Globally, of the top ten causes of death, three of them or almost 16% are from infectious diseases1. Among infectious diseases, respiratory illnesses account for 3.9 million deaths annually, while HIV/AIDS accounts for 2.5 million deaths annually and Tuberculosis for 1.7 million deaths annually1. Ongoing research helps us understand ways to identify new and emerging respiratory pathogens, helping to make it faster to detect and manage subsequent illnesses.
Deborah Steensels, Resident at Erasme University Hospital
Deborah Steensels of the Erasme University hospital – ULB is studying respiratory illnesses. She studied Pharmaceutical Sciences from the Catholic University of Leuven (Belgium) in 2009, then obtained a Masters degree in Clinical Biology (hematology, chemistry and microbiology) in 2011 and a Masters degree in Hospital Hygiene in 2014. She finished her specialist training in 2014. Since then, she works as a resident at the microbiology department of the Erasme University hospital.
She is principally responsible for the infectious serology, virology and molecular microbiology departments. She is an active member of the Belgian Society for Microbiology and Infectious diseases. She combines these activities with a PhD focusing on the identification of respiratory pathogens using a syndromic approach and has developed a customized respiratory TaqMan® Array Card. We sat down with Deborah to understand the differences between conventional and molecular methods for pathogen detection and how their Custom TaqMan® Array Card is helping them with pathogen identification for research projects.
Conventional methods vs. molecular based methods – Out with the old, in with the new
SWATI: Could you tell me a little bit about your research?
DEBORAH: When I arrived at the Erasme-ULB hospital in September 2014, there was already a will to improve the lab’s strategy for respiratory diseases, in particular for the viral pathogens. We still use many conventional methods with a limited pathogen coverage, limited sensitivity and/or long turnaround time. So we were looking to improve our strategy moving forward to a molecular based method covering more respiratory pathogens with a higher sensitivity and a shorter turnaround time.
SWATI: That’s great. Can you tell me a little bit about traditional methods that are currently being used in some of the labs?
DEBORAH: For respiratory viruses, we perform direct fluorescent antibody (DFA) tests and rapid viral culture for all samples, conventional viral culture with two supplemental cell lines (A549 and MRC-5) for bronchoaveolar samples and then of course we also perform bacterial and fungal cultures. With DFA we search for Influenza A and B viruses, Respiratory Syncytial Virus (RSV), Parainfluenza Viruses (PIV) 1, 2, and 3, Human Metapneumovirus (hMPV) and Adenovirus. Rapid viral culture allows detection of the same viruses except for hMPV and conventional viral culture is only done for BAL samples to additionally detect Herpesviruses (CMV, HSV, VZV). So you see the scope, the number of different pathogens that we are able to identify with our traditional screening methods is really low. Going to a molecular based method that allows a syndromic approach would allow us to look for more than 30 different respiratory pathogens at once which is huge and which would be an enormous gain in the future.
SWATI: It must speed up your time in terms of looking at six pathogens at one time versus 24 or more, right?
DEBORAH: Yeah because now-a-days everybody works in a sort of “test-based” way and with that I mean that the clinician does his clinical examination and based on his differential diagnosis, if needed, he will ask for a test. If the test comes back negative, he will go further and test for other pathogens. But that means that it takes more time to get some understanding of the situation and a syndromic method in the future would be a huge step forward.
SWATI: Viruses can be very similar to each other especially when they tend to evolve. So what does specificity look like for these conventional methods, such as antibody or viral cultures compared to molecular techniques?
DEBORAH: I think specificity is not really an issue for conventional methods. I think the main problems are the limited number of pathogens that are covered and the sensitivity because not only do you need very experienced lab technicians who read these microscopic images and it’s really not easy I can tell you. Your sample quality is also really decisive for the sensitivity.
Taking a multi-panel approach – Pathogens to associated phenotype
SWATI: So how does a multi panel approach help to guide your current understanding of the problems of pathogen and possibly associated phenotypes?
DEBORAH: As discussed, the number of pathogens that can be detected will increase significantly using a syndromic panel and there will be a great learning curve not only for microbiologists, but also future clinicians interpreting data. There is just not enough knowledge in the literature on the prevalence of some of these pathogens in asymptomatic patients. If you test you and me for instance, we might find a Rhinovirus or whatever virus and this information on “carriage” in healthy persons is crucial because it’s going to help us interpret the results. We are already working on a research study that goes in that way because it’s really something that is missing in the literature and I think our current understanding is only going to explode once we begin routine use of these large panels.
SWATI: Absolutely. Yeah and I agree with you about, asymptomatic individuals because a lot of the times, even with infectious diseases someone might be infected and there’s a spectrum of phenotypes where someone can show an extreme phenotype versus another that shows none. So how do you understand that spectrum in general?
DEBORAH: Of course, and that’s why we also focus on immuno-compromised individuals because basically they have (theoretically and it is also proven that they have) a bigger chance of developing an infection. A basic virus that normally causes like a sniff for two days in healthy individuals can go down to pneumonia and even death in an immuno-compromised individual.
SWATI: Absolutely, because, their immune system is compromised so there’s no way for them to fight back, but we need to understand that scenario so that’s certainly very important.
Studying respiratory pathogens – What does your research focus on?
SWATI: Can you tell me a bit about your inclusion criteria for your research?
DEBORAH: We chose to concentrate on samples from immuno-compromised adults. That is the population where the most gain is to be expected because of the reasons mentioned before. We collect information on phenotypes, type of immuno-suppression; did they have a flu vaccination or not and so on.
SWATI: So when you collect samples what are you exactly collecting and what does the quality look like of the samples?
DEBORAH: Collections are from nose-throat swabs, nasal pharyngeal aspirations and broncho alveolar samples as well. Sputum samples are actually accepted for bacterial culture, but not for viral identification and not for molecular based methods.
About the quality of the sample, so first of all that depends on the type of sample and then also on the method that is used. Sample quality is very important for traditional methods but is less critical for molecular techniques because they just look for DNA and/or RNA present in the sample so the bacteria, viruses or fungal pathogens can already be dead when you still identify them.
Interpreting the results – Bringing in a multi-disciplinary approach with several experts
SWATI: Right, right, right. It’s looking for the presence of these specific pathogens
DEBORAH: Yeah, of course that also makes the interpretation of results from molecular techniques somewhat more difficult because it’s not necessarily true that if you find DNA and/or RNA of a certain pathogen in your sample that it means that the pathogen is alive. It could also be colonization or even contamination, but as mentioned before, that is something for which a large experience has to be built. Also, every single result has to be seen in the light of other results and collected information, etc. So it’s very important to have interaction and collaboration of experts from several different fields to interpret the research results.
SWATI: Absolutely. I mean I think that’s really critical and it’s nice to hear that there are several different types of scientists or experts in the field involved in kind of delineating if a specific infection exists or not.
DEBORAH: It’s a multi-disciplinary approach if you will, yeah, it should be a team that’s really collaborating closely too.
SWATI: To make that call.
DEBORAH: Yeah.
Why use more than one probe when detecting a pathogen – Case studies
SWATI: So, we hear about antibiotic resistant bacteria all the time and virus evolution. Since microbes that may be constantly evolving, with these molecular techniques or panels that you use, how are you able to change and are you able to detect the microbes that are evolving?
DEBORAH: Your question is actually very interesting and I think there are several answers. First of all I believe multi target identification, which means including several gene targets for one pathogen is important. So that means, when we want to detect the Influenza A virus for instance, not to include only one but maybe two, three or four distinct PCR probes. I can refer to a review we have published to highlight the importance of multi-target testing in molecular microbiology. Not only did we review the cases published in the literature, but also several cases of false negatives that were discovered in the lab where I was working at the time. Using only one target for one pathogen has proven to be dangerous because these microbes and especially RNA viruses change constantly with a risk of false negative results2.
One example, which really made me hallucinate the first time I read it, was the case of a Chlamydia trachomatis. Actually in Sweden they saw a drop in the prevalence of this pathogen. It’s a common cause of STD, so they thought our young people are protecting themselves when having sex. But in fact, years later, they found out that the strain had mutated and was therefore missed by an important commercial method that was used in most labs there. So the mutated version of the pathogen went unnoticed (and untreated) and spread throughout Europe.
So yeah that is the first part of my answer on this question, microorganisms genetically change as Darwin never sleeps. But what is also important is that “new” pathogens will surely be discovered in the future. It was only around 2000 that the Human Metapneumovirus was discovered. So maybe in 2017 another important respiratory virus will be discovered and it’s really important to have a method that allows great flexibility that can rapidly be adapted to include these newly discovered pathogens.
SWATI: That’s interesting. I’ve certainly heard about multi target identification, but these examples show you the true value of having the ability to use such an approach.
DEBORAH: Yeah. Of course you have to be practical. It is a similar approach we take in our panel that we are developing in collaboration with Thermo Fisher Scientific, of course we cannot do multi target identification for each and every target because that would limit the number of pathogens that we could include on the panel. So it’s a really balanced choice and of course for the most important respiratory pathogens we will include more than one target. Another major lesson from this review is the importance of knowing your assays and to monitor your assays to ensure quality results – do they still work or are we not missing anything? So that’s really a continuous effort that has to be done, in order to ensure a good detection.
What about detecting multiple viruses/organisms? – From assays to data analysis
DEBORAH: With our conventional methods very few to none of the multiple organisms present in the same sample are detected. But multiple organisms are routinely reported using multi pathogen molecular assays. Detecting multiple pathogens that may be present in the same sample is definitely also a major advantage of this multi pathogen method.
SWATI: What if some of these microbial strains have a very similar genetic background?
DEBORAH: Oh yeah, of course. Well actually even molecular methods are not always 100% capable of distinguishing some genetically closely related strains. For instance Rhinoviruses and Enteroviruses are really, really genetically similar, so I think the most important thing here is to know your assays and microbiology and to know that there are limitations. And so if we have let’s say a really strong signal that comes up for a Rhinovirus assay in our panel and then only a very weak signal in our Enterovirus assay, we know it’s a Rhinovirus that is basically a little bit picked up by the Enterovirus assay. So it can be really helpful to have the Ct values and this raw data to interpret your results in cases of multiple organism detection.
What does the workflow look like? – Scaling up and workflow
SWATI: And so getting back to your samples and your work flow how long is the workflow and how many samples can you run at a given moment?
DEBORAH: Well for DFA it takes about two hours, rapid culture takes about up to 48 to 72 hours and conventional culture takes up to three weeks, but the TaqMan Array Card (TAC) technology on itself takes about one hour from card set-up and data analysis. However, a separate extraction of the DNA and/or RNA is needed.
SWATI: Why did you use or want to use the Taqman Array Card for your research? What was important to you when you were choosing such technology?
DEBORAH: Well actually, when I took on the project of going for a new laboratory strategy for respiratory testing I started with a large comparative study of the many molecular systems available on the market. The main criteria were the number of targets, open versus close system, turnaround time, technical complexity and cost. The main reason for choosing the TAC technology was the great flexibility. For example the choice of pathogens, the choice and number of key targets per pathogen to be included on the card, that’s just really great because the composition of the card can be chosen in relation to the samples we’re studying. Since we work with lot of samples from immuno-compromised individuals, the panels that were available commercially are not necessarily always looking at the pathogens that we are looking at for our specific population groups.
Deborah Steensels with her team at Erasme University Hospital
Another advantage is the spatial separation of the 48 reactions, which allows assays to be easily changed without the need for extensive re-optimization and verification of highly multiplexed assays. So this could also aid in the rapid inclusion of novel or emerging pathogens or mutated pathogens, which is also really important I think. The technology uses Real-Time PCR which generates a semi-quantitative Ct value for each separate target, so this is also as I said an important tool to have an adequate and correct interpretation of the results and particularly in cases of co-infections. Then you add the simplicity of the workflows – you don’t need robotics since the reagents for each assay are pre-aliquoted in the reaction wells and since the nucleic acid extracts only need to be added once. Once we decided this technique was the most suitable for our lab and the samples we’re studying, we contacted our colleagues from the St-Jan Brugge-Oostende hospital who were already working with this technique and they kindly welcomed us in their lab to show us their routine and they enabled us to get familiar with the technique using their respiratory TaqMan Array cards. We used these cards, which were developed and verified by Cambridge and Bruges for our first study that was published last year. We learned to work with the technique. We were even more enthusiastic after having done this study and after seeing the results and seeing how easy it was to perform. I think there’s a great interest from different labs on this kind of technique.
Creating a database of all verified research assays – Stopping the reinvention of the wheel
DEBORAH: Now you have to know that not every lab has the possibility and has the know how to be able to develop their own assays and do a large study to verify the assays, which is not only time consuming, but also expensive.
I think the creation of a database for many different infectious emblems with verified assays from which customers could choose to create their own cards, would be very interesting for many labs and that would open the accessibility of the technology. So that’s my kind of a dream.
SWATI: So the ability to customize.
DEBORAH: If I could choose where it was going, it would be the creation of a large database that takes together all of these verified assays, not only the assays that we are collaborating on, but also those from other labs that are working with the technology and for other infectious syndromes such as gastro-enteritis, STD, etc. That would just be enormous because it would be sad to repeat all of the work that some of our colleagues have done and that is really good work, you know.
SWATI: Absolutely. I think, you know, at the end of the day you want to help propel research as fast as possible without having to reinvent the wheel again and again. So it would certainly be helpful.
DEBORAH: Yeah, exactly.
Training and application – Setting up the assays for routine use
You were talking about how your collaborators helped you understand the technology and let you play with it, how complex was the introduction of these technologies into your current workflow at your institution? Was it easy to integrate them or did it take a bit of time or were there any other limitations or challenges that you had to face?
DEBORAH: Based on our experience during the last two winter seasons, the introduction of the technology is not complex at all. As said earlier, you possibly have to adapt to your extraction workflow, but doing this card technology is really simple and I don’t see any problem in integrating or introducing it. We try to keep the price as low as possible for each sample, that way we can study more samples.
SWATI: So what’s next for your work?
DEBORAH: Well actually we’re now finalizing two other large studies using the TACs that were developed and verified by the Cambridge University Hospital and the hospital in Brugge. One of them is under review for publication and the other one is a really large epidemiological multi-center study so it will take a bit longer to write all of it down, but all of the results are there. And then of course last but definitely not least we are working on the development and verification of our own respiratory TaqMan Array Card in collaboration with Thermo Fisher Scientific and we’re now finalizing phase 2 (analytical verification of the assays in TaqMan Array Card format), so we hope that we will be ready for upcoming winter.
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References:
1] “The Global Health Policy Center.” Infectious Diseases. Center for Strategic and International Studies, n.d. Web. 17 Aug. 2016.
2] Steensels D, Vankeerberghen A, De Beenhouwer H: Towards multitarget testing in molecular microbiology. Int J Microbiol 2013, 2013:121057.
*For Research Use only. Not for use in diagnostic procedures
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