New diagnostics for paediatric lung infections

Progress on breath testing

+++ Rats help sniff TB +++

+++ Bees:the newest land mine sniffers? +++

+++ Dogs Smell Cancer in Patients' Breath, Study Shows +++

Complexity of the problem Available technologies Smell prints Specific volatiles

Dealing with breath Organism specific volatiles Problems of contamination Environmental Food Upper airways

Recent developments

Volume of a normal breath 0.5 l Moles in a breath 0.022321429 Molecules in a breath 1.34357 x 1022

Breath constituents Molecules 78% N2 1.05 x 1022 20% O2 2.69 x 1021 1.9% H2O (80% relative humidity) 2.55x1020

400 ppm CO2 (0.04%) 5.375 x 1018 5ppm CO (0.0005%) 6.71875x1016 500 ppb for 150 VOC’s 6.71875x1015 500 ppt for 100 unknowns 6.71875x1012 >500 unknowns at femtomolar range (10-15) 6.71875x109

XX at attamolar range (10-18) 10,000 ??

YYY at zeptomolar (10-21) 10 ?

1. Electronic nose 2. Identification of bacterial specific volatile

organic compounds Mass spectrometry GCMS Selective ion flow-tube mass spectrometry Electrospray ionisation

Online system that uses an array of sensors to detect a pattern of VOCs or ‘smell print’ representing the mixture of volatiles in the

Does not identify specific compounds and requires prior exposure (training) to a particular smell (pattern) in order to recognize it.

The sensitivity of most sensors has not reached the levels of other techniques.

Sensitivity can be improved by pre-concentration eg SPME or Tenax before they are desorbed onto the instrument giving a real-time readout.

Sensitivity - ppb

VOC profiling for both host-derived oxidative stress volatile metabolites (alkane and alkane derivatives) and possible markers of M. tuberculosis (cyclohexane and benzene) has shown promising results

A study of patients from the UK, USA and the Philippines referred for the investigation of tuberculosis found C-statistic for the profile for smear-positive disease was 0.76, sputum culture 0.68 and chest radiography 0.66.

Performed in a population preselected for a high risk of tuberculosis

Advantages Extremely sensitive Can do full scan or peak Enables high certainty of identification of

compound by fragmentation patterns

Disadvantages Relatively slow Requires breath collection and pre-concentration

and then sample injection – not online Low mass molecules not well seen

12

Properties – Mol. wt. =138

distinctive mass fragmentation Precursor – linoleic acid

lipoxygenase

Figure 2. Pathway of 2-PF synthesis

Figure 1. Mass fragmentation of 2-PF

2-PF -a VOC- is a product of linoleic acid peroxidation

Peroxidation can occur by Energy UV light and photosensitizers

Metal ions Fe3+, Cu2+

Reactive oxygen species (ROS)

A. fumigatus (15) 2+ A. flavus (9) 1+ A. niger (5) 1+ A. terreus (6) 1+ Fusarium spp. (10) 3+ S. apiospermum (6) 1+ C. albicans (7) nd R. arrhizus (4) nd Ps. aeruginosa (5) nd Ps. fluorescens (5) nd B. cepacia (5) nd S. aureus (7) nd S. pneumoniae (7) 3+ M. catarrhalis (6) nd E . coli (6) nd L. pneumophila (6) nd.* H. influenza (6) nd **

Medical mycology

Case 1. Mr B An 82 year-old Caucasian male with moderately severe

chronic obstructive pulmonary disease and tobacco smoking Limited stage small cell lung cancer on cytology from fine

needle aspiration and CT scanning of his chest palliative chemotherapy consisting of dose-reduced

carboplatin (AUC5) and etoposide (80mg m-2). Febrile neutropenia and aspergillus cultured from sputum

and pleural fluid and Cunninghamella sp. from sputum Treated with amphotericin and then posaconazole

Top row: Positive breath sample from a case 1 of invasive aspergillosis Bottom row: Negative breath sample from the same patient after two months

False positive attributable to ingestion of soy products Verified on soy challenge studies

Subsequent studies showed intermittent false positive tests in normal subjects

Environmental air intermittently positive for 2-PF. Probably due to activated carbon in the atmosphere which binds environmental 2-PF

Organisms particular problematic in cystic fibrosis

Best chance to eliminate Ps. aeruginosa from children is when they are first infected.

Could we develop a simple, repeatable, child friendly screening test that would identify early colonisation with Ps. aeruginosa

What about a breath test? Ps. aeruginosa know to produce a grape like

smell – 2 amino acetophenone Tryptophan is a precursor – very low levels in

peripheral blood

2-Aminoacetophenone detected in 4/78 samples tested in vitro: corn chips and canned tuna (high pmol mol−1) and

egg white and 1/3beers (low pmol mol−1). No 2-aminoacetophenone detected in the CF

medication or cosmetics tested. 28 of 30 environmental air samples were

negative for 2-aminoacetophenone (below 50 pmol mol−1).

A challenge study with ten healthy subjects Levels of 2-aminoacetophenone were immediately elevated after corn chip consumption, but after 2 hr returned to baseline

(mainlib) Benzoic acid, 4-methoxy-, methyl ester30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

0

50

100

3841

5053

64

77

92

107

119 123

135

166

O O

O

NIST Library Full Scan Spectra

(mainlib) Benzeneacetic acid, methyl ester10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

0

50

100

15

18 29

39

4151 59 63

65

7789

91

105 119

150

O

O

(mainlib) Methyl nicotinate10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

0

50

100

15

27 39

51

59 66

78

93

106

109

137

OO

N

(mainlib) 1,1'-Biphenyl, 2-methoxy-10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

0

50

100

1527

3951

57

63

65 69

76 89

98102

115

118128

141

152

155165

169

184

O

Methyl phenylacetate (MPA)

Methyl p-anisate (MP)

Methyl nicotinate (MN)

o-Phenylanisole (OPA)

Anaglyptus subfasciatus Cryptomeria twig borer

Pieris rapae -Small cabbage butterfly

Methyl

phenylacetate

Methyl, p-anisate Methyl salicylate Methyl

nicotinate

O-phenylanisole

Volatiles from cigarettes (N=8)

Cigarettes# 0(0%) 3(38%) 1(13%) 8(100%) 0(0%)

Volatiles in breath before and after smoking a cigarette (N=13)

Before smoking 2(15%) 0% 7(54)%* 0%** 0%

5 minutes after

smoking##

5(38%) 0% 12(92%)* 6(46%)** 0%

# Sampoerna A, Samperna Mild Menthol, Djarm Black, Djarm No Filter, Gudang Garam, Lucky Strike, Winfield Green &

Holiday Blue ## Port Royal (3), Holiday Blue (3), Pocket Edition (1), Park Drive Blue (3), Winfield Blue (1), Horizon (1) & Freedom (1)

Fishers exact test: *P = 0.073 & **P = 0.014

Number of

samples

analysed

Methyl

phenylacetate

Methyl, p-

anisate

Methyl

salicylate

Methyl

nicotinate

O-

phenylanisole

Volatiles in environmental air Environmental

air N=30

1(3%) 9(30%) 30(100%) 0(0%) 0(0%)

Volatiles in breath samples of healthy subjects Morning

N=20

7(35%) 0(0%) 14(70%) 3(15%) 1(5%)

Afternoon

N=20

6(30%) 0(0%) 18(90%) 4(20%) 0(0%)

Fasting

N=20

7(35%) 0(0%) 17(70%) 3(15%) 0%

Non-Fasting

N=20

4(20%) 1(5%) 17(85%) 2(10%) 1(5%)

Repeated

samples from 8

subjects over 12

days (N=96)

17(18%) 2(2%) 41(43%) 7(7%) 0(0%)

Total

N=176

41(23%) 3(2%) 107(61%) 19(11%) 2(1%)

Compared four collection receptacles

Silanised glass bulbs

Tedlar® bag SupelTM inert bag SupelTM inert foil

bag

MP was detected in all new bags tested

2012 International Breath Analysis Meeting; A Scott-Thomas

30 Tedlar; MPA = PI 22000, MN – PI 3000, MP = PI 3000 & OPA = PI 37000 Error Bars – Standard Error of 4 replicates

0

20000

40000

60000

80000

100000

120000

140000

MPA MN MP OPA

Peak

Inte

grat

ion

Markers

1 Bulb 5 Bulbs 10 Bulbs 20 Bulbs 30 Bulbs

Each breath spiked with 5ul 10ppb Error Bars – Standard Error of 4 replicates

0

20000

40000

60000

80000

100000

120000

140000

MPA MN MP OPA

Peak

Inte

grat

ion

30 Breath & Tenax 1 Breath & SPME

* p-value < 0.0001

* p-value = 0.0007

* p-value = 0.0212

Advantages On line Good sensitivity for low molecular weight

compounds

Disadvantages Time of flight mass only Less sensitive than GCMS for many compounds

Quantification of hydrogen cyanide (HCN) in breath using selected ion flow tube mass spectrometry—HCN is not a biomarker of Pseudomonas in chronic suppurative lung disease Jack Dummer1, Malina Storer2, Sharon Sturney1, Amy Scott-Thomas3,

Stephen Chambers4, Maureen Swanney1 and Michael Epton1,5

J. Breath Res. 7 (2013) 017105 (8pp)

HCN is produced by Ps. aeruginosa Reported as biomarker for lung infection Also produced by salivary peroxidases Potential for contamination at oral cavity

In healthy volunteers a significantly higher end exhaled HCN concentration was measured in oral exhalations compared to nasal exhalations

No differences between exhaled HCN concentrations of subjects colonized with P. aeruginosa v non colonised

Breath HCN is not a suitable biomarker of P. aeruginosa colonization.

Mouse model Inoculated by intra-tracheal route Control – PBS Organisms – S. aureus, Ps. aeruginosa Sample collection Intubation of mouse trachea Mechanical ventillation Collection of 5 l of breath in tedlar bag

Breath analysis by SESI-MS

1. Breath is an extremely complex mixture 2. Current technology adequate to demonstrate

breath testing is possible 3. Some important pathogenic organisms produce

volatiles that are detectable in breath 4. In vitro testing does not necessarily predict

what will be produced in vivo as nutrition different. Animal models offer useful insights as nutrition of organisms approximates human conditions.

5. Profiling contains more information that single volatile detection and will probably be more robust

6. Clinical studies needed to minimise confounding from environment, food and upper airways

7. Many basic issues have been identified and can be overcome.

8. Understanding Host volatiles will be important in the diagnosis of infection rather than colonisation

Amy Scott-Thomas Shrawan Bandhari Mona Syhre Jennifer Scotter Malina Storer Jack Dummer Michael Epton John Pearson David Murdoch

Health Research Council of New Zealand Cure Kids Lotteries Health Otago Innovation University of Otago Department of Pathology, UOC

Bacterial species Ps. aeruginosa, Burkholderia cepacia, Escherchia coli,

Haemophilus influenzae, Legionella pneumophilia, Moraxella catarrhalis, Pseudomonas fluorescence, Staphylococcus aureus and Streptococcus pneumonia

Assay Headspace by GC-MS Results

2-pentylfuran A. fumigatus 2-AA detected only in headspace of Ps. aeruginosa

culture Methyl p-nicotinate, methyl p-asinate acid, methyl

phenylacetate o-phenylanisole from Mycobacterium tuberculosis