New diagnostics for paediatric lung infections Progress on breath testing
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