Lecture 14- Quality assurance when doing antimicrobial susceptibility testing

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Antimicrobial resistance - theory and methods

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Technical University of Denmark (DTU)

Antimicrobial resistance - theory and methods

570 classificações

The course will cover the topics related to antimicrobial resistance with basic definitions and overview on antimicrobials their use and the emergence and spread of resistance. The course will guide you through the concepts and the importance of resistance spread and dissemination and how that happens. It will show you how bacteria become resistant and which mechanisms they might use for this. And as part of the course you will also receive some training in methods for antimicrobial susceptibility testing (AST) and detection of specific resistance in the microbiological laboratories with the basic methods available and with focus on the obtention of good quality results which can be interpreted and used for different purposes. Additionally, it will show you how to use genomic analysis tools to analyze whole genome sequencing data to detect resistance genes (and or other genes of interest) in a simple and easy way using online tools freely available. In the new new version an additional module including detection of specific resistance mechanisms was added. After this course you should be able to: 1. Describe the most important families of antimicrobials and mode of action 2. Understand the basic concepts of antimicrobial resistance from several perspectives (clinical, research and microbiological) 3. Enumerate and describe how bacteria can become resistant and the mechanisms that may be involved in that process 4. Describe how antimicrobial resistance emerges and spreads around the world including concepts of antimicrobial resistance transfer, selection and dissemination 5. Enumerate the methods used for antimicrobial susceptibility testing (AST) 6. Compare dilution and diffusion methods and know the basic techniques of agar disk diffusion, broth dilution and agar dilution methods 7. Have detailed theoretical knowledge on how to perform the main methods in a laboratory 8. Know the basic concepts about analysis and interpretation of results of AST, including different breakpoints, cut-off setting and their applications. 9. Understand the importance and related concepts related to quality management and quality assurance method standardization, applied to AST 10. Relate the information obtained in this course with real cases of resistant bacteria spreading in patients, the community, animals or the environment 11. Relate the phenotypical results with results from genotyping using molecular techniques for detection of resistance mechanisms 12. Understand the concept and be able to apply genomic analysis tools used to detect resistance genes and other relevant genes from Whole Genome Sequencing (WGS) data (with demonstration of selected online tools) Disclaimer: Please note that the guidelines and methods referred or links included in these materials are updated when the videos lectures are produced and before the course is released, however these might become outdated with time.

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Module E- Quality assurance

This module is dedicated to Quality management and Quality assurance (QA/ Quality control (QC) procedures and use of QC strains to assure the qulaity of results of antimicrobial susceptibility testing: importance, elements and principles.

Lecture 13- Quality management in Antimicrobial susceptibility testing9:45

Lecture 14- Quality assurance when doing antimicrobial susceptibility testing22:49

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Lina Cavaco
Ext Associate Professor
Copenhagen University- ICMM /Statens Serum Institut - Department for Bacteria, Parasites and Fungi
Pimlapas Leekitcharoenphon
PostDoc
Research Group for Genomic Epidemiology, DTU Food
Rene S. Hendriksen
Professor, PhD
Technical University of Denmark, National Food Institute, Research Group of Genomic Epidemiology
Valeria Bortolaia

Hello everyone. My name is Lina Cavaco,

and I'm going to present to you the second lecture of this module on quality and

quality management in performing MIC and susceptibility testing in the laboratory.

This presentation is more focused on the procedures,

and what we can do about the quality assurance in practical terms.

So, as an outline,

and we all bring again the importance of doing

such procedures and making sure that things go right,

the importance of standardization.

Then we will focus on the standards available and the in-house procedures,

or the quality assurance procedures,

that should be implemented in the laboratory so that we

make sure that the results are quite all right.

As a final part,

we will look into the quality control,

with control strains and some final considerations

about how we can use the quality assurance in the susceptibility labs,

and be sure that the quality is right as we expect and results are reliable.

So, just to make sure that we are thinking of the importance of this,

well there's many parts of the laboratory work and

the microbiology labs where the quality assurance is quite important.

But here in susceptibility testing it's where it's

actually one of the parts that is most important because we have quantitative results,

we have results that are maybe leading up to treatment use.

Then it's really important that the results are reliable.

Also for the monitoring,

of course they should be reliable,

especially when lives of people are in our hands,

then it's really important.

So, quality assurance and quality control are really essential here.

Of course we want to test if the bacteria are susceptible for an

anti-microbial and therefore we don't want other things to interfere.

We want microorganism's reaction to the drug to be the thing that we are seeing.

If we put in other issues and other issues with the plates, with the reagents,

with the media, then we might be seeing results that are due

to other things and not the reaction we want to see.

So, in our world of susceptibility testing standardization is really a big key,

because there are so many factors that can affect the results.

If we are just naming a number of them, there is inoculum,

the media, and many of them are really difficult to control,

but we need to have procedures to control them all or at least we should

then make sure that that control is in other hands if we,

for example, buy some of these products already made.

But then there's also when you do the tests and

when you read the interpretation, the reading,

then it's also very important that we still have

a control because then we think it's done, it's ready,

and we have seen many times that it's failing just here where somebody is

distracted and the results looks totally

wrong because they just made a mistake in the very end.

So, all the procedure is important because mistakes can happen in all the steps.

If we are doing susceptibility testing,

we might use it different methods.

If you are doing dilution methods, these apply.

But if we are doing diffusion methods,

even more apply because there's more factors,

especially regarding the diffusion in the agar.

If you're using disk diffusion,

this depth, and dryness, and the growth rate,

it could be even more complicated to standardize

a diffusion method than it is to standardize a dilution method.

However, because it's cheap,

it's practical, it's fast,

some people would still choose this one and it's perfectly fine if you have the control.

So, if we want to have reliable of reproducible data,

we really need to rely on the standards and follow them all the time.

When we are thinking of standards and we have had this discussion with many laboratories,

it's really to follow the whole procedure and to check all the steps.

Because many times people follow the standards as they are following

a guideline and they change little things

here and there and they think they get to the same result.

But if they have changed here and then adjusted there,

then they are not following anything anymore and it might be that they get

the right result by chance but it is difficult to say that they are in the right track.

So, we have both standards and guidelines.

If we are thinking of standard,

they really describe the methods that we should use in very, very detail.

If we are following quality assurance systems we are following ISO standards,

we also have the clinical standards

from the United States and the European Committee Standards.

You can follow one of them,

and if you follow one of them you follow all the parts of that standard.

You cannot just take one part from this one,

part from that, and in the end you have a mix.

So, when you're choosing the method,

you choose the standard and you stick to it.

If you are doing the quality control and quality assurance procedures,

you want to monitor things.

You want to monitor that the results are precise and they are reliable,

that all the reagents are working like they should,

that the people who are doing this are doing it right.

They are following as they should.

If the strain that you are using as control are testing this with known susceptibility,

you can have an idea if you are deviating to one side or the other,

or if you're in the right track.

So, one of the things that are really important in this is to have the right material,

to have the right reference strains for example,

so that you can test that your procedures are fine.

Of course it depends on which test strains you have,

if you are testing gram negatives in general,

you would use E. coli strain.

If you using testing Campylobacter bacteria,

you would be recommended to use this Campylobacter strain.

If you're testing gram positives,

you might want to use the Enterococcus faecalis strain that is referred here.

Also, this strain is quite important to check the media if you have

a possibility to have some inhibitors to trimethoprim and sulphonamides in the media.

You can have an idea by checking this strain.

Then you have additional strains which you might use or not.

There is pseudomonas which is quite important for

detecting the cation concentrations in the media,

and you might need others.

It depends on the test strains that you normally test in your labs.

So, these are the basis panel that you should have, and these strains,

they need to be maintained and subcultured in a very,

very specific way that is described.

So, it's not only just having the strains.

It's to keep them in the right way,

to subculture in the right way and to keep them

and check them every time you test or every week,

depending on the system that you use for the quality control,

but you need to have the strains and to make sure that you are using it well.

So, you need to also,

when you are doing these checks,

these quality control ranges checks,

you are doing it for the antimicrobial you are testing.

It might happen that for some antimicrobials,

you don't have the ranges.

In that case, if you don't have them in

the table you cannot really make them up but you can still

keep the data in your laboratory and follow up if something is

deviating from what you usually have you would notice.

So for all the tests that you do with these strains,

it's a really good idea to keep the databases and follow well.

I tested this day, this day,

this day, I got this results.

If suddenly the strain is giving strange results,

you know when it started to give strange results.

It might be something wrong with the strain,

it might be something wrong with the media or with the test.

So, that's really important.

Also, in regards to reference material as such,

you need, as I mentioned,

not only to have them but you have this subculture and

maintenance guidelines and you can see that under the CLSI for example,

where the strains must be

used for the testing in a certain way and kept in a certain way.

You also have some strains that are to be used with this diffusion and not with MIC.

So you'll also need to make sure that the strains that you use at quality control,

and the ranges that you use for quality control are the right ones.

For example, if you're testing,

now it's not here on the slide, but for Campylobacter,

if you're testing, you have two temperatures and two time slots that you can test.

If you test at 37 degrees for 48 hours,

then you have a different range for testing the higher temperature in a shorter time.

So, you need to check the ranges also that you're using the right ones.

Another big important issue is also for the whole procedure is a good idea

to check the control strains in terms of

colony counts so that you make sure that the inoculum,

the dilutions that you are making for the test,

are the right ones.

You check this based with the quality control strains.

You can also check with some of the test strains but at least this one should work fine.

Again, in relation to the use of the quality control strains,

each time you make any change,

you should check them.

Each time you introduce new media, you should check.

Each time you change something in a method, you should check.

So, you should always check the quality control ranges.

This should be done, well,

each time you have a new batch of media or new reagents or so on.

But if you have a quality control procedure in your laboratory you can do it every day,

or you can do it every week and this is described

under the standards how you can do that.

So, just to browse up on that,

the test per day would be used when you are not

testing these trains very frequently when you don't have test strains that frequently.

But you can also test from daily to weekly.

If you have tested it daily for a long time,

for 30 times for example,

and you have documented it,

then you can go once a week.

But it has some of the procedures here that you can read.

If something is out of range, you can,

if more than one, you need to check the corrections.

If you have satisfactory product performance,

then it's fine with weekly, and then,

of course, you need to still check if there's anything out of range.

So again as I mentioned it is a very good idea and

it's described both in the ISL and then the CLSI to make these inoculum checks.

This is because all the standards are made in

such a way that you should have a correct amount in the test,

otherwise the test is not really valid.

Of course there's some relatively big ranges here,

but the design of the standard is done so in a way that you

have for example in the final cell number in the wells,

when your test should be around this amount and it could vary from this to this.

So in that range it should be.

So in the hundreds of millions of cells per milliliter is still a big number,

but it should be around there.

I'm sorry not the hundreds of millions,

it starts with a hundreds of millions but then it goes to 100,000 or 200,000 to 800,000.

So, when you do this,

you are doing dilutions and then again

dilutions and there are some mistakes that can happen.

The first is when you do this dilution in

a cell line or in water and you adjust with the densitometer,

there's some calibration issues that can go on here,

and you want to check that this is right so that you get more or less this amount.

This is approximately this 100 million number here.

Then you have a second dilution.

This is quite important that the labs check and validate

in their labs how it works out with this second dilution.

This is what we use more or less in our lab

but it could be different if you have a different system,

a different validation so that you get more or less to this amount per milliliter.

Then in the end of the well,

you have this concentration because you are putting in this amount in each well.

That's also this part with the outer inoculate you need to check.

Of course, to have these and to pipette and make the dilutions correct,

you also need the system to check your pipettes and to check

your autoinoculator so that you make sure that the volumes you have there are right.

That was for pipetting and broth and so on.

But if we're thinking of antimicrobial disk diffusion and antimicrobials as such,

we need also to have some considerations about where we

get the antimicrobials and the disks with antimicrobials from.

Normally we buy these things.

So if you are using antimicrobials for doing MIC testing,

they might come in the panels already,

in the ready to use panels where we don't need to think very much, they're there already.

If you are doing agar dilution,

we might have the antimicrobials in powder where we

will need to think where they come from and are they good enough.

If we have them in this,

we need to make sure that we keep them in the right way,

and that we have it stored in the right way.

So, there would be some consideration on how to keep antimicrobials because in any case,

if it's in the panels or in the powder or in the disks, antimicrobials can degrade.

So they can lose activity if you keep them wrong.

So, also when we do the results and we do the test we should validate

the performance by checking with

the quality control so that we still make sure that the media is good,

but also that the antimicrobials are acting as they should.

But also, when we have done most of

the procedures in the test and we're just putting it in our incubator,

we're still not really relaxed,

that is just going in and the next day we take it out,

we still have to keep a look into the incubator itself.

Of course depending on which tests and it could

also be different temperatures if we're doing specific tests,

we need to check the ranges of temperature and that we are in the right ranges.

We have to document how the temperature is going

on and we might have to change the temperature ranges if

we are testing specific organisms that have a different temperature or

atmosphere requirement where we need also to check those things.

We need to document all of that,

the temperature, the incubation,

the time of the incubation and so on.

If we need to play with the gases that are in the incubator,

we also need to document all of that if we are doing generator system

or putting in gases into an incubator to have the right mix of gases.

So, that's more or less the procedure and we are almost getting the result.

So when we get the results and we still have results outside of the range,

that they are strange, that are not what they should be, what happens?

That's really a question that we should take to ourselves is what happened?

What did go wrong?

Things that are easy to do is to switch something,

to contaminate something is not very difficult in a lab where things go on around,

but it can definitely happen sometimes and we should do things to avoid it of course.

The growth could be not so good and then we'd need to check the media or the strain.

There could be also things with the incubation and things with

the time and maybe we have to use new media or something like that,

so we need to check all the sources.

Here, I'm not being extensive,

I have an et cetera there because there are many sources of errors.

We have seen lots of cases and our labs

in our network normally ask us questions and we have seen

everything from the common switch of a strain to

having the wrong reading tables and putting the right results into the wrong boxes,

to having errors with the inoculation where they had just too much or too little in it,

there's a lot of things that can go wrong.

Every time you have a mistake,

you need to go back and check everything

like a detective and make sure that it goes well.

Also when you have an idea what it is,

you need to test it again.

Well, if it was the media then I do it with

the new media and let's see what happens and so on.

So also, now that I mentioned the media,

we normally have a good idea that the Mueller Hinton is a good media and of course

it's the one defined in the standards that

is okay batch to batch but we still need to test.

So, if we have this media cation adjusted already bought,

or if we do it ourselves it depends,

but we still need to check and check with the control strains.

If we need to use supplements,

we also need to either buy them from

a reliable source or have a good procedure to make them.

So, also when we do diffusion and dilution,

we need to check all the procedure again and again.

Of course, if the quality control show some deviations, we don't read.

So when we want to read the results of the test strain,

we check that is everything in agreement?

Is the control strain in the right range?

If it is not then we don't even care about the test strains.

If it is all right then we can read the test values again.

As I mentioned, the troubleshooting is really important.

If there is something wrong,

then we need to retest and check.

It could also be a good idea to check over

time because sometimes it's only out of range today,

but it has been deviating towards that limit for sometime before.

So we can also see if that was

since we changed the media or since we did something rather different.

Quality assurance procedure that I have mentioned before is this ring trials which are

basically tests that are done with

the same strains in different laboratories and then compared.

Its a external quality assurance and this is really

interesting because it gives so much information to the participants.

First it gives the idea of their results are good.

It makes also the opportunity to test some strains that they normally would not have in

their labs because then interesting strains and new resistance would we send around,

and they can really evaluate and check.

If they have mistakes,

they would also get some help to check on these deviations,

to check on the problems and to correct them.

That's what our work is actually all about,

is to organize these and also to give

this information and this help to laboratories most of the time.

Of course, if you are keeping all documented and you can trace back what you have,

you might likely find the mistakes.

Since you are using this quality control all the time,

you can also review back and check,

"Well, from this point in time to that point in time,

did we have good results?

Are the results reproducible for this period or a long time?"

You have graphs and you have data.

You can also check with the standards.

If you have diffusion test data,

you can also have this additional considerations but

you have probably taken them into account already.

All of this helps you to have the good results in the end.

There are some considerations here regarding the limits and the ATCC strains but that

is data that you normally have in the lab that you

would check when you are doing the tests.

So, just as a final considerations,

as I said before,

quality management and quality assurance does

not help that you don't make mistakes once in a while.

We are humans and we end up making

some mistake even if you are really good at the procedures.

But when it detects,

it can help them prevent.

If you don't have it,

then you might have mistakes and never notice and sometimes it might be too late.

Thank you very much.

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