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                         Asked by others and by ourselves, our thoughts on how to practically implement Solution has shaped the project and forced us to tackle the problem from many angles. We needed a method by which we could expose the catheter, and bladder, to our anti-microbial proteins, and in a safe manner. Safety was a key consideration when designing delivery methods. We looked at a range of different approaches, including a variety of different catheter coatings, a semi-permeable membrane inside the catheter and encasing our bacteria within <a class="definition" title="sodium alginate" data-content="Anionic polysaccharide is distributed widely in the cell walls of brown algae, where through binding with water it forms a viscous gum.">sodium alginate</a> beads. For more information, check out our <a href="https://2015.igem.org/Team:Oxford/Safety">safety</a> and <a href="https://2015.igem.org/Team:Oxford/Design">design</a> pages.
 
                         Asked by others and by ourselves, our thoughts on how to practically implement Solution has shaped the project and forced us to tackle the problem from many angles. We needed a method by which we could expose the catheter, and bladder, to our anti-microbial proteins, and in a safe manner. Safety was a key consideration when designing delivery methods. We looked at a range of different approaches, including a variety of different catheter coatings, a semi-permeable membrane inside the catheter and encasing our bacteria within <a class="definition" title="sodium alginate" data-content="Anionic polysaccharide is distributed widely in the cell walls of brown algae, where through binding with water it forms a viscous gum.">sodium alginate</a> beads. For more information, check out our <a href="https://2015.igem.org/Team:Oxford/Safety">safety</a> and <a href="https://2015.igem.org/Team:Oxford/Design">design</a> pages.
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Revision as of 14:57, 18 September 2015

Practices

Overview

Coordinated action is required to minimize emergence and spread of antibacterial resistance (ABR). The World Health Organisation have recently (May 2015) endorsed a global action plan to tackle antimicrobial resistance.

The plan sets out 5 objectives:

  1. Improve awareness and understanding of antimicrobial resistance
  2. Strengthen surveillance and research
  3. Reduce the incidence of infection
  4. Optimize the use of antimicrobial medicines
  5. Ensure sustainable investment in countering antimicrobial resistance

Our work is focused on the first two WHO objectives. In our project, we are developing the use of bacteria as living therapeutics to provide an alternative to administering antibiotics, thereby addressing objective number two. However, ABR cannot be overcome with research alone in view of the fact that misinformed antibiotic use contributes a large part to the increasing worldwide acquired ABR.

In line with the first objective of the WHO global action plan, to improve awareness and understanding of ABR, our project relies on a three way conversation between the team, the public and experts. This influences every aspect of our project, from our choice of application to the details of our delivery system. The emphasis of our Policy and Practises is to take an iterative approach to developing our idea, continuously taking input from the public and healthcare professionals, so as to ensure that our project could become a realistic method to overcome ABR.

The practices approach of our project has been inspired by the Royal College of General Practitioners TARGET Antibiotics Toolkit. TARGET stands for: Treat Antibiotics Responsibly, Guidance, Education, Tools.1 Our guidance comes from healthcare professionals and the public; in terms of education we have worked on increasing awareness of ABR and synthetic biology; and our tools are based around using engineered bacteria as therapeutic agents. The targeted nature of our solution is achieved by building the project around our practices, constantly feeding back and forth between the two.

We want to make our website as accessible as possible to all readers, regardless of their level of expertise. Words with a dotted underline will show a definition when you click them.

Discourse

Approaching the public

From the offset we wanted to tackle a problem raised to us by the public. We sent out an initial questionnaire to the public to ask what big problems they wanted solving and to hear about what they thought about synthetic biology. We took the questionnaire to schools, to the streets and to our friends. Examples of their suggestions for the applications of synthetic biology included bacteria which:

  • Remove carbon dioxide from the atmosphere
  • Target and kill cancerous cells
  • Help treat Alzheimer's disease
  • Produce energy
  • Sew up holes in clothes
  • Produce teeth glue
  • Indicate how long someone has been dead
  • Combat antibiotic resistance

Antibiotic resistance

Of the responses to our initial survey, 40 of the 86 were related to Medicine and Health. Inspired by a talk given by Professor Dame Sally Davies: “A ticking time bomb: the infectious threat of antibiotic resistance” and the public responses, antibiotic resistance stood out to us as an issue we could tackle with synthetic biology. To confirm that we were on the right track, we approached the public again with the question: To what extent do you feel that antibiotic resistance is a problem that needs addressing in society today? This graph clearly shows that, according to the general public, antibiotic resistance is an important problem that needs solving.

Urinary Tract Infections (UTIs)

It was our team member George Driscoll’s work at the Hornsey UTI clinic in London which helped us to select UTIs as a specific cause to focus on. Due to the unaesthetic nature of the infection, it often receives less attention with regard to research. Having worked in the clinic, George knew first-hand that UTIs are a problem for a significant proportion of the population, and that resistance to the antibiotics used to treat UTIs is growing. In fact the clinic’s primary treatment method is long, high-dose courses of multiple antibiotics to overcome long-lasting infections despite the clinic’s awareness that their treatment choice may lead to further resistance.

UTIs are the most common healthcare-acquired infection. Among UTIs acquired in the hospital, approximately 75% are associated with a urinary catheter.2

Interaction with nurses

To learn more about catheter associated UTIs (CAUTIs), we visited the Churchill Hospital in Oxford. Our first visit to the hospital was to the outpatient clinic during which we spoke with Jan Harris, one of the nurses on the ward. Jan explained to us the current treatment of UTIs. The protocol for treatment is to first take a urine sample to test for the presence of bacterial colonies. If the dip test comes back as positive, the patient is treated with a wide spectrum antibiotic, after which an antibiotic treatment specific to the type of bacteria identified may be given. Whether or not a catheter is fitted with prophylactic antibiotic treatment is the doctor’s decision.

Up to this point, we weren’t fully aware of the potential seriousness of UTIs. Jan told us about a case of a person getting septicaemia as a result of a UTI. The patient had received antibiotics for seven days and had come back for a check up. Their urine sample was clear and but, despite this, the patient rapidly went into septic shock. This life threatening infection is caused by the uropathogenic bacteria rising up the ureter to the kidney. If a patient becomes septic the catheter has to be removed or CAUTI can be fatal. This shocking example made our project feel very relevant and we took a lot from this initial conversation. We went on to investigate the pros and cons of the current methods of CAUTI treatment, and compare these to what our solution could offer. Following this meeting, the design of the catheter became an integral part of our project.

Laura Evans, from the Adams Ward (geriatric) in the John Radcliffe Hospital, also talked to us about the current treatment of UTIs, informing us that the use of antibiotics is often ineffective and therefore infections are recurrent. We asked Laura the following questions.

What happens when a catheterised patient tests positive for a UTI?

“Whether or not the catheter is removed if a patient tests positive for a UTI depends largely on the reason that the catheter has been fitted. In most cases, the catheter remains fitted and the patient is treated with a large dose of antibiotics.”

Is antibiotic resistance a problem?

“Yes, particularly on this ward. As we treat elderly patients with recurring infections, the uropathogens they succumb to are often resistant to antibiotic treatment. As a result, we have to try different combinations of antibiotics, but in many cases this does not stop recurrences of the infection.”

Conversation with academics

In collaboration with Oxford University Scientific Society, we organised a public talk “Biofilms: role in health and disease, recalcitrance and in vitro modelling” by Dr. Andrew McBain, Biofilm Research Group, Manchester Pharmacy School. This talk helped the team understand the role of biofilms in causing infection and why they are resistant to antibiotic treatment. During his talk, Dr. McBain explained the role of biofilms in infections and how they confer ABR. He talked to us about persister cells and their importance in the recalcitrance of biofilms to antibiotic treatment. Hence, in the project design, we looked at agents that specifically target these sessile, metabolically inactive persister cells and decided on Art-175 as one of our antibacterial agents.

Professor Timothy K. Lu, from the Synthetic Biology Group at MIT recommended that we use endogenous anti-biofilm agents rather than designing them bottom up. He said that our idea to use synthetic biology as a means of fighting UTIs and ABR was “interesting” but raised to us the the problem of our bacteria being subject to resistance mechanisms depending on the agents used, but that we could overcome this by exploring combinatorial therapies.

During the summer, we also gave two talks, one at the termly Corpus Christi College Biochemistry talks and another to a group of alumni from the Oxford Biochemistry department. Again, the feedback we received in terms of our project idea was positive and our idea was described as “ambitious but achievable”. Max Crispin, Oxford Glycoprotein Therapeutics Laboratory raised the issue of whether the proteins we planned on secreting would be immunogenic. We explored this, and similar issues, in a report we wrote about the urinary biome. Another question we were faced with concerned the issue of whether our solution would make fungal infections more likely. This issue is addressed in the Futher Considerations section.

Return to the public

Having investigated the feasibility of our idea, we approached the public again to seek their opinion. The idea working on paper is one thing, but having the support of the public and the patients who would be using our solution is another aspect entirely.

We asked the public, “If you had the option of using two treatments for an infection, where one was antibiotics and one was bacteria that were designed to cure the infection, which would you choose?”

The majority (70%) of the people we surveyed said that they would take the advice of their doctor on this. This reinforced to us the importance of nurses and doctors backing our project idea. However, encouragingly, of those who wouldn't take the advice of their doctor, our treatment was favourite with just under half saying they would prefer the designed bacteria (48%), compared to 40% who would prefer antibiotics.

We also asked, “If your doctor recommended a treatment for an infection, which involved the use of bacteria that had been engineered to treat the infection, would you use it?”

Our survey showed that if a doctor recommended our treatment, only 6% would not consider it. This is very encouraging data for our project, but again highlights the importance of gaining support from doctors, because without their backing, the use of engineered bacteria as therapeutic agents will not progress from primary research. See the full results to this and our other surveys here.

We had very similar feedback when we spoke to patients. During a visit to the Bedford Ward at the John Radcliffe Hospital, we spoke with one of the catheterised patients there called Mavis. She admitted to having had UTIs in the past, but said she had not contracted a UTI since having a catheter fitted. We found that patients like Mavis could end up using the same catheter for up to 10 weeks. This enforced the importance of being able to keep our bacteria alive for a sustained period of time. When we asked her about treating infection with bacteria, she said she would be happy to, if it had been recommended to her by a doctor. She said, in her view, it is not dissimilar to using antibiotics.

Return to nurses

During this visit we were able to talk to Laura Evans again, this time to ask questions specifically about our project.

Our project involves designing a catheter that prevents the formation of a biofilm on its surface. What do you think of this idea?

A catheter like that would be useful, but it depends on how long your catheter would work for. Patients can have a single catheter fitted for 3 months or longer. Catheters are also removed for other reasons, for example if they become blocked. Catheter blockage is a common issue for patients suffering from UTIs.

Both Laura’s and Mavis’ response regarding how long a catheter remains in place inspired us to research how we could keep our Solution bacteria alive, the results of which can be found in design. Jan Harris, Churchill Hospital, raised the issue that our solution does not tackle intracellular bacterial communities (IBCs) and community UTIs. Jan made the point that UTIs are not just contracted through catheterisation, and it is important for us to also consider community based UTIs. IBCs and community UTIs are complex issues to solve but we have explored the potential of commensal bacteria as a treatment approach (See Commensal Bacteria).

Feedback from doctors

"Years from now we will treat most infections with bacteria and not antibiotics."

Professor James Malone-Lee
Barlow Professor of Geriatric Medicine

Our interaction with the public highlighted the importance of gaining doctor’s support for our project. With this is mind, George went back to Hornsey UTI clinic to speak with Dr. Sheela Swamy and Professor Malone-Lee, the consultant clinician as well as the principal investigator of the laboratory associated with the clinic. The Professor’s feedback on our project was extremely positive. “Biofilms have a profound effect on antibiotic resistance and so bacteria that can penetrate through biofilms strikes me as a very sensible project direction.”

Dr. Sheela Swamy, Hornsey UTI clinic, said:

“We get through so many buckets of antibiotics here, if your product could prevent infections or reduce our reliance on those antibiotics that is hugely appealing as a practitioner.”

“It will be really great if we actually have bacteria engineered to achieve biofilm and cellular penetration and a bactericidal effect will be a total seller. That may be the answer to the Chronic UTI if we can get it to work.”

The doctors at the UTI clinic left us with a few points to bear in mind as we are currently focused on only killing Escherichia coli and Pseudomonas aeruginosa:

  1. The bladder biome is complex with many culturable and nonculturable pathogens. What role each of these have and what concentration they need to be in to cause UTI symptoms is unknown.
  2. Uropathogens listed in literature are not the only organisms responsible for UTI as they are also present in normal controls yet don't cause symptoms.
  3. Bacteria are opportunistic and a skin commensal if in the bladder can cause symptoms, and we have had many women with pyuric UTIs even though they only grew a Staphylococcus so you may have to try develop something that works on a wide spectrum of bacteria.

Speaking with Patients

During his time meeting with Dr. Sheela Swamy and Prof. Malone-Lee, George was invited to attend a pair of clinic sessions in which chronic urinary infection patients came to see doctors and change their treatment plan or give samples for urinalysis. During these sessions George sat in on 8 consultations and interviewed 6 urinalysis patients to gain an understanding of their symptoms and the way their infection is currently managed. George was also given the chance to interview each of these patients and learn what they thought about antibiotic regimes, catheter-based delivery and our synthetic biology solution to their infections. From the 14 interviews conducted George drew these particular conclusions from his conversations with Hornsey’s patients:

  • That comfort levels with catheterisation are reliant on previous experience with catheterisation.
  • That antibiotic use often has side effects that significantly decrease the quality of life of patients often through nausea but also induced faecal incontinence.
  • Most patients are comfortable with the idea of GM solutions to infection given the backing of the practitioners in the clinic.

Delivery

“Is there a material for the catheter or its coating that is suitably permeable to this mystery molecule(s) yet entirely able to contain the source of microbes without any leaks?”

Professor Jef D. Boeke
Director, Institute for Systems Genetics
NYU Langone Medical Center

Asked by others and by ourselves, our thoughts on how to practically implement Solution has shaped the project and forced us to tackle the problem from many angles. We needed a method by which we could expose the catheter, and bladder, to our anti-microbial proteins, and in a safe manner. Safety was a key consideration when designing delivery methods. We looked at a range of different approaches, including a variety of different catheter coatings, a semi-permeable membrane inside the catheter and encasing our bacteria within sodium alginate beads. For more information, check out our safety and design pages.

Commensal Bacteria

We thought further about the discussions we had had at Hornsey UTI clinic and were inspired by what Prof. Malone-Lee said to us:

“Biofilms have a profound effect on antibiotic resistance … bacteria that can penetrate through biofilms strikes me as a very sensible project direction.”

At the clinic they talked to us out using competing species of bacteria to replace the common uropathogens.

“Patients keep taking probiotics while being treated with antibiotics in our unit and they don't seem to mind ingesting 20-40 billion acidophilus bacteria without a second thought.”

Dr. Sheela Swamy

With these conversations in mind, we went back to the drawing board. Our data showed us that we needed a high local concentration of anti-biofilm and antimicrobial agents to be able to break down a biofilm. We thought about how we could create these high local concentrations and came up with a new solution: to introduce free living bacteria into the urinary tract. Whilst we are aware that current GMO regulations would not permit this, we have explored the possibility of it becoming an option in the future.

Many people perceive the use of GMOs to be unethical and this has far-reaching impacts on society, such as the enaction of anti-GMO legislation. To explore these ethical questions and find out the public’s opinion on our project, we sent out a questionnaire to over 150 people, asking: If your doctor recommended a treatment for an infection, which involved the use of bacteria that had been engineered to treat the infection, would you use it? We also asked this question to a number of medical professionals during our visits to hospitals and clinics.

Whilst the majority of feedback was very positive, we did encounter some valuable criticism, as shown in the graph below. Most medical professionals we spoke to had positive responses, though one nurse had reservations about our idea.

This proportion of negative feedback, although small, highlighted to us that our dialogue with the public needed to be improved. We believe that, through improving people's understanding of our project, we can convince pessimists that genetic engineering is now a force for good. This also led us onto holding talks to student groups, which you can find in the Outreach section below. Nevertheless, this is very encouraging data for our project, and again highlights the importance of gaining support from doctors for this to become a realistic treatment option.

We also approached the public later on with a variety of different delivery option for them to choose from, including the idea of introducing free-living, genetically engineered bacteria into the urinary tract, alongside different catheter-associated treatments. 44% of the 50 people we asked were most happy with the free living bacteria, due to its effectiveness at treating the infection. This was by far the most popular option, followed by 20% opting to have the bacteria sealed within a catheter, since this balanced efficiency with safety.

The public’s biggest concern for this option is side effects from the engineered bacteria colonising the urinary tract. However, over 2/3 of the people who raised this concern said that they would be happy to use the method if they got a clear explanation of how it worked from a health care professional. Additionally, the concern was outweighed by the public’s opinion of having a safe kill switch, and that this treatment is the most effective. Again, full results can be seen here.

Safety Considerations for Free-living Bacteria

For the idea of introducing our engineered bacteria into the human body to be feasible, there need to be sufficient safety considerations in place. Whereas for the physically contained options, ensuring that if the bacteria escape, they die is important, it is especially important to make sure that molecular containment is in place for the free-living delivery option.

We have created theoretical kill switches for each of the situations that our engineered bacteria might find themselves in, to allow us complete control over when they die. Since the kill switches are based on the lac operon, if there are any concerns about the action of the bacteria, we can kill the whole population immediately, by introducing IPTG through the catheter. Furthermore, the kill switch is also sensitive to other elements which could be present (AI2 for free-living bacteria, valine for bacteria in media) to allow extra degrees of control. The kill switch options were another aspect we surveyed the public on. The results were that they were happy to use them, provided their healthcare professional ensured that it was safe. For more details, check out our safety and questionnaire pages.

Outreach

In an attempt to improve the general public's understanding of Synthetic Biology, we used a variety of approaches. We thought we could help certain members of audience, who represent the 6% of pessimists we found in an earlier survey, to fully understand the concepts of genetic engineering, and realise the incredible gains that can be made from projects like ours in the field of synthetic biology.

We asked the public: Which of these do you think is the most common infection acquired in hospitals?

Most of the public (45.5%) thought that MRSA is the most commonly acquired infection in hospitals, showing that they think that antibiotic resistance is a problem. However, with UTIs coming in quite far behind this (19.5%), it seems that the public are not aware of the risk of getting UTIs in hospital due to urinary catheterisation. This could be due to the fact that UTIs are advertised less than MRSA, but since this is an important issue, we looked into educating the public through sessions with summer schools and school students, as well as getting in touch with local media, such as BBC Radio Oxford, and promoting our project across social media. We believe that this has helped raise awareness of problems associated with catheter-based infections, not only in the urinary tract, but also with infections in other catheterised parts of the body.

BBC Radio Oxford

We were invited to talk about antibiotic resistance on BBC Radio Oxford. We went on the Kat Orman's morning show, alongside Monty Python's Michael Palin, and spoke about the increasing threat that antibiotic resistance poses, and what we're doing to combat it. We were asked questions about why we chose this area to pursue, whether our project was likely to be lincensed, and about the basics of synthetic biology.

Mabel and Helen in the studio with Kat Orman!

We answered this by using the analogy of computer hardware: if your computer hard drive is broken, you can buy a new model from the shop without worrying about whether it will fit or not, and it's the same with stretches of DNA sequence that you can stick together. We were also asked about our outreach and the efforts we were making to change the mindset of a society that demands to be prescribed antibiotics.

Finally, Kat ended by asking if we thought we could change the world with our project. After a little nervous laughter, we gave our answer: hopefully! Our section begins 1hr 6mins into the 3 hour show. Click here to have a listen!

UNIQ Workshop

We met with 40 prospective Oxford students to teach them about Synthetic Biology. The students had in interest in Biochemistry but knew nothing about iGEM.

We hammered home the key message of Synthetic Biology - that we achieve more progress by expanding a registry of standardised biological parts - through a 15 minute introductory presentation on BioBricks.

We then split them into groups and gave each one a mentor from our iGEM team. We worked through questions to test their understanding in a tutorial style and asked them to explain the constructs of previous iGEM teams. They finished by presenting their findings to each other.

Duke discusses the central dogma with UNIQ Summer School

UTC Oxfordshire

A couple of us gave a presentation on antibiotic resistance to a class of GCSE students from UTC Oxfordshire (a local school specialising in science) at the Natural History Museum in Oxford, The Pitt Rivers Museum.

Our talk covered the discovery of antibiotics, the advantages of them (including their use in laboratory work), how they work, and how bacteria can evolve to gain resistance to them, as well as concepts such as horizontal gene transfer and the consequences of antibiotic resistance on our everyday lives. It also covered our project outline, and pros and cons of Solution, showing how it should help combat antibiotic resistance.

At the end, we held a discussion between the students and our team about antibiotic resistance, and their perception of the concern. We also asked how they would feel about using our engineered bacteria, and the response was positive, with most of the students saying that if their doctor recommended the treatment, they would be open to using it.

Bang! Magazine

An article was written about us and our SolUTIon in Oxford University's science magazine, Bang! Read it here.

Further Considerations

Fungal Infections

“What about fungal infections?”

This question was asked following a talk we gave to Oxford alumni medical professionals. It was suggested that eliminating the bacterial population inside the urinary tract would increase the chance of the patient acquiring a fungal infection. Up to this point, we had not considered UTIs that are caused by fungi. The most common cause of fungal urinary infections are species of Candida. Infection with Candida usually occurs in patients with urinary catheters, typically after antibiotic therapy, although candidal and bacterial infections frequently occur simultaneously. We then attended a talk by Dr. Haribabu Arthanari that discussed multidrug resistance in Candida, highlighting Candida as the second most common cause of fungal infection globally.

For a comprehensive treatment of UTIs, our solution also needs to target fungi. The most common species of Candida involved in infections, particularly in the urinary tract, is Candida albicans 3. Unfortunately, according to a fungi specialist (Professor Neil Gow, University of Aberdeen), “there is no fungal equivalent to bacterial exotoxins”, so anything we use to sense fungal virulence must be a constitutive part of the fungal cell. Additionally, if we use a hyphal cell wall protein (as suggested by Professor Gow), this would only allow our bacteria to sense C. albicans.

If we were to use our current system and adapt it to target fungi, we would propose incorporating a system into our bacteria that senses hyphal wall protein 1 (HWP1), a protein used for hyphal development and yeast adhesion to epithelial cells, which is only present when the Candida are virulent 4. We would then use our chassis to produce an antifungal agent that would target biofilm formation as well as breaking down the virulent fungal cells. An example of such an antifungal agent is the Echinocandin class of antifungals. The problem with this is that it is very complicated to engineer bacteria to produce these agents, so a better solution for using our current system to target fungi as well as bacteria would be to coat the catheter in pre-made Echinocandin to prevent Candida biofilm formation. 56

Treatment Pros and Cons

Treatment Pros Cons
Antibiotics Effective against many common infections Not effective against biofilms
Easy to administer Bacteria can develop resistance
Few side effects Some can result in side effects that exaggerate initial symptoms
Inexpensive Can cause severe allergic reactions
Catheter coated with silver nanoparticles Bacteria cannot develop resistance There can be serious side effects
Noninvasive in those who would otherwise be having a catheter fitted anyway Catheters support biofilm formation which can lead to recurring infections
Botox Increases comfort by lessening symptoms of incontinence Does not treat underlying infection
May need to have a catheter fitted which increases incidence of infection
Long term effect unknown
Invasive
Methenamine hippurate Bacteria can’t develop resistance Have to take doses every day to prevent infection
Requires additional supplements for effective treatment (Vitamin C, Cranberry supplements)
Relatively new treatment which hasn’t been tested in children, the elderly, or breastfeeding women, so this limits the number of cases it can be prescribed in

Social Media Summary

Twitter

  • 396 tweets
  • 495 followers
  • Retweeted by: The Wellcome Trust, the Royal Society, FRONT nurses (leading Research Nurses from across the UK), The Guardian Science, The Economist, and lots of other iGEM teams!

Facebook

  • 321 likes
  • 5.0/5 star ratings
  • 6 reviews, all very positive

Instagram

  • 24 posts
  • 21 followers

References

  1. TARGET Antibiotics Toolkit http://www.rcgp.org.uk/clinical-and-research/toolkits/target-antibiotics-toolkit.aspx
  2. Catheter-associated Urinary Tract Infections (CAUTI), Centres for Disease Control and Prevention http://www.cdc.gov/HAI/ca_uti/uti.html
  3. http://www.cdc.gov/fungal/diseases/candidiasis/
  4. http://www.sciencedirect.com/science/article/pii/S0882401014000369
  5. Morris MI, Villmann M (September 2006) "Echinocandins in the management of invasive fungal infections, part 1". Am J Health Syst Pharm 63 (18): 1693–703
  6. Morris MI, Villmann M (October 2006). "Echinocandins in the management of invasive fungal infections, Part 2". Am J Health Syst Pharm 63 (19): 1813–20.