[MUSIC] Hello everyone, welcome to another lecture of this course and actually with this lecture, we start another module. We have so far focused on antimicrobials, on antimicrobial resistance, and now we are going to give an insight how this is tested. And how do we find out that they actually are resistant in the laboratory, for example. My name is Lina Cavaco, and I work with the reference testing of antimicrobial resistance as the European Union Reference Laboratory and also for the WHO, for the World Health Organization on antimicrobial resistance. So we focus a lot on the testing and how we can find out that they are really the resistance ones that we are looking for. On this lecture, we are going to have an overview of how we test and why we test, what is the importance of testing. How it is in terms of clinical testing and testing for research, for example, and about the importance of having standards and doing it in the proper way with all the standardization that is needed and how we actually measure resistance. And, look at from the measurement, from the laboratory point of view and also from the genetically point of view. And also how to, we present the data qualitative or quantitative, depending on the methods that we use. And then we have some lecture focusing the actual methods in the rest of the module. So why do we do this? Well, in principle, we do this because we want to predict something. We want to predict, for example, that the person that is sick can be treated. We want to predict that the treatment works. We also want to predict what happens in the environment, what happens in resistance in our country, or in the world, or in a certain area. We want to detect that resistance is arising. A new one, for example. And we also want to compare between different areas if we do different things. And another very big important thing while we are collecting all these data because we want to do something about it. And if we do something about it if we do an intervention. We want to know how it was before, how it was after we acted on it, and did it really solve the case, or was it better, or was it worse so that we can prevent what happens. So the major objectives for testing are actually to use these data and these data should be good and should be reliable. We are testing, for example, isolates against antimicrobials. And we want either for research or for the patient's sake, look at this from in the laboratory. Of course, in the laboratory it's not the real thing. It's not the patient but we want to predict how it work. We will have to take an account that some drugs are different from others. We have to take also in account that bacteria different from others. So we always have to look at the combination of bacteria versus drug because it could be very different from one bacteria to the other or from one drug to the other and the combination of the both of them. So, when we are testing we look at resistance patterns and this would be helping us to choose the treatment. It could be that the certain pattern, let's say a strain that is resistant to five different antimicrobials. And all the strains that are related to that strain would also have the same pattern. It could also help us on typing. For example, for identification of the species, if we know that one species is always resistant to a certain antibiotic. We can also have an idea that it could be that species. We can use it for research projects. Of course, there's many research projects and areas going on antimicrobial resistance. That's where we work on and we work also a lot on monitoring and surveillance and that's where we want to know trends. We want to know in the country. We want to know in the world. We want to know what is going on and how it's spreading, how it's get worse, or how it's getting better. Both in humans or in animals it could be different from the objectives that we have. So, well we'll do a test and by doing a test we will also need to do some disclaimers. Testing is a guideline for the treatment, it's not that the absolute truth. We might be some factors that we cannot deal with in the person, and it can behave in a different way. If we give it into a person or an animal than when we do it in the laboratory. But the patient response will be the last answer to this, if the patient is treated then it went good, if the patient doesn't get better then it went bad even though the test might have said something different. So, when we are, for example, in a clinical laboratory, the obligations that we have, while even though there's this limitation is to give as much information as we can and the best information that we can. So, we want to give the best clinical information towards the test that we are doing. We want to provide the best antimicrobials that are effective for that case. Which doesn't mean that it's only the best ones, we might need to control also the use of some drugs that should not be used. So we actually also think about inappropriate use and we should think about that we should give the ones that are indicated for that case and not too much data. And we want also to reduce the emergence of new resistance. So we want also to check on what is going on and maybe avoid some antimicrobials so that the resistance doesn't grow worse in that area. So when we do the testing, we are in the lab, we need to do something. We have to follow methods. And these methods are very standardized. We have the American and the European standards for example, that really are to be followed. Totally, if we change anything in the standard, we need to validate why we are doing those changes and validate how we are changing and the standards take up the whole method, very, very detailed. So the principle is to standardize so that we can compare between the laboratories and we can get the best results. This standardization goes all the way. We are using culture media because we are growing bacteria in the laboratory and we are using the media that is defined in the standard. We are supplementing, if necessary, as defined in the standard. We are putting in some bacteria which are also defined in terms of how much, how dense the culture should be, and how many bacteria should go in the test. And we are doing the test very, very strictly under the standard conditions. Also to make the standard work, we also need some control and for that we use some control strains that have known results or known ranges of results where we check that our test is going well. And in the next presentations, I'm going to go in much more detail about the testing, but this is really the general things about how we do testing and how we have to standardize. Again, in terms of standardization given a historical perspective. Well, we have evolved using different kinds of tests, and we have maybe more in the past but still, used a lot of this diffusion tests, which is an easy and practical and cheap test. We are moving more towards dilution tests, which are more quantitative and more precise for obtaining results, and we are evolving towards having much more laboratory and credited with quality assurance really critically checked so that we have the results as good as it can, as reliable as they can be. And we also have contacted in networks between laboratories so that we are doing the things similarly not only in terms of the standards we use but also in terms of which drugs we test, and so that the results between countries can be compared and having some information that is available for everyone. So standardization is a very big issue here. Everything is very sensitive, all the methods are sensitive to any small change. So, if we change something it will influence the result. And here are some factors that influence the result, that's why we have to control them quite strictly. Small things like the inoculum, or the pH, or a little bit on the time or the temperature will make a change, so we have to be very strict about them. Also, if we are doing, for example, this diffusion, we even have to think of extra factors because we are working with agar, and there's some depth of the agar and dryness and the growth is different. So we have even to take more measures. But we are going to talk about it more in specific when we are talking about the diffusion methods. So the essential things here in the methods is, well, we have some back here an inoculum that is standardized so that we have the right amount of bacteria in the test, and then we have some medium where it's growing, where we have to check the conditions of the medium. And then we have, when they are in contact, when the bacteria are growing in the medium, then we have to check temperature, and conditions, and the gases, and we have to do quality control with quality control strains that have expected results and ranges. And when we do it in the lab, we have to measure it in some way. We could do it in several ways. Now we are talking mostly about the phenotypic way, the way of actually growing the bacteria in the media and looking at the result. But we could also do it molecularly. A lot of our labs are doing molecular tests, either by PCR or by genomic sequencing, and it can look directly there for resistance determinance. So we can look directly in the genomes or doing tests for detecting the resistance genes. It's not very practical yet, but it's really getting there. And actually, nowadays it is one of the main ways we do it. Now when we have new resistance coming up and we just had the example two weeks ago where we have a new gene coming up, the first string with it was not testing the strings, not taking them all up to the freezer. No, we checked genomes. And in that way, in one weekend, we tested 3,000 strands. We would never be able to do that in doing tests in the laboratory. So when we have the information, when we have the data, when we have the possibility to do molecular tests really very practical, very quick. When we don't, and then the clinical information that we also need the phenotypic test, we do phenotypical testing also because it is complementarily to the molecular. Also because it gives the clinical information that we need for the patient, really in vitro, also because the molecular testing sometimes there are genes that are there but not expressed. So here we actually see the effect. And we can have both qualitative test that give a classification, if it's resistant, or intermediate, or totally susceptible. Or we can have the quantitative classification of the results where we have actual concentration, a minimum inhibitory concentration attributed to a certain drug to that species that we have. So when we do this testing, we have the methods, as I've told you there's a lot of standardization. There's the standards and we choose which methods. We still have some methods to choose, and we choose them based on how easy they are, how flexible, what we need, what we want to do, sometimes we need to do some automatic systems that could be available to make it faster to make more tests. It could also depend on how much it costs and how much technical abilities we have, and so on. But, these methods are basically based on three different variants. Well, two big variance which is diffusion and dilution, and then in the dilution could be in broth and agar. And, they have this phases, and they will be developed from there. All of them have standardization going on, and if we get qualitative or quantitative resistance results depends on the methods we used. If we use qualitative, what we want to know, is it resistant? Is it susceptible? Or do we get a result that is kind of intermediate? And here, we want to make a decision for the treatment basically. It's difficult to compare because it's qualitative, we don't have so much security with the methods, unless we use some calculations of these diameter zones. For example, that I can show you ahead. For the quantitative, results we get a number, we get a concentration so it's much more precise. And this information can be shared and compared between laboratories. So when we choose this minimum inhibitory concentration which is the lowest antimicrobial concentration that will inhibit that particular species or that particular strain actually. We are saying that that concentration is the MIC and that should be similar in another lab. [COUGH] So it is actually the basic measurement of the effect of the drug in that particular string. So how do we do this? Well, we have the qualitative way, the first method that was discovered by Kirby and Bauer, and it is basically a method that is going on on a petri dish with an agar, with a solid medium on it. And there is an agar, and you spread a layer of the bacterial innoculum. That is spread in a certain standard way. And then you put on these disks that contain antimicrobial on paper. So it contains the drug. The drug will diffuse. This will be incubated in an incubator from one day to the next. And when you go there the next day and look at the plate, you see this. You see the growth of the bacteria in the layer and you see some in addition around the disks. And what you would do is to go on, measure the diameters, and that's your inhibition zone diameter. These inhibition zone diameters can be looked up on tables. And for a certain antimicrobial and a certain species of bacteria, you have a table that tells you, is it resistant, is it susceptible, or is it something in between. And that's your qualitative result. When you're doing dilution methods, you get a quantitative result. So, you have media. Which is liquid for example, in wells, in microtiter plate which have about 5200 microliters of liquid media. Or in tubes which is a larger volume, but it's a similar approach. And you have here a range of concentrations of the drug. And you put the same amount of bacteria and then you read the by growth or no growth. Here we would see growth, here we would see no growth. And here it's no growth, and the turbid ones have growth. You can do sort of the same, instead of doing in tubes, on agar plates. So, the concentration of the antimicrobial is on plates. You have plates for each concentration, and then you spot on the bacteria that you want to test. If they grow, it's growth. If they don't grow, it's no growth. And you have the same spots on every concentration so you can also measure your minimum inhibitory concentration. You have something in between, which is this E-test approach. Which is a diffusion test but because it is a very special one, you have a strip where the antimicrobial drug is on. But the drug is on a gradient so that the highest concentration on this side and the lowest on this. And when it diffuses, it causes an ellipse. And it's made in a smart way that you actually can read the result in the scale as an MIC result here. So this is kind of a mixed approach. It's not a very standard method but it's quite used. However, it's quite expensive. So this was an introduction to the methods. We'll have some detail lectures on the actual methods themselves. Thank you very much. [MUSIC]
