Daniel Romero-Alvarez comes from earlier training in Ecuador as a medical doctor, but has become fascinated with the ecology and biogeography of diseases. Here, is presents his doctoral dissertation at the University of Kansas, in the Department of Ecology and Evolutionary Biology, on the distribution and ecology of the bacilli that cause leprosy, and their likely role as a zoonotic source of leprosy infections. Daniel’s web page is at http://www.romerostories.com/.
Okay uh thank you very much to for the introduction uh hello everybody thank you very much for being here uh my name is Daniel Romero Alvarez I am from Ecuador and today I am going to uh talk about what can be the summary of my entire research which is unveiling the
Zop and environmental potential of leprosy transmission um Jus stopped working again perfect okay so whenever I am start talking about infectious diseases I love to uh show this painting which is a portrait of our society in 1562 this painting is called the Triumph of death and was painted by Peter brel
That is also called The Elder and it’s a portrait of our society because regard L of social status economic position infectious diseases are always there hunting human Societies in this painting you can see the symbol of death which are these white skeletons um which is the classic symbol of infectious diseases and this
Agent can take the form of many pathogens and in this particular talk it will take the form of microbacterium lepre which is the pathogen causing Leos so in this changing World anthropogenic alterations of the ecosystems allow pathogens to move from Wildlife to human societies and this is happening because pathogens live
Naturally in ecosystems in Wildland but whenever there is a unthought deforestation excessive agriculture unthought mining then then pathogens need to find other sources to live and then that is why they jump into us we understand now that approximately 70% of all infectious diseases are from a zoptic
Origin so in this talk I am I am going to go through these Five Points first I will set the stage of it is currently known about leprosy then I will walk through the three chapters of research and then we will finish with some interesting conclusions so in order to
Set the stage I will present you the three main protagonists of this story which is the disease leprosy the pathogens causing it and what we know about transmissibility first character the disease so leprosy is a very ancient pathology it it was recorded since Biblical times and depending on the
Immun immunological factors of the host it can manifest itself as a very mild disease with very easy to treat lesions right like for example hypop pigmentations on the skin or it can manifest in the severe form that is called lepromatous leprosy which is uh the form of the disease that we know
From popular culture right the one that is very disabil dating for the patient the one that can make a patient be blind that can make the patient to lose fingers popular symbol of lepr um so although we know that Immunology is uh it has something to do with the this
Manifestation it is not clear whether a particular patient is going to develop a very mild form of the disease or a very severe form of the disease in this map published in 1891 you can see the distribution of what we understood was Ney at that time um the arrows the
Arrows here are pointing important places that are showing high prevalences of leprosy so you can see that it’s mainly concentrated in Tropics um however look this arrow in Norway so totally a different ecosystem during this time Norway was the place with the higher incidents of leprosy um and as a
Matter of fact leprosy was discovered there like the pathogen CA from leprosy so this map it’s is showing what we know about the disease nowadays in 2022 by the World Health Organization and you you can see that some of those arrows still highlight the places where the majority of leprosy cases are reported
Today which is Brazil India and Indonesia and interestingly interestingly we don’t have more cases in Norway if you talk with a medical doctor about leprosy and this happened in Ecuador like hey what do you think about the incidence of leprosy in this don’t they they answer like ah but
Leprosy has been eradicated and this that is very interesting because at the end there is no awareness of how this diseas is still prevalent even here right in 2022 around 136 patients were reported in the US so second character of this story the pathogen here I am talking about microbacterium
Lee this pathogen has certain characteristics that make doing research on it very difficult among them it is an obligate intracellular vasilos so that means that it cannot survive outside its host and different to all other microbacterium species it’s the only one that is a noncultivable bacteria on microbiolog microbiological medium
Meaning that you cannot do microbiology experiments to actually understand super important features of about pathogen such as transmission Dynamics in order to understand how this bacteria is transmitted or how it work in act in real life we have relied on animal models specifically infecting armadillos and also infecting mice and that is the
Only way the bacteria has been studied until today the other two important features is that is the slowest growing bacteria among the myobacterium genus so it slows really it grows really slowly super um the replication time can go from 12 to 14 days which makes the pathogen super super low to grow and
Then all the microbacterium genos including microbacterium Lee has this extremely thick cell wall composed with molic acids and that is why it’s called microbacterium and this protects the bacteria against normal antibiotic treatments so because you cannot call fure um micro Lee in microbiological medium we started understanding some
Important features of the pathogen uh when the genomic when the Genome of myobacterium Lee was published and this study published in 2010 collected around 200 strains of microbacterium Lee across the world and compared them among each other and then they they discover three important points M Lee has the smallest genome in
The family which 3.27 million base pairs and these 200 strings share a similarity of 99.995% across the entire world however we have leveraged the very small differ of 0.005 to subclassify the bacteria in forest meat types and also for Te of tyes then in in 28 this happened two Mexican patients died from
Leos and when they when they when the researchers try to find the causing pathogen they realized that instead of microbacterium prev the agent causing leprosy they found a microbacterium with 99.1% difference with micro Lee after five years of this publication the Le ology field accepted that leprosy is
Also caused by a new agent myobacterium lepromatosis and it is also something that usually nobody is aware so leprosy is caused by two pathogens right with the rest of discovered mum leprosis surprisingly enough this bacteria was found causing leprosy like illness in redos spals in the British
Is so the third main character of this story what we know about transmission classically we understand leprosy as a humano human transmitted pathogen from infected individuals to susceptible individuals however starting in the 80s uh armados in Wildlife were found to host myobacterium Lee and a super important study
Published in 2011 demonstrated that the same strain that circulates in Armadillos was also circulating in human beings and they of course their test the possibility of this xonotic transmission of leprosy as I mentioned before myomatosis was found and infecting red spirels and red squirrels have like they are transmitting the bacteria between
Themselves so they have established a cycle of infection in their population however they receive the pathogen from humans this has also happened with non-human primates uh involving Su Mavis Maks and chimpanzees uh but they have also received the bacteria from humans and also we know
That if you go to a Le leprosy Colony for example and you collect soil samples or water samples then you will find the microbacterium Le there but not a dead bacteria a living rep replicative organism so the environment might serve as a reservoir as well so however from where the bacteria
Is coming from originally in 2021 one this very interesting study uh followed two populations of chimpanzees that develop leprosy one population was hosting the microbacterium lepr Trin that was expected for Africa and that was also culating among humans there however the other population was hosting an Trin that was totally unexpected in Africa
That was was never discovered there so potentially they got it that population of Chim is from an environmental source so notice the plot that is depicted with your left um this blood is fundamental to this talk because of three things first it was published on the
90s uh that is 10 years after the world F organization introduced a fantastic antibiotic regime that can actually fight deposit very well however although you can see this decreas on prevalence of human infected patients right the bars at the bottoms are showing that the detection of new
Cases so the incidence of new detection case rate was never affected we had a very good antibiotic regime to trade leprosy but new cases were showing up uh and this of course was published in the is but this is the situation that we can see until
Today um so that is super s um one of the things uh that put me into this Research b is that we do not know how leprosy is actually transmitted what we knowe is that if someone gets the disease it has to be exposed for a very
Long time to the source and this very long time is something it’s a parameter that we don’t know it can be weeks it can be months um theoretically these pathogen can be airbone transmitted or contact transmitted because a large concentration of the bacteria can be
Found in the air mucosa of the hosts and also on the ulcers on the damage of the skin then we know that armadilo is a risk factor so people manipulating the animal it’s actually it has a higher risk of infection and also the environment is doing doing something that we don’t know
So as you saw this information has been accumulated in the last 10 years and here you can see the manual of the World Health Organization which is a guideline to control leprosy in the world and it’s called toward zero leprosy and you might expect that all this information has
Been included in the manual right the science and public health work together and that is not the case if you read this manual in detail you will find that they only focus on human train visibility that they barely touch the topic of zoonotic infection in southern us it is extremely surprising that
Across the manual they never mention the presence of these other agent causing leprosy myobacterium lepromatosis and of course they don’t say a word about environments so that this lack of knowledge in Public Health Organization and the lack of understanding of transmission is the main reasons why this research exists so
We start with chapter one Museum specimens here you can see well what we accept as the main host of M is the nine banded armadillo which is Theus and in pink you can see its distribution this armadillo can live from Ur to Central us and notice this picture these are m road
Kill um was was found in in m in sou Park potentially some of you actually saw the armadilo and potentially this is the first site of NB armadillo here in Lawrence um so across its distribution nine banded armadillos infected with myobacterium Lee has been found in the
United States in Mexico very recently in Colombia in Brazil and in Argentina so how can we test whether other n banded armados populations are infected so that is when we decided to uh look information about the presence of armadilo tissues in open databases specifically Beret and arctos and then
We noticed that these 10 museums were hosting tissues of armados we asked for the tissues send those the tissues we managed to gather 159 armad tissues from at least seven species and you can see in the maps that is kind of geographically comprehensive uh in compassing multiple regions of
South America uh because these tissues were not intended to be a part of an infectious disease project so the tissues were super heterogeneous we have liers we have SPS we have lungs um and they were preserved with different methods right Theo Frozen stuff like that so A Very heterogen heterogenic sample um
The oldest sample came from 1974 the newest came from 2017 in red are the positives that we found the majority of positives were found in 2013 but more importantly here is the geographic distribution of positives and negatives in total from the 159 tissues 122 belong to nine banded armadillos and
From them we found 18 positiv 16 in North America and two in South America and these South American identifications of microbacterium in armaros are the first um realizations of the presence of the bacteria in Bolivia and parag in armados from those countries um overall then we said that
The prevalence is of 14.8% of micro ACR this something um so as I was mentioning before mic PR can be classified in types and subtypes uh from these uh 18 positives we were able to identify 13 at the species level so M um five were able to be identified at
The sub type level so sample 3i and we were able to find two gen and when this happened I was very kind of disappointed right because from my te samples only two genomes I was kind of but I was working with the word expert here and this information will come in candy
Later um you can see there what we accept is the philogenetic tree of around 300 genomes of myobacterium in colors are different branches the genomes that we collected belong to the branch 3i and they cluster together with other genomes of armadillos and with other genomes of myobacterium preing
Humans it was super nice to find that at least one of our genomes from an armadilo collected in 1996 um was also clustering here demonstrating the presence of this zootic pathway of transmission at least 30 years before the paramone publication in 2011 um yeah so This research was published
In the emerging infectious disease journal and there is a podcast that you can hear about this research if you are interested and also I published a past a scientific communication in the conversation and also in Spanish in my blog so you can go ahead and review so I
Am from Ecuador so the next logical question is if the armados are living there what is happening in my country so Ecuador is a beautiful place it’s divided in three ecore regions by the andian mountain range the coast the andian mountains and the Amazonian region and Rural communities in eador
And multiple places of Latin America are using the nine banded armadillo as a cultural item right to create these box and also to create musical instruments and they are also using the armadillo as a protein source and during this work I was able to taste the nine band
Armadillo and I don’t like it at all potentially people like it but I don’t um so in order to obtain armadillo tissue from different regions in Ecuador we put together a network of hunters and communities that were willing to work with us in order to uh see if they were
Hosting microbacterium Lee so you can see uh in the map of eador the all the points where we managed to find armad right and because there there is no information about there is no so many Publications about the taxonomic status of armadillos in Ecuador so we also Al applied molecular tools to actually
Differentiate the species that were collecting this was also very important because at the end this was not a a scientific expedition going to collect mama is what it was the hunters like willing to okay we want to eat armadillo this weekend so they collected it and
Then they call like hey we rather than Aro so we didn’t know almost nothing about the potential species that was collected so here are some results uh about the armadillo species that participated in this study we found 40 nine banded armadillos and they are represented by the orange
Triangles well red reddish triangles um we found one cavaso centralis represented by the yellow spot in the northern part of Ecuador and also another species Theus pastasi in the Eastern side of Ecuador represented by that cross and then we found at least six armadillos that were not able to identify using this molecular
T and then what about the distribution of microbacterium Lee here you can see the positives in red and the negative in blue you can see that the there’s a lot of positives first all of those positives are distributed across the country and then we found that half of
The n b armados were positive for M the only theas species was positive for and then two of six of the unknown species were also positives and then this gave us an overall prevalence of 47.9% basically a lot of armadillos are infected in both research and I have
Forgotten to mention this in chapter one and chapter two we found a zero prevalence of the other pathogen of microbacterium elatos and I believe that there was in the slid that I totally forgot to mention so remember the other detail that I I I highlighted before so here we found 23 positives
Armad and these were not Museum specimens meaning that they were not old samples they were really fresh we collected these samples between 2021 and 2022 so you might expect that it was super like straightforward to obtain uh uh the sub taxonomic levels of microbacterium however we couldn’t do it
So we couldn’t identify types and we couldn’t identify genom here is when I understood that the fact that in the previous chapter we obtained two genomes were actually something to celebrate a lot so even with fresh samples getting full genom of micm is a nightmare so
Super sad there the the start of this project was intended to find the Zone notic Link in Equador of leprosy transmission but we failed about that however we are still trying to get new micos and trying to actually complete this research um so with all the information that has been gathered
Through chapter one and chapter two we decided to use ecological tools to understand the potential distribution of myobacterium Le so this slide is very important because during the making of chapter 3 I discovered this curious paper published in 1981 that was quoting This research from 1910 right M Lee was discovered in
1973 and as far as 1910 Dr s in this second leprosy Congress in beran Norway suggested that potentially micro was transmitted by indirect sources not necessarily from other humans so this idea is Super O um and regarding what we currently know about indirect transmission environmental transmission of micum Lee
This fantastic study published in 2014 demonstrated that being an obligatory intracellular pathogen microbacterium Lee found that it can leave super happy inside amas and maintain its vence Factor even if the amoeba is exposed to very unfavorable conditions right and also this paper basically isolated my
And put it in pet dishes and then just leave it there to see what happens right so they put the Petri dig in the freezer and then they expose the bacteria to minus 20 c0 degrees and also they just leave the petri dish for five months in
In humid conditions and then in both studies they isolated the bacteria again infect mice with it and then the mice developed what we expected for a leprosy infection so the bacteria was able to survive outside the host and was keeping their virence factors so then a lot of Publications
Especially in India and French buana have also tested the presence of micro Sol water um as a replicative bacteria right so there’s a lot of Pap that was the point of this um and something very curious also to share regarding these indirect sources of micro pre transmission is that in vitro it has
Been proved that micro pre can live in THS and they can be horizontally and vertically transmitted so MTI can transmit micro th and then they can also transmit micro to RAB so myep might be everywhere okay so then uh in order to develop to understand the distribution of M we put together some
Ecological niche models the core behind ecological niche modeling is what we understand as the Hutchinson Duality which means that a particular point in geography can be represented by multiple points but but M multiple environmental conditions being temperature humidity um basically indexes and on the contrary one point the environment let’s say 23
CI degrees can be manifested in multiple points in geography that is why it’s called a duality and this fantastic plot demonstrates that very gracely so you can see in the map of South America that there are three cities there Kito wakil from Ecuador and Rio deeo from Brazil
You can see that wakil geographically speaking is super far from Rio caneo but if you transform this map on temperature and precipitation axis then you can see how wakil and rioo are very close together so in order to put together these models we rely on two experiments
In each of them we use these basic three strategies we use as Geographic points the identifications of microbacterium Lee in the cus species we use as environmental variables determinant of temperature humidity soils and vegetation and then we use three algorithms com schools one class support Vector machines and maxent
And the first question that we say so something that is very challenging with these models is to actually evaluate them and test if they are actually doing what they are supposed to do so we have this very interesting independent data set of points which was the collection
Of samples from Ecuador so basically we calibrated the models from identifications of microe arados in the Americas and then put together the models and see whether they can predict what we found already in Ecuador and the these are some results so u comvex schools were able to predict only 25% of our
Observations um one class for Vector machines were able to predict 75% of our detections and maxen was able to predict 100 perable detections uh in order to gather the majority of useful information across the models we put together an emble which is the last panel and theoretically future research can be
Directed there something that I like to mention here is that Maxin is showing a 100% of detection predicting that the entire country is good for the bacteria which I believe is kind of not informative because we are losing a lot of specificity right if something says that everything can be happening then
It’s like saying nothing so for me a good a for me the one class super Vector machine is kind of a good balance but whatever okay so then uh the second experiment was doing the same thing but the question was not only U focus on Ecuador but focus on the entire world in
Order to do that uh we well I put together what I like to call the most comprehensive set of points where myobacterium Le has been identified in nonhuman sources which you can see here and all the characters that have been in a way or in another discussed during
This talk are present here so in West Africa you can see the chin Banes that we talk at the beginning um you can see the red squirels in the rage is right and you can see also um the environmental isolations in Northern India and what we did here is to
Calibrate the model with the red points and then project the models to the world and see if they can anticipate the Blue Points so some results in ah okay so in these plots in order to complete what we were doing we also put together models using the entire set of points so the
Models calibrated with the cus occurrences are in red and the ones calibrated with the ones developed with all the points are in Gray so one algorithm was able to predict 48% of points the other 52% of points the other 57% of points so they were not doing that great job um I
Learned through this particular mix exercise how maxent which is a very popular algorithm to do these models extrapolates vast so if you compare the plot you can see how Maxim is like predicting like largely um okay so as we did in eador we put together the information that all these
Algorithms were agreeing with in order to suggest what what is the potential distribution of micum Lee nonhuman sources and as I mentioned before the models were not doing the best job ever however some interesting things that we can learn from this distribution is that remember that in chapter one for the
First time I found the presence of my Bolivia and Paraguay so the model suggests also areas of Bolivia and parai um to be good sources of nonhuman for nonhuman leprosy so this is a second line of evidence suggestions that we should do some research there um sadly the models failed to predict the
Presence of the red squirrels infected and also the environmental sources in India and also they fail to do a good prediction in Indonesia so uh some conclusions regarding all these research so first ecological D models in absence of information are doing a good job in terms of informing a
Evidence guide decision to see where we can find this where we can find nonhuman sources of leers however potentially the models are are are failing because we don’t have enough information aboutum and this is basically a consequence of lack of awareness of leprosy beinging other places rather than only in
Humans so then uh we saw this map before thanks to This research we added the blue triangles on the map um we found this 14.8% in chapter one of prevalence of mum mados 47.9% of microbacterium Lee in Ecuadorian armados and thanks to the two genomes that we found we anticipate that
The potential zono transmission of leprosy was happening in the US at least 30 years before it was accepted and then it is super important to mention that across this study we never found microbacterium lepromatosis the other agent of so remember that PL um we as I mentioned before we knew
That this stable detection of new cases is happening until now and as as we have discussed throughout this talk there is at least 20 years of evidence demonstrated that leprosy is a is a non-specific human pathogen that it can Lees Beyond human beings however this information is not included in my view
Public Health institutions are having this bail on their eyes that do not allow them to include research on the development of their guidance and this is happening because the huge gap between research and public health and only by reaching that Gap closer is that guidelines will be actually useful for Stuff
Um only by accepting that leprosy transmission is a very complex phenomena Public Health institutions will be able to actually develop guidelines to control the disease and avoid further suffering to the people affected by thanks [Applause] uh what do we do now do we have