Diagnosing the fungal infection talaromycosis earlier to save lives
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Diagnosing the fungal infection talaromycosis earlier to save lives
Talaromycosis is a fungal infection found in Southeast Asia and is life-threatening for people living with HIV and other immunocompromising conditions. The disease has non-specific smouldering symptoms and spreads to multiple organs before diagnosis is made. Current diagnosis is made by culturing the fungus from patient specimens, but this is a process that takes anywhere between 5 days to four weeks, delaying diagnosis and making treatments less effective. Dr Thuy Le, of Duke University School of Medicine in the US, has found a new way to test for talaromycosis, with the aim of diagnosing the disease earlier and saving lives.
Talk like an infectious diseases researcher
Antibody — a protein created by the immune system which protects against foreign substances, including bacteria, viruses or fungi
Antigen — a substance unknown to the body which causes the immune system to produce antibodies
Assay — a laboratory procedure to measure the presence of a substance
Culture — the process of growing cells in laboratory conditions
Fungal pathogen — a fungus which causes disease in humans, animals and plants
Human immunodeficiency virus (HIV) — a virus that damages cells in the immune system, making it harder for the body to fight disease and infection
Infectious disease — a disease caused by organisms such as fungi, bacteria, viruses or parasites that can be passed on from one person to another, from animals or insects to humans, and from the environment to humans
Protein — a large and complex molecule that performs essential functions in the body, including the support of immune responses
Fungi are everywhere, and can be used in food, medicine, and even building materials. However, fungal pathogens, or disease-causing fungi, cause a variety of human diseases, ranging from mild athlete’s foot (an itchy skin infection between the toes) to life-threatening diseases like talaromycosis.
Talaromycosis is an invasive fungal infection found in Southeast Asia, caused by breathing in a type of fungal pathogen called Talaromyces marneffei. Healthy people are unlikely to develop the disease, but for individuals with a weakened immune system caused by medical conditions, such as human immunodeficiency virus (HIV), it can be fatal. Up to one in three individuals with talaromycosis will die of the disease, even after receiving treatment.
Unfortunately, early symptoms are non-specific, often mild and difficult to spot, and methods of diagnosing talaromycosis take so long that the disease has often spread throughout the body by the time a patient has been diagnosed. “As with cancer, the earlier you diagnose talaromycosis, before it becomes disseminated to multiple organs, the better the treatment outcome for the patient,” says Dr Thuy Le of Duke University School of Medicine. Dr Le is developing several rapid non-culture-based tests to make an early diagnosis of talaromycosis, with the aim of reducing the number of deaths caused by the disease.
How is talaromycosis currently diagnosed?
Due to its initially vague, non-specific symptoms, patients do not seek care for talaromycosis until the disease has reached its advanced stage. In addition, the disease is diagnosed by a slow process called culturing. Culturing allows microorganisms to multiply in a nutritious media over time and reach a level that can be observed by eyes, and can be identified in a laboratory with the help of a microscope and some chemical reactions. This process is relatively fast for most bacteria, which take one to five days to identify. However, in the case of the fungus Talaromyces marneffei, this process is substantially slower. “It can take up to a whole month to grow Talaromyces marneffei in culture, so the diagnosis is often not made until talaromycosis is already at an advanced stage, when treatment is least effective,” says Dr Le.
What is Dr Le’s team doing differently?
“Aside from the decades-old culture method, we apply methods that are used for many other pathogens but that haven’t been used for this disease,” explains Dr Le. “We look for specific protein antigens being released by this fungus during infection, or the fungus DNA itself.”
Many pathogens release large amounts of protein antigens that fight against the immune response of the host (the person or animal with the disease). The host’s immune system responds to the antigens by producing antibodies and triggering other immune cells to fight back. To find specific protein antigens being produced by Talaromyces marneffei, the researchers followed laborious but logical procedures. Firstly, they isolated total ribonucleic acid (RNA) from a culture of Talaromyces
marneffei and made copy nucleic acid (cDNA) from the RNA. They then made a library of different segments of the cDNA (~ 1 million) and introduced them into the bacteria Escherichia coli, which created around 50,000 proteins that the cDNA codes for.
To screen this library for protein antigens produced by Talaromyces marneffei, the team injected Talaromyces marneffei into an animal model, such as a mouse or rabbit. The animal responded by producing natural antibodies against Talaromyces marneffei. The team then took a blood sample from the animal and screened it against the 50,000 Talaromyces marneffei proteins to find out which proteins react to the antibodies. The protein the researchers found is called Mp1p. Dr Le explains, “This particular protein antigen is expressed in abundance in the cell wall of the fungus and is excreted in the circulation during infection, so it is a perfect target for a diagnostic assay. We cloned the Mp1p protein, injected it into an animal model to produce specific antibodies to this protein, and then cloned the antibodies and made them in bulk to produce an antigen detection assay for Talaromyces marneffei.”
When a blood or urine sample from a patient is added to the mixture of cloned Mp1p antigen and cloned antibodies, Dr Le can see if the patient has the protein and, therefore, the infection. “The technology involved in this assay development is very conventional; it’s not rocket science! But the hard work that goes into applying the systematic screening of novel protein antigens produced by a pathogen, and using this protein antigen as a diagnostic marker for disease, makes this work innovative and impactful,” says Dr Le. “We can now detect this Mp1p protein, which we know is unique to this pathogen. Diagnostics need to be sensitive and specific, and make accurate predictions.
We’ve achieved these. We have found that detecting the Mp1p antigen is more sensitive than using conventional blood culture methods. It is also much faster, with results within six hours compared to the culture method of up to one month, and we can detect the infection up to four months before a culture can.” Being able to diagnose talaromycosis so much faster and earlier allows early treatment that will undoubtedly save many lives.
Created in BioRender. Brown, L. (2024) https://BioRender.com/h61p998.
Collaboration
The success of Dr Le’s work relies on collaborating with many people. “We partner with Professor Kwok-Yung Yuen from the University of Hong Kong and many researchers from academic institutions across Southeast Asia, as well as commercial partners to produce new tests. Our work to validate these tests for clinical use takes an entire ‘village’ of clinicians, nurses, coordinators, regulatory managers and hospital sites,” she says. “Most importantly, without the patients who volunteer to participate in our research, we wouldn’t be able to advance the diagnosis of this disease and make this level of impact. As researchers, we often get the spotlight, but the research participants are the true heroes who deserve credit. They allow us to advance the field, and I take this opportunity to acknowledge all research participants globally for helping us advancing diagnosis and treatment of human diseases.”
What are the next steps?
Reference
https://doi.org/10.33424/FUTURUM562
For Dr Le, the work continues beyond the laboratory. “We have established that this protein antigen can be used to make early diagnoses,” she says. “The next step is to develop a rapid point-of-care test, such as the lateral flow test for pregnancy or for COVID-19 that can be used at the bedside or in the clinic.” Dr Le is working with IMMY, a fungal diagnostic company in the US, to get approval for one of the tests they are working on from agencies such as the Food and Drug Administration in the US and the European Medicines Agency in Europe. Dr Le adds, “Once the assay is approved for clinical use, our work still doesn’t stop. We need to engage with policy makers, clinicians and the public to implement the assay. We will continue to work on extending its applications. Can we use it, for example, to screen asymptomatic people at risk of contracting this infection, such as people with advanced HIV? If they have the protein, should we treat them pre-emptively with antifungal drugs to stop the disease from developing? The research continues!”
Dr Thuy Le
Division of Infectious Diseases and International Health, Duke University School of Medicine, USA, and the Tropical Medicine Research Center for Talaromycosis at Pham Ngoc Thach University of Medicine, Vietnam
Fields of research: Infectious diseases, medical mycology, epidemiology, clinical trials, and global health
Research projects:
• NIH R01: Making an early diagnosis of talaromycosis – an approach to reduce morbidity and mortality in advanced HIV disease in Southeast Asia
• NIH U01: Tropical Medicine Research Center for Talaromycosis in Vietnam
• NIH R01: Development, clinical validation, and readiness for implementation of a novel Mp1p D4 point-of-care test for rapid diagnosis of talaromycosis
• NIH SBIR: Development of a Point of-Care Lateral Flow Antigen Test for Talaromycosis
Funder: US National Institutes of Health (NIH)
About infectious diseases research
Specialising in infectious diseases research can lead to a varied and rewarding career, working to improve our understanding of the pathogenesis (development) of pathogens. The goal of this research is to improve the diagnosis, treatment and prevention of a range of diseases caused by viruses, bacteria, fungi or parasites. Conducting laboratory research and working directly with patients will give you a real opportunity to make substantial impact in the field, while helping to educate and train others.
Dr Le believes it is also important to take research out into the real world. “We go beyond the laboratory and research hospitals to implementation science because, otherwise, our impact would be limited to academic publications,” she explains. “As researchers, we can make an impact beyond a single individual patient, at the population level, by engaging policymakers and healthcare sectors in our work.”
Dr Le is incredibly passionate about her work and about doing everything she can to reduce the number of deaths caused by talaromycosis. “Beyond making an early diagnosis in people who already have symptoms of disease, can we prevent the disease from happening in the first place? Can we be proactive, screen for the disease in high-risk people, and prevent the disease progressing by giving pre-emptive treatment?”
These larger goals involve multidisciplinary partnerships and strategic collaborations. “We need to collaborate with health economists to analyse whether such a screen-and-treat strategy is cost effective,” says Dr Le. “Does it save lives and money compared to waiting for people to come in for treatment? Screening costs money and may not be beneficial at the population level from the health system perspective. We need to collaborate with social scientists to conduct qualitative studies to find out who’s willing to pay for screening – an insurance company, policy makers or the patients themselves?” These questions need to be answered for a real and bigger impact to be made.
Infectious diseases research will always be relevant and important. Dr Le says, “There will always be new and emerging pathogens, and clinical problems and research questions to be answered. Other fields of medicine continue to progress because of advances we have made in infectious diseases, antibiotics and vaccines, and which have allowed humanity to survive deadly pandemic infections.”
Pathway from school to infectious diseases research
Build your skills and knowledge in biology and chemistry, and other relevant subjects such as mathematics, physics or psychology, as these are likely to be required for further study in medicine.
Learn about biostatistics, coding, data management and data visualisation, as these will help with analysing results.
During university, you can help with research projects being conducted in your department to get direct experience of the methodology and processes of medical research, data collection and analysis.
Explore careers in infectious diseases research
Learn more about the work of Dr Le and her colleagues working in the field of infectious diseases at the Duke University School of Medicine: medicine.duke.edu/divisions/infectious-diseases
Follow the work of the Tropical Medicine Research Center for Talaromycosis in Vietnam, established by Dr Le: tmrc-network.org/research-centers/vietnam
The Infectious Diseases Society of America and the International Society for Infectious Diseases have a range of articles about infectious diseases research and career paths on their websites.
The National Health Service in England has a helpful webpage about careers in infectious diseases.
Meet Dr Le
My research career was nurtured during my Infectious Diseases Fellowship at Yale University and my PhD training at the Oxford University Clinical Research Unit (OUCRU) in Vietnam. Since then, I have established the Tropical Medicine Research Centre for Talaromycosis in Vietnam. We aim to advance a pipeline of new non-culture-based diagnostics and test new treatment strategies for this neglected disease. We are building research capacity for Vietnamese researchers and collaborators to collectively make a difference in the field.
Combining my experience of laboratory work and understanding of basic molecular microbiology methods with my PhD in epidemiology, my knowledge of public health, and my clinical work in diagnostic treatment and prevention has allowed me to build a research programme that is comprehensive, highly translational, interdisciplinary and impactful.
My work has been inspired by my patients, my desire to make a positive impact in the world, my education, training, travels and exposure to the world. I grew up in Vietnam and received my college, medical and graduate degrees in the US and UK. I always wanted to return to Vietnam – where there are less resources, researchers and funding – and bring what I’ve learnt from the US and UK to a place that needs that knowledge the most. Being able to return, with my education and expertise, and continue to improve health and tackle diseases in my country is very gratifying for me.
You have to work with people to achieve greater things. This involves inspiring people and creating win-win situations for everyone involved.
Impact and inspiration come with time – they are benefits that accumulate through persistent hard work and seeing how that hard work makes an impact in the field, rather than one ‘shiny’ research paper or a big grant. I am very proud of the first-ever clinical trial for treatment of talaromycosis that I led in Vietnam. It took seven years from the conception of the idea to publishing the results, but those results changed treatment guidelines for talaromycosis globally. Afterwards, I was invited to write clinical guidelines for the disease, and only then truly began to feel the impact of our hard work.
I’m already a leader in the field, so my goal now is to train the next generation of researchers to amplify the work that we have started.
Dr Le’s top tips
1. Be grateful for mentorship and build mentorship relationships. That applies to any field, not just medicine or research. The more people supporting and accompanying you on your career journey, the more pleasure you have and the better chance you have of being successful.
2. Seek exposure to global health issues and to resource-limited settings. You can’t appreciate the need for solving problems unless you’ve been exposed to them. Get out there!
3. The path least travelled can be very gratifying.
Do you have a question for Dr Le?
Write it in the comments box below and Dr Le will get back to you. (Remember, researchers are very busy people, so you may have to wait a few days.)
Learn how infection biologists are identifying new strategies to fight bacterial infections:
www.futurumcareers.com/a-new-paradigm-in-antibiotic-research
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