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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 1 Figure 3. Aspergillus under a light microscope Rhys Thomas 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 2 Rhys Thomas 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. 3 Rhys Thomas Nuffield Foundation 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* 4 Rhys Thomas Nuffield Foundation 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. 5 Rhys Thomas Nuffield Foundation 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 6 Rhys Thomas Nuffield Foundation 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. 12 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. 7 Rhys Thomas Nuffield Foundation 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. 8 Rhys Thomas Nuffield Foundation 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 9 Rhys Thomas Nuffield Foundation 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 10 Rhys Thomas 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. 11 Rhys Thomas Nuffield Foundation 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 12 Rhys Thomas Nuffield Foundation 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 13 Rhys Thomas Nuffield Foundation 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. 14 Rhys Thomas Nuffield Foundation 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 15