No shit - a new way to study diarrhoeal disease

Diarrhoeal disease is awful. I don't think I have to tell anyone that. 

According to the WHO:

Key facts

• Diarrhoeal disease is the second leading cause of death in children under five years old. It is both preventable and treatable.

• Diarrhoeal disease kills 1.5 million children every year.

• Globally, there are about two billion cases of diarrhoeal disease every year.

• Diarrhoeal disease mainly affects children under two years old.

• Diarrhoea is a leading cause of malnutrition in children under five years old.

Behold the wonder of the intestine

 The major causes of disease are infectious agents, namely the likes of noroviruses or rotaviruses. (very good wikipedia article here) But also bacteria, parasites and non microbial assaults. These pathogens enter our bodies via the oro-faecal route where they are able to infect the lining of our intestinal tract. Here they exert their biological effects and manipulate their resident tissue to aid in their replication and spread in the general population. In areas with poor sanitation this is why these diseases are such massive killers. They are acutely adapted to this way of life.

We are somewhat out of our grasp when dealing with - and studying - these organisms. In many cases (noroviruses especially) we cannot even grow the viruses in the lab. Many groups use mouse noroviruses and immunocompromised mice but of course this really isn't optimal for human viruses - especially when pathogenesis and drug development/vaccine studies come along.

So, if we cannot grow them how can we study them? Plus even if we did have the ability to culture them we lack accurate model systems of the human gut to even discover anything worthwhile about the way they grow.

But imagine if we had the ability to study human viruses in the lab, which had been themselves grown in the lab, with human tissue which had also been grown in the lab. 


Enter the human intestinal organoid.  

An intestinal organoid a la Spence et al 2011

An organoid is a structure that resembles an organ. It is not an 'organ' itself taken from a body - it's constructed in the lab to function as one. But organs are pretty complex and none less so than the gut. Its complexity resides in the physical (three dimensional) and biological (cell type/gene expression) planes. So how can we build this 'awesome' work of evolutionary engineering ourselves?

Easy.

You just copy mother nature.

Briefly, the NIH-approved embryonic stem cell line WA09 (originating from the WiCell Research Institute and obtained from the Baylor College of Medicine Human Embryonic Stem Cell Core) was cultured using feeder-free conditions. Stem cells were split at a high density, and, once they reached 85 to 90% confluence, cells were treated for 3 days with a series of differentiation media containing activin A to begin differentiation into definitive endoderm. Definitive endoderm was then treated for 2 to 5 days with growth factors Wnt3a and FGF4, leading to formation of hindgut spheroids. Once spheroids spontaneously detached from monolayers, they were collected, embedded into matrigel (BD Biosciences), and supplied with media supplemented with intestinal growth factors (Wnt3a, R-Spondin1, Noggin, and epidermal growth factor [EGF]; all supplied from R&D Systems). Spheroids matured into intestinal organoids over the course of ~1 to 2 months before they were used for experiments.

It started off like this

Anded up like this

Grown in this way you can 'easily' generate what effectively looks and feels like a human intestinal epithelium (these things also contain some underlying mesenhcymal tissue). They can even be kept alive for over 3 months and the stem cells from which they derive can be frozen and re-animated any time to set up more and more organoid cultures.  


So do these organoids allow viral replication? And can we use them to understand how these viruses infect and cause disease? Well the short answer is yes.


A U.S group from Baylor College of Medicine in Texas were able to cut open the spherical structures and infect them with rotaviruses, even clinical isolates of the virus (OA paper here). In this case the virus needed access to the inside of the structure, the part the resembles in insides of our gut and the place where the cell it likes to replicate in are found. These viruses bound to cells, got in and began replicating and generating new viral particles which could go on and initiate a whole new round of growth. This a whole lot better than using primary monkey kidney cells to isolate and grow the virus or using lab adapted strains.

Organoid structures could be infected with rotaviruses

This was only a preliminary observational study showing a proof of concept that this technique which had previously only been done using mouse stem cells, could work for humans and that they could be used for infection studies. What they didn't show was that rotavirus infection of the organoids bore any resemblance pathogenically to infection in humans. I'm sure this is the next step. What also would be of great use would be to see whether other non-culturable but important human pathogens could be grown this way - I'm thinking noroviruses. What is neat about this work is that we have the ability through recombinant gene vectors to knockdown or over-express any gene we want. The initial paper documented this in 2011. And this is being done with an NIH-approved stem cell line - imagine what could be done with healthy or 'diseased' stem cells.

One problem with this lies in the way they were infected. In order to access the inside of the spherical structure the organoid was cut open using a tungsten needle - an extremely sharp instrument. Who know what kind of effect this would have on nearby cells? But I guess without it this work could not be done and this was probably the safest way of doing so.


Another issue is - like the majority of other in vitro models - there is not immune system component to the structure. And of course the innate immune cells residing in tissues would have a wide ranging effect on the subsequent spread and infection kinetics of the virus. However, you could imagine that in the future it may not be too difficult to add these in to the system. 

Stacy R. Finkbeinera,, Xi-Lei Zenga,, Budi Utamaa,, Robert L. Atmara,b,, Noah F. Shroyerc,, & and Mary K. Estesa,b (2012). Stem Cell-Derived Human Intestinal Organoids as an Infection Model for Rotaviruses mBio, 3 (4) DOI: 10.1128/mBio.00159-12

#microtwjc 5(?) Microbiology of the built environment - the toilet edition

This weeks microbiology twitter journal club is:

Microbial Biogeography of Public Restroom Surfaces

Gilberto E. Flores¹, Scott T. Bates¹, Dan Knights², Christian L. Lauber¹, Jesse Stombaugh³, Rob Knight^3,4, Noah Fierer^1,5*

1 Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Computer Science, University of Colorado, Boulder, Colorado, United States of America, 3Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, United States of America, 4 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States of America, 5 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado, United States of America

have a look at the abstract here (emphasis my own):

We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla: Actinobacteria,Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: those found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with hands. On toilet surfaces, gut-associated taxa were more prevalent, suggesting fecal contamination of these surfaces. Floor surfaces were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associated bacteria, especially the Propionibacteriaceae, dominated surfaces routinely touched with our hands. Certain taxa were more common in female than in male restrooms as vagina-associated Lactobacillaceae were widely distributed in female restrooms, likely from urine contamination. Use of the SourceTracker algorithm confirmed many of our taxonomic observations as human skin was the primary source of bacteria on restroom surfaces. Overall, these results demonstrate that restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with clear linkages between communities on or in different body sites and those communities found on restroom surfaces. More generally, this work is relevant to the public health field as we show that human-associated microbes are commonly found on restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touching of surfaces. Furthermore, we demonstrate that we can use high-throughput analyses of bacterial communities to determine sources of bacteria on indoor surfaces, an approach which could be used to track pathogen transmission and test the efficacy of hygiene practices

Here's my thoughts of this (briefly) 

As you can see from this paper certain American restrooms harbour a massive diversity of bacterial species (only bacteria were looked at - I wonder what kind of viruses are here). The majority of which have a strong association with the human species; others come from soil or water. Most of all the diversity of bacteria in the restrooms come from our skin (no surprise there really). 

You might ask: "So what?", "Who cares?" or "Why bother?" 

Is it just because you had access to some fancy machine and 12 bathrooms?

I have to disagree with this viewpoint: This is only the beginning.

 In order to fully understand the microbiology of the built environment and how it effects the human population residing in it we have to start somewhere - and deep sequencing or bacterial diversity is a damn good place t start. (after all we spend the majority of our time in doors - and even when we're outside it will probably be in or in close proximity to major population centres like cities). 

If you don't believe have a look at this site.

This falls into a larger investigation looking into the microbial diversity found across different environments, such as the office (this groups follow on paper) or nursery. We can study 'healthy' environments and we can study 'unhealthy' environments. Potentially there are important microbiological differences between the two. By determining the baseline microbiome we could in theory begin to specifically alter its appearance to enhance the human experience of the build environment for health, environmental or economic reasons. 

What's the difference between one kid with a fever and one without?

A paper out last month goes a little way to answering a question I have had for a while: what are the major causes of fevers in children?

Imagine this: your child goes to the doctor, they have a fever so they might get given antibiotics or they might be sent home for bed rest because of a non-descript  'viral' infection. But what is that non-descript virus? and how is it causing feverish disease? Maybe if we knew exactly what was doing this we may have some chance to prevent it.


Fevers are an annoyance and in some cases can even be life threatening. Often times they are your bodies way of helping clear an infection. Your body will 'see' and detect infectious (and potentially dangerous) micro-organisms like viruses or bacteria and set off a chain reaction of chemical signals resulting in a rising temperature. But fevers may also be caused by a range of other ailments: arthritis, cancer or basically anything that causes inflammation. 

This paper in PLoS ONE suggests that we should delve into those children's viromes to see what is making them sick. Using next generation sequencing of nasopharyngeal swabs as well as plasma blood samples from febrile and non-febrile American children, they attempt to tease apart they causes of their illness. They also looked with PCR to detect viruses more specifically. This allowed them to essentially cover all viral bases in a comprehensive manner and hopefully detect what was truly in there. 


However, this approach is only based on looking for viral genetic sequences and no search for the actual virus was carried out. This means that those viruses detected could may never have infected the person, could have come directly from the environment or had been recently cleared by the immune system. WIthout virus isolation we cannot be sure to what extent a viral sequence is associated with a certain condition.

The viruses they found by PCR and deep sequencing.

In some cases this knee-jerk reaction to infection actually can damage you and can even result in seizure and death. Diagnosing the cause of fevers and treating early in thus a major concern and of the major issues of fevers is that they can be set off by a multitude of pathogens, the majority of which we have never really studied in much detail. Hence this group spending their time and money investigating it. 


Abstract:

Why did they do the work:

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF.


Methods:


We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. 

Comparing viruses seen in febrile versus afebrile kids. Spot the differences?

Results:


An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. 


Conclusions:


These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.

Points to consider:

As I said previously, the group only looked at viral sequences. No viruses were isolated or grown from these samples which makes it a little complicated in drawing conclusions based on this data. But it is extremely interesting and worthwhile and perhaps in some instances may give us a less biased view into the virome.

We all harbour viral sequences. Even the 'healthy' control populations when you look at their upper respiratory tract and circulation, indicating that the issue of fever versus no fever may be more complicated than expected. Maybe some people are much easily able to withstand disease progression from an infection.

They only looked at plasma and at the upper respiratory tract. Many other parts of the body could be sampled in the future to more comprehensively investigate how the virome of a particular is associated with any particular condition or disease. The digestive tract, urogenital tract or blood cells (not plasma) could and should be investigated.

Causation cannot be determined from this study. And they even say that this was not the aim of the investigation. To determine causation that a particular virus caused fever in kids one would have to fulfil Koch's postulates.

Fever could have been associated with bacterial exposure/infection yet this was not looked at here. Perhaps this may have been more obvious to the diagnosing clinician and antibiotics would have prescribed.

In conclusion, this is an interesting paper but it is in a sense quite preliminary. They have defined their methods and proven they can detect a wide range of viral sequences in clinical samples. But at the minute it is rather difficult to conclude anything else from this. I look forward to further characterisation of the viromes of multiple other tissue samples and even the characterisation of the viruses they identified. Perhaps they will represent entirely unknown disease causing viruses.



Kristine M. Wylie1*, Kathie A. Mihindukulasuriya1, Erica Sodergren1, George M. Weinstock1, Gregory A. Storch2 (2012). Sequence Analysis of the Human Virome in Febrile and Afebrile Children PLoS ONE, 7 (6) : 10.1371/journal.pone.0027735