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How many bacteria/viruses are actually removed by the body’s natural defenses while we are sick?

March 10, 2015 Leave a comment

The answer to this question is completely dependent on the context of infection. For instance, when you brush your teeth, you introduce bacteria from your mouth into your blood stream and become temporarily bacteremic. Fortunately, the immune system clears these bacteria quickly. In this daily event, thousands of bacteria enter the bloodstream. After brushing, bacteria in the blood were below 104 cfu/ml, as estimated by PCR, and exact numbers are nearly impossible to quantify (Lockhart et al. 2008).

In contrast,  Ebolavirus replicates to ~107 – 108 virions/ml in the blood of non-human primates (Bente et al. 2009). The average person has ~5 liters of blood. Very conservatively, an Ebola survivor will have cleared ~5 x 1010 virions from just the blood. That’s 50 billion virions.

Keep in mind that this is also just a snapshot of a moment during infection. Viruses are constantly propagating, and a single virally infected cell can produce thousands of viral particles in a single day. It’s possible that over the course of a 2 week infection, 1011 – 1012 virions may be produced and cleared (it’s probably much higher). While this number is staggering, it somehow pales in comparison to the magnitude of the U.S. debt, which currently stands at ~$18 trillion, or $1.8 x 1013.

Geisbert et al. 2004 Nat. Med.

Geisbert et al. 2004 Nat. Med.

I also estimated the rough number of virions from the liver and spleen, two organs with significant ebola titer, and the total is nowhere near the magnitude of virions in the blood.* Additionally, ebola virus is being shed through mucosa and other bodily fluids. People generate ~ 4 liters of mucus daily in the gastrointestinal tract. I could not find an accurate estimate of Ebola viral titers in the mucus, but it’s unlikely to be near the levels of that in the blood. Granted, mucus could still contribute significantly to viral titer. There’s also the possibility that other organs harbor high titers of ebola virus, especially the gut.

*Approximately 104 – 105 virions/gram were detected in the liver and spleen using a mouse model for Ebola (Bray et al. 1996). Given that the average mass of a human liver is 1.5kg and a spleen is 140g (Molina et al. 2012), and using conservative estimates, about 1.6 x 107 virions from the spleen and liver alone, over 1000 times less than that from the blood.

What’s a Superbug anyways?

March 5, 2015 Leave a comment

About 23,000 deaths due to approximately 2 million infections by antibiotic resistant bacteria occur each year in the US. Additionally, failed antibiotic treatments result in an estimated $20 billion spent on health care. The bacteria that cause these deaths have been called “superbugs.” Most recently, 7 deaths from “superbugs” at a UCLA hospital were spread from patient to patient through endoscopes that were not yet FDA approved. The media has been using the term “superbug” for years now, but what does it even mean?

In the mid 90s, the term “superbug” was used sporadically in peer reviewed journals. Notably, these articles highlighted emerging antibiotic resistance in strains of E. coli, Streptococcus, Enterococci, and importantly Staphylococcus. Methicillin resistant Staphylococcus aureus (MRSA) emerged only one year after the first use of methicillin as an antibiotic in 1959. MRSA is now one of the most widespread pathogens and common causes of hospital acquired infection. As a result of methicillin resistance, one of the antibiotics most widely used to treat MRSA in the 90s was vancomycin.

In 2002 in Michigan, one of the first reports of vancomycin resistant MRSA, now dubbed VRSA, came to light. An article in Nature used to term “Superbug” to describe the vancomycin resistant organisms. Now superbug is commonly used by the media to describe any variety of antibiotic resistant bacteria. Unfortunately “superbug” is not descriptive, and has been used to headline deaths by not only MRSA, but also antibiotic resistant strains of Klebsiella pneumoniae, Clostridium difficile, Acinetobacter baumannii, Mycobacterium tuberculosis, to list a few of the dozens.

antibioticmechanismsThere are several classes of antibiotics, each which exert their antimicrobial action in a different way. Similarly there are several different mechanisms by which antibiotic resistance occurs. For example, aminoglycoside antibiotics are able to bind to and block the bacterial ribosome, which effectively inhibits or alters bacterial translation, resulting in cell death. Some bacteria have acquired genes that encode proteins that can bind to and modify these aminoglycoside antibiotics. Modification of the aminoglycoside renders it inert, and unable to kill the cell. Several classes of antibiotics are shown in the figure above, along with a few mechanisms of resistance. “Superbug” doesn’t describe the type of bacterial infection, nor does it inform about the mechanism of resistance.

Scannell et al. 2012 Nat Rev Drug Dev

Scannell et al. 2012 Nat Rev Drug Dev

The problem

Only 12 FDA approved antibiotics have been developed since 2000. In the 90s, 20 antibiotics were FDA approved, and 43 in the 80s. The steady decrease in antibiotic development is a result of several compounding factors.

1) A new drug takes an estimated 10 years to develop from scratch. Most drugs fail at some point between initial discovery, in vitro testing, animal testing, and clinical trials.

2) Drugs are expensive to develop. If you want a profit you make a drug that people take for life, not week like most antibiotics. This ties into point 3.

3) Antibiotic resistance is often detected within years of its initial use in the clinic. Therefore the time an antibiotic is actually relevant and useful is relatively low compared to drugs that treat arthritis or cardiovascular disease.

4) The low hanging fruit have been harvested, and nobody wants to pioneer the more difficult work. A majority of the antibiotic research presented at conferences, such as ICAAC, deal with betalactamases. The first antibiotic to be discovered, penicillin, is a betalactamase antibiotic. Dozens of investigators flocked to study the betalactamases after penicillin’s discovery. Graduate students and trainees from those labs then went to start their own labs, doing research on betalactamases. And now 50 years down the line, despite having a dozen classes of antibiotics, a majority of the antibiotic-focused academic labs study betalactamases. It’s easy to study betalactamases because it’s what we’ve been doing for half a decade. Moving forward is easier when the foundation is already set.

The solution

A recent article in Wired emphasized the need for more spending in the field of antibiotics. Only 1.2% of all funding from the NIH goes toward antibiotic resistance. The NIH budget has remained stagnant in the last decade, with a whopping 0.5% increase for 2015 to a grand total of ~$30 billion. To put this in perspective: The total federal budget in 2014 was ~$3.5 trillion. We don’t just need more money going toward antibiotics. We need more money going toward research in general.

Measles: Thinning the Herd

January 22, 2015 Leave a comment

CDC Measles51 confirmed cases of measles. Really? Measles? John Franklin Enders first isolated measlesvirus in 1954, and immediately began work to develop a cure. In 1960, Enders began to test his measles vaccine, and a year later he announced that the vaccine was 100% effective.

Now, more than half a century later, we have a problem. There hasn’t been a failure with the vaccine, or in the scientific process. The measles vaccine is still ~100% effective. We continue to dive deeper into molecular mechanisms of disease and come up with clever cures. A paper published just two days ago demonstrates stem cell therapy as a treatment for multiple sclerosis.  However, we do have a social problem that unfortunately bleeds into global health.

It’s interesting that a lot of diseases have been well characterized and would not be an issue if not for social dysfunction. Take polio for example. The polio vaccine is extremely effective. There are enough doses of polio vaccine to go around and in fact the WHO actively sends teams of vaccinators to the last three countries where polio is endemic: Pakistan, Nigeria, and Afghanistan. Despite vaccine efficacy, there are uneducated radicals opposed to vaccination, and often these groups are violently hostile. Efforts to eradicate polio have been undermined, and it’s because of social problems. In the U.S., we may not have violent extremists opposed to vaccination, but we do have major social issues.

herd-immunity1. We take for granted herd immunity (or ignore it completely).

People hear about the 0.01% chance of adverse reaction to a vaccine, in contrast to the low odds of contracting measles in the U.S., and choose not to vaccinate their children. As far as I’m aware, there is no scientific fix for ignorance. The only real solution is education.

2. Ignorance often trumps scientific evidence.

Let’s start with Andrew Wakefield. In the late 90’s, this guy published a fraudulent paper that drew a link between vaccines, autism, and gastrointestinal disease. The paper was disproven, and after an investigation, many signs of misconduct came to light. Sure, fraud happens, and it would have been okay if not for what happened next.

Normally the conclusions of a disproven paper are disregarded. But the torch had already been lit and the anti-vaccine movement had begun. Jenny McCarthy used her public position to advocate the anti-vaccine movement, claiming her child developed autism due to vaccines. People empathize with anecdotes. (Please allow this brief interruption for an introduction to the Jenny McCarthy Body Count: Deaths attributable to the anti-vaccine movement) The torch is now a wildfire. Again, we have an example of social dysfunction that could be effectively fixed with education.

Sometimes it takes a disaster to develop a fix. Unfortunately, this problem can’t be solved with any technology or scientific advancement. Alas, social science may be relevant, thanks to measles.

How Hantavirus Hurts

December 19, 2014 Leave a comment
Click me to get to the reference page

Annual Hantavirus cases. Blue bars are # of cases. Black line is case fatality rate. CDC

There have been 6 confirmed cases of hantavirus pulmonary syndrome, and 3 deaths caused by Hantavirus in New Mexico this year. Hantavirus pulmonary syndrome is characterized as severe flu-like symptoms during the early stages of disease, which further progress into shortness of breath and accumulation of fluid in the lungs. Between 11 and 48 cases of hantavirus pulmonary syndrome occur annually in the US. Despite the low frequency of Hantavirus infections in the US, the case fatality rate of hantavirus is roughly 40%. Hantavirus is endemic in most countries, and circulates in wild rodents, which is then transmitted to humans through contact with rodent feces. Transmission usually occurs after breathing in aerosolized feces. Interestingly, there has only been one reported case of person-to-person transmission of Hantavirus in South America. Evidence suggests that person-to-person transmission does not occur with Hantavirus in the US.

There are several species of Hantavirus, of which the Andes virus is the most well characterized and prevalent in South America. In the US, an isolate of Hantavirus from the Four Corners region was cleverly named Sin Nombre virus, which in spanish translates to “No Name virus.” In 1993, an outbreak of Sin Nombre Hantavirus in New Mexico resulted in 24 confirmed infections, and 12 deaths. Hantaviruses belong to a family of viruses called the Bunyaviridae that are segmented single-stranded negative-sense RNA enveloped viruses.

Click me for a link to the paper

Hantavirus replication. Vaheri et al. 2013

The genome of Hantaviruses consists of three segments of single-stranded RNA appropriately named the S, M and L segments for their respective lengths. The genome encodes at least four proteins, the nucleocapsid protein, two envelope glycoproteins, and the RNA-dependent RNA polymerase that is required for replication of the RNA genome. Hantaviruses are able to enter cells via binding to host cell B2-integrin proteins that are normally responsible for cell-to-cell adhesion. It is curious that while Hantaviruses are able to replicate in lung epithelial cells similar to influenza, Hantavirus infection alone does not kill these epithelial cells. This brings up many questions regarding Hantavirus pathogenesis, which is somewhat addressed in a recent paper by Braun et al.

A subset of immune cells called Natural Killer (NK) cells are important for recognition and removal of virus-infected cells through the production of enzymes that kill infected host cells. NK cells are part of the innate immune response that are activated during early infection, and have the capacity to recognize when cells are infected through binding specific cell surface proteins. More specifically, NK cells can distinguish between normal healthy conditions and aberrant expression of these cell surface proteins that signal to inhibit or activate NK cell functions. Additionally, NK cells can be activated by interferon or other stimulating cytokines such as IL-12, 15, and 18.

Infection with Hantavirus leads to increased numbers of NK cells, which makes sense in the context of infection. Braun et al. observed that this population of NK cells is also activated, expressing increased levels of CD69, an activation marker, in peripheral circulating NK cells. In addition, granzyme and perforin, two effector molecules of NK cells that are released to kill cells were also upregulated. This indicates that NK cells proliferate and are activated in the context of Hantavirus infection.

Importantly, NK cells are unable to respond to Hantavirus alone, and Hantavirus is also unable to infect NK cells. In contrast, NK cells are able to recognize Hantavirus-infected epithelial cells that respond to Hantavirus infection by producing IL-15. However, the NK cells that are activated by Hantavirus-infected cells are unable to kill Hantavirus-infected cells and are more efficient at killing uninfected cells.It has also been reported that the Hantavirus NP protein can inhibit the activity of granzyme, thus blocking death of Hantavirus-infected cells. This strange effect may partially explain Hantavirus pathology.

Lymphocytes

Diversity of lymphocytes and their development. Janeway’s Immunobiology

Normally, activated NK cells are inhibited by healthy cells due to regular levels of cell surface proteins that tell the NK cell to deactivate. However, NK cell activation by Hantavirus infection leads to nonspecific killing of host cells. This cell death may contribute to vascular permeablization that occurs during Hantavirus infection. While this model may explain some Hantavirus pathogenesis, it is highly simplified as NK cells are a small subset of the immune system. It is also improbable that NK cells are the sole mediator of Hantavirus pathogenesis.

There are currently no approved antivirals or vaccines that provide protection against Hantavirus infection in the US. While the translational aspects of virology are important, it is often the basic science that leads to breakthroughs. This paper describing the interactions between NK cells and Hantavirus doesn’t directly provide a vaccine. However, like all basic research, it betters our understanding of Hantavirus pathogenesis which is necessary to make advances in potential Hantavirus therapies. I would also argue that we learn more through basic research than through applied research although the effects of basic research may take longer to materialize.

Categories: Science Tags: ,

Your odds of contracting Ebola vs. Chikungunya

August 12, 2014 1 comment

Sure, Ebola is scary. However, the issue with Ebola is primarily a social one where misinformation, a lack of infrastructure, and general government mistrust have thrown the epidemic out of control (I suggest reading a historical perspective on Ebola response and prevention). Unless you’ve recently traveled to west Africa to eat bushmeat or treat Ebola patients, there is little cause for concern. You are very unlikely to contract Ebola, but recently your odds of getting Chikungunya have significantly increased.

On June 17, 2014, the CDC announced the first case of Chikungunya acquired in the continental US. Previously, Chikungunya in the US had only been identified in travelers coming from the Caribbean where over half a million people have contracted Chikungunya (Caribbean Chikungunya Cases Climb 8%, top 500,000). In late 2013, Chikungunya was introduced to the Caribbean from Africa or Asia, where the disease has been endemic since its discovery in the 1950s. This rapid spread of Chikungunya is particularly alarming, and has been heavily influenced by the increase in international travel.

They're gonna have to change this map.

They’re gonna have to update this map.

What’s Chikungunya?
Chikungunya is a disease caused by chikungunya virus, which is transmitted by Aedes mosquitos. Infection by Chikungunya may result in a fever, rash, insomnia, headache, and joint pain. While Chikungunya is rarely fatal, it is incredibly debilitating, causing symptoms for weeks to months. Chikungunya also presents with other symptoms such as leg swelling and ocular inflammation. Chikungunya pathology is not well understood, and as a result antivirals and vaccines have not been developed.

800px-Aedes_aegypti_feeding

Avoid Aedes mosquitos.

Ebola is not likely to cause a pandemic.
Ebola is one of the deadliest diseases on the planet, but with the proper infrastructure can be controlled. Additionally, the likely reservoir of Ebola is in bats, which are often eaten as bushmeat in villages in Africa (Africans still eating bushmeat despite Ebola). 1. Here in the US we rarely come in contact with or eat bats. 2. Even if we were more bat oriented, the Ebola reservoir is likely specific to animals in Africa. 3. Education plays a major role in the spread of the disease. African tribes are notorious for rejecting help from Doctors without Borders and the like (Superstitions play a role). 4. Ebola transmission requires contact with bodily fluid such as blood, vomit, or diarrhea. It is not a respiratory infection and close contact with a patient is unlikely to cause transmission.

In contrast, Chikungunya transmission is nearly impossible to avoid. Aedes albopictus and Aedes aegypti, reservoirs of Chikungunya virus, are invasive species of Asian tiger mosquito that are now firmly seated in the US. Importantly, on August 7th, Chikungunya was found in mosquitos in the US. The West African outbreak of Ebola is certainly devastating, but Chikungunya is far more widespread and is transmitted with relative ease. So while it is important to be mindful of the current state of Ebola, overlooking other potential outbreaks may have devastating consequences.

Prokaryotic pow-wows and tackling antibiotic resistance

August 13, 2013 Leave a comment

Drug development cost: ForbesAntibiotic resistance is and will continue to be a key issue in the years to come. Many pathogens have been dubbed “super-bugs” as a result of their ability to persist in the presence of multiple antibiotics. Some bacterial pathogens, like totally drug-resistant tuberculosis, MRSA, Klebsiella pneumoniae, and Acinetobacter baumannii are of particular interest because of their clinical prevalence and ability to quickly acquire drug resistance.

One primary concern with antibiotic resistance is the lack of new drug development. The median cost of developing a new drug is $4.2 billion dollars (forbes article), and without proper antibiotic stewardship, a drug may only be useful for 2 or 3 years. That’s not much return on investment. Big pharmaceuticals will not dump money into a drug without the prospect of long term revenue.

However, there are novel ways to tackle the issue of antibiotic resistance without the billion bucks. The most heavily researched mechanism of antibiotic resistance is mediated by a class of enzymes called betalactamases. 80% of the research in the field of antibiotic resistance has been focused on betalactams, and we seem to have forgotten that there are several other classes of antibiotics that we can play with. I discussed the problem of such heavy focus on one antibiotic here: Gripes with antibiotic resistance research.

Bacteria have gained resistance to every class of antibiotics including aminoglycosides, tetracyclines, quinolones, and polymyxins. Each method of resistance is typically granted by an enzyme, and these enzymes should be new drug targets. If we can inhibit an enzyme that grants resistance, then by coupling the new drug with an old antibiotic, we can reutilize obsolete antibiotics.

Another method that has recently gained traction is the inhibition of virulence factors. Most bacteria are not virulent, and incapable of infecting humans. Remember, the human body is an incredibly harsh environment for a microbe. However, those that are virulent typically carry genetic determinants that give them the ability to colonize a host. For example, Escherichia coli is a commensal bacteria of the human gut, but some strains have gained the ability to produce toxins or adhere to particular mucosal surfaces, making these strains virulent. The development of new drugs to inhibit virulence may be able to prevent disease. I briefly described one such method in Dealing with antibiotic resistance through anti-virulence factors, where inhibition of a toxin leads to survival of mice and protection from disease.

One process that can be considered a virulence factor is quorum sensing, a mechanism for bacteria to detect population density. Bacteria excrete peptides that they also have receptors for, and when the concentration of bacteria increases, so too does the excreted peptides. When the population reaches a particular concentration, gene expression changes and may induce virulence factors. Pseudomonas aeruginosa and A. baumannii are opportunistic human pathogens, and through quorum sensing are able to coordinate the formation of biofilms, a virulence trait. Inhibition of the quorum sensing may inhibit the progression of disease, and a method for discovering drugs that can inhibit this system is described in this paper published this month in PNAS: A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases.

The growing issue of antibiotic resistance is critical to global health, but the problem is still just a pea under a mattress and not enough attention has turned its way. Translational research and drug development are absolutely integral to advancements in the biomedical sciences, but basic research will give insight to novel drug targets and molecular mechanisms that we can utilize to cure disease.  The pre-antibiotic era and the days where a common strep-throat or UTI would kill you are gone. However, without progress in research, stewardship, and activism, we could certainly regress.

The unfounded fears of Influenza research

August 10, 2013 Leave a comment

Tweety has the fluAs if the H5N1 transmission papers weren’t talked about enough, now the likelihood of similar research with H7N9 has lead to more unfounded fears. The first report of a possible human to human transmission of H7N9 came out a few days ago, and rather than instill fear that scientists will “create a deadly virus just to prove they can,” this possibility of an epidemic should highlight the importance of studying such a pathogen.

This has been harped on by every scientist I know, but it apparently still needs to be said. Even if we tried to engineer a more virulent and transmissible virus, we would never be able to out-craft nature. Natural selection will take place, and an animal-to-human or human-to-human transmissible virus will eventually pass into humans, and it is our job to get ahead of it and find out where it is going. There isn’t a single pathogen we could engineer that nature wouldn’t be able to do better.

Maybe more importantly, nature does not stop playing the game even if we decide to quit. It is Jumanji, and we are the kids playing the game. Even if we want to leave all the horrors of the game behind, we can’t. We are but pawns in nature’s game of chess.

The value of understanding mechanisms behind a potential pandemic virus is immeasurable. In the highly possible scenario, where a few thousand people come down with serious illness caused by influenza H7N9, the worst possible thing we could say is “well you’re screwed because we stopped looking for a cure.” Even in the most extreme circumstance, where a lab strain is somehow released that is highly transmissible and virulent, the best way to combat the threat is to do more research. 

Biosecurity is something every lab takes very seriously. As researchers, we take precautions to prevent both samples from getting contaminated, as well as ourselves from getting infected. Scientists are humans, too. We would rather not get seriously ill, and thus we have measures to prevent exposure to pathogens.

The fear mongering will never end because layman-shocking sensationalized headlines sell. But like all trolls, they get off on your attention. So give them none, and leave the scientists and the ferrets to save your lives. After all, “it is a curious feature of our existence that we come from a planet that is very good at promoting life but even better at extinguishing it.” – Bill Bryson in A Short History of Nearly Everything.

Categories: Science Tags: , , ,