an

individual patient with an

infectious disease

OR

Responding to a

world–wide pandemic

“The rapid and

accurate establishment of a

microbial cause is

fundamental to quality care”

Syndromic approach of molecular diagnosis

Syndromic approach refers to diagnosing a constellation of signs and symptoms with a distinct clinical entity, with varied etiology and is diagnosed by assays based on molecular technique which detects from the panel of organisms commonly responsible for each syndrome .

Clinical diagnosis of the syndrome with a confirmed definite etiological diagnosis given by the laboratory are both important and complementary.

Lab should offer a test which can clinch the diagnosis faster by testing for myriad pathogens from a single sample with a single test – “Syndromic approach”

Case 1 – Non-Syndromic approach A 5 year old male child admitted with c/o

mild fever and severe headache for 2 days

Vomiting three times -1 day

Generalised seizure once

Investigations: Both CSF and blood was sent to the lab

Blood- TLC- 8500, N-75, L- 25, E-0. M-0

Hbgm%-12

CSF-Cytology: cell count-100/mm3, all lymphocytes. Biochemical parameters: protein : 50mg/dl, Sugar: 100mg/dl,

chloride- 120mmol/L

Microbiological investigations

Microscopy- Few RBCs and lymphocytes seen. No organisms seen

Culture: No organisms isolated.

Bacterial antigen test negative

CSF PCR for HSV & VZV -Negative …( 48 hours)

PCR for enterovirus done the next day – positive (72 hours)

Antibiotic Treatment

Patient was put on Inj. Ceftriaxone 1 gm I.V. 12 hrly and the patient improved after 48 hours

There was a debate whether to continue antibiotics….

Patient was lost to follow up

Case 2 – Syndromic approach A 2 year old male child was admitted with c/o

Mild fever for 2 days

Respiratory distress – 1 day

Chest examination revealed: Occasional creps and bilateral ronchii

X-Ray chest was normal

Patient was treated symptomatically

PCR of nasal swab for respiratory panel was sent and was positive for RSV-A (48 hours)

Antibiotics stopped on the third day. Patient improved and discharged in 5 days.

Advantages of syndromic approach in molecular Diagnostics

Testing for multiple causative pathogens implicated, in a single

tube sample saves time

The pathogens which are tested for are a combination of

viruses, bacteria, yeasts

Time taken to reporting is usually 24-48 hours.

Multiplex PCR Facilitates syndromic approach –reduces the

number of tests to just a single test from a single sample

Respifinder® RG Panel from QIAGEN : Multiple pathogens-

18 viruses and 4 atypical bacteria

•Mahoney et al in his

study coverted all

paediatric respiratory

virus testing to a more

expensive IVD cleared

multiplex molecular

method from a protocol

of DFA and viral culture

•Resulted in cost

savings of > $ 500,000

per year or $291 per

case

Syndromic approach to respiratory infections

A confirmatory laboratory test can aid clinicians differentiate between

bacterial and viral infections for appropriate prescription of anti-

microbials

So the right diagnostic test should be offered for best patient

management and outcome

12.27 hours reduction in identification time

0.4 days reduction in duration of antibiotic use

0.2 day reduction in inpatients length of stay

Rogers BB et al. Impact of respiratory panel test on patients outcomes. Arch

Pathol Lab Med 2015;139(5):636-649

Impact on CNS infections

Detected positive

results in 15 of the 48

smear negative CSF

samples

Viruses detected –

HSV 1(3),

EBV ( 8),

Enterovirus (1),

S. pneumoniae -2,

Cryptococcus (1)

Field of clinical microbiology is currently in transition

Though conventional techniques like

culture still remain the “Gold standard”

In the setting of infectious disease

emergencies & syndromic approach we

look forward to a technology that is -

Rapid

Facilitates Accurate & early

detection

Optimizes antibiotic therapy and

Clinical outcome in a timely manner

Participates and helps indirectly in

antibiotic stewardship

Improves patient care

Characteristics of a Detection System

Qualities we look for in a good laboratory test

♣Sensitivity ♣Specificity ♣Simplicity

Sensitivity means that the test must be able to detect very small amounts of target even in the presence of other molecules.

Specificity: the test yields a positive result for the target molecule only – no cross-reactions

Simplicity: the test must run efficiently on a routine basis and should be user friendly with a short TAT

14

Molecular Diagnostics

Uses for nucleic–acid based tests in Infective

diseases

Non-culturable microbes

HCV Hepatitis B virus Quantitative monitoring of patients on anti-viral therapy

Fastidious, slow-growing agents Mycobacterium tuberculosis Legionella pneumophilia

Test of choice in detecting highly infectious agents & can prevent

minimum handling Francisella tularensis Brucella species Coccidioidis immitis

Uses for nucleic–acid based tests

Differentiation of antigenically similar agents:

for detecting specific virus genotypes

HPV Type 16 and 18 associated with human cancers from low risk strains like 6 and 11 cause venereal warts

Testing for drug resistance: HIV & M. tuberculosis

Organisms present in small volume specimens Intra-ocular fluid Vesicle /blister fluid

Molecular typing to identify point sources for hospital and community-based

outbreaks

Uses for nucleic –acid based tests

Certain target population whose antibody tests are negative

Helps in detection of early infections of viruses by decreasing the window period

Nails the diagnosis in intriguing cases

Disadvantages

Expensive

Too sensitive? Results should be clinically correlated

Differentiation between infection and Disease

False positive Results:

Laboratory contamination

Inter sample contamination

Types of nucleic acid molecular techniques

Direct probe testing / Hybridisation techniques and Microarray

Sequencing methods: Pyrosequencing, Next Gen sequencing

methods

Amplification methods – used to improve the sensitivity of the

nucleic acid testing technique

Target amplification (PCR, TMA, NASBA)

Probe amplification (LCR, SDA)

Signal amplification ( bDNA technology, Hybrid capture)

Combinations of the above

Direct Probe assays

Digene Hybrid capture assay, is a hybridisation assay

available and FDA cleared for detection of N. gonorrhoea

and C. trachomatis

A DNA probe complementary to a specific sequence of N.

gonorrhoea / C. trachomatis in a single clinical specimen

(urine/ urethral swab)

Turn around time – Same day.

Sensitivity: 71.6%, Specificity: 99.7% ( CDC)

Variant Direct Probe assays Microarray assays: Multiplex detection by hybridisation

probes. Multiplex PCR prior to microarray analysis

increases sensitivity Eg: ResPlex II assay by QIAGEN for

respiratory viruses.

Sensitivity – 95%, specificity – 98%

Sensitivity increases if combined with PCR.

Used for Determination of resistance

Amplification Methods In vitro production of large quantities of desired sequence of

DNA into billion copies (exponentially increased) by thermal

(PCR) and isothermal ( NASBA, TMA) methods

RT-PCR: For RNA viruses - a cDNA copy of RNA is made using

reverse transcriptase (RT), cDNA then acts as template for PCR,

hence reverse transcription PCR or RT-PCR

Specificity of the amplified region can be targeted by specific

primers

Modifications of PCR

Nested PCR – Extremely sensitive uses dual amplification

primers, contamination reduced by using two different annealing

temperatures. M.pneumoniae detection ( Minerva Biolabs, Berlin).

Real time PCR: In 1993 Higuchi first described real time PCR

which is usually a quantitative assay for any amplifiable DNA

sequence

Multiplex PCR - Tandem PCR assays testing for all the common

respiratory viruses along with fastidious bacteria causing

pneumonia can be detected in one run eg. Respifinder® RG Panel

from QIAGEN – Cost effective and decreased TAT

Real Time PCR ( Thermal amplification)

Led to significant enhancement in diagnosing and characterisation of

infectious disease

Replaced conventional PCR

Requires less manipulation

More rapid

Closed tube format ( decreases risk of contamination)

Highly sensitive & specific

Greatest impact : Provides quantitative information (monitoring

therapy and prognosis)

Multiplex Real time PCR Detects of more than one target in a single PCR

reaction tube - A multi-pathogen test

Favors high through put : Ease of detection multiple

pathogens in a short time by Multiplex PCR a high

sample throughput with report in 5 -6 hours

Favours automation : Increases Sensitivity,

specificity and TAT

Multiplex PCR Respiratory Panel Panel: 21 panel & 33 panel

Sepsis Panel : 28 pathogens

Meningitis Panel: panel 10 and 14

Gastro-intestinal Panel: 10 panel – 38 organisms panel

Transplant Panels –

ACE (Adenovirus, CMV, EBV)

BCE ( BKV, CMV, EBV )

Other multiplex Panels (microbial q PCR array) still RUO

Targets 16S rRNA gene & fungal ribosomal rRNA gene sequences

UTI – 12 bacterial pathogens

Bacterial vaginosis -42 bacterial and fungal pathogens

Antibiotic Resistant genes – 87 antibiotic resistance genes belonging to

Fluoroquinolone macrolide, streptogramins, lincosamide, tetracycline, vancomycin, erythromycinbeta-lactam and aminoglycosides,

SGRH experience with multiplex Respiratory panel All symptomatic pediatric patients admitted in our hospital within the age

group of > 1 month to < 16 years of age, suffering from acute respiratory

infection as stated in the standard case definition of WHO were included4.

TOTAL NO. OF SAMPLES -232

0

50

100

150

200

POSITIVE NEGATIVE

50

78.4% positive for respiratory viruses

182

Monthly Distribution

0

5

10

15

20

25

30

35

40

oct'15 nov'15 dec'15 jan'16 feb'16 march'16 april'16

Respiratory viral infections are an important

cause of hospitalization in pediatric population

under two years of age

64%

36%

Gender Distribution

male

female

63%

26%

11%

Age Distribution

1mo - <2yr 2yr - <5 yr 5yr - ≤16 yr

0

20

40

60

80

100

120

140

1-5 days >5-10days

> 10 days

47

20

115

Days of hospital stay and admissions in ICU and Ward (n=182)

0

20

40

60

80

100

120

PICU ward

71

111

Total viruses isolated N=254

0

20

40

60

80

100

120112

40

20

14 13 12

12

7 6 4 3 3 2 2 2 1 1

Co-morbidity (n=61)

34%

66%

associated illness

not associated with illness

0

5

10

15

20

25

30

35

congenitalheart disease

malignancy H/Orecurrent

resp.infections

others

30

15

10

6

Percentage Co Infectivity

0 50 100 150

> 2 viruses

2 viruses

single virus

43(23%)

5(0.02%)

134 (73%)

Automation Automated extractions,

purifications systems, pipetting

robots are freeing individuals

from baby sitting the assays

reduces sample handling and

contaminations

Ensures high sensitivity &

specificity

Analyses more samples with

higher throughput

Future Directions- Automated systems

Fully integrated and easy to use modular systems Eg: QIAsymphony SP/AS instruments from QIAGEN

Ensures exceptional product safety

Minimized the risk of cross contamination

Integrated UV lights enables decontamination of work table

Flexible processing of high range of samples

Can process 96 samples in one go

Automated platforms Smaller integrated user friendly analyzers : Eg: GeneXpert

MTB/RIF Real Time PCR system

Rapid platforms Eg: bioMerieux- Biofire Film array’s

Respiratory Panel detects 17 viruses and 3 bacteria in less

than 2 hours time. Other Panels also available: sepsis and

Meningitis panel etc.

Game changers GeneXpert MTB/RIF assay

simultaneously detects DNA

of Mycobacterium

tuberculosis complex and resistance

to rifampin (i.e. mutation of the rpoB

gene) in less than 2 hours

Sample extraction, amplification and

detection are all carried out within this

self-contained cartridge.

It is available in a one, two, four, or 16-

module configuration

GeneXpert(Cepheid Inc,

Sunnyvale, USA).

Automation in multiplex PCR – A Smart & Small Foot

print

FilmArray is a PCR multiplex system which integrates steps

from extraction to detection.

• Easy – Two minutes of hands-on time

• Fast – Results in about 1 hour

Syndromic approach for the benefit of patients

and healthcare facilities

20 targets

Respiratory Panel

27 targets

Blood Culture Identification

Panel

Gastrointestinal Panel

22 targets

• 3 bacteria • 17 viruses

• 19 bacteria • 5 yeast • 3 antibiotic

resistance genes

• 13 bacteria • 5 viruses • 4 parasites

One system. Many applications.

Meningitis Encephalitis

Panel

14 targets

• 6 bacteria • 7 viruses • 1 fungus

Launched

Respiratory Panel CE & FDA-Cleared

43

Sample : Nasopharyngeal Swabs

Viral Adenovirus Coronavirus 229E Coronavirus HKU1 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 RSV Bacterial Bordetella pertussis Chlamydophila pneumoniae Mycoplasma pneumoniae

20 pathogens

Blood Culture ID Panel CE & FDA-Cleared

Gram + Bacteria:

Enterococcus spp.

L. monocytogenes

Staphylococcus

S. aureus

Streptococcus spp.

S. agalactiae (Group B)

S. pyogenes (Group A)

S. pneumoniae

Antibiotic Resistance:

mecA

Van A/B

KPC

Gram - Bacteria :

A. baumannii

Enterobacteriaceae

Enterobacter cloacae Complex

E. coli

H. influenzae

K. oxytoca

K. pneumoniae

N. meningitidis

P. aeruginosa

Proteus

S. marcescens

Fungi:

C. albicans

C. glabrata

C. krusei

C. parapsiolosis

C. tropicalis

Sample : Positive Blood culture

27 pathogens

GI Panel CE & FDA-Cleared

Bacteria: Aeromonas Campylobacter Clostridium difficile (Toxin A/B) Plesiomonas shigelloides Salmonella Vibrio Vibrio cholerae Yersinia enterocolitica Diarrheagenic E. coli / Shigella E. coli O157 Enteroaggregative E. coli (EAEC) Enteropathogenic E. coli (EPEC) Enterotoxigenic E. coli (ETEC) Shiga-like toxin-producing E. coli (STEC)

Shigella/Enteroinvasive E. coli (EIEC)

Protozoa: Cryptosporidium Cyclospora cayetanensis Entamoeba histolytica Giardia lamblia Viruses: Adenovirus F 40/41 Astrovirus Norovirus GI/GII Rotavirus A Sapovirus

Sample : Stool resuspended in Cary Blair

22 pathogens

Meningitis / Encephalitis – CE & FDA APPROVED

Bacteria:

E. coli

H. influenzae

L. monocytogenes

N. meningitidis

S. agalactiae

S. pneumoniae

Fungi:

Cryptococcus

neoformans / gattii

Sample : Cerebral Spinal Fluid

14 pathogens

Viruses:

Cytomegalovirus (CMV)

Enterovirus

Herpes simplex type 1 (HSV-1)

Herpes simplex type 2 (HSV-2)

Human herpesvirus 6 (HHV-6)

Parechovirus

Varicella zoster virus (VZV)

Platforms we use at SGRH Time line ( 2002-2015)

GeneXpert MTB/RIF : Real time PCR fully integrated system – 2015

QIAsymphony RGQ – For most of the viral parameters – 2014

Chiron Procleix target capture system – Blood bank samples ID-

NAT test – 2011

NucliSens EasyQ® for Real time NASBA HIV quantitation – 2009

which replaced NucliSens Reader (CMV pp67 assay from 2002)

COBAS® TaqMan® 48 Analyzer for hepatitis B & C quantitative -

2008 & Lightcycler® 2.0 – 2008

GenProbe - ( TMA) for M.tuberculosis complex detection – 2003

Molecular assays at SGRH

4

8

10 11

13 14

15

17

0

2

4

6

8

10

12

14

16

18

2008 2009 2010 2011 2012 2013 2014 2015

New tests added /year

New tests added /year

Sample size – Viral Molecular diagnosis

626 930

1486 1608 2042

2931 2673

4300

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

2008 2009 2010 2011 2012 2013 2014 2015

Sample size

Sample size

Good Practices – Enables Good results.

Advantages of molecular assays especially syndromic approach is appealing

but should be matched with Stringent Quality control and rigorous validation

practices to maintain nucleic acid integrity

Contamination Control: automation has helped us immensely

establish a unidirectional work flow from a DNA-free area for reagent

preparation, to a sample processing area, to area where amplification and

detection

Positive and Negative Controls always to be incorporated

EQAS samples should be run at regular intervals

Molecular Results: Always should be correlated clinically

• Molecular diagnostics have proven to be a vital & valuable tool •With its impact on clinical and economic outcomes and its broad coverage on infectious disease….

•It’s a “Silver lining” that can help us win the long drawn war against

microbes & anti-microbial resistance