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Meet the Guinea Worm

Screen Shot 2016-03-14 at 10.48.20 PM.pngGuinea worm disease is caused by a parasitic worm found in stagnant waters of Africa. It has been known since early recorded history with possible mentions in the Bible and a definite reference (along with a treatment method for removing the worm that is still used today) in the Ebers Papyrus, an ancient scroll written around 1500B.C.
Presently, only four countries, Chad, Ethiopia, Mali and South Sudan have reported cases of the disease. The worm, Dracunculus medinesis infects its (human) host through the drinking of unfiltered water inhabited by the larval forms that have been eaten by “water fleas.” When a person drinks water containing these water fleas, the larva are released from their insect host while in the stomach and burrow through the digestive tract into the body cavity where they grow into adult worms. After fertilization, male worms die in the host, but female worms can grow up to 2-3 feet long.

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A section of the Ebers Papyrus

The insidious nature of the beast is the way that it manipulates the host (human) when it is ready to release larvae. At this time, the worm will burrow to the surface of the skin (usually on the foot) where it will cause a blister. When the blister erupts, it causes a painful burning sensation that is somewhat alleviated when immersed in water.

Whenever the blistered area is immersed, the worm will eject a milky liquid containing millions of larvae into the water to repeat the life cycle. During this time, the pain can be disabling and the blisters are easily infected with bacteria.
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As the worm emerges, it can be captured and wound around a twig, etc. Pulling the worm will result in a break that worsens the condition, but if attended to, the worm can be removed a bit at a time as it emerges from the blister over the course of days to weeks.
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Since the 1980s eradication of the Guinea Worm has been aggressively pursued by a number of organizations, most notably the Carter Center, founded by President Jimmy Carter. The Carter Center has coordinated the efforts of the Nation Ministries of Health in affected countries, with the World Health Organization, The US Centers for Disease Control and Prevention, and UNICEF. Together, these organizations provide education to at risk populations, water filters, and pumps to obtain uninfected groundwater.
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These efforts have reduced the number of Guinea Worm Disease cases from 3.5 million in 17 countries in1986 to just 22 in four countries in 2015 (provisional total). Importantly, humans are the principal host of Guinea Worms, therefore, if all cases of infection can be eliminated for just one year, this should lead to complete eradication of the organism.

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Posted by on March 14, 2016 in Uncategorized

 

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HCV, briefly

Viral Hepatitis comes in a number of flavors, named HAV, HBV, HCV, HDV, and HEV (not to mention any subtypes). HCV, identified as recently as 1990, is a serious form of Hepatitis causing cirrhosis of the liver, chronic infection, and often hepatocellular cancer. Prior to 1990, the most common way to become infected was through transfusion with contaminated blood. However, after identifying the virus, tests became available to prevent this form of passage, leaving the primary mode of transmission being sharing of needles between IV drug users and sexual contact.
Unlike other viruses (HAV), few people ever clear HCV and, instead, become chronically ill. This may, in part, be due to the inability of the body to generate protective, neutralizing antibodies. Those antibodies that are produced are mostly usable only as markers of disease. Symptoms of disease include fatigue, nausea, vomiting, loss of appetite, abdominal pain, jaundice, dark urine, and clay colored feces. The CDC definition of a case is: (sorry this isn’t clearer)

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Serum alanine aminotransferase (ALT) levels greater than 400 IU/L indicate hepatocellular damage. This enzyme is normally found only in
liver cells, but is released into the blood when these cells are injured. Normal ALT should not exceed 60 IU/L, providing a fairly clear altmeasure of cell injury.This can be seen clearly below as serum levels of ALT spike with symptoms of disease and then return (however not down to normal, ‘healthy’ amounts) to lower levels following resolution of symptoms.

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There is no vaccine against HCV, so the best way to avoid it is to avoid contact with blood or other bodily fluids that may be contaminated.


Don’t forget to check out my comments on the novel ‘Rosemary’s Baby’ on my other blog. I highly recommend this book to anyone who enjoys thrillers / light horror (a la Stephen King).

 
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Posted by on April 12, 2015 in Uncategorized

 

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Congressional hearing on Vaccination

schuchatIn the wake of several months of nation wide fear of ebola,  a new outbreak strikes home. One can only wonder whether the timing of these events will have a lasting effect on the country.

as a side note, it’s interesting to hear Dr Schuchat, the Director of the National Center for Immunization and Respiratory Diseases at the CDC, bring up the number of annual deaths attributed to influenza in the US as between 5-30,000. Because of the way deaths are reported, it is actually tricky to get a very accurate number for this, but a CDC study attempting to do so sets the range as falling between two recent extremes of  “3,349 in 1986–87 to 48,614 in 2003–04.”  For perspective, the 2014 / 15 outbreak of ebola in Africa has claimed just over 9,000 lives.

Despite the high annual mortality of flu, only about 40% of American adults get vaccinated each year.

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Measles has a track record of much higher mortality rates than flu, and is much more contagious. The WHO warns, “[t]he highly contagious virus is spread by coughing and sneezing, close personal contact or direct contact with infected nasal or throat secretions.

The virus remains active and contagious in the air or on infected surfaces for up to 2 hours.”

In 2004, Perry and Halsey summarized what we, as a population have forgotten, “Before the introduction of measles vaccines, measles virus infected 95%–98% of children by age 18 years, and measles was considered an inevitable rite of passage.”

Measles is associated with a number of complications, including pneumonia (either directly as a result of measles or from another agent) which is associated with the majority of deaths attributed to the disease. Over the years, as medical interventions have improved, the number of measles-associated fatalities has dropped from “One hundred years ago in Scotland, the measles case-fatality rate was 30–40 deaths per 1000 cases. In the United States, mortality from measles decreased from 25 per 1000 reported cases in 1912 to 1 per 1000 reported cases in 1962.”(see figure below) Nevertheless, it remains a dangerous disease capable of causing a number of complications and death. (all those deaths from flu mentioned above, flu kills only about 1.4 to 16.7 deaths per 100,000 persons.)

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Perry and Halsey conclude:

Measles vaccination is one of the most cost-effective health interventions ever developed. Without the vaccine, 5 million children would die each year from measles-assuming an estimated case-fatality rate of 2%–3%. Without measles vaccination, the costs of caring for those with measles in the United States would be ~$2.2 billion annually, and the indirect costs would be an additional $1.6 billion. Each dollar spent on measles vaccine saves $12–$ 17 in direct and indirect costs.

With this in mind, here is the full video broadcast of CSPAN’s coverage of the the hearing on childhood vaccination:

http://www.c-span.org/video/?324253-1/hearing-childhood-vaccination

 
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Posted by on February 11, 2015 in Uncategorized

 

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Virus, Vaccine and Passive Antibody Therapy

The immune system is a many-layered construction that protects the body through barrier defences, additional non-specific responses including phagocytosis and chemokines, an antibody-mediated humoral response capable of neutralizing viral particles, and a cellular response for eliminating infected cells.

Ebola: Disease and Response

mapEbola is a viral disease first identified during a first appeared in 1976 in two simultaneous outbreaks, one in Nzara, Sudan, and the other in Yambuku, Democratic Republic of Congo.  It is reasonable to suspect that Ebola has infected humans prior to this time without being identified specifically. This is a reasonable assertion because, like the first, all subsequent outbreaks have occurred in remote areas of Western African countries that are largely isolated. Although infamous for its lethality, this remoteness has proved self-limiting in terms spread.

The current epidemic has defied these rules resulting in escape from the remote areas of West African villages to larger population centers, and for the first time ever, even resulting in at least one case presenting in the United States. (citation)

In general, although viral infections are not treatable by classical antibiotics, vaccines against these types of organisms have been largely successful. Although it is impossible to know exactly why a specific vaccine works, it is reasonable to assume that a humoral response (i.e. mediated by antibodies) is involved in most cases as antibody titer correlates well with protection.

I the case of Ebola, there is data regarding the type of immune responses mounted by patients who have survived the disease compared to those who have not. Baize et al report that “early and increasing levels of IgG, directed mainly against the nucleoprotein and the 40-kDa viral protein, were followed by clearance of circulating viral antigen and activation of cytotoxic T cells” in survivors of disease. While “fatal infection was characterized by impaired humoral responses, with absent specific IgG and barely detectable IgM.” Again, this supports the idea that an effective humoral response is key to protection.

More evidence of the centrality of the humoral response comes from data published by Villinger, et al (citation) showing that “IL-6 levels are unusually low among fatal cases.” They suggest that this points to a deficiency of the endothelial cells that produce this cytokine leading to failure to protect. An alternative explanation may be that macrophages, which are key targets of ebola infection – and are producers of IL-6, are also failing to respond appropriately due to their involvement as targets. This leads to an obvious defect in immune response as IL-6 supports the growth of B cells and is antagonistic to regulatory responses (i.e. regulatory T cells).

If antibodies are so important to response, what are the targets of these antibodies and what issues are there related to this response?

Ebola Virus:

Eboal5Ebola has only one known surface protein found on virions and infected cells. It is presumed that this protein, a ‘sugar-coated’ glycoprotein (GP), is what enables virions to adhere to target cells, a vital first step in the infection of host cells by animal viruses. As neutralizing immunity against viruses is presumed to be a result of the opsinization of viral particles by antibody, the Ebola GP is the obvious target of these antibodies. However, there are still a number of epitopes (regions of the protein to which immune reactions develop) on the GP protein to which antibodies bind. And, furthermore, two versions of GP are made, one in the viral envelope (membrane) and one that is secreted from infected cells. Together, this means that there are a lot of different spots for antibodies to bind, and some spots may be better for protective immunity, while others have no protective effect at all.

Vaccines against ebola are currently being developed with the hope of bringing these to affected areas to either prevent – or at least control- outbreaks at their source. The benefits of developing an effective vaccine include actively inducing life-long immunity.

A second method of fighting disease is to treat with previously generated antibodies in a way that the virus is neutralized, but life-long protection is not induced. One way of accomplishing this treatment is by harvesting serum from patients who were infected, but survived the disease. This has obvious limitations logistically and there is insufficient data on these treatments to know whether they were actually helpful in treating patients. Another way to transfer this sort of ‘passive’ immunity is by making large amounts of a single antibody in cell culture. These ‘monoclonal’ antibodies are highly standardized and can be produced in very large quantities.

A number of monoclonal antibodies targeting different epitopes on the Ebola GP have been developed and show protective effects when administered after viral exposure (i.e. therapeutically). One example of this kind of therapy is ZMapp  from Mapp biopharmaceutical. In studies with animals, they found that “a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge.”

Treatment of Ebola patients with Convalescent Serum

Treatment of Ebola patients with Convalescent Serum

I’ve written before in this space about one of the challenges that antibody treatment against ebola. Because ebola infects macrophages as one of its targets, and because one of the jobs of macrophages is to clear opsonized (antibody-coated) particles, ebola appears to have co-opted this function as a mechanism for penetrating and infecting cells. This characteristic is termed Antibody-Dependent Enhancement (ADE) of infection and has been shown to increase the infectivity of the embryonic kidney cell line, HEK-293, in vitro (Takeda et al 2003). Reportedly, the mechanism for this enhancement is via the complement protein, C1q, and receptors on the host cells.

Together, these data beg the question of whether antibody treatments, such as ZMapp, or vaccines leading to humoral responses will be helpful or harmful in the treatment and protection of patients.

“On 11 August, a group of experts convened by WHO reached consensus that the use of experimental medicines and vaccines under the exceptional circumstances of the Ebola epidemic is ethically acceptable.” So, we may find out the answers to these questions much sooner than we would otherwise expect.

 
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Posted by on November 5, 2014 in Uncategorized

 

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Polio Does Not Go Gentle into that Good Night

While, as recently as 1988, there were as many as 350,000 cases of Polio per year, there were only 417 cases of polio recognized in 2013. At that time Polio was endemic in just three countries.

Old age … burn[s] and rave[s] at close of day.

So far this year there has been nearly 3 times the global number of cases of Polio as there was last year at this time.

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Though wise men at their end know dark is right

It’s not often that scientists will rally around the idea of intentionally pushing an organism to extinction, but this is exactly what disease eradication is – and it is the ultimate goal of all vaccination programs. The eradication of Smallpox has been hailed as one of the great successes in modern medicine. In fact, the philosophical arguments against making eradication the goal are not meant to subvert eradication, but actually to prevent complacence in monitoring programs and to avoid wasting money on tracking down ‘one last case’ of disease.

This said, a goal of the World Health Organization does have an endgame strategy for polio and plans on having it eradicated by 2018.

Rage, rage against the dying of the light.

Despite our best efforts, The Polio Eradication Initiative, in its May 2014 Special Alert, reports, “WHO Director-General Margaret Chan declared the recent international spread of wild poliovirus a ‘public health emergency of international concern,’ and issued Temporary Recommendations under the International Health Regulations (2005) to prevent further spread of the disease as the high season approaches.” This declaration has the weight of international law for the 194 signee countries.

Curse, bless, me now with your fierce tears, I pray.

Obstacles to final eradication remain largely human and political. Thinkprogress.org’s Hayes Brown reports that, “Pakistan has been the epicenter of attacks on vaccination programs, following the revelation that the CIA in 2010 used a fake vaccination campaign to hide their intelligence gathering efforts to locate Osama bin Laden. Since then, the Pakistani Taliban have now come to see all health workers as suspect and prime for targeting”

War, especially civil war, has led to countries such as Somalia, Pakistan and The Gaza Strip of Israel becoming reservoirs for disease and expanding the number of countries with wild polio.

ImageNevertheless, Even as polio does Rage against our efforts, it is still much reduced and there is no reason to give up hope that a combination of political and medical interventions will be sufficient to add polio to that short list of diseases that are ‘forever gone.’

 
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Posted by on May 6, 2014 in Uncategorized

 

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An Epidemiological Method: Using RFLP to Identify Strains of Pathogens

An excellent classroom resource for a case study in epidemiology is presented by the CDC. This study walks students through an outbreak of E. coli O157:H7 in Michigan.

The purpose of this study is to provide student investigators with the opportunity to walk through the procedures and rationale behind investigating the etiology and to develop experiments testing hypotheses generated by the students.

I am using this exercise as an end-of-semester project for my microbiology students to work through collaboratively now that we have completed our discussion of Paul Offit’s Vaccinated.

The study begins:

PART I – OUTBREAK DETECTION

 

Escherichia coli O157:H7 was first identified as a human pathogen in 1982 in the United States of America, following an outbreak of bloody diarrhea associated with contaminated hamburger meat. Sporadic infections and outbreaks have since been reported from many parts of the world, including North America, Western Europe, Australia, Asia, and Africa. Although other animals are capable of carrying and transmitting the infection, cattle are the primary reservoir for E. coli O157:H7. Implicated foods are typically those derived from cattle (e.g., beef, hamburger, raw milk); however, the infection has also been transmitted through contact with infected persons, contaminated water, and other contaminated food products.

Infection with E. coli O157:H7 is diagnosed by detecting the bacterium in the stool. Most laboratories that culture stool do not routinely test for E. coli O157:H7, but require a special request from the health care provider. Only recently has E. coli O157:H7 infection become nationally notifiable in the U.S. Outside the U.S., reporting is limited to a few but increasing number of countries.

In the last week of June 1997, the Michigan Department of Community Health (MDCH) noticed an increase in laboratory reports of E. coli O157:H7 infection. Fifty-two infections had been reported that month, compared with 18 in June of 1996. In preliminary investigations, no obvious epidemiologic linkages between the patients were found.   The increase in cases continued into July.

Students are then asked a number of introductory questions and then presented with the following problem:

Compare the DNA fingerprints in Figure 2 from seven of the Michigan E. coli O157:H7 cases. Each isolate has its own vertical lane (i.e., column). Controls appear in lanes #1, 5, and 10. Which Michigan isolates appear similar?

This question requires some background in DNA Fingerprinting (aka Restriction Fragment Length Polymorphisms, or RFLPs), which I want to take some time to explain.

As the source material states, The purpose of this test is to identify common strains of organisms through their DNA banding pattern. “Different DNA composition will result in different PFGE banding patterns. Bacteria descended from the same original parent will have virtually identical DNA and their DNA fingerprints will be indistinguishable. Identification of a cluster of isolates with the same PFGE pattern suggests that they arose from the same parent and could be from the same source. “ (emphasis mine).

The method involves two core techniques. First, DNA from the target organism must be isolated and cut with one or more restriction enzyme(s). This will create a number of DNA fragments, where the precise number and size of fragments is determined by the sequence of that organism’s DNA.

As an example, let’s imagine a 10,000 base pair (bp) chromosome that we intend to cut with the restriction enzyme, EcoRI. EcoRI recognizes and cuts double stranded DNA at a specific sequence of 6 bases.

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Figure: DNA cut by the Restriction Enzyme, EcoRI. A. DNA sequence with EcoRI recognition site highlighted and cut pattern illustrated. B. Enzyme binds to DNA at the recognition site. C. DNA has been cleaved.

On average, this enzyme will cut a random sequence of DNA every 4096 bases (this can be estimated by 4 raised to the power of n, where n = the number of bases in the enzyme’s recognition sequence , or 46 = 4096 in this case.) In our example, this suggests that a 10,000 bp chromosome will have two EcoRI sites by random chance.

The circular chromosome should be cut twice by this enzyme, resulting in two fragments of DNA (see note #2, below). Let’s say the two bands are 4000 bp and 6000 bp.

We can see these two fragments by running them through agarose, which works as a molecular sieve, to separate the two fragments by size

How does this work?

DNA is a negatively charged molecule with that charge spread uniformly across the length of the fragment. Therefore, there is no difference in charge between our two fragments, except in proportion to their length. This means that as they run through the sieve, the only difference between the molecules comes from their lengths. As any sieve, smaller objects go through easier, while larger ones are held up.

ImageThe result is that the two fragments will appear as distinct bands on a gel, with the smaller fragment running farther through the agarose that the larger. (here, the smaller band at the bottom of the gel has migrated farther toward the positive electrode)

If someone new were to become infected with this bacteria, we could isolate it from them, digest the DNA and get the same banding pattern. A closely related bacteria may have one additional EcoRI site. This would result in one of the two bands being cut into two smaller fragments, meaning that the two strains could be easily distinguished.

Back to the question posed above…

Given this, examine the following compilation of samples. Controls appear in lanes #1, 5, and 10. Which of the remaining isolates appear similar?

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Definitions:

  1. Restriction Enzyme or Restriction Endonuclease– an enzyme that can recognize and cut DNA.
  2. Recognition Sequence – the sequence of bases that a restriction enzyme recognizes and binds to.

 

Notes:

  1. In my example, we are using the restriction enzyme, EcoRI, to cut DNA from E. coli. As the name suggests, EcoRI actually derives from E.coli, where it functions as a defence against invading DNA, i.e. a virus. In order to do this successfully, E. coli will either not have any EcoRI restriction sites in its own DNA, or it will protect them by methylation so that the enzyme does not destroy the host’s own DNA. I am ignoring the possibility that the DNA we are dealing with in our experiment may not be cleavable with this enzyme.
  2. Also note, that bacterial chromosomes are circular, rather than linear – interestingly, this means that they are not actually ‘chromosomes’ at all. Again, let’s ignore this.
 
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Posted by on April 18, 2014 in Uncategorized

 

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epidemiology

I’m looking forward to discussing a bit of epidemiology in my Microbiology class on Tuesday. I’ve been looking at the CDC’s Epidemic Intelligence Service website and a package of case studies obtained from them and I think we can manage at least some of the broader questions without losing valuable time that I am itching to discuss immunology.

While researching, I came across this site by Google that uses search engine terms as a method for tracking flu worldwide. The data is used to create a map like the one shown below.

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Previous years’ information yielded these data illustrating spikes in flu during the winter months last year.

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Posted by on November 8, 2013 in Uncategorized

 

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