229: Gram-Negative Cocci: Beyond the Plate

0:00Hey there, MicroFriends! Welcome to another episode of Lestog Micro, as always, I am so happy that you're here with me. And if this is the first time you're listening to Lestog Micro, well, thank you for choosing it as one of your podcasts. And I know you're tuning in because you want to learn more about Gram-negative coxize, so let's go ahead and jump on it. So this is the third episode of a series about Gram-negative coxize. And the goal of this series is to make us a little bit stronger when it comes to Gram-negative coxize. Sometimes, you know, you can be scary, identifying them in the lab, especially when you see a Gram-stain. You might be a little hesitant to call it, right? So at least by making sure that we have as much knowledge as we can, as much repetition as we can, and that is going to help us build confidence, which is something that's very important when you work as a microbiologist, right? You have to be confident, of course. You have your knowledge, your education, repetition, and those things, you know, they build confidence.

1:02And that's something that you have it. And of course, I'm not saying that you never question anything, of course, you know, if you're not sure about something, please consult with a more experienced tech, consult with a supervisor, a director. But if we don't have confidence, then that means that we're going to hesitate a lot on our day-to-day work, and that's going to make for a very long day. So this series, it's meant to help us with that, you know, to learn more about Gram-negative coxine. And for the benefit of those that are tuning in for the first time, the first episode focused on the Gram-stain and the different general and species. And I also went over some sites where you can expect to see Gram-negative coxine. Of course, you know, we have two major genera, which are Moraxela and Nasiria. And we know that there are some species within this general that are Gram-negative rots. So we are focusing on the Gram-negative coxine ones. For example, Nasiria gonna Ria, Nasiria meningitis, other Nasiria, such as Lactamica and

2:03Sica, and then, of course, for Moraxela, Moraxela, Catarralis. And we also focus on some anaerobes, so we talked about Vivalnela, and we also mentioned some other species. And with those, you know, we are not spending as much time as with Nasiria gonna Ria, for example, and Moraxela, Catarralis. Because Nasiria, Moraxela, they have some very significant diseases that we wanna make sure we spend some time on. Okay, and that was episode one. And then on the last one, we focus on median testing. And at the same time, going over some concepts that if you're preparing to take your certification as a medical lab scientist, either like ASCP, maybe AMT, so these concepts are very important in all because you might get some questions. And this type of examination is always good as you're reading things, making those connections where you have those case studies, and you look for those classic things. So for example, we say Hockey Park, you think Moraxela, Catarralis, things like that.

3:04So talking about the media, we went over some types of media that are helpful to isolate organisms like Nasiria gonna Ria and Nasiria meningitis, one of them being modified by the year Martin, which is selective and enriched. And they enriched portion, you know, it's a chocolate auger. And the selective portion, it's made of antimicrobials. For example, we talked about colestine, trimethyperin, vancomizing, and niestatin. So make sure you go ahead and review those. And one thing we talked about, you know, speaking about examinations, we talked about Nasiria gonna Ria needing carbon dioxide to grow. And sometimes, you know, the transport of the samples to the lab can be challenging. Sometimes, you know, how microbiology labs are nowadays, where you have like a centralized lab that does most of the micro for other hospitals. So sometimes the transport time, you know, it can be the lane. And this may affect the chances of you recovering this organism, if that's what you're looking for. So there's a system that we talked about called JEMPEC, JEMBEC, which is a system for transport.

4:10So those two episodes are packed with information, so definitely go back and check them. Okay. So by now, you know the media that the different gram-negative coxide grow on. You also know the sources where you can potentially find these organisms. And this is something that's very helpful because by knowing these sources, right, you can expect some of them in some areas, like if you're looking at a respiratory sample and you see gram-negative coxide, you know, that's expected because non-papagending Nasiria is part of the respiratory floor. And in other sources, even though they might not be there all the time, but at least if you have a particular source and you see them, you're like, okay, you know, this organism when it is seen, you see it in this area. While there are some Nasirias, like non-papagending Nasiria that are part of the respiratory floor, there are some Nasirias that are pathogenic. For example, Nasiria is going to be. And in the lab, you can get cultures just to rule it out. And it can be from many sources. Based on what we have discussed, sources are typically genital, throat, and eye.

5:14And with this type of culture, you get a chocolate plate and a modified theory of martin. Now remember just because something is growing on modified theory of martin, it does not mean that it is Nasiria going to be here, or Nasiria meningitis. The gram stain is always your best friend. If you perform one and see gram-negative coxide, you still need to sub it to media where you can properly identify it. And that brings us to today's episode, in which I am going to be mentioning some instruments and tests, and it does not mean that I am endorsing these products, but they are commonly used in the lab. So by now you have a gram-negative coxide. You look at it on the plate and you are confident it is a gram-negative coxide. Maybe you're not, right? We've been talking about how daunting it can be. But in this scenario, you know what it is. You went for it. You're confident. This is what it is. Now it is time to prove it, which means it is time to identify it. Okay, so let's go ahead and start with the easiest one.

6:15And those of you that work in the lab, you know that it is easy to identify a more acceptable catarallus. You don't even need bite-tank or moldy tongue. You see the gram-negative coxide, the colonies slide around the auger, oxidative-spositive, you do what? A catarallus does, right? And that's it. Once that reaction is positive on that disc, you can go ahead and call it more acetyl-catarallus and then you're done. That catarallus disc is a very easy test. It doesn't take a long time. So let's go ahead and talk about tests or instruments and identify methods that take longer. So of course you have your classic bite-tank NHKAR. It identifies nice, you're going to read, meningitis, syka, lactamica, senioria, and more acetyl-catarallus. And those of you that work in the lab know that one of the drawbacks of this card, compared to other methods, is that it requires a large amount of organism. And we have talked about this in the podcast before. For this card, you need a suspension of 2.7 to 3.3 McFarlane standard.

7:20That is a heavy inoculum, so you definitely need more colonies. And that means if you have the organism mixed with others, you might have to isolate it first so you can have enough to run it on the system. So bottom line results take longer than other methods. One thing that is mentioned for this card is that NYSIRIA, Flavessence, or Mikosa, might be misidentified as NYSIRIA's syka. But it mentions in the package insert some tests to rule them out. Then you have the rapid NH system. And those of you that have been listening to for a while, I have talked about the system, especially when it comes to anaeropes. There are several rapid tests like this. You know, there's like a panel for anaeropes, which is commonly used in the labs, which is the rapid ANA panel. There's one for chorini bacterium. We used to have one in the lab before for like enterococcus. So these panels are, you know, they're small. You can hold them in your hand. And they have like a series of wells and you make a suspension and then you put specific

8:22amounts in the wells. And then you incubate for four hours. And at the end of that time, then you observe for different colors. Some of the wells you have to add reagents and then observe for color changes. And then based on that, you have positive or negative reactions, which is going to give you an numeric code. And then you go into the software for this test and you enter that code and you get an ID. And this rapid NH panel, you know, it's a small panel for nice area homophils and morax cell. So you have that numeric code. You put it in the system in the software and then you get that ID. For example, nice area gonorrhea, nice area meningitis, morax cell like it around us. Now the test, the test takes four hours. So it is something that you can set it up in the morning and then a lunchtime reading, which is what typically people do, right? They go over, especially when it comes to anaeropes, you look at them. You set up your test in the morning and then a lunchtime read them.

9:23And for small laps, you know, I think this makes sense, you know, based on space, the cost of having something like mallet off. But what I don't like about this test is that people play with the numbers, meaning you get like an unidentified result or you get a weird ID and then you go back to your test and you go, okay, well, this was a positive, less sort of negative or even when you're looking at the test and you're not sure, maybe you put like a star next to it, either positive or negative. So you play with those interpretations to change the numbers. And sometimes you get an ID. So it's too subjective. So that's why I don't like about it. But that is my opinion. And then, of course, there is mallet off, which we did some episodes here in Lestog Micro. So I invite you to check out episode 112 and 115 to learn more about mallet off MS. And in the nutshell, right, you have a slide, you put your sample there, you add a substance

10:27and then a laser is pointed at the sample and then that substance, what's what it's going to do? It's going to ionize the molecules. And then the molecules, they travel through a tube and then, you know, the time of flight is measure and an spectrum is produced and that is compared to a database of no inspector and you get an ID. Of course, with mallet off, you get a result so fast and then you have a larger database of organisms. You need less organisms, you know, like one or two colonies is enough. So unless it's very mixed, if you are able to see the colonies and you have enough, you know, isolated ones. You can go ahead and do the moldy and get your ID. And with this database of organisms with moldy, something to keep in mind is that in this database, you have some organisms that have been clear by the FDA and some haven. So what I mean by clear, they have been clear for clinical diagnostics and those that have not been clear, they are called nonclane.

11:28And those of you that use moldy, you see that the organism has like an N in parentheses next to the name of the organism. And this is basically that they haven't been validated. So but that is something that you can do in your lab. So you get that ID and then you test that organism via other methods and then you get an X number of samples and then you are able to utilize that ID. So all you have to do is a validation, right? Some organisms are more difficult than others, right? Because systems like the Vitic and things like that, you have limited databases. So you might have to send some maybe through gene sequencing, you might have to send them to another place for that. But other ones that you see daily in the lab like Eric, you know, that's one that shows us nonclane and you can do a validation because Vitic can identify it. So that's a little bit easier than other organisms. Anyway, that N in the parentheses for nonclane is not going to go away, but once you have done your validation and you have a list in your workspace, you can go ahead and release

12:31that ID. Something very important to remember is that when you are doing this validation or you have a sample that you're running it, you get a nonclane organism and then you decide to run it in another platform to confirm that ID, you have to make sure that that organism that ID isn't the database of that other instrument. For example, if you go to the Vitic and then you check a specific card, like a specific identification card, go over and check the list of organisms that it can identify. Because if the organism that you're trying to confirm, it's not in that system like that Vitic card, you can get an an erroneous ID. Maybe you can get another species, but maybe you can identify it as something else altogether. You have to make sure that that particular organism is on that database. By now we have discussed how to identify them once you have grown them in the lab. You went through the putting it on media, you grew it, you isolated it, you identified

13:38it, and you're good. How about from direct samples? Well, if you work in the micro lab, you know that a lot of testing for NICERA gonorrhea is done via molecular methods. NICERA meningitis, as we have discussed is an organism that is seen in meningitis, which is very critical. So if we have a meningitis panel in the lab, like a PCR panel, it makes sense to have NICERA meningitis in this panel. And I will talk about this in a few minutes. But let's go ahead and talk about some tests where we can identify gram-negative coxide without culture. Like for example, from a cerebral spinal fluid, where the clinical presentation looks like meningitis, so the provider wants to rule out an agent of meningitis, or a blood culture where you see this gram-negative coxide, or another source where the doctor wants to rule out something like NICERA gonorrhea. So let's talk about those tests. And that is worse in dromic panels and nucleic acid amplification tests really come into play.

14:43So let's start with blood, right, definitely in the micro lab. You have a blood culture instrument, either like a virtual or a BDFX. And then very typical, right, that bottle is flag as positive, you do a gram stain depending on what you see, you set up one of these blood culture identification panels. One example is the biofire BCID2 panel. And in this panel, you have multiple targets. So one panel, you have bacteria, you have fungi, and resistant markers. And in this panel, one of the targets is NICERA meningitis. So if it's pressing, you can get that ID. So before in the case of blood, because in the lab, you know, you played it, you do a culture book, before you do this and recover the organism, you can have that ID and give it to the physician. So they can start treatment. And there are other systems that do this as well. For example, the EPEX blood culture ID panels. And this ones, you know, you do the tech, the same, you know, like a bacterial pathogens,

15:48fungal pathogens, resistant markers, but in the case of the EPEX, you have different test panels, one for gram positive, one for gram negative, one for fungi. And a little example of a system, of a test system is the verigen from Diosaurin. Same thing, you have like a gram positive panel, you have a gram negative panel. So these systems, they are very helpful because you can get an ID right away, but something to keep in mind that for this instrument, for this system, you, you know, you test them on a blood culture that has already been flat positive. You know, it's not like you collect it in the blood culture bottle and then you go ahead and run the test, right? You have to put it on the instrument first. So the EPEX and the verigen, they have targets for nicer young meningitis. Now, let's switch to cerebral spinal fluid. A great example is the bio fire meningitis in ciflinus panel where you take the CSF, right?

16:49You collect it, you take the CSF, you load it into the cartridge, and then the system tests for a panel of a bacterial viral and fungal causes of meningitis. So this panel has a target for nicer young meningitis. And of course, you have other bacterial pathogens that are seen in meningitis. You have streptococcus like a lactease, streptococcus pneumonia, listeria, monocyteogenase, chymophilus influenza, e-coline. So this is a widely used panel and it is very helpful in the identification of organisms that are seen in meningitis. And this panel can detect that meningococcal DNA from CSF, even if the patient already received antibiotics and the culture might end up negative, which is something to keep in mind when you're doing molecular tests like PCR and things like that, then you have to keep in mind that the organism might be non-viable so you might not recover in a culture. Okay, so before we continue going, let me talk a little bit about nucleic acid amplification

17:50test or NAT. And basically for the nucleic acid amplification test, you extract the nucleic acid, you amplify a specific target using a method like PCR or transcription mediated amplification, and then you detect that amplify a product with a probe or a fluorescent signal. And then if that target is present, the signal lights up and the instrument calls it positive. And this is something that happens in a closed system, which is very cool and allow this test that I'm talking about sometimes, some of them are quite small and they are closed systems. And of course, the advantages of this type of test is that, you know, there is sensitivity and speed, you know, you don't need viable organisms. And you can get answers in about an hour or two instead of waiting, for example, 24, 48 hours for a traditional culture. And that is huge when you're dealing with an extreme meningitis in CSF or a nice year going to rea and a genital side, let's say, I mean, something like meningitis is very

18:51critical. So you're getting that identification so quick instead of 24 to 48 hours, you know, it's very important. I wait, you know, the physician, the health care provider can start treatment. Now of course, with that is the trait of that, as I mentioned, right, you're using the sample and not an organism that you have grown in culture. So you're not going to get those up to abilities from that. So if you're looking for some specific drugs, you know, some specific antibiotics, then you have to do a culture and test for that. Okay. So now that we have a better understanding of what nets are, then we can see how helpful they are. And I mentioned, you know, I talked about E-plags, I talked about biofire and things like that and multiple targets. So with these, you know, nucleic acid amplification tests, you have this endromic panels where you're having, you know, multiple targets, as I mentioned, you know, with meningitis, like, nice, you're meningitis, it was influenced, allisteria.

19:53With the blood cultures, you have the resistance markers, you know, with some of these panels, you have things like Van A and Van V for vancomycin resistance and Mk and Mxc for oxygen resistance and KPC and NDM for carapena resistance. So these tests, you know, they are very helpful. They provide quite a deal of information and, you know, we talked about blood and we talked about CSF and then we have this other nucleic acid amplification test for like a nice year going to read and chlamydia and those are very classic and I'm sure as you're listening you're familiar, you know, at some point in time in the lab, you have tested one of this, like nice year, chlamydia panels, you know, they come together and you test for both. And some examples are like the systems like Optima, CoA, Abbott, GeneXper, BDMAX, which can detect nice year going to read directly from urine and mucosal swabs.

20:55And those are usually validated for genital rectal and pharyngeal specimens. So they are very common in the lab. And then we have something like a joint fluid or another type of body fluid for those you might have to do like a culture or maybe if you're thinking of a disseminated infection, then maybe try to find like the source of the portal ventry. And based on that decide, you know, which test is right for that. Okay, so let's go ahead and do a quick recap, right? So when we want to identify gram-negative coxine, typically if you have grown them in culture, you have things like bi-tech NHKAR, rapid NHK, you have molded off, which I know we have been focusing on nicerium or acela. But for molded off, you know, with molded off, you can identify viral nail also. And then when you have samples like a blood culture or a serious spinal fluid or another source where you are thinking of this organism and you want to rule it out or you need to rule it out, then you have things like blood culture ID panels that can identify nice year

21:59meningitis, you have the meningitis panel for nice year meningitis, and then you have those chlamydia, nice year, you're going to re-unoclake acid amplification test that let us detect going to coquel DNA from the colicicides. Okay, so by now we have gone over testing identification. So next thing we'll be talking about the different general and species of gram-negative coxine in a little more detail. And that is what the next episode is about. Well, there you have it. I hope you enjoyed this episode. As always, I enjoy sharing this information with you. So please stay tuned. You know, this is content that's very important for those of us that work in the bench. And you know, even if you have your experience, a good refresher always comes in handy. So thank you for listening. And speaking of listening, of course, you can find us on any podcast platform. So if you go to Apple's Spotify, I'm a some music, good pods. You can find us there. So please

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25:10As always, please continue bringing that passion to what you do. It's so important. You do such great work. So as always, stay motivated, stay safe, and of course, continue talking micro until the next time. Bye.