Prof Miles H Beaman FRACP FRCPA FACTMMedical Director

Western Diagnostic PathologyClinical Professor

University of Western Australia University of Notre Dame Australia

Why order microbiology tests? Traditional Microbiological Techniques STI diagnostics

In service monitoring New test validation

Gonococcal NAAT

Interlaboratory validation Trichomonas NAAT

Respiratory Infections Respiratory Pathogen PCR

Pandemic experience Respiratory Viral Panel Respiratory Atypical Bacterial Panel

GI infections GI viral panel GI bacterial panel C. difficile testing

Epidemiological insights

• Non-NAAT methods Malditof

Is an antibiotic clinically indicated?

i.e they are NOT antipyretics

Have appropriate specimens been taken?

What organisms usually cause this syndrome?

Which regimen will best cover these organisms?

What is the optimal duration?

Choose initial empiric regimen

Modify regimen according to cultures

9/9/2014 4SAM-2 The Science Behind

0

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50

8-39 y ≥ 40 y All

Sulfa

No Rx

2/69

Mort

alit

y (

%)

Adapted from Evans GM. Lancet. 1938;2:14-19.

27/10011/68 6/31 16/32 8/100

Age:

Alvarez-Lerma F, et al. Intensive Care Med. 1996;22:387-394.Ibrahim EH, et al. Chest. 2000;118L146-155.Kollef MH, et al. Chest. 1999; 115:462-474.

Kollef MH, et al. Chest. 1998;113:412-420.Luna CM, et al. Chest. 1997;111:676-685.Rello J, et al. Am J Respir Crit Care Med. 1997;156:196-200.

0 20 40 60 80 100

% Mortality

Initial adequate

therapy

Initial inadequate

therapy

Luna, 1997

Ibrahim, 2000

Kollef, 1998

Kollef, 1999

Rello, 1997

Alvarez-Lerma,1996

Specimen arrives in Laboratory Day 1

Microscopy (same day) Plate out on relevant solid media

Need to warn if TB, fungi suspected (slower growth)

Day 2-5 Pick colonies after growth obtained

Day 3-6 Identification and sensitivity

Serology First sample often negative Need follow up sample 2-6 weeks

Neisseria gonorrhoea

Chlamydia trachomatis

Trichomonas vaginalis

Mycoplasma genitalis

Ureaplasma

9/09/2014 8

fastidious organism toxic sampling equipment inhibition of growth by body secretions susceptibility of organism to antimicrobials poor organism survival with prolonged transport or

suboptimal specimen storage transport media originally devised to specifically

support the maintenance of the gonococcus

80-95% sensitivity for acute symptomatic gonorrhoea 50% (or less) sensitivity for asymptomatic females

WDP experience central Australia- 25% culture yield from PCR+ urine

9/09/2014 9

Methods of amplification Polymerase Chain Reaction (PCR)

“in-house” cppB gene porA gene

AMPLICOR putative cytosine DNA methyltransferase gene

Strand Displacement Amplification (SDA) ProbeTec

multicopy pilin gene-inverting protein homologue

Nucleic Acid Sequence Based Amplification (NASBA) Nuclisens

16S rRNA sequence

Transcription Mediated Amplification (TMA)

Aptima 16S rRNA sequence

9/09/2014 10

9/09/2014 13Cook, R. L. et. al. Ann Intern Med 2005;142:914-925

Sensitivity of nucleic acid amplification tests

for Neisseria gonorrhoeae in women and men

799/1859 culture results available [43%]

418 Urines cultured: 83 culture positive [20%]

8/83 PCR neg

culture pos false negative PCR rate 10%

381 Swabs cultured: 50 culture positive [13%]

9/50 PCR neg

culture pos false negative PCR rate 18%

9/09/2014 15

• Absolute specificity for target

organism is achieved through

targeting of unique sequences

• Sensitivity increased through

“biological amplification”

• Each bacterial cell contains:

– One or several copies of DNA

– Up to 10,000 copies of

ribosomal RNA

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Target Capture technology is designed to:

Virtually eliminate false negatives by removing inhibitors before beginning amplification

Simplify sample processing

Allow the use of large sample volumes

Accommodate numerous specimen types

Allows testing of urines and swabs in same run

9/09/2014 16

DIS+ DIS-

APT+ 57 0

APT- 1 501

9/09/2014 17

New test validation: Aptima NG

Sensitivity = 48/59=81.4 %

Specificity= 100%

PPV=100%

NPV=97.9%

9/09/2014 18

• DIS+ DIS-

•AMP+ 48 0

•AMP- 11 501

Culture insensitive, specific

antigen EIA improved sensitivity

Molecular testing PCR

sens 64.1-98.4%,spec 92.9-100%

TMA sens 88.5-100%, spec 99-100%

SDA sens 85.7-93.1%, spec 92.6-97.6%

9/09/2014 19

9/09/2014 20

• DIS+ DIS-

•APT+ 53 0

•APT- 2 585

DIS+ DIS-

AMP+ 48 0

AMP- 7 585

9/09/2014 21

Prevalence 3.4% (330 asymptomatic men)

Sensitivity Specificity

Lab 1 (PCR)

72.7% 100%

Aptima

90.9 99.4

Lab 3(PCR)

90.9 99.7

9/09/2014 22

NT

Total TV Results 13952

Total TV POS 1423 Percent TV POS 10.2%

Total Chlamydia Results 18557

Total TV POS 977 Percent CHP POS 5.26%

Total Gonorrhoea Results 18557

Total TV POS 603 Percent GON POS 3.25%

Total Gonorrhoea & Chlamydia Results 18557

Total CHP & GON POS 602 Percent DUAL POS 0.9%

WA

Total TV Results 8932

Total TV POS 707 Percent TV POS 7.9%

Total Chlamydia Results 40705

Total CHP POS 2610 Percent CHP POS 6.42%

Total Gonorrhoea Results 40705

Total GON POS 602 Percent GON POS 1.48%

Total Gonorrhoea & Chlamydia Results 40705

Total CHP & GON POS 162 Percent DUAL POS 0.4%

Viral “URTI” Coryza, pharyngitis, laryngotracheobronchitis (croup)

Influenza Syndrome Bronchitis

Acute Viral 90% (Mycoplasma, Chlamydophila, Bordetella)

Chronic (COPD)

Community-acquired Pneumonia (CAP) 50% aetiological diagnosis

Sputum 35-50% Blood 20-30%

S. pneumoniae 20-60%, H. influenza (4-15%), other gram neg (7-18%), Staph. aureus(2-10%)

Bronchopneumonia Lobar pneumonia Atypical pneumonia Immunocompromised pneumonia Aspiration Pneumonia

Nosocomial pneumonia gram neg, Staph. aureus

Active v Flu A and B

Treatment (reduced 1.5 d symptoms) and prophylactic (infection reduced 30-50%, illness 67-84%)

Zanamivir inhaled

Oseltamivir Oral

Mild GI SE

National Pandemic Flu Plan

Benchmark lab TAT 48 hr

All specimens to central reference labs

Private Sector advised strategy faulty

public sector likely to be overwhelmed

Testing at the coal face advised

Pathwest Influenza RT 29/04-09/10/09

0

1000

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6000

7000

8000

Apr-09 May-09 Jun-09 Jul-09 Aug-09 Sep-09 Oct-09 Total tests

29/04 to

09/10/09

A/H1N1 PCR

Influenza PCR

Influenza Serology

Total by all Test Type

0

50

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ALERT DELAY CONTAIN PROTECT RESTRICTED*

Pandemic Category

*restricted testing by reference laboratory

Tu

rn a

rou

nd

tim

e (

hrs

)

• Beaman MH and Leung M. Pandemic Influenza testing at the coalface. Med J Aust 2010:192;102-4.

Referring specimens require as much effort as performing the tests

Hospitals have limited isolation facilities

Bottle neck to care if slow Lab TAT

->WDP in-house respiratory PCR

Only refer positive specimens for typing

Beaman and Leung MJA 2010:192;102-4

Influenza A and B

RSV

Parainfluenza 1-3

HMPV

Adenovirus

(rhinovirus, bocavirus,enterovirus desirable for paediatrics)

Note: Medicare funds 3 tests only

TAT 2010-11 median 33 hr

(P/W 99 hr)

2011-13

Flu

A

Flu

B

RSV HMP

V

Para

1

Para

2

Para

3

Ade

no

N Total +

NT 16.4

%

4.2 2.9 1.1 0.4 0.3 0.5 0.5 1105 290 (26.2%)

DAR 17.1 2.76 3.08 1.06 0.11 0.11 0.43 0.64 941 238 (25.3)

AS 12.2 12.2 1.83 1.22 1.83 1.22 1.22 0.00 164 52 (31.7)

WA 11.7 7.4 5.9 2 0.6 0.3 1.2 0.5 4706 1392 (29.6)

Mycoplasma. pneumoniae

Legionella pneumophila

Legionella longbeachae

Chlamydophila. pneumoniaeBordetella. pertussis

Myco Chlamy Legion PJP N Total +

NT 7.27% 0 0 0 55 0.27 %

WA 3.75% 0 2% 2% 400 7.75%

384 well plates

Second leading cause of infectious mortality 4 billion/episodes/yr 2.2 million deaths/yr

Greatest burden young children in developing countries (regions)

10% longer than 2 weeks Foodborne

US 76 million/yr 325,000 hospitalisations 5000 deaths Salmonella, Campylobacter, Listeria

Waterborne 32.8 million cases/year Cryptosporidium, cholera

Person-to-person Shigella

Norovirus 1 million clinic attendances/year 200,000 deaths developing countries 64,000 hospital admissions developed countries Outbreaks on cruise ships, nursing homes, military camps Food and water, person to person

Rotavirus Affect all ages Major cause diarrhoea young children Most severe illness <2years US 410,000 Doctor visits 272,000 ED visits 70,000 hospitalisations 60 deaths Vaccine reduced disease burden

TOT(%) NT DAR WA

SPEC 283 106 94 177

NEG 189 62 (58.5%) 50 (53.2) 127 (71.8)

NORO 21 (7.4) 1 (0.9) 1 (1.1) 20 (11.3)

ROTA 28 (9.9) 22 (20.8) 22 (23.4) 6 (3.4)

ADV 33 (11.7) 10 (9.4) 10 (10.6) 23 (13)

NORO+ADV 1 (0.3) 0 0 1 (0.6)

NORO+ROT

A

2 (0.7) 2 (1.9) 2 (2.1) 0

ROTA+ADV 9 (3.2) 9 (8.5) 9 (9.6) 0

TOTAL(%) 92 (33.2) 44 (41.5) 44 (46.8) 50 (28.2)

no. detected

PCR MC&S

Dientamoeba 47 0

Blastocystis 58 3

Giardia 12 6

Entamoeba 0 0

Cryptosporidium 8 0

Salmonella 13 10

Shigella 7 0

Campylobacter 34 21

Yersinia 2 1

negative for all 318 458

total 499

Table 1Table 2

% positive

PCR MC&S

Dientamoeba 9.42% 0.00%

Blastocystis 11.62% 0.60%

Giardia 2.40% 1.20%

Entamoeba 0.00% 0.00%

Cryptosporidium 1.60% 0.00%

Salmonella 2.61% 2.00%

Shigella 1.40% 0.00%

Campylobacter 6.81% 4.21%

Yersinia 0.40% 0.20%

TOTAL 36.27% 8.22%

0.00%

2.00%

4.00%

6.00%

8.00%

10.00%

12.00%

14.00%

16.00%

18.00%

20.00%

Ent Gia Die Cry Bla Yer Cam Shi Sal Aer

NT FPM prevalence

0.00%

2.00%

4.00%

6.00%

8.00%

10.00%

12.00%

14.00%

16.00%

Ent Gia Die Cry Bla Yer Cam Shi Sal Aer

WA FPM prevalence

Hospital

Community

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

0-5: 5-10: 10-15: 15-20: 20-25: 25-50: 50+ 0-5: 5-10: 10-15: 15-20: 20-25: 25-50: 50+

% p

os

Age

WA C. difficile %pos

PCR

pos dll

Ent 1 1 0.03%

Gia 231 35 3.36%

Die 957 142 13.88%

Cry 76 8 1.06%

Bla 1189 29 15.38%

Yer 22 8 0.38%

Cam 465 21 6.14%

Sal 127 27 1.94%

Shi 63 15 0.98%

Aer 153 52 2.59%

tota tested 7919

MC&S

pos

Ent 0 0.00%

Die 0 0.00%

Bla 11 0.09%

Yer 3 0.02%

Cam 445 3.09%

Shi 4 0.03%

Sal 230 1.59%

Aer 26 0.18%

tota tested 11698

ELISA

GIARDIA 209 2.65%

CRYPTO 84 1.08%

total 7664

Matrix-AssistedLaserDesorption /IonisationTime Of FlightMass Spectrometry

Generates molecule mass spectrum from a specimen (including whole organisms)

DNA, proteins, peptides, sugars

Polymers, dendramers, other macromolecules

These molecules tend to be too fragile and easily fragment using more conventional ionisation methods

Compare spectra to reference database

Microbiology application: Rapid & accurate classification of unknown organisms

Genus, species (strain) level

Instrument consists of 3 components:

Specimen ionisation chamber

Where laser vaporisation of specimen occurs

Time of flight mass analyser

Particle detector

Result in 5-10 minutes!

Shimadzu – BioMerieux

NAAT testing important component Pathology

Improved sensitivity cf. culture

False negatives and positives possible

Pre- and post-introduction monitoring essential

Malditof useful adjunct in ID and TAT

Automation proceeding in all areas