Category Archives: The more you know

Notes from the Field: Expired Influenza Vaccines

In what will undoubtedly be ammo for the antivaxxer movement, the latest issue of Morbidity Mortality Weekly Report from the CDC reports the ongoing surveillance of vaccine administration for seasonal influenza is not perfect. The seasonal influenza vaccine comes in two forms: an inactivated virus formulation that is injected, and a live, attenuated virus formulation that is administered nasally. Both forms of the vaccines are generally widely available in late summer/early fall, and are recommended for the general population of the US for everyone over the age of 6 months. The inactivated vaccine has an expiration date of June of the following year, and is contraindicated for use at that time, mainly because of the lack of protection that it will offer to novel influenza strains that will have arisen by that point. The live attenuated vaccine on the other hand has an expiration date of about 18 weeks (4.5 months), and should be disposed of at that point even if the flu season is still going on. Since the flu season generally runs from November through March, it generally should not expire during this time, however if the vaccine is produced but not administered earlier in the season, stockpiles of expired vaccine may accumulate.

Epidemiologists from the CDC analyzed data from the national Vaccine Adverse Event Reporting System (VAERS), from 2007 through 2014. Of reports using live virus vaccines, approximately 18% of those reports indicated that expired vaccine had been administered to patients, and the vast majority of those reports did not document any adverse health events. The most likely outcome due to administration of any expired vaccine is a lack of protection against season flu. Consequently, revaccination with a valid dose is recommended to maintain protection against flu. The CDC recommends that all health care providers be aware of the significantly shorter shelf life of the live vaccine, and to be aware of return and replacement options from vaccine manufacturers.

Food allergies and our normal microorganisms

Peanuts_with_skinA news article that made the rounds through the popular press this week caught my eye: “Commensal bacteria protect against food allergen sensitization,” which appears in the early access section of the journal of the National Academy of Sciences. I have been a big fan of this type of research for a while now. The basic premise is this: our modern lifestyle has potentially begun to diminish the numbers and variety of microorganisms that live on our bodies in the absence of disease (the normal microbiota), and as a consequence, benefits that these benign organisms can confer to us are being lost. So far, loss of diversity of the normal microbiota have been correlated with a long list of ailments including potentially autism and cancer.

An opinion piece in this week’s Nature warns against drawing too many conclusions from these studies, and suggests that over reporting of some of them by the press reinforces the need to ensure that the public understand the distinction between “correlation” and “causation”–these concepts are frequently confused, and the distinction is sometimes not clear. Indeed, the editorial in Nature suggests that reporting of microbiome analysis and human disease should be tempered by asking 5 questions:

  • Can experiments detect differences that matter? Characterization of microbiomes is generally accomplished by sequencing very highly related genes, and this analysis may hide real differences.
  • Does the study show causation or just correlation? Many of the cases of a disease association with certain microorganisms may be the result of conditions in the body becoming favorable for the microbe, meaning the disease caused the microbes to alter.
  • What is the mechanism? Demonstrating causation is important, however without an explanation of how a change occurs, it is not sufficient.
  • How much do experiments reflect reality? Many of the putative effects of the microbiome on health involve germ free mice; that is mice that have been raised to have no normal microorganisms of their own, as this makes interpreting the effects somewhat easier. However, mice and humans are not the same, and the microorganisms that live on each are not the same.
  • Could anything else explain the results? Many things can cause disease, and other factors should be considered and tested.

With this in mind, I read the article on food allergies linked up at the top. The authors carried out the study to address the hypothesis that the normal microbiota of the gastrointestinal tract are able to guide adaptive immunity at this site. The intestinal tract of animals hosts an incredible variety of organisms in the absence of disease. The immune system needs to be non-responsive to these organisms, as well as to all of the food antigens that enter the digestive tract. Immune cells in lymphoid tissue along the digestive tract modulate signals between the microbiota and the epithelial barrier of the digestive tract, which helps to prevent an ongoing inflammatory response, and thereby promote a homeostatic relationship between the microbiota and the host.

The researchers first experiment was to treat neonatal wild-type mice with an antibiotic regimen prior to weaning to eliminate intestinal microbiota, then sensitized by gastric administration of Peanut Antigen (PN). Three weeks later, the mice were challenged with the antigen and allergic responses were measured a day later by collecting blood. Control mice had essentially undetectable levels of allergic responses, while antibiotic treated mice showed highly elevated levels of IgE. Analysis of the bacteria from feces of mice at the same time intervals also showed that the antibiotic treated mice had lowered levels of fecal bacteria, and greatly diminished diversity of fecal bacteria. Specifically, members of the prokayotic phyla Bacteriodetes and Firmicutes, present under normal conditions, were essentially absent in the antibiotic treated mice. These bacteria were replaced with members of Lactobacilli, a result consistent with another recent report examining changes in the microbiota of antibiotic fed mice. The results outlined above were achieved using outbred mice strains housed in pathogen-free, but not germ free conditions; therefore this study addresses one of the critiques above with the use of outbred mice.

This paper was also significant, in that the authors also propose a mechanism for how the immune modulation occurs. Recolonization of antibiotic fed mice with a group of Firmucutes from genus Clostridium (the major genus of the Firmucutes from normal mice), prevented the allergic response produced by peanuts. Dissection of the intestines from these animals indicated that specific T cells involved in adaptive immune regulation are more prevalent in Clostridia colonized mice. Additionally, mice colonized with Clostridia in comparison to germ free mice and control mice exhibited high levels of an immune cytokine Interleukin 22 (IL-22). The authors propose that IL-22 (induced by the presence of Clostridia) causes the intestinal epithelial barrier to be reinforced, reducing the permeability to dietary proteins. To address this possibility, they then measured the levels of food allergens in the bloodstream after intragastric gavage. Colonization by Clostridia resulted in significantly lower levels of these allergens in comparison to germ free mice, supporting this hypothesis.

The major conclusions of this paper support the important role of the benign normal microbiota in promoting health. Their model argues that tolerance to food antigens is aided by the presence of those antigens along with specific components of the normal microbiota. To translate this work to human therapies, the role of Clostridia needs to be confirmed in humans. Indeed, other work has shown that Clostridia species isolated from human feces do induce the same immune regulatory cells discussed above when transferred to germ free mice, suggesting that they may be playing similar roles in both species.

Brushing your teeth is still controversial

A pretty graph

All time BIO230 blog stats since mid-2011. The two peaks to the right are due to two days only, November 30 and December 1, 2013.

Thanksgiving Break is generally a quiet time around the BIO230 blog site, as the major visitors are spending time away from the Internet and Microbiology to spend time with their loved ones. Imagine my surprise to open my email over break and see that there were multiple comments to the blog! Student engagement! Discussion! Microbiology talk even when a grade isn’t on the line! However, when I scanned the comments in moderation, I didn’t recognize any of the names, and furthermore they were all for a posting that I had put up almost 3 years ago. What had happened was this: @joedevon who is tech writer and developer based in California came across my posting from 2011 describing the competing commensal relationships between different Streptococcus species in the human mouth.

In the article, I was trying to point out that the web of interactions between microbes is complex, but we could conceivably tweak the interactions in our favor to promote good oral health. @joedevon found my posting via a Google search, and posted the link to the web headline aggregator Hacker News. This lead to an approximately 1000-fold increase in the number of people visiting the blog. The previous best day was in November 2011 on a day that had both a lab report due and a bonus opportunity on the blog, and there were about 120 hits from students that day and I have never come close to that number a second time. November 30, 2013 had almost 12,000 people, several of whom left comments on the blog.

Two commenters remarked about the tenacious nature of biofilms, and the difficulty of removing them. From Craig:

There’s a perfectly good, and reasonablylow-tech, way to break up dental biofilms: using irrigators like Waterpik, or similar gizmos made by Panasonic, Phillips and others. These things are really needle-jet pressure washers that blast apart biofilms that toothbrushes or floss can’t touch, on even the most unexposed dental surfaces.

and from Clay:

Green Tea is the best thing you can do other than floss and brush and maybe use an H2O2 mouthrinse. Green Tea basically disolves the plaque, and does so very effectively indeed.

Biofilms are indeed difficult to remove, and the act of physically brushing does indeed work to break them apart.

Several commenters remarked on the hold that Big Dental has on Western Society, offering insights into the controversy that public health measures bring. From Transfire:

This has been worked out before at the university of florida. It has been possible to all but eliminate cavities for ten years, but $ talks, cures walk.

and these from  GogglesNinetynine and Smokes:

There is little evidence that consumption of fluoride increases tooth strength or promotes enamel growth. This is junk science that is forced on citizens because the “nanny” knows best.

…google truth about water flouridation.. it destroyes our teeth…fortunately there are companies now that started making toothpaste without flour in it…

I would point out to Smokes that gluten-free toothpaste is very important for our friends with Celiacs disease. Wait, I don’t think that’s what he meant. I’m sorry guys, but there is a phenomenal body of epidemiological data supporting that small amounts of fluoride promote dental health. The conspiracy argument suggesting that the money involved in prophylactic fluoridation campaigns is somehow lucrative just doesn’t hold water. Look, your average dentist will make far more money from extensive oral reconstruction than he or she does with a twice yearly polishing.


Biofilm (Photo credit: AJC1)

One commenter who passed my moderation test actually spoke to the topic which the original blog post was about; that is, is it feasible to tweak the complex interaction of microorganisms in the mouth to our advantage? Here is a link back to my conversation with Jonathan in the original post. His comment about the diffusion barrier that a biofilm presents is an interesting concept to think about. I think that small molecules might easily pass in and out of a biofilm, however larger molecules might have more of a problem.

This then shows a difficulty with one approach I put forward 3 years ago, where the use of an enzymatic mouthwash to dissociate biofilms that have formed requires that the enzymes have access to the biofilm material. If the biofilm represents a diffusion barrier for large molecules, the enzymes in the mouthwash would need to chew up the biofilm from the outside in, which might not be the most efficient method. Regardless, the tried and true methods of biofilm dissociation likely remain the best options for the foreseeable future.

BONUS: for those that have read down this far, list an organism (at least to the Genus level) that is part of the normal microbiota of the human oral cavity, and include a link to where you got that information. UPDATE! I have finished adding points into Blackboard, so I am calling time, thank you for playing!

Epidemiology Cracking Criminal Cases

New Picture (7)Courtney Gladstone (12 Micro) found the discussion of epidemiology very illuminating, and found this article from Scientific American. Criminals should be scared to know that Courtney is on the case! Here is what she has to say about it:

Hearing cases about outbreaks of different viruses or diseases in hospitals or other health care facilities is not completely uncommon, it happens. Most of the time these outbreaks are completely accidental or careless mistakes by health care providers. However, sometimes these cases of outbreaks are criminal. That’s what happened in a small town in New Hampshire, when four cases of Hepatitis C appeared to be linked in some kind of way. That’s where forensic epidemiology comes in and saves the day.

When looking at the strains and genetic codes of the Hepatitis C in these four patients, the genetic codes were almost exactly the same. Jose Montero, an epidemiologist that worked on this case said that this virus mutates so rapidly that it just had to have come from the same person. The first step to solving this problem was figuring out the similarities of all of these people infected. These people had all been to the Exeter Hospital’s Cardiac Catheterization Laboratory, three of them being patients and one of them being employed at this location. After constructing an evolutionary tree of these viruses and sequencing genomes it all went back to the one who was an employee at this lab, David Kwiatkowski, as the beginning piece of this horrible puzzle. Investigators believe that he injected himself with one of the needles and uses it on these patients without any sterilization. Kwiatkowski later plead guilty before the trial could reach an actual jury.

This however isn’t the only happening of this event. Fernando González Candelas of the University of Valencia helped to retrace an even larger outbreak in Spain, where an anesthesiologist was suspected of spreading HCV to hundreds of patients.  This case was similar to the one mentioned previously in that the genetic codes were all similar to the one of the anesthesiologists strain of HCV (Hepatitis C). What’s even more amazing is that with this epidemiology Candelas and his colleagues were also able to estimate when a person became infected to confirm that the infection occurred while that person was under the doctor’s care. The scary thing about this virus is that people can have it and not even know it and spread it without their knowledge, in this case infecting two hundred and seventy-five people.

Forensic epidemiology is extremely important in everyday life. Although it may seem as though in class this is all just information to just get tested on and move on, this kind of information can help aid in stopping criminal acts. Who would have thought things you learn in microbiology could help solve a criminal case!

Salmonella from breast milk!

Via the New York Times, a story to add to the list of things that have led to Salmonella outbreaks recently. A study published this week in the journal Pediatrics details a growing problem with breast milk sold or donated via websites that has been found to be contaminated with high levels of potentially pathogenic microorganisms. Researchers obtained 101 samples from a popular Internet milk-sharing website, as well as 20 samples of unpasteurized breast milk which had been donated to a more traditional local milk bank.  Most of the Internet samples (74%) were contaminated with Gram negative bacteria, whereas 35% of the banked milk were contaminated. Additionally, contaminated Internet samples had at least 10-fold higher levels of bacteria in comparison to the banked samples.  Internet samples were also contaminated with cytomegalovirus 20% of the time.

The authors conclude that the high level of contamination reflects the overall poor attention to “collection, storage, and shipping practices” with very fundamental lapses in aseptic technique. Although the websites typically prominently display collection criteria, because there is no oversight in the process, it is unclear what level of compliance is actually followed. Local milk bank resources on the other hand have strict guidelines, and the milk is pasteurized prior to distribution. Some advocates of the websites maintain that pasteurization itself diminishes the benefits of expressed breast milk relative to infant formula, however there doesn’t appear to be any concrete evidence that such benefits exist to use unpasteurized milk when pasteurization is easily accomplished. Comments in the Times article from Kim Updegrove, president of the milk-bank association, further underscore issues with these unregulated Internet sites, including the possibility that recipients might be getting cow’s milk or formula from an unknown Internet source.

Seasonal flu vaccine and Guillain-Barre: no correlation!


I do not smell BS here, but I do smell much Science!

I spotted this article in the Health Section of the New York Times; an extensive retrospective study published in the journal Clinical Infectious Diseases has found no correlation between receiving the seasonal influenza vaccine and developing the serious neurological condition Guillain-Barré Syndrome (GBS). This finding gave me great joy, and also gives me an opportunity to link back to one of my favorite Rage Stroke®-inducing BIO230 postings. The possibility for correlation arose from the 1976 Swine flu vaccine, in which the Centers for Disease Control and Prevention reported a slightly increased risk for developing GBS following receiving that vaccine, with the risk being approximately 1 case of GBS per 100,000 vaccine doses. This measurable increase led to a moratorium and reformulation of the vaccine.

An extensive investigation by the Institutes of Medicine (IOM) confirmed the increased risk of developing GBS with the 1976 influenza vaccine, and although several theories were put forth to explain the potential correlation, the cause remains unclear. One possibility hypothesized in the IOM study linked above suggests that the clear association between developing GBS and infection with the bacterium Campylobacter jejuni might be to blame. Campylobacter is an ubiquitously distributed organism, however it does infect chickens, and eggs are used to produce the influenza virus used to produce the vaccine. The increased incidence of GBS with that year’s vaccine therefore might have been due to contaminating Campylobacter antigens present in the killed vaccine preparation.

The study described in the New York Times article recounts an extensive retrospective analysis of patients from a California health care system, and data examined covered more than 30 million person-years worth of medical records. In that data set, 415 cases of Guillain-Barré were observed for a total annual incidence of approximately 1 in 90,000 people in the population, a number that is very much in line with what is reported by the CDC. Out of those 415 confirmed cases, 25 of them had a reported influenza vaccine in the 6 weeks prior to developing GBS symptoms. Most of the cohort had received a flu vaccine at some point in their medical history, but had additionally had a large variety of other vaccines. The researchers recognize that the very limited number of GBS cases in relation to the large number of records examined limits the statistical power of their analysis, however no apparent correlation between first receiving a seasonal influenza vaccine and then developing GBS could be demonstrated.  The researchers further affirm that during the 1976 outbreak there was a causal link between the swine flu vaccine and developing GBS, but as vaccine formulations have been modified, no further link exists.  The much more likely culprit causing Guillain-Barré Syndrome is an underlying infection, most likely due to Campylobacter, which can be easily prevented by properly cooking food and washing your hands.

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