dr. sanghamitra datta consultant dept. of clinical ...€¦ · microbial cause is fundamental to...
TRANSCRIPT
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
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