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  

http://www.biorad-ads.com/qpcr_tutorial/content/

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