Download The Impact of Diagnostics of Fungal Infections on Antifungal Usage

Rhys Thomas
Nuffield Foundation
The Impact of Diagnostics of Fungal Infections on Antifungal Usage
Synopsis
During my 4 week placement at the Department of Medical Microbiology in
the School of Medicine, Cardiff University, Heath campus, I investigated
whether new molecular techniques can improve diagnostics for diseases such
as aspergilliosis caused by a fungus called Aspergillus, and bacteria capable of
causing diarrhoea and/or vomiting. I gathered results to determine if results
were reliable and accurate, and could improve diagnosis leading to improved
patient management. I had
help from my provider Prof.
Rosemary Barnes, Dr P. Lewis
White and Mr Michael Perry.
Introduction
Some fungi cause destruction
and disease, others are used
to make medicines and
decompose dead material.
Figure 1. Mould on a piece of bread caused by fungi
Every day our immune system
(body’s defence system) defends against fungi and other invading organisms to
prevent diseases they may cause. Fungi are found in many environments, at
any climate, from inside your
socks (causes athletes foot) to
growing on old bread. The
most known fungi are the
mushrooms. I have studied
briefly on fungi and other
organisms in GCSE and AS
level Biology. I used the
Figure 2. Aspergillus in a human lung tissue
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Figure 3. Aspergillus under a light
microscope
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Nuffield Foundation
internet and published articles to gather information on molecular techniques
for identification of fungi.
Antifungal drugs are used to treat fungal diseases such as aspergillosis and
candidiasis. They are amongst the top ten most expensive drugs hospitals buy
today. The majority of the patients who use these antifungal drugs are those
with a weak immune system and are at a high risk of developing a lifethreatening illness due to invasive fungi. Doctors frequently give antifungal
drugs to patients who show symptoms of general infection because they are
afraid it might be a fungal infection; the reason it is difficult to diagnose, and
delays in treatment lead to an unacceptable death rate of between 50-90%.
Figure 4. An electron micrograph of
Aspergillus
Colour
For my project, I will be focusing on one of the most
invasive fungi in the world; Aspergillus. Members of
the Aspergillus family grow in areas where there is a
lot of oxygen and areas where there’s a high amount
of salt and sugar, and naturally digest living and dead
material (leaves, bones etc.). New molecular
techniques provide an improved approach of
detecting a fungal infection by frequent testing of at
risk patients, and the costly (and toxic) drugs are
targeted to the patients with evidence of infection.
The molecular techniques I would be assessing are
an ELISA and PCR specific for Aspergillus. ELISA
(Enzyme-Linked Immunosorbent Assay) is a sandwich
test where antibody coated wells are used to detect
an antigen (galactomannan) specific to Aspergillus,
then using a second antibody coated with an enzyme
capable of generating a colour reaction when the
Aspergillus antigen is present.
Figure 5. A picture to explaining the ELISA technique
The colour is recorded and the intensity is
proportional to the amount of antigen present in the sample. It is compared to
a control sample containing a known amount of antigen and expressed as a
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Nuffield Foundation
ratio index. An index that is equal to or higher than 0.5 is regarded as a
positive.
PCR (Polymerase Chain Reaction) is a test that makes millions of copies of an
organism’s DNA/RNA from just a single strand so it can be detected even when
present only in tiny amounts. At the end of the reaction, probes labelled with
different colours are used to identify DNA of the particular organism which
cameras detect and then upload it to the system. This way, scientists can very
rapidly find out what organisms were in the samples even if they are dead
and/or not
capable of being
grown in the
laboratory.
Aim
The aim of the project is to determine if the current index used to determine
Aspergillus ELISA positivity (0.5) is optimal, and whether index values below
this are an early sign of infection that could be used to improve diagnosis and
patient management. Information will be gathered using the system database.
If evidence shows that the new method improves diagnostics, it will allow
doctors to prescribe antifungal drugs earlier and more accurately, which will
potentially save money by preventing disease and limit patient toxicity from
the drugs. It is hypothesised that the new molecular techniques will deliver
more accurate results and in a shorter period of time than the older
techniques. The research will concentrate on ELISA results just below the
accepted index of positivity (ie specimens giving indices 0.3 and 0.4) to see if
they are clinically useful and could allow earlier diagnosis of invasive disease.
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Part I: Data analysis
Method
As part of the routine diagnostic service patients were tested twice weekly by
PCR and ELISA. Using Microsoft Excel and Microsoft Access, access was gained
to anonymised data for these tests from patients at high risk of Aspergillus.
The method of analysis was to determine how many patients had risen, but
negative ELISA result (0.3-0.4 index) which later became positive (index >0.5)
on repeat testing with the ELISA. These patients were then categorised as
having proven, probably or possibly aspergillosis using clinical information. For
patients that did not achieve a clinical diagnosis of aspergillosis (unclassified)
PCR data was checked to see if this supported the ELISA result, as this would
indicate an early stage of infection and will support the idea that new
molecular techniques improve accuracies in diagnostics.
Out of 557 patients, 6 of these patients were proven aspergillosis, 47 patients
had the probable disease and 21 possibly had the infection. 483 patients were
unclassified. Definitions of these categories are;
Proven - demonstration of Aspergillus hyphae in diseased tissues and culture
of the mould in the laboratory (See Figure 2.)
Probable – specific signs on an x ray suggestive of aspergillosis combined with
isolation of the fungus and or a positive ELISA in a patient with known risk
factors of fungal infection
Possible - signs on an x ray suggestive of aspergillosis in a patient with known
risk factors of fungal infection but no other markers of disease
Unclassified - there are 3 ways of categorising the patients that could not be
classified using the above definitions. They are:
Risk factors, non-specific clinical features and negative/ not done mycology
tests
Or
Risk factors, no clinical or no -specific features but positive by ELISA or PCR*
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Or
Risk factors, no clinical features and negative/not done mycology but positive
by both PCR and ELISA*
*Please note: PCR is not currently included in the consensus definitions used to
define a case of proven, probable or possible aspergillosis.
Results
With the ELISA
A total of 149 (26.8%) patients gave an indeterminate 0.3/0.4 results in the
ELISA.
With the 6 proven patients all had sub threshold ELISA results at some stage, 4
(66.7%) of these started with a 0.3/0.4 in the ELISA tests and later increased
above 0.5; the rest (33.3%) started with 0.5 or higher and but indices fell on
treatment. All patients had positive PCR results.
Out of the 47 patients with the probable aspergillosis, 42 (89.4%) had 0.3/0.4
in their tests. In 16 patients it was the first antigen marker, with 14 (29.9%)
subsequently rising above the positive threshold. 3 patients started with a
positive threshold and never dropped below 0.5. 41 patients out of the 47 had
positive PCR results.
All of the 21 possible patients by definition had no positive ELISA results but 7
patients gave a 0.3/0.4 ELISA result. In 4 patients this remained negative and 3
patients only a single specimen was sent for testing. 16 out of the 21 possible
patients had positive PCR results.
As for the unclassified patients, 106 patients had positive results, 24 (5.0%) of
them first had 0.3/0.4 threshold recorded prior to being positive. A further 71
having 0.3/0.4 indices but never crossed the positive threshold. There were
105 single patient tests with 95 of them not having a positive index and 10 of
them having an index 0.5 or above. 211 of the unclassified patients had
positive PCR results.
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In summary
Patient population Galactomannan ELISA
First index
0.3/0.4 Rising to
0.3/0.4
true positive
Proven n=6
4
4
Probable n=47
16
14
Possible n=21
7
0
Unclassified n=483 95
24
Diagnostic
significance
18/27 (66.7%)
24/95 (25.3%)
ELISA index
Patients who went on to develop proven or probable disease were
approximately twice as likely to have a subthreshold (0.3/0.4) index prior to a
positive ELISA test than patients who did not develop likely aspergillosis
(Proven/Probable 20/53: 0.378 compared to unclassified 95/483: 0.197). The
probability of developing proven, probable or possible aspergillosis after
having a 0.3/0.4 ELISA result was 22.2%, compared to 13.3% prior to the test. If
a patient had 0.3/0.4 ELISA that was then followed by a positive (>0.5 index)
ELISA result the probability of aspergillosis was 42.9%.
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Proven 1
Proven 2
Proven 3
1 3 5 7 9 1113151719212325272931333537394143454749515355575961636567
Sample number
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Each graph below and above, shows the ELISA index on the Y axis and the
number of tests on the X axis for the 3 proven patients. There is no general
pattern for the progress of the disease. The graphs show that the ELISA indexes
are variable: an increasing index could represent disease progression, whereas
decreasing index values could represent the response to therapy.
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10
ELISA index
8
Proven 4
6
Proven 5
4
Proven 6
2
0
1 3 5 7 9 1113151719212325272931333537394143454749515355575961636567
Sample Number
Comparing results with the PCR and ELISA tests
148 PCR results were recorded for the 6 patients proven of aspergilliosis. 35
(23.6%) of these were recorded as detected, 113 (76.4%) were recorded as not
detected. Out of the 35 detected PCR tests, 29 of them had an ELISA index less
than or equal to 0.4; the other 6 had detected with an ELISA index of 0.5 or
above. 4 patients started with a negative threshold to a positive threshold with
all of them having a positive PCR.
There were 840 PCR results recorded for the probable patients. 173 (20.6%) of
the PCR tests were detected and 667 (79.4%) were not detected; 36 out of the
173 positive PCR results had a positive ELISA index with 137 of the PCR positive
results had a negative ELISA index. In regards to the ELISA results, 845 (83.8%)
of the tests were not detected, while 163 (16.2%) of the tests came out
positive. Out of the 13 tests that increased from a negative to a positive
threshold, 12 of them had a positive PCR.
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43 (27.7%) PCR results were positive for the possible patients with 112 (72.3%)
negative results; all of the 43 positive results had negative ELISA indexes. There
were 0 tests detected by the ELISA and 175 not detected. 6 patients with
possible aspergillosis had a 0.3/0.4 ELISA index as well as being PCR positive at
some stage.
Finally, for the unclassified patients, 2,398 PCR results were recorded, 447
(18.6%) were detected with 1,951 (81.4%) not detected. 44 out of the 447
positive PCR results had a positive ELISA index with 403 having a negative ELISA
threshold. 189 (6.3%) ELISA tests had a positive threshold, 2,774 (93.6%) not
detected.. 24 patients had tests that increased from a 0.3/0.4 to a positive
threshold by ELISA, 16 of the 24 had a positive PCR. 86 patients had a positive
PCR with an ELISA index of 0.3/0.4 at some point during testing.
Conclusion
Looking at the results, I believe that the new molecular techniques are reliable
and accurate. The fact that the new molecular techniques are reliable,
accurate and diagnose diseases in under 2-3 hours I believe that all hospitals
should invest in equipment such as this. Although the equipment is expensive,
it may save money on the long term. A raised but still negative index of 0.3/0.4
may be an early indicator of aspergillosis. The fact that all of the 6 proven
patients had a 0.3/0.4 in their ELISA tests and a positive PCR shows reliability
and accuracy with the equipment. In summary, if the new molecular
techniques are utilized in the future there will be improvement in diagnostics,
it will save money and it will save lives. Positive results in patients who were
not categorised as having aspergillosis may represent early infection or
undiagnosed disease.
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Evaluation
I believe that if my work is continued, it should be repeated and be
reproducible in order to finally be approved for diagnostics. I believe that more
tests should be performed for each patient especially if the first ELISA
threshold is 0.3/0.4, as there were some instances where patient had a only a
single test which may have progressed to a positive result. More people should
be trained to use the equipment which would then allow more diagnostics and
tests to be made, only a few were trained in using the ELISA equipment..
Patients in the unclassified category should be further investigated and be
examined under post-mortem in order to find out whether Aspergillus was
present in the body as predicted by the positive molecular tests. If found then
the molecular techniques should be used in order to accurately come to a
conclusion whether it is the best way to diagnose a patient.
Part 2: Practical Techniques
Initially, I was meant to evaluate the new molecular diagnostic assay called the
PNA-FISH (Peptide Nucleic Acid – Fluorescence In Situ Hybridization). It
incorporates a fluorescently labelled probe that is added to positive cultures,
making a specific organism to show a specific colour. Scientists look at the
stained culture under a microscope and identify the organism just by observing
the colour and shape. Although the
assay is very expensive, it is more
accurate and reliable than the other
diagnostic equipment. It is also a very
rapid way of identifying fungi, and
could change the antibiotic therapy
received by the patient. This would
have complemented the previous
project evaluating the impact of
Figure 6. A culture growing on a petri dish
molecular diagnostics. However, it
could not be performed due to a fault in the filter on the lab microscope that
was not compatible with the fluorescent labels. In response to this problem, I
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was able assist another clinical scientist (Mr Michael Perry) in using the PCR
equipment to identify the organisms responsible for diarrhoea.
Various organisms can cause infection in the stomach which causes symptoms
such as diarrhoea. Once a patient has been diagnosed with a stomach disease
by a doctor, a sample of the patient’s faeces may be sent to a laboratory to be
tested. Tests include using faecal cultures and the previously mentioned
molecular technique PCR. As I was inexperienced in working with complicated
and expensive machinery, my supervisor provided me with a sound knowledge
of the molecular details and how to use the equipment safely and correctly,
the internet and articles will be used to obtain information on the diagnostic
techniques.
Current Faecal diagnostics involve performing cultures on a petri dish,
consisting of an agar gel which encourages the organism to grow in colonies.
Different antibiotics are placed on the petri dish which kills the unwanted
organisms that can mask the pathogen. Depending on the organism, it may
take up to a week in order to find results. When the organism has had a
sufficient time to grow, large clusters will form on the plate. A swab is taken of
the clusters and placed on a glass plate under a microscope; the organism can
be identified by their shape and colour or by other
tests.
PCR (Polymerase Chain Reaction) is a test that
makes millions of copies of an organism’s
DNA/RNA from just a single strand so it can be
detected. It involves cycles of different
temperatures that allows an organism’s DNA/RNA
to break and then to be replicated using various
chemicals and enzymes. The enzyme responsible
Figure 7. Bases in different
for duplicating the DNA/RNA is polymerase; it is
formations (sequences)
attracted to the DNA/RNA when a primer
(synthetic DNA) is attached to a specific base sequence. The polymerase
attaches to the DNA using the primer; it begins to move along the strand and
replicates the DNA/RNA. On the strand, bonded chemicals can be attached to
another piece of synthetic DNA/RNA (the probe) which binds to the DNA/RNA
of the organism depending on the arrangement of the bases. The 2 bonded
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Nuffield Foundation
chemicals are called fluorophores and quenchers, and when bonded no signal
is visible. As the polymerase moves along the DNA/RNA strand, it meets the
probe and begins to denature (break) the bonds. This then leaves the
fluorophores and the quenchers to become separated and a coloured light is
given off (different colours for different organism targets). At the end of every
cycle, the different colours are detected by a camera and uploaded to a system
to be viewed. This way, scientists can find out what organisms were in the
samples and thus take action for treatment for the patient.
PCR testing has the capacity to improve diagnosis of faecal pathogens by
detecting more cases and providing quicker results compared to the current
approach. This work was being performed in preparation for the NATO summit
in Cardiff, which involves insuring a laboratory can respond to a public health
emergency such as an outbreak of an infectious disease such as diarrhoea or
vomiting.
Methods
Faecal culture results were retrieved from the system database
All molecular processing was performed by the Eppendorf pipetting station
and the LC480 which is a real time PCR platform under the guidance of Mr
Michael Perry.
The pipetting station takes individual samples of up to 46 patients’ faeces and
places them in strips with reaction wells. The reaction wells contain ingredients
to detect the organisms such as the polymerase, primers and the probe. There
are two types of strips; strip A has the ingredients in the reaction wells to
detect Salmonella, Shigella and Campylobacter. In strip B, the reaction wells
have the ingredients to detect verotoxin-producing Escherichia coli (VTEC),
Cryptosporidium and Giardia. After each sample is put into individual reaction
wells on both strips, the strips are then taken to be centrifuged before being
placed on the PCR platform. A centrifuge is a machine that spins samples at
high speed and forces the liquid in the strips to sink to the bottom. The sample
is then placed on the PCR platform to be amplified and results are shown on
the system at the end of the cycle.
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Figure 8. Typical real-time PCR results
After PCR results had been generated, they were uploaded into Microsoft Excel
along with the results from the faecal cultures of the same patients for
comparative purposes.
Results
There were 718 patients, 331 of them were Male and 387 Female. There were
306 General Patients, 336 hospital In-Patients and 76 Out Patients.
The results of the organisms detected from the patients’ faeces are expressed
in a table below.
Campylobacter
PCR technique
50
Faecal Culture technique
38
Salmonella
3
2
Shigella
3
1
VTEC E. coli
2
0
Cryptosporidium
2
2
Giardia
7
6
Total
67
49
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Results above show that the PCR has identified more organisms than the faecal
cultures.
With the campylobacter there were 641 negative PCR results, of these 1 was
positive by faecal culture. Of the 26 Campylobacter PCR positive results, 12
were negative by faecal culture.
The PCR identified 3 Salmonella species with only 1 negative in the faecal
cultures.
As for the Giardia species, the PCR identified 7 with 2 negatives in the faecal
cultures. The faecal culture identified 6 Giardia species.
The PCR identified 3 Shigella species with 2 faecal cultures having negative
results.
Conclusions
The PCR test generated more positives than standard laboratory methods.
The results for the molecular technique demonstrate acceptable accuracy and
reliability. After more tests, scientists should be able to use this technique in
place of culture and doctors might be able to prescribe accurate treatment and
detect outbreaks of disease much earlier than if it was diagnosed with the
faecal cultures permitting improved infection control.
The molecular techniques used, despite appearing complex, were relatively
easy to perform and could easily be performed by scientific staff with greater
experience
Acknowledgements and Personal Reflection
I would like to thank you for the members of staff who helped me complete
my project and provided me with experience and skills which will benefit me
greatly in the future; Prof. Rosemary Barnes, Dr P.Lewis White and Mr Michael
Perry. I have learnt that scientists are not only looking for new drugs to treat
patients, to further their knowledge on other organisms or how to reduce the
chances of the disease spreading internally and externally, but to also find new
ways in making diagnoses which will make a knock-on effect on whether the
patients would need treatment and why. The area and equipment used has
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fascinated me and has encouraged to one day pursue the career in scientific
research. I believe the practical side of the project and the guidance and
support from staff helped me understand the topic and learn a lot more on
fungi. I believe this will benefit me when next year in my A levels as it is part of
the biology curriculum. I believe the department met my expectations; with a
friendly environment and dedicated lab specialists using advanced equipment.
References
1.
Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F, et al.
Defining opportunistic invasive fungal infections in immunocompromised
patients with cancer and hematopoietic stem cell transplants: An international
consensus. Clinical Infectious Diseases. 2002;34(1):7-14.
2.
De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et
al. Revised definitions of invasive fungal disease from the European
Organization for Research and Treatment of Cancer/Invasive Fungal Infections
Cooperative Group and the National Institute of Allergy and Infectious Diseases
Mycoses Study Group (EORTC/MSG) Consensus Group. Clinical Infectious
Diseases. 2008;46(12):1813-21.
3.
Barnes RA. Directed therapy for fungal infections: focus on aspergillosis.
Journal of Antimicrobial Chemotherapy. 2013;68(11):2431-4.
4.
Barnes RA, Stocking K, Bowden S, Poynton MH, White PL. Prevention
and diagnosis of invasive fungal disease in high-risk patients within an
integrative care pathway. Journal of Infection. 2013;67: 206-14.
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Bibliography
Readers interested in the topic may find more information on these websites:




www.aspergillus.org.uk
www.fluorogenics.co.uk
www.nhs.uk/conditions/aspergillosis
www.medicalnewstoday.com
Many articles can be found on the Royal Medical Society.
www.rsm.ac.uk
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