nucleic acid amplification testing in clinical microbiology
TRANSCRIPT
Nucleic Acid Amplifica.on Tes.ng (NAAT) in Clinical Microbiology
Lourens Robberts, PhD, D(ABMM), FCCM Director & Clinical Microbiologist
Newfoundland & Labrador Public Health Laboratory 2012
Tradi.onal microbiological culture-‐based techniques, in retrospect, are .me consuming, labor intensive and oNen lacks sensi.vity.
Case Presenta.on { 61 year old ♂ { EtOH abuse { Pneumonia { COPD
x An.microbials x Bronchodilators x Cor.costeroid
Progression
x Day 7 { Tonic-‐clonic seizures { Febrile (39°C) { Mild nuchal rigidity { Neurological exam normal { Mental status
x Somnolent, confusion, disorienta.on, ina[en.on
Laboratory inves.ga.ons CSF WBC
ñ 34 (97%) Glucose
ð 67 mg/dl Protein
ñ 63 mg/dl
{ Microscopy and culture
x Viral encephali.s
Figure 1.
Fluid a[enua.on inversion recovery coronal MRI sec.on. Day 4 of neurological symptoms.
Test Bacterial Viral Fungal Tubercular Opening pressure Elevated Usually normal Variable Variable
White blood cell count ≥1,000 per mm3 <100 per mm3 Variable Variable
Cell differential Predominance of PMNs
Predominance of lymphocytes
Predominance of lymphocytes
Predominance of lymphocytes
Protein Mild to marked elevation
Normal to elevated Elevated Elevated
CSF-to-serum glucose ratio
Normal to marked
decrease Usually normal Low Low
CSF Findings during Central Nervous System infec.on
HSV infection of diploid human fibroblast cells
1 2 3 4 5
1 2 Shell vial
4h LightCycler PCR
Test turn around time
HSV PCR
Real-time PCR Amplification Curve
__POS CNTRL __NEG CNTRL __PT A __PT B
Real-‐.me PCR Mel.ng Peaks
__POS CNTRL __NEG CNTRL __PT A __PT B
HSV-‐1 Tm = 54°C
HSV-‐2 Tm = 68°C
Source
%
HSV genotype detec.on
1 2
Mayo Clinic, 2009
0
10
20
30
40
50
60
70
80
HSV-1 HSV-2
HSV-1
HSV-2
0
10
20
30
40
50
60
70
80
Male Female
HSV Genotype Frequency All sites
(2005 – 2011)
Male/Female Genotype Frequency All sites
(2005 – 2011)
Newfoundland & Labrador PHL, 2011
Management
x Acyclovir 10mg/kg; tds; 10 days x Complete recovery x Neurological and mental status normal at 3 months
x Expedient iden.fica.on and appropriate an.viral therapy
Summary
N Procedure :LP vs. Brain Biopsy 8 Accuracy :ñ Sensi.vity 22% � TAT :4 hours
Virus CPE Development (days) Adenovirus 4 – 7 Cytomegalovirus 7 – 10 Enterovirus 2 – 5 Herpes simplex virus (HSV) 2 – 5 Influenza 3 – 5 Metapneumovirus 10 – 12 Respiratory Syncytial virus (RSV) 3 – 5 Varicella-zoster virus (VZV) 4 – 7 Human Immunodeficiency Virus (HIV) Requires 2 weeks, leukocytes Hepatitis C virus No culture method Human Papilloma Virus (HPV) No culture method
TradiLonal culture-‐based methods: increased TAT
Bacteria Incubation time (days) M. tuberculosis 21 - 35 N. gonorrhoeae 72h C. trachomatis Cell culture 48 – 72h
Pneumocystis jiroveci No culture method
Nucleic Acid Extraction and Purification
• DNA/RNA of high quality (intact) • Free from proteins and cellular
material • Ability to recover small quantities of
pathogen/target DNA from large quantity of specimen material/host DNA
Silica filter-based spin-column
Magnetic bead-based purification
NAAT Relies and u.lizes the structure and sequence of nucleic acids in DNA or
RNA
5ʹ′ 3ʹ′
Science, like nothing else among the ins.tu.ons of mankind, grows like a weed every year. Art is subject to arbitrary fashion, religion is inwardly focused and driven only to sustain itself, law shu[les between freeing us and enslaving us.
Polymerase Chain Reac.on (PCR)
5ʹ′ 3ʹ′
Thermus aqua+cus (Taq)
To u.lize NAAT to detect and iden.fy an organism one has to find within the DNA/RNA sequence specific sequences that are specific for the organism/target of interest
Iden.cal
Different
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PCR cocktail ingredients Function Primer 1 (oligonucleotide) Targets a DNA sequence of interest
(3′ - 5′) Primer 2 (oligonucleitide) Targets the same DNA sequence on
the complementary strand (5′ - 3′)
Probe Anneals to specific target sequence, reporter dya
DNA polymerase (Taq) DNA replication MgCl2 For Taq to function Deoxynucleotide Triphosphates (dNTPs)
A, T, G, C for Taq to build the new DNA strand
Buffer and H2O Template DNA Specimen / organism under
investigation
Ingredients for a basic PCR
25 °C
94 °C
55 °C
72 °C 72 °C
20 – 30 cycles
4 °C
Final elonga.on
Denature
Anneal
Elonga.on
Thermocycling
DETECTION Agarose Gel Electrophoresis
1. ds DNA (nega.vely charged) travels in an electric field with the current (thus from nega.ve to posi.ve terminals)
2. Agarose Gel – molecular sieve. The agarose separates the DNA molecules according to size. When an electric field is applied to the sample the larger DNA strands will migrate propor.onally slower than the smaller strands, and thus the smaller strands will reach the end of the gel first. The size of the strand is measured employing a sample with known size standards.
3. Sizing the PCR product confirms that the amplicon (product) is what was expected.
4. Referred to as “end-‐point PCR” (versus real-‐.me PCR)
Incorpora.on of Ethidium Bromide enables detec.on of the DNA strand since ethidium bromide intercalates with dsDNA and fluoresces under UV irradia.on. Ethidium bromide is both a carcinogen and a mutagen! Other safer stains are available.
Ethidium bromide intercalates with dsDNA
Agarose gel electrophoresis
DNA Ladder/marker
100
400
Real.me PCR
Exonuclease ac.vity of Polymerase enzyme
Lab Operation Quality Assurance
PCR amplicons (amplified product of PCR) can act as a template for its own PCR, therefore special care is needed to prevent the transfer of previous amplicons into PCR reac.ons. Amplicons number in the billions in a 25 μl reac.on. Opening a completed PCR reac.on tube in a lab area where PCR reac.ons are being set up can cause cross contamina.on and lead to false-‐posi.ve results Special precau.ons are needed in PCR laboratories to prevent amplicon contamina.on
UNIDIRECTIONAL WORKFLOW OK
NOT
Documentation and Controls
Primary Specimens
Lab Number (e.g. YY-### / 14-001) Name/initial or other second identifier (L R) Specimen type: U, BL, TISS, SPUT Date Collected: 2014-01-20
14 -002
14 -003
14 -005
14 -004
Lab # Initials Spec Date Type Storage 14-001 L R 2014-01-20 U A-01
Database
NEG Prep: 01/06/14
POS Prep: 08/06/14
Lab No Initials
BIL-00123 L R
BIL-00124 R K
14JAN20 01 Resp1 ##/name
234-456-1 01/02/15
L R
Secondary Specimens
Nucleic acid extraction: 14-001-E1 L R
14 -002 -E1
14 -003 -E1
14 -005 -E1
14 -004 -E1
Lab # Initials Spec Date Type Specimen DNA Extr
14-001 L R 2014-01-20 U A-01 B-017
Database
PCR Master Mix
PCR Lab
On the System: ?Were are results stored Create Folder for your experiments: e.g. BILLC Save data files for easy retrieval: e.g. BILLC 14Jan20 01
PCR Worksheet….
BILLC 22/08/2014
LC 2.0 10
BIL-00123 L R
NEG Extr Prep: 01/06/14 Extr: 02/06/14
BILJan01 M J
14JAN20-01 PB CM
22/08/14 23/08/14
28
92
POS Prep: 01/06/14 Extr: 02/06/14
NEG H20
BIL-00124 R K
BIL-00129 M J
35
33
32
N
N
92
92
93
+
+
+
+
Lab # Extr BILLC Result 14-001 14Jan20 14/08/22 D 14-002 14Jan20 14/08/22 N
D = Detected N = Not Detected
QC acceptability • Negative controls
ü No Cp ü no melting curve
• Positive control ü Tm range 60.7°C ±2.5°C ü Cp of Positive control ±3 cycles of mean as tested for lot of
Positive control
• Internal Control • If absent – specimen invalid (possible inhibitory)
• Repeat that specimen on next run
• If present – specimen valid (not inhibitory)
• Quantitative standards ü Tm – agree with standards ü Cp - ± 1 cycle of standards
QC failures
• Quantitative standards – ≥1 standards fail, report negative specimens
(if Pos control OK). Positive specimens should be retested
• Negative controls – Extraction control failure: Repeat extraction – PCR control (H20) failure: Repeat only PCR – Repeat testing gives same results –
environmental swipe test AND DECONTAMINATE
QC failures
• Positive control – Tm out of range: repeat PCR reaction – Cp >4 cycles from mean value AND melting
curve on Positive control OK – Repeat PCR reaction (not extraction)
– Repeat using same Positive control and another valid control (new batch/aliquot)
– Acceptable if both are positive – If both are invalid – consult management
BILLC Prep: 01/06/14 Extr: 02/06/14 35 2 92 1
POS Control NEG
NEG Extr Control POS
NEG H2O Control POS
Instrument Error Code ###
DOCUMENT EVERYTHING
TRACEABILITY IS KING