Malaria Laboratory Diagnostic Testing

So hello again, for those of you that are just entering. My name is Tamara McMillan, and I'm a consultant with Guidehouse supporting CDC's OneLab initiative. A couple of notes before we dive into this webinar. If you're having any technical issues throughout, feel free to email OneLab inbox for support at [email protected]. Again, that's OneLab at CDC.gov. If you have any questions throughout the session, insert them into the Q&A function. We will have a Q&A session at the end of the presentation where we'll do our very best to not only review all, but then do our best to answer all of those questions. Note, the link to the live caption is also being posted in the chat as well. If you're utilizing the closed caption, ensure that you have that window open in addition to the Teams webinar. So now, on to our agenda. We will begin discussing some new OneLab resources. Then we'll go through some introductions, and we will have a 10 to 11-minute Q&A session at the end with our speakers for today, where they'll review and we'll do our best to answer those questions. Today's session will also end with a discussion of upcoming events that we would love to share with each and every one of you. Now, on for our chat box rules. We will be leaving the chat feature open for your engagement. When utilizing the chat, please keep in mind the appropriate rules of engagement. Feel free to use the chat to connect with others by reacting to what you're hearing, sharing experiences, and asking questions to fellow participants. If you have questions for the presenters, please, I'm going to stress that, please use the Q&A function because that's where our team will be pulling and looking for questions to give to our presenters to answer. Do not use the chat. We will not be looking at the chat. Additionally, please engage with respect and professionalism. Any inappropriate language, improper conduct, and other forms of discrimination may result in removal from the webinar. Please ensure your comments are relevant to the topic, and if a moderator gives you some direction regarding the chat behavior, please comply accordingly. Lastly, please notify moderators if you're experiencing any technical difficulties or observe any disruptive behavior. So now, I will introduce our new One Lab Network lead, Dr. Sonia Strider, and afterwards, she will share some of our new and relevant resources and then introduce our speakers for today. Dr. Sonia has been with the CDC for 31 years. She is a public health professional with a multidisciplinary background in health education, workforce development training, laboratory science, and public health programs. Her experience bridges scientific and programmatic perspectives, focusing on translating public health and laboratory knowledge into effective education, training, and program strategies that strengthen workforce capacity and advance population health. Dr. Sonia? Hello, everyone. Hello. I am awfully glad to be here. Thank you for the warm, warm welcome. We have a lot to discuss today, so let's just get right into it. I can't wait to hear what we have about the malaria and testing and parasitic disease. So it is an awesome, awesome, awesome event today. Yes. Next slide, please. So I just want to remind you that we have all of these resources available. So now available is the Packing and Shipping Dangerous Goods Sample Training Material. The materials are included. They include an e-learning and job aid to support general familiarization and training on packing and shipping dangerous goods, including Division 6.2 infectious substances and dry ice, and they are intended for U.S.-based clinical and public health laboratory professionals. You can find this on our OneLab REACH network, or you can scan the QR code here at the bottom of the slide and be able to go right to the information. Next slide, please. And I want to make sure I give the disclaimer here. The subject matter experts and presenters wish to disclose they have no financial interest or other relationships with the manufacturers of commercial products, suppliers of commercial services, or commercial supporters. And let's hear who we have today. I am so pleased to welcome Dr. Brian Raphael, who is the Chief of the Laboratory Science and Diagnostics Branch within CDC's Division of Parasitic Disease and Malaria. The branch conducts assay development, evaluation, and testing for parasitic disease diagnostics and to inform domestic and global public health programs. Dr. Raphael has been at CDC for over 20 years, working to develop and implement tests for infectious diseases with a focus on parasitology since 2022. He will be discussing the importance of ensuring rapid malaria diagnostic testing and opportunities to implement and sustain this testing in clinical and public health laboratories. Joining him is Dr. Sarah Sapp, who is a biologist and the section lead of the morphology unit, also known as DPDX, within the Division of Parasitic Disease and Malaria here at CDC, specializing in clinical parasitology and the morphological identification of parasites. The DPDX service provides direct diagnostic assistance for parasitic diseases globally and domestically as well as educational resources for laboratories. Dr. Sapp earned her doctorate in veterinary parasitology at the University of Georgia and has served as a parasitology expert at CDC since 2019. She is passionate about workforce development and the preservation of diagnostic parasitology capacity in public health and clinical laboratories. Welcome, Dr. Raphael and Dr. Sam. All right, we have a lot of content for you all today. So first, we're going to talk about some malaria basics with the biology and disease. Then we're going to get into some diagnostic strategies, including our three major tools we have here, and then tie that all together with an updated algorithm for diagnosis. Many of us are probably familiar with these basics of malaria, but just as a quick reminder, malaria is a disease caused by a genus of parasites called Plasmodium, which are intracellular protozoa that infect red blood cells. This is vectored by a number of mosquitoes in the genus Anopheles worldwide, and again, causes a potentially fatal febrile illness. This is a widespread public health problem that affects over half of over half of the world's population resides in an endemic area. So the current global status of malaria, you can get a sense of how widespread this is. While there's been a great deal of progress made in the elimination of malaria from many parts of the world globally there are still 282 million malaria cases and over 600,000 deaths globally and this was the status as of 2024 particularly many of these deaths and cases come from sub-saharan Africa here in the United States we experience about 2,000 malaria cases per year due to travel with the exception of some very uncommon local transmission events if you see the asterisk here. This indicates that there were 11 such local transmission events in 2023 through 2024. Some of you all may remember that from the news. We have four major human infecting malaria species with a fifth that is sometimes included in this group. And these include Plasmodium falciparum. This species is responsible for the majority of such fatal and severe cases and occurs all across that zone of malaria in different countries that we just presented. Plasmodium vivax is less likely to kill the patient, but apparently, I'm told, is still not a fun infection to have. This can cause serious disease. The most important fact about Plasmodium vivax is that it is the predominant species responsible for malaria outside of Sub-Saharan Africa. We have Plasmodium ovale, which is clinically similar to P. vivax. but is quite geographically restricted to West Africa and a few other parts of the old world. And finally, we have P. malariae, which was the first malaria species described in humans, hence the name. This does not typically cause as severe of disease, but is associated with some specific complications. It also occurs broadly, but is less common than other species of malaria. And now we have our fifth that's sometimes included. The reason that this is sort of on the outside here is that it is a zoonotic species. Humans are not the natural host for Plasmodium knowlesi. This normally infects macaques and other non-human primates. This is fairly restricted to Malaysia and other parts of Southeast Asia. And it can cause very, very severe disease in humans if it is encountered. We're parasitologists so we love our life cycles. This is a lot of information but we want to kind of focus in on the human side of this indicated by the blue arrows. So if we start at the top here a mosquito infected with plasmodium sporozoites will bite a human. The sporozoites then proceed to invade liver cells first and then they undergo a round of replication in the liver fairly silently. And once that is complete, the young parasites are released into the bloodstream. And this really begins where we're going to talk about malaria, this erythrocytic cycle or the human blood stages. So these young parasites that are released invade red blood cells where they start to develop into an immature trophozoite or ring stage. From this point, to personify a parasite a little bit. It has two choices. It can either proceed through an asexual development cycle where the trophozoite matures, then forms a schizont where asexual reproduction occurs. You get the production of merozoites or daughter cells. Eventually this destroys the red blood cell and they are dumped out into the bloodstream and start all over again. The other choice that it has is to proceed through sexual development and form gametocytes. This is the form that will be taken up by mosquitoes. It cannot replicate further. Like playing the board game of life, there is no going backwards from that stage. Once you have the gametocytes, that is the end of transmission. Importantly, we want to focus on this stage because this is responsible for the clinical signs, and this is where we're going to see the diagnostic forms that we're going to talk about today. Briefly, a little more background on the clinical aspects of malaria. So very classically, this disease is associated with periodic fevers and chills called paroxysms. And this is due to the timing of the destruction of red blood cells by the parasite. The different species vary in the timing of this. For example, P. falciparum, vivax, and ovale are about every three days. Malaria is every four days, and P. knowlesi actually has a 24-hour cycle. Note that some of these classical patterns may not be as evident in travelers, but this is our classical presentation of malaria in the endemic countries. Naturally, also occurring with malaria is a whole constellation of unpleasant somatic symptoms, and we do want to underscore through this presentation, and we hope you take this home today, that malaria is a medical emergency, particularly when we talk about Plasmodium falciparum. This species can cause complications like cerebral malaria, where infected red cells become stuck in the small capillaries of the brain, blackwater fever in response to an overwhelming destruction of red blood cells, cerebral malaria, and more. Understand that this is not just unpleasant. These patients can go with severe infections can go from sick to dead in 24 hours. The deterioration is quite rapid. And so if you have an infection that's considered severe, which is a parasitemia of greater than 5%, and this percent refers to the percentage of red blood cells infected by the asexual form of parasite, which is responsible ultimately for those clinical signs. In such infections, this requires immediate intervention with IV artesunate. Oral medications are not sufficient and don't act fast enough to save these patients. So all in all, time is of the essence. That being said, say you encounter a patient who had been to an endemic area and has a severe febrile illness. You've got to ask yourself two questions. The first being, is it malaria? Malaria is not the only tropical disease that can present with a febrile type illness. The next question, if that's established as a yes, is, is it Plasmodium falciparum? Again, this species being associated with the more severe outcomes and death, it is a medical emergency, no matter the species, but particularly for this species, P. falciparum. At that point, if that's established, the next course of action would be to determine if you have that greater than 5% parasitemia. by doing a um a parasitemia count to inform what kind of treatment and what route of administration would be needed but what if your answer to either of those questions was perhaps a no um this still matters you don't want to throw your hands up and walk away um without a detailed answer um some special considerations for other species uh include for example for P. vivax and P. ovale these species can cause relapses through a particularly adapted liver stage called a hypnozoite so unless this hypnozoite stage is treated with particular drugs these infections can relapse months even years after the initial infection P. malariae doesn't typically cause again severe disease but sometimes it can appear to to go away when really it's lurking around at such a low level, and then it will recrudesce years later. And what if it's not malaria at all? What if it's babesiosis? Babesia is a parasite endemic in many parts of the U.S. It causes a malaria-like illness. Looks a little bit like it microscopically as well. This is particularly important to rule out because the drug treatment strategy will be different than malaria. Malarial drugs are not effective against Babesia. So those are some heavy-hitting questions. How do we go about answering them? We have three tools available to us primarily for the diagnosis of malaria, and those are rapid antigen testing, microscopy, and molecular testing. And as we walk through this presentation, we will discuss the relative uses and virtues and interpretation of each, and I am going to jump into microscopy. So this is 2026. Why are we still looking at blood smears through a microscope when we have all of these new fancy toys available to us like NGS and you pick your other favorite testing platform? Well, there are still advantages to microscopy, some pretty significant ones. First, the turnaround is quick versus PCR and some of our newer methods. A smear can be prepared and read within hours very, very easily and not have to be sent off or, you know, subject to further processing. What's also great, no proprietary kits, no analyzers. All you need is a microscope, some staining reagents, and a good analyst. So the cost aspect is beneficial as well. By virtue of just putting eyeballs on this slide, you can do many things, such as distinguish which parasite stages are present, such as what asexual stages are present. Are there gametocytes present? And you can also rule out other blood parasites. This also allows you to count the parasitemia accurately by being able to visualize the parasite and count only the asexual forms responsible for the clinical illness. And if you're successful, that's a efficiency, that's a real efficient outcome. You've reduced the need for a follow-up PCR. So what goes into a microscopic diagnosis of malaria? Obviously, at the top here, we have what does the parasite look like across its four developmental life cycle stages that we discussed, the ring, the trophozoite, the schizont, and gametocyte. Another aspect is the appearance of the infected red blood cell. So all of these malaria parasites, each species can produce particular changes in the infected red cell that can be diagnostically useful. And the third aspect are extrinsic factors, such as a travel history or history of transfusion. We want to caveat the use of these external factors, simply because travel history is not always accurately reported, nor is it always available to the laboratory. How many times do you have a submission form that all it says is, please test for malaria? there's no there's no context there's no background you don't know where this patient's been and also but if you know it say it might help you push you one way or another on a diagnosis for example if you knew that a patient had not had been to say South America had not been to West Africa had a parasite that sort of looked like Plasmodium ovale but a little bit like vivax that might that travel history may push you in the direction of vivax and all those things come together to create our gold standard. So now we're going to talk about some technical aspects of microscopy. When we refer to blood smears or blood films, those terms are synonymous by the way they can be used interchangeably. We talk about thick smears and thin smears. The difference between these two specimen types is more nuanced than just the amount of blood that's used. For example, the thick smear has more blood, but this blood is simply air dried and not fixed in methanol because when it is stained, this will lyse the red blood cells and create the thick smear that can be examined. Because there's a lot more blood and because the red cells are lysed, this enhances visibility of the parasites and the sensitivity. So really think of the thick smear as your screening tool. This is going to be the first one in this set that will be reviewed typically. Unfortunately, a drawback of thick smears is that the parasite morphology is usually not ideal. And because the red cells are lysed in the staining, you're not going to have that visual available to you. On the other hand, the thin smear, this is where a small amount of blood is spread thin into a sort of monolayer called the feathered edge at the distal end of the smear. This allows you to view individual red cells and parasites within. thin smears are fixed in methanol prior to staining to stabilize the red blood cells keep everything looking exactly how it did you can think of this thin smear as sort of the gold standard of the gold standard this is where your species id is likely going to be made the drawback being less blood it's less sensitive this is a nice visual i like to show highlighting the differences between the thick and the thin smear. These are prepared from the same blood sample that is P. falciparum positive. On the left, we have a thick smear. You can see numerous ring stage parasites throughout these dots with this little crescent cytoplasm around each. You can see that overlaid kind of on this cloudy sponge painting kind of looking background where the red cells have been lysed. And you can see just how many parasites there are. compared to the thin smear on the right while we can see the individual red cells you can see some parasites within just just you can appreciate how many fewer parasites are visible now this is a pretty high parasitemia infection so yes you see you see numerous parasites even in the thin if this were a light infection um looking at the thin the thick versus the thin can make a really really big difference in your chances of detecting that infection what about staining so we have a few options available to us when we stain for blood parasites the first being the Wright and Wright Giemsa type staining these are bread and butter hematology stains you might see these in big hospitals used in an auto stainer setup an advantage of them is that they are suitable for that use and they can be this can be performed very quickly only about one to ten minutes depending on the protocol and you know this this is appropriate for blood parasite ID although it's not preferred the Wright stain alone can kind of produce a bit of a substandard staining on a lot of Plasmodium parasites in the example here you can see a schizont of P. ovale that's been right stained and this blue cytoplasm that you can see it looks a little bit washed out and pale. However, oftentimes this is still identifiable on the smear by a good microscopist. Many diagnoses of malaria are made from the hematology smears. On the other hand, we have our classic Giemsa stain and this is our preferred blood parasite stain. However, it takes longer to stain, usually 45 to 60 minutes versus Wright and Wright Giemsa. So this is pretty much much restricted to specific blood parasite test orders. The beautiful thing about Giemsa is that the pH conditions can be altered, which influences the appearance of certain characteristics that are useful for diagnosis. The chief one in malaria being the stippling of the red blood cell. So if the Giemsa stained pH is adjusted to 7.2, this will highlight the feature called Schüffner's dots or stippling, which is produced exclusively by P. ovale and P. vivax. So being able to observe that may really help you identify those particular species. I want to include this slide because this is a hot topic that gets attention pretty predictably about every five to ten years with Ebola and Marburg outbreaks. Recall I mentioned earlier that malaria is not the only tropical disease on the block that can present with a febrile-type illness. Unfortunately, some other potential mimics are these high-consequence viral hemorrhagic fevers. Screening of blood, hand preparing and screening of a blood smear to rule out malaria in these potential patients creates a real biosafety hazard for the laboratorian. So over the years, a modified staining protocol has been developed for thin smears in these cases that will eliminate any virus that might be present in that blood. The detailed protocol can be found on the DPDX website in the link, but I wanted to just highlight that the main difference between it and a standard Giemsa stain is a longer exposure of the thin smear to methanol before staining. and I want to highlight also that a thick smear protocol is under development. We've done all this hard work at the bench and now what do we do? We get to read our slides. So what goes in to a reading a malaria slide? A parasitologist at the bench has to be able to do three things. First, they have to recognize objects of interest as they scan the slide. Two, they have to determine if these objects are are the genuine article are they plasmodium are the other blood parasites or are they potentially artifacts and three they have to be able to accurately identify the species by examining and comparing all of these diagnostic features now this is a big topic i cannot teach you how to accurately identify plasmodium uh species and blood smears from a 40 minute presentation. This requires a lot of blood, sweat, tears, eye strain, whatever you'd like to call it at the microscope, a lot of self-study and practice to be able to gain competency and keep it. So what we hope to do here is create sort of a starting off point. Should that be of interest, should you want to hone your skills, you'll know where to start. So I just want to show you what this might look like the best that I can without a live demonstration. This is a simulated field. Looks like something you might see as you're scanning a blood film, but I wanted to simulate this to show you all of these stages that you might see. So, our three steps. First, recognize object of interest. I've already done that for you by creating this figure. In reality, the analyst would be doing this as they scan through multiple, multiple fields on this slide. second is recognize that these objects are plasmodium artifacts or something else the easiest way that i like to teach how to go about that is to break this down by those four stages so we have our ring stage parasites here that looks like a you know a it looks like what it's named a ring stage you have a little crescent of cytoplasm that's bluish um with a sort of magenta chromatin dot and we see various stages of of development in those ring stages we have For trophozoite, that's the asexual stage that the ring develops into. It becomes larger with more cytoplasm, more chromatin. The trophozoite may proceed to a schizont stage, that dividing stage, where you're going to see those nuclei of the individual daughter cells start to become visible. And then finally, we have the sexual stage, if that young parasite has proceeded down that road. and again, these are going to look large, bluish cytoplasm. You're going to see some granules of the parasite's pigment or hemozoin as those brownish granules there. All right, well, what about this other stuff? So, this might catch an analyst's eye, but a trained analyst will quickly know that those are essentially normal blood elements, such as a white blood cell. Platelets, in particular, can be a confusing artifact if they are stuck on top of a red blood cell, because that superficially may look like a parasite in a cell. And then you have things like debris and other contaminants on the slide. And now we have to accurately identify the species. And this is the real meat of it. Again, very, very large topic. I can't teach you how to do this in 30 minutes, but I want to give some examples of what we might be looking at here. Something like what stages do you see? What's predominant? What do you see a lot of? For example, Plasmodium falciparum, this PF panel here. Typically, you're going to see mostly ring stages, maybe a few trophozoites. Very uncommon to see schizonts. Versus P. vivax, where it's quite normal to see a mix of all four stages on one smear. The general appearance of the parasite. So this is the real subjective one. You're going to start to see a lot of adjectives thrown around. Things like sturdy, delicate, amoeboid, compact. Any particular characteristic shapes or forms present. Shown below is a P. ovale ring stage, which is classically a sturdy or robust parasite and large ring stage. Compare this to Plasmodium falciparum below, which creates small, delicate rings, oftentimes with that characteristic double dot form, or also called head phone. We have a characteristic that's a little more objective, as in the number of merozoites or daughter cells in a mature schizont. So if those are found and counted, that can be quite useful. For example, P. malariae produces very few, typically about six to eight. Compare that to P. vivax, which often produces more than 13, all the way up to 24. So if you saw that stage, potentially a very useful feature to be able to assess. The shape of the gametocytes are a really important one and a very useful one for ruling out or in P. falciparum. The question, you've got two options here. You have crescent-shaped or round. Plasmodium falciparum, you can see on the left here, produces that crescent or banana-shaped gametocyte. And it is the only human plasmodium that does so, making that a very, very useful stage to observe. And finally, I mentioned the appearance of the infected red cell. We're going to be looking at things like the size relative to the uninfected red blood cells. Is it larger? Is it smaller? Is it distorted? Do we see stippling present? Our examples here, we have P. falciparum. This doesn't typically impact the size of the infected red cells so much, pretty much the same as its surrounds. P. malariae often presents the smaller infected red cell. And then finally, P. vivax and P. ovale produce enlarged, distorted shapes, and if they're stained under the correct conditions, you may see that stippling starting to show up, and we can see that quite well in these examples. So pivoting back to this simulated field here, we confirm that this is P. falciparum based on number of those type of traits, such as the equal size of the infected red cells versus the uninfected cells, mostly rings, some of those double dot forms. And again, that crescent gametocytes highly useful here. So that's just a taste of what it might, this process might look like. How would you continue to gain skills in your laboratory? DPDX offers many diagnostic resources, including bench aids. These are very useful documents to have. We go over technical procedures such as how to prep smears, how to stain, how to do parasitemia counts. And in addition to that, we also have comparative morphology charts. Here's an example, again, breaking down those species by these diagnostic stages. And we also have, you know, illustrations and photographs as well. These are very useful to have as you're learning to read smears and understand plasmodium, But also highly useful to have just as a reference for even if you're trained, if you need to remind yourself of certain things. And we also offer diagnostic services, including telediagnosis and conventional slide review. To really quickly highlight our telediagnosis service, this is where we can accept images in lieu of a physical specimen, provide a review, and respond to you, the submitter, directly with our identification. This is handled as a specific test order in the test directory. To request this, you need to email us directly at dpdx.cdc.gov for a secure upload link. We like to provide our initial identification by email, sometimes that same day, sometimes next day. We may at that point request additional specimens be submitted for further testing, but oftentimes it can reach an ID from images alone. I want to just emphasize really quickly here before I transition to Brian that telediagnosis is a reference diagnostic service. It does not negate the need for rapid review of blood films in a hospital lab or in a clinical lab setting. We intend this, again, as a reference service where we confirm equivocal slides, provide additional confirmation of species, purposes like that. but again if this is desired please reach out to us directly so that we can guide you through this process right and i'm going to hand it now over to brian to conclude our presentation and talk about our molecular and rapid testing thank you sarah so now that you've heard the exquisite importance of parasite morphology for for malaria identification i want to go over just some of the additional considerations one needs to include in malaria diagnostics. On the left-hand side, there are some implementation challenges to microscopy. This includes sort of a high level to train and build competency in the laboratory scientists. When laboratories don't receive a lot of samples, competency can deteriorate over time, and it's important to offer 24-7 coverage of this service, and that can be very difficult depending on workforce and staffing needs. There's also some other constraints and important considerations around the use of microscopy. One is the need to really test samples very rapidly for the best morphological identification. So anytime specimens sit around or take a long time to get to the laboratory, morphology identification can suffer. There are concerns about the sensitivity of morphology. For instance, in low parasitemia situations, morphological identification may may not adequately detect malaria in all situations, and one needs to keep an open mind, especially when there's a high clinical suspicion of malaria. And lastly, there can be difficulty, and this is very frequently associated with experience and competency around complicated, difficult cases, cases of mixed infection, and we'll go over a few of those kinds of cases. So how do we sort of mitigate some of these issues. And there are some rapid tests that can help us mitigate these potential limitations of microscopy. Let's start with rapid antigen testing. So we'll take another tool out of the toolbox. And for rapid antigen testing, there are many different rapid antigen tests. These are lateral flow devices globally. But in the United States, there is one assay that's FDA cleared, and that's shown here. This assay, and I won't go through sort of the instructions for use, but you can see on the actual device itself that there are multiple lines, a control line, a line that's specific for Plasmodium falciparum, and then a line for other Plasmodium species. This can be a very important and useful test in a clinical setting. It offers rapid turnaround. I think it's about 15 minutes. You can use either venous blood draw or finger prick blood samples can be used in this assay. It's simple to perform. It's rapid to perform. It can be used while other laboratory tests are pending, and it can even provide a presumptive result for Plasmodium falciparum or other Plasmodium species that can be used to initiate treatment. There are some disadvantages to um to the next slide there are some disadvantages that i want to highlight some disadvantages include um the fact that all presumptive tests with a rapid antigen test need to be uh confirmed by um by microscopy or another method okay we're going to move straight to molecular testing, which is another opportunity to conduct some rapid testing in addition to microscopy. Next slide, please. So when could molecular detection of malaria possibly be used? There are several circumstances that I can go through here. Microscopic examination of blood smears may not be satisfactory for species-level identification. So at the bench, there may be a question between two different Plasmodium species. And molecular identification can help establish which one it is. Blood smears may not be immediately available for reading, and a molecular test could be useful. In addition, because of the sensitivity issues around microscopy, a blood smear may be read as negative, but clinically there may continue to be high suspicion for malaria, and a molecular test may be indicated in such situations. There could be situations with possible mixed species infections where there's multiple Plasmodium species in the sample or even other parasites such as Babesia in endemic areas that can frequently have similar morphologies. And there can be several other special circumstances where a molecular assay is desirable. Next slide, please. So some of the things to consider for molecular detection of malaria include what types of testings are appropriate for clinical diagnostic testing. In the U.S., there's an FDA cleared assay. An example would be film array panels where malaria is on a multi-pathogen panel, and there might be multiple markers that can be used to detect malaria at a molecular level. And then there's several laboratories, generally in reference laboratories, that have developed laboratory-developed tests. I do want to mentioned a few things to keep in mind when considering these types of tests. What are the targets on the molecular assay? Are there pan-Plasmodium species targets, or are there species-specific targets? And it's important to review the assay limitations. Some assays may have difficulty distinguishing different species that may be present in a mixed Plasmodium species infection, where one Plasmodium species could be at a much higher level or infection level than another. Some assays might not distinguish between all assays. You might find that there are targets that include both Plasmodium vivax and Plasmodium ovale, or Plasmodium knowlesi may not be detectable in the assay. And then finally, it's really important to consider that generally these assays are not appropriate for parasitemia calculation. And parasitemia calculations continues to be critical for clinical monitoring of patients with malaria. So it's important to also use a morphological assay to calculate parasitemia. Next slide. CDC has developed a malaria PCR laboratory-developed test that detects the five different species that we've talked about in this webinar. The specimen that's acceptable for this particular assay is whole blood EDTA treated. And the platform that we validated in our laboratory was the QuantStudio 5DX real-time PCR system. Next slide, please. This assay uses two triplex reactions shown here. These are both multiplex reactions. You can see the markers have different fluorophores for each of the targets. One of the reactions also has an internal process control as well included to monitor the assay. Next slide. A quick review of the performance specifications is shown here. This assay was shown to have very high sensitivity for the correct species with clinical samples with known species identification, 100% specificity for samples that were negative clinically for malaria. The specificity with respect to cross-reactivity, just a notable situation with Plasmodium brazilianum, did cross-react with Plasmodium malariae. You can find that that is actually expected because of overlap of the target in those two species. The limit of detection was less than a single parasite per microliter of whole blood. This procedure itself may be requested from CDC by contacting parasiteslab at cdc.gov. Next slide. So let's put everything all together. Here's an updated algorithm for malaria diagnostic testing in the U.S. So it starts with microscopy. Microscopy plays a central role in malaria diagnostics, but if available, our other diagnostic methodologies also consider. So microscopy plus rapid diagnostic testing on the left-hand side of this algorithm, you can follow the solid arrows for required steps and dotted arrows for those situations that are indicated in specific circumstances. When a rapid diagnostic test is positive for a Plasmodium marker, you still need to confirm those results by microscopy using a blood smear to determine the species and also to determine the parasitemia. Even when you have negative results, you still want to confirm results with microscopy. For molecular testing, you want to also review a blood smear to calculate parasitemia and determine species, if that's still a concern. With a negative result, it may not be necessary to conduct microscopy, but if there's a high clinical suspicion, microscopy is still warranted. And what about with microscopy itself? Always report the results to determine species, parasitemia, and determine if molecular testing is still needed. In those situations where one needs to confirm species, consider mixed infections. What if the sample's negative at the first read, but malaria testing has been ordered? It's important to repeat microscopy every 12 to 24 hours up to three times with a negative sample. Next slide. So some of the key takeaways. Malaria is a medical emergency. We discussed how time is of the essence and outcomes are important to drive accurate diagnosis and treatment of patients. It's important to consider the multiple techniques for malaria diagnosis, everything from microscopy to rapid diagnostic antigen tests and molecular testings, consider all of their strengths and weaknesses, and use well-defined testing algorithms to understand the use and interpretation of these tests. And then finally, it's important to really maintain preparedness for lab capacity and proficiency. There are many services offered, resources, and technical support available to clinical and public health laboratories in blood parasite identification, and we've offered a few resources here. One of the key things that we'd like to leave you with is the importance to rapidly report results to drive patient care. Next slide. So with that, we want to thank you, and I think we do have a little time for questions. I think there's one more slide. Here is some contact information for Sarah and myself. Our email addresses are here, as well as contact phone numbers. There's the DPDX website, which has all of the wonderful resources that Sarah has mentioned during her part of the talk. and you can also email dpdx at cdc.gov for diagnostic services resources around microscopy training and so forth and then of course parasites and lab at cdc.gov for other types of testing including molecular identification for malaria thank you very much thank you thank you this was so informative. I believe we do have a few questions. Of course, we always get the question about the slides and the audio recordings, and they will be available. It will be posted on the website within two weeks of today, so you can check back to the REACH website at reach.cdc.gov backslash one lab test, and you will be able to find the presentation from this recording in about two weeks. Let's see what other kinds of questions we have here. Are you guys ready? I think we're ready. You may switch between Sarah and myself, depending. Okay. So we have a couple questions about, I was trying to get some of the good ones here. for, so here's one, for a modified thin smear protocol to inactivate ortho Ebola viruses and ortho Marburg viruses, can any Giemsa stain formula be used? Or do we have to use the one listed? The example is with two drops, 5% Triton X-100 to 100 rather. Or is it just the 15 to 30 minute in 100 percent methanol that will inactivate the virus that was a lot let me know if you need me to repeat that, that's fine and that that's a good question um so in that context this refers to a generic Giemsa stain. This could be something you make in in lab something you buy commercially but at the end of the day a Giemsa stain is is a is a standard formula so the brand itself doesn't matter um the inactivation with that protocol is taking place in in the methanol, not in the actual Giemsa reaction itself. So hopefully that answers your question. So the answer is, you know, this is basically a generic Giemsa stain protocol, plus the modification with the extended time in methanol. Wonderful. Thank you. So here's another. The sickle cell anemia trait hamper the ability to accurately ID the Plasmodium species, and how can this be mitigated? All right, I'll try to answer that. That's a really good question. So personally, we've had maybe a couple of those cases come through where we have sickle cell, we have like a congenital ovalocytosis. And in those cases, it's interesting. You can still see parasites that are recognizable, although again, the review of that thin smear may be a little more complicated. In your thick smear, since you're lysing the red cells, that may not be a factor but um yes you can still identify these it just it just may be a little more challenging um and you may want to do something like reach out to a reference lab uh hint hint DPDx telediagnosis um if you're having trouble with a particular uh specimen and i have one more i think we have time for just one more um is there a determining factor that would lead to gametocytes gametocytes forming versus the asexual stage or is it random? So this is again going to vary a lot by the parasite strain, the species, even the host. But in the context of clinical diagnostics, when we look at a case that is only gametocytes, that often is an end-stage infection. So after treatment, you may actually see only the gametocytes remaining. That's usually the last one to leave the party. And again, those are dead end. They're not clinically relevant anymore. And so that's, again, an important interpretation of that smear and in the reporting. That's why, again, being able to report down to parasite stages is useful. But yeah, in terms of the determination between asexual and gametocytes, that's actually still a very much a question under investigation from a biology standpoint. Well, I think that's all the time we have for questions. If we did not get to your question, you are welcome to email them as well, and we'll see what we are able to answer. However, that is all the time we have for questions right now. Next slide, please. 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