[MUSIC] Hello everyone, my name is Lina Cavaco, and I'm going to give you a lecture on another module, this antimicrobial resistance course and this module is dealing with how to make sense out of the data, how to interpret what you obtain in the resistance testing. So I told you about a series of methods, how we do them, how we get the results but here is really how we make sense and how we interpret what is going on with that bacteria, with that drug in real life so to say. So in this lecture we are going to look at the applications of the results that we obtained, so we try to do all these tests and we obtain some results, we want to see where we can apply them, a little bit about the standards that we are using, and the procedures and the quality assurance around this. And really, how you read these results and what it means. So here we are going to look really, on these readings, and when you have end results, how you actually use it and how you check this is right and not something that is a mistake source, and how you actually use this further in the world by interpreting and by checking the factors that are in this interpretation. And in the world, how you actually use the results in the end. So, we start having bacteria and having antimicrobial drugs and we want to make this into an interpretation that gives some idea about what is going on in resistance. As I mentioned, the resistance mechanisms are there, we are just checking in the lab how this works. So in the real world we have people that are sick or animals that are sick maybe. It's not always the case that we have a patient in our hands that we are, but we are going to look at the possibility of the treatment to work or not. So we are going to look if there is an indication from the lab tests. If the drug is going to work in real terms, in the patient. But we also have the other real world, which is the smaller real world, the genetic part where the real results we got from the testing might give us good indications of which resistance mechanisms are there. And these resistance mechanisms are there in the genome of bacteria or in plasmids, and by doing these tests we are much closer to get an idea of what the genetic background is. And this is really exciting for researchers because we really can see in a result, sometimes, well, this is an MRSA strain. If we are testing a gram-postiive, a staphylococcus and it's showing this resistance, definitely it must be this. And or if we are testing a gram-negative for example, an ecoli and we show this pattern of resistance. This is in famous ESPL producing a bacteria that has an extended spectrum and this is really important for the world and we are going to fight it. So we are going to look up end result in some perspectives, also because these resistance determinants have some influence about the treatment of the patients, because then we can also decide better which drugs to use if we also have the genetic part but it plays together. But when we are sitting in the lab, we see the microbiological part. So, that's the part we're actually seeing when you do the tests, we are reacting antimicrobials and bacteria together and we are checking what is the ability of the microorganisms to survive or to be killed in this concentration. So if this bacteria is resistant, it's able to survive in higher concentrations than its friends. Then the ones that are in the same group or in the same species. So of course we cannot interpret if we don't know the species, for example. We would be looking at the resistance as the ability to survive in these conditions. So we kind of interplay all the three slides of this when we look at results. And sometimes we only have this, sometimes we are actually so fast that we only have the genetic part that we immediately have sequenced and checked. And we have this and we don't know what the real world effect. And sometimes we have this because we're in a hospital or in a clinic. So, that's why we use standards. Because if it didn't have standards, we could make all the tests of the world, but we couldn't compare anything. And we couldn't see what means what. And these standards give us the method. So that we make it in the same way. And by making it in the same way, it makes sense that it's resistant, susceptible or intermediate. It also means something for the patient if we use it as a clinical. And if we have a certain MIC, it means something in the genetic terms or in the clinical terms, it means something in the end that we can say, well, if the MIC is that high, maybe it's this genetic mechanism. Maybe the patient will get well with the treatment and so on. So we need to interpret these results very carefully and very concerned about how they are going to be used. So that we want to get it right. It's very, very important before even reading the tests to do some controls, some checks. We need to check that we do it in the right way, that we observed all the standards. We need to check that the culture is pure. If we have a contamination in the culture, then we are just not testing the right thing. We also need to check the controls. If they are not growing in the medium, nothing can really be checked. And we also need to check for possible contaminations, which could be taken from these purity controls. And we need also to check the skips, because there could be something wrong with the the media or the panels or some contaminations making these skips for example. So before even reading we have to check all these things, and we also need to check the quality control strange ranges. I didn't write it here but that's an interpretation of the quality control and checking if they're in the right range is very important before you check the other strains. So for the quality control you also do this procedure and check the MIC. If you look for the growth and you check your control growth. And you check the lowest concentration that gives no growth, you'll have your MIC and you can read it. You read it both for the test strains and for the quality control strains, so that you make sure that your results are in the right range. Similarly, in the micro broth essay, well it's just on the panels you would be reading, I just explained how to read. We look for skips in this case where you have two skips you might think of repeating the test. You would read where there's no growth. Actually this part should have been here, then. And here, you have no growth, that means that the concentration is below or equal there. And here it's because the concentration of the MIC is above this. When we have a little different growth from the control wells we might think that there might be a contamination and think of repeating this test as well. And when the reading is Growth, no growth, then you have an MIC that's quite easy to read. So you check the whole panel, because in the same panel you will have different drugs, and if this happens with one of the drug, you might need to repeat the whole panel again. So, to make sure that you have the right result. Also, with the agar dilution you would be reading your results, checking the growth. Now, I don't have the growth control here, but you check your MIC for example here you don't see growth here. So, the MIC would be this 8 for most of the strains that are here. Actually, yeah, you cannot see most of them there, but it's most, for most of them it's 8. So, you got that result. And you're pretty sure that the result is right. If you exclude any contaminations, if you excluded any skips and repeated the test, it had to be repeated. You have a result. You have an MIC for example from the dilution tests but you still have this insecurity that when you do dilution, you haven't had the whole gradient. So you report the result you got. You report your 16 for example. But you know that the MIC is somewhere up or down from here between 8 and 16 because that's where you can detect by eye where the growth is but you know there's some limitations. Again, as I showed before, you also know there's some limitations if you test the same strain many times. You might one dilution up and one dilution down. This is also important when you are comparing your results with other laboratories and testing the same strain. It might be that I have a result here and you have a result there and another person have it there. And actually we are testing exactly the same in the end. Again, I didn't show so much about Etest because it's a commercial method. But we also have a reading here where this ellipse has this diffusion pattern around this gradient that is higher in concentration here and lower here. And where the scale is intercepted here, this gradient strip is intercepted here with the ellipse and that's where you read your plates. If you have done exactly as the provider and this is as a commercial method you followed the provider's rules, you probably have an MIC reading here. This is just an example of a very resistance strain. So where the growth is all over, there's no ellipse at all. So the only thing you know, that it's above the concentration that is highest here. So as I mentioned when you read the results and you want to interpret, you need really to check if there's anything that looks wrong. If it looks wrong, it's probably wrong. You also need to check something else. You have read your results. You have the results. Is something there that never happens? If it never happens and it happened today, it's likely to be a mistake or it is a very new thing that has never been seen and you're the first in the world to see it, which is good. But don't jump up with excitement in the first minute because you need to check it. Sometimes the unusual things are just a mistake, either because you just got the species identification wrong and you need to check that. Or maybe you are very lucky and you got a new resistance, a new gene, or a new pattern that has never been seen before. But also check when the results look fine in the test strains but the quality control strain looks out of the range. Then you just shouldn't trust the results at all, and you should repeat if the quality control strain is outside of range, if there's not good explanation for that. In case of any doubt, the good thing is to re-test. Of course if you're re-testing and you have the same media and the same material, you can get the same result, but you can also ask a colleague, try to check with other media or try to check with another plate. Well we have, in this case we have lots of experience of getting phone calls, emails about things that go wrong. And we try to figure out and it's like detective work. Because many times this things that we are not thinking of that when wrong. It could be as simple as writing the results in the wrong table. [LAUGH] It could also be a, identification issue or a contamination or something else. But the strangest ones I've seen, and it's an error on the result table. And it's just wrong because it doesn't fit with the pattern. But once you have that, and you have rechecked and checked, and you're really sure about the results,then you can actually interpret. And when you do this, you look at the standouts, you look at the tables, and then you can use different cut-offs or a clinical breakpoints. And this decision needs to be made consciently because the purpose of the data is different. If you have monitoring data, imagine you are here in Denmark and you want to know for the monitoring program that we have in place for many years. How is it with resistance going on in the country? Then, we want to know as much as possible what is the resistance level. And we want to use break points that are very low so that every resistance is noticed. If we want to check this for a patient, we take this clinical break points which are break points that tell us something about the clinical effect of the drugs. So if the drug is used in a treatment would it work, would it not work? Maybe sometime this this is a little bit higher than this, epidemiological cut-offs because sometimes a strain could have a little resistance mechanism and still the drug could be used for that patient but sometimes it's just the same. It might be coincidentally the same. And I'm going to explain more what these are separately. So when we are testing and we want to be successful in it, we need to follow the standards. We also need to train the persons that are doing it. You need to check, you need to do the quality control. And it's very advised that we do it with the quality control tested in the right conditions and periodically. And that we also check it with external labs so that we have a ring trial or a test where several labs are testing the same strains to make sure that we get the same results, for example, to be sure. And if there is anything that goes a little bit off or if the control strains are outside of the ranges, that we need to make something about it. Another thing that is really important in the labs is to keep the data that you are doing. So that when you get the tests you keep a record of everything you are testing. If it's the control strain, keep the data, maintain databases, check the quality control. And check if something is deviating or something is going off. Or something is getting higher than expected. Check all the time because if you're testing today and it's high. Well I don't know if it was high yesterday. That doesn't help you so much. But if you have the data for a certain period, you can really see the differences. And if something is unusual, well, check, because it could be that you are very lucky, but it could be also a mistake. So, the standards, as I mentioned many times before, are there to help us. And they describe all the methods. They describe everything that you need to do. And we are just having some slides really focusing a little bit more on the standards themselves. So here I brought up the most important ones, the American ones, the CLSI, that are available, well they are not available on the website. The actual standards, you need to buy the as a manual or in PDF files. But they are definitely purchased and they are updated periodically. So you need to have the latest version of them. But they have standards for different methods. This diffusion, dilution. They also have some for the veterinary islets which are different from the human islets. And the supplements that have the tables for the interpretation. So you have this material that should be available in the laboratory where you are working. And then we also have the European standards, which are from this group that is the EUCAST. It's the European Committee for Antimicrobial Susceptibility Testing, and they have decided to keep every document that they have online, available free. For everyone, they have both the methods where they have for example, define the method for this diffusion, where all the methods and guidelines are available online, and they also have databases with the diameter distributions, qc tables. They have all this information, and they have the database for MIC values and MIC distributions. And from these distributions, they have defined epidemiological cutoffs, clinical break points, etc. So, you have this material available. I will just focus a little bit more on what our epidemiological and clinical break points at the later, at these slides. So, now we are still in eucast, and we will just define what is this epidemiological cut-off. I have spoken about it in another presentation, but I haven't defined it yet. So imagine that you have tested a large number of strains of the same species, and these have been tested in several laboratories. So it's not results only from one laboratory that give this. It's at least from three laboratories, or even more. And you have in these tables that are available in ECOFF. All the results that are collected. And you have by MIC, how many strains have had this MIC? If you take the blue strains, they are the susceptible ones, the wild type. And these red ones are the non-wild types, so strains that have some resistance mechanism. So the epidemiological cut-off is set up by the committee, this [COUGH] and is set up so that it's separates the wild type from the non-wild type. And it's basically based on the MIC levels. It's not taking into account anything to do with the patients or the animals to be treated, it's based on the bacteria and these MIC values. And we are normally using them to really define these two groups. Because if, for example, in monitoring if we want to know what is going on. For example here in Denmark, we want to know how many strains are susceptible to Wild Type and we want to know how many are Non Wild Type so that we can also see if this number is increasing or if the percentages of these are increasing in a certain species, for example, a long time. So here it's for monitoring purposes, these are very, very important. Then we have also the same, but not for MIC, for this diffusion. Also on the ECOFFs page but then it's a bit inverted because the higher the diameter the more the susceptibility and not the resistance, so it's the opposite. We also have the strains that are susceptible as blue, so they have larger diameters and the strains that are resistant and therefore have smaller diameters. And they can be separate by an ECOFF similar to the MICs, but then it doesn't correspond as a concentration. It corresponds to a diameter zone. And that is also on tables, on distributions, on the website there. And this principle is the same. We could also use this for monitoring. However, if we think some results are not exactly black and white, because here I have an example which is quinolone resistance. Quinolone, as an example, Ciprofloxacin is one of the quinolones. Fluoroquinolone, actually. In the gram negative entereobacteriaceae where we have strains that have a very low MIC, they are fully susceptible. We have strains that are fully resistant because they have these mutations in their chromosome and we might have many of these mutations or a cumulation of these mutations and not only just one. And you have some strain that have just one mutation or an additional different resistance caused by a gene that are here in the middle. So they are kind of difficult to separate. But then the ECOFFS are designed more to separate as much as possible and you might need to test additional drugs sometimes to be able to separate. So sometimes it's not enough to test one drug. So, but what we want, in the end, is to have an idea what happens. Now, a little bit different concept. It's not epidemiological cutoff anymore. It's clinical breakpoints, and when we say resistant, intermediate, susceptible, we are thinking about the patient or the animal with sickness. So we want also to take in consideration what happens in the body of that animal or that person when you do the treatment. So we actually want to see if there's a treatment failure possibility, if the treatment might be possible but tricky, or if the treatment is successful. So here we need to take some other things in consideration when doing these breakpoints. And actually, what we need to take in consideration is not just the effect of the drug on the bacteria, but we also need to take in the pharmacokinetics of the drug in the body of the patient or in the site of infection. So, both it takes in account in vitro activity of the drug and the MIC levels and the results that you get from there, but then it takes also the Pharmacokinetics. How does the drug distribute in the organism? How much is there available? How much is absorbed? How is it distributed into different organs? Does it get to the infection site and so on? And this phamacodynamic indexes that has also to do with the effect of the drug. So, some of the parameters are pharmacokinetic, like I showed here, what is the concentration that you can expect to have, what is the time that the drug is above the MIC, for example. That is pharmacodynamic and pharmacokinetic because we are taking the MIC into account. And how much of the effect can be seen in this area under the curve, where the drug is above the MIC. So we are calculating this to have an idea of the success of the treatment in the end. And of course, some pharmacologist will do these calculations and will try to look up, well, what would be the right MIC to get this success with this drug that has these pharmacokinetic properties? So, they do then know the pharmacokinetic properties from the drug in that particular species when given in that way to the animal or to the person. And then they will calculate back what is the MIC that you need. And with that, they will check and confirm some results from clinical trials and they will define these breakpoints. So in that, when they do this collection of data and check, they will have these breakpoints that are clinical, that define if the resistance is susceptible or intermediate. So, in the end when you do your own testing, when you interpret with these either ECOFFs or the clinical breakpoints, you want to collect data. You want to make sure that's if it's the doctor they get the right results, if it's the veterinarian and so on, that they give the right treatment. Or your country gets the right data in the monitoring. And you want the data to be of good quality. So, your laboratory should check your own results over time and check that things are going well, and check that things are not behaving differently. And if something new happens, you also want to be alert. You want to confirm. You want to check and it's a great success when you find a new thing because then you are really on the right track. Because you have, you know what is available. You know when you find something new then. So, in any case, we also need to take some disclaimers. Because, well, it's biology and something react differently. But also some things, when you are using this in clinical terms from the doctors or the veterinarians you also need to take some decisions. It's not just looking at the results and prescribing a drug. Sometimes you don't even need a drug. Sometimes you need to check that, well, for this species of there's no worth to give this drug because of natural resistance. And you also need to know what the patient is suffering from and what is the story and how much treatment they had before and so on. So, you need to avoid problems with resistance, problems with the toxicity, you need to avoid any strange results. And you might also be sure that, well, you're not giving the last resort drug for something that can be treated with another one. So the doctors need to make some decisions from the result that they got from the lab. So it's not that the doctor can just use your results from the lab. The doctor needs to decide also on several other things before they decide what to use. So, just to summarize in the end, we are talking about the lab and the results and what you obtain. You are finished with the results, where does this go? Well, one of the main things where this go is to doctors and veterinarians. It's to the hospitals and the patients for the treatment. It's for the world in terms of knowing what is happening globally. And, well, pharmaceutical companies need this data as well, to decide what they are doing about the drugs they have and to decide maybe on some of the strategies. And the politicians, they also have something to say and they will manage some of these big issues down here. So, thank you very much for this lecture. [SOUND]
