Thank you very much and for inviting me, and it's been really nice to visit the campus and it's very, very impressive. My mother is an artist, so the aesthetics is really so pleasant to look at and I think it changes the whole atmosphere and it's great. I'm at the University of Calgary and I'm going to talk a little bit about our work in Africa and also in Asia, and focus a little bit on this trial that we did. First, I want to acknowledge that I live and work in the Treaty Seven lands of the Blackfoot Confederacy, the Tsuut'ina First Nation and Stoney Nakoda, and the city of Calgary is also home to the Métis Nation of Alberta, district five and six. Malaria in pregnancy is a big problem, and this slide pretty much captures that half of the world lives in malaria endemic areas, and over 30 million women are at risk of malaria and many are exposed during pregnancy.
We focused on this population for this trial because it's a vulnerable population. We're very interested in the impacts of malaria and pregnancy. Some of the issues that arise for pregnant women with Malaria in pregnancy include anemia, fetal loss, preterm delivery, low birth weight for the neonate, small for gestational age and susceptibility to severe malaria. A big issue, and the World Health Organization has prioritized it as a major concern. Africa, of course, is a focal point for malaria. Most of the malaria in the world, falciparum malaria occurs in Africa. Prevention of malaria can be done in two ways. One, on the top there is intermittent preventive treatment for pregnancy. What that essentially means is, chemoprophylaxis for malaria during pregnancy. Now, there's some advantages to that. It's easy to give drugs, you can give it broadly, there's little side effects, but there's no coverage often in first trimester when malaria has its biggest impact because pregnant women don't come to the clinic necessarily in their first trimester, they may come later.
Drug resistance is an issue and the public health infrastructure in low middle income countries isn't great for that. The other approach is to screen for malaria and treat. There, you may have less issues with drug resistance because it's not empiric treatment, it's targeted treatment, less side effects, parasite specific treatment. More recently we are running into an issue where the rapid diagnostic tests are not as effective because of deletions in the gene that they detect. The parasite is very smart, it's figured out how to evade our diagnostic tests. The test often lack sensitivity in terms of analytical sensitivity, and asymptomatic infections are often not detected. If you are not symptomatic, you may not come to the clinic, you may not get tested. In fact, a lot of malaria, even though we think of it as a devastating lethal infection, is actually just under the surface and, especially in pregnant women, can cause asymptomatic infection.
What we wanted to do, given that background, in Ethiopia, is to screen and treat for malaria using a state-of-the-art technology which we call LAMP. Loop Mediated Amplification and Isothermal Amplification method targeting the DNA as opposed to looking down a microscope or detecting antigens using rapid tests. We wanted to specifically understand the impact of asymptomatic malaria because we know already that those low-level infections are flying under the radar and often not detected with traditional diagnostics to understand what the impact was on the mother and the child. Here is a picture at one of the rural health centers where we implemented this technology. There you can see the picture, and one of my co-investigators, Banchamlak, who lives in Bahir Dar in Ethiopia. We implemented this technology using good GCP, good clinical practice, good laboratory practice, trained approximately a hundred staff at eight different health centers across the country and worked closely with a company called Human DE and Eiken Chemical Company in Tokyo.
Human DE is based in Wiesbaden, Germany, and Eiken is in Tokyo. The technology developed essentially is through those two companies and it's a CE-marked product, and there you see the registration of the trial. Here is a little bit more about that. This is the instrument that you saw in that picture and some of the protocols which might be familiar to us as scientists here, especially at NEB, involving DNA extraction. Fairly simple method of gravity flow column and then visual detection of this isothermal reaction using fluorescents on this instrument. One of the challenges with the settings we work in is that it's very, very resource limited, and so even electricity can't be taken for granted. This is powered by electricity, but we had backup generators and we had to implement Kodak batteries and things like that just to make sure that these instruments didn't stop in the middle of a reaction when we're doing this diagnostic.
For those of you not familiar, this is Ethiopia and these are the different health centers that we implemented this technology across the country. They were chosen strategically, if you will, because of the different transmission levels. We want to understand how the technology impacted a low-level transmission in the highlands versus high-level transmission in the lowlands here on the South Sudan border, which again, very challenging environments, there's a lot of instability both in terms of famine, war, et cetera, you name it, there were challenges during this trial, so we're very happy to have completed it. This is the trial design. The inclusion period often is early second trimester, women were enrolled into the trial with consent, of course, and followed through to delivery and past delivery out to 28 days. We wanted to understand, again, as they came to the antenatal clinics, how they were doing.
This is essentially the trial design. It's very important to point out there's no chemo prevention in Ethiopia. They don't use chemo prevention during pregnancy because that would have been a big confounder to our diagnostics. If everyone was getting drugs, we wouldn't really understand the value of the diagnostics. Very difficult to do, very challenging to convince folks to come back to the clinic and follow and adhere to the trial, so we did have some loss to follow up, which is understandable in those environments, as you can imagine. Inclusion criteria, well, you have to be pregnant. We made sure of that by actually using a Vancouver-based technology called Clarius, which is an ultrasound. They don't have ultrasounds in those health centers, they rely on more traditional diagnostics. We implemented that and actually trained midwives on how to use ultrasound, and so that was a big leapfrog, really, in technology there.
These are some of the exclusion criteria, which is, if you were recently treated, we didn't include you at high risk in third trimester. This is what it looks like, the diagnostic protocol, you were either randomized to traditional diagnostics, which is microscopy and RDT out here, and you are only tested if you are symptomatic. That's the standard of care as per the Ministry of Health in Ethiopia. The intervention in this case was to screen asymptomatic and symptomatic, so you didn't have to have a fever or have any symptoms, we'd still screen you. We have the addition there, you can see, of the isothermal LAMP technology in the intervention arm. We already knew LAMP was more sensitive, we'd already done the studies, we already published that, but what we really wanted understand was, was there a difference in outcomes if you had that technology to detect malaria and you were treated.
Our outcomes were based on the impacts of malaria in pregnancy, which is birth weight and weight gain, and other things like preterm delivery, anemia, hemoglobin levels, pregnancy loss, maternal hemoglobin and placental malaria. These were some of the outcomes we measured in the women out to delivery. Some pictures, this involved a lot of training as I said, and this is a post-doc in my group, Claire Kamaliddin, who was instrumental in some of the field-based implementation and working in the clinics, training folks on GCP and GLP. Of course, this was a clinical trial, everything was documented along the way in terms of the health records and so on and so forth. Of course, we had to enroll women into the trial. A lot of young folks in terms of the average age, and they all got routine antenatal care with the addition in the intervention of this isothermal technology. We implemented, as I mentioned, this Clarius ultrasound-based technology.
This is what more traditional malaria diagnostics looks like. Gustav Giemsa invented the Giemsa stain over a hundred years ago. We still use that for diagnosing malaria, in fact, that's the recommended diagnostic tool by the FDA and CDC. It still involves microscopy. This is the stain, you can see where slides are stained and et cetera. These are some of the conditions that are there. We came along with this instrument and, as I say, we implemented it. That's what those reactions look like on the far right. You can see the fluorescence of a positive reaction at the far right there, and then two negative reactions where you don't see fluorescence. That's how you interpret a malaria result in the field. We implemented birth weight, as I mentioned, as an outcome, we had to get it right to three decimal places.
We actually implemented these instruments, these powered digital weighing scales, and here you can see it being calibrated. That was also critical and something that wasn't available. During the process of this, we implemented a lot of new things that help. Here are some of the results. We enrolled approximately 2,400 women, and as you can see, they were randomized and weighted more towards the LAMP arm, and statistically we wanted to see if there was an impact. It was a two to one ratio. We had obviously some loss to follow up as I mentioned, but we did capture a fair amount of outcomes-based data in terms of birth weight and neonatal weight. Here is the punchline. After a large trial over three years and a lot of heartache and work, we did see a signal in terms of the newborn weight at day 28 and the weight gain in the first 28 days to significance.
In terms of the other outcomes, we didn't see a significant impact statistically between them, but that weight gain was a critical outcome difference in the LAMP arm with significantly improved weight gain in the first 28 days, which is important and our primary outcome. In more detail, if you look at the weight gain in the first 28 days by percentile, you can see there there's about a 200 gram weight gain at the 50th percentile in the LAMP arm. There's something happening there in terms of improvement, let's say in the outcome for the neonate. More analysis here in terms of multivariable regression. We are trying to sort of now isolate and understand what is influencing this weight gain. The most significant thing we find is that being in the standard of care arm was detrimental in terms of weight gain.
The other things we think were influential, such as maternal age, transmission setting, anemia, gravidity is important. If you've had many pregnancies, you are generally protected from malaria. These things didn't seem to influence that weight gain, but being in the standard of care arm did. When we zoom in a little bit more, that impact on weight gain was seen more in the low, moderate transmission areas versus the high transmission areas. You are probably wondering, well, what about the diagnostics, right? I mean, these are the outcomes, but was there impact in terms of detecting malaria? The answer is yes. In the red, a lot more malaria cases were detected with LAMP than RDT across the board at all of the sites. The question is, are these true positives or are they false positives? What's going on? Jack Burke Gaffney, who's in the audience here, did some work in Ethiopia with discrepant resolutions or taking the discrepants and doing an independent method to verify, which was in fact correct.
You can see, in terms of specificity in the yellow, they're all comparable, but in terms of sensitivity, LAMP is more accurate and detecting quite considerably more accurate, in fact in detecting those malaria cases after discrepant resolution. When we tried to understand where are those additional cases being detected, it was in the asymptomatic infections, the ones that we thought were low level, was indeed where there was greater detection here compared to RDT and microscopy. Moreover, those asymptomatic infections seem to be occurring in the low moderate transmission settings versus the high. We don't have time to get into malaria epidemiology and all that sort of thing, but that sort of makes sense that that's where they occur.
I presented this data to a diagnostics company a few years ago or a few months ago, and they said, well, those cases you missed in the standard of care arm, can you go back and prove that you missed them? Did you actually miss them? We kept all the blood samples and we went back to the standard of care arm and asked the question, what about those folks who were undiagnosed, did they do poorly compared to you? That's what this is, and you can see in the undiagnosed, we went back to those samples, conducted LAMP on them, fished out those ones that we missed, those 88 patients there, and you can see there was more placental malaria significantly than in the ones that were detected by LAMP. The story is, you could probably get the picture now that what's sort of happening there.
In summary, LAMP seems to be showing more sensitivity, there's weight gain, obviously advantages there. It was feasible to implement, for the most part. In Ethiopia anyway, these data do support a sensitive method to detect malaria and preventing malaria in pregnancy, so all together, I think a nice story in the use of nucleic acid-based detection for malaria. I also wanted to just give a plug for some work we are doing in the Philippines, in a similar vein using LAMP technology. This is Ryan Chaffee, a graduate student in my group, doing some training on LAMP at the University of Philippines Diliman. We are focusing here on a project to look at antibiotic resistance in Philippines lakes. Different target, not malaria this time, but looking at antimicrobial resistance, again, using LAMP based technology in a one health approach. Looking at humans, we have collaborators looking at the veterinary sector and in the environment.
Here, this is actually technology developed in my lab. This is not the instrument you saw before, but engineers in my group have developed a similar instrument. Here you can see the tubes of where LAMP reactions occur, detecting different highly drug resistant genes that cause resistance to penicillin based drug, beta-lactam and carbapenems, and we've been using that technology there and conducting studies. Here you see some positive reactions for those targets. We've also developed a cell phone app to take some of the subjectivity out of interpreting these results and using some machine learning algorithms.
Biniyam Mazgebo, a postdoc in my group has enabled this software to detect the positives and also tell you what antibiotics not to use if you are positive for certain resistance genes. As we speak, we are implementing this at a hospital called Colombo, adjacent to some of the lakes because what's happening is a lot of the resistant bacteria, in this case, E. coli, end up in the hospital effluence and go into the lake, and then it's recycling through in the farms and so on. The intervention we are looking at here is to implement this screen for these resistance genes and then implement certain infection control measures that might reduce the transmission of these bacteria.
That's an ongoing project and it's sort of modeled on what the WHO advocates for global surveillance of these resistant E. coli. A lot of people to thank here, for the LAMPREG work, my co-principal investigators, Dr. Mekonnen Teferi at the Armauer Hansen Research Institute in Addis Ababa, and also I mentioned Banchamlak and also Delenasaw are the leads from the Ethiopian institutions we work with. In ARPHILAKE, I mentioned Ryan, but Ricardo Castellanos, he is a postdoc in my group, he is leading that project together with some of the other folks mentioned there. The funding for the LAMPREG trial was through Grand Challenges Canada and The Bill and Melinda Gates Foundation institution, FIND, based out of Geneva, Switzerland, and also the Canadian Institute for Health Research. Thank you very much. This is actually the origin of the Nile River starts in the part of Ethiopia where we work, and some of the team members there are at one of the waterfalls off the Blue Nile. Thanks.
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