Phylogenetics: Finding The Smoking Genome

By Mark Esposito, Weekend Contributor

maeso-hepatitis-valencia_thumbBespectacled Juan Maeso led a fairly mundane life as an anesthetist in the Spanish coastal town of Valencia. All that changed in 2007 when Maeso was convicted of serial murder. A morphine addict, Maeso had been skimming the painkiller meant for his patients and then using the same compromised needle to inject them. Over a decade, 275 patients contracted hepatitis-c (HCV)  and four of them died from complications from the disease. A Spanish court sentenced Maeso to 1,933 years in prison but the sentence pales in interest to how the murderous soporifist was finally caught.

A fascinating article in the journal Nature details the laboratory hunt for the killer with all the twists and turns of an Arthur Conan Doyle story. Led by researchers at the University of Valencia, the work involved analyzing and categorizing 4200 viral sequences to backtrack to Maeso’s particular strain of hepatitis-c. The process known as phylogenetic forensics has been successfully used to track down the origins of such infamous cases as the 2009 anthrax-laced heroine scare in Europe and the case of Bruce Ivins, a microbiologist at the US Army Medical Research Institute, strongly suspected of sending anthrax tainted letters to Senators in 2001. Ivins committed suicide before charges were placed.

Phylogenetic forensics involves concepts taken from traditional evolutionary-biology augmented with modern sequencing technology. Basically the process works this way, scientist look at rapidly mutating viruses like hepatitis-c in various individuals noting small differences in the genomes. The comparison of genome differences leads to the creation of a series of predecessor and successor genetic markers which comprises a family tree of the virus.“What we are doing is a virus genealogy,” says Oliver Pybus, who studies evolution and infectious diseases at the University of Oxford, UK.

Graphically, the process works this way:

The process allows scientists to determine if two or more infections are closely related and what their relationship is to one another – either parent or off-spring. The results paint a path back to the originating virus. The results of the hunt are not definitive due to the myriad of mutations and the very small differences being compared.  As Anne-Mieke Vandamme, an evolutionary geneticist at the University of Leuven in Belgium, warns “You can never prove guilt.”

But you can link various individuals to an outbreak and from that information determine the likely origin. Such was the case of Dr. Richard Schmidt, who, in 1998, was convicted by a Louisiana court of attempted second-degree murder. Schmidt was charged with injecting his former girlfriend with HIV- and hepatitis-c carrier (HCV)-tainted blood, and allaying her concerns by telling her that he was giving her a vitamin B12 shot. Scouring hospital records, detectives determined that Schmidt has taken a blood sample from a patient but had not turned it over to the hospital lab on the night in question. When police interviewed the patient they determined that he was an HIV as well as a HCV carrier. Virus DNA collected from Schmidt’s patient and the victim matched almost perfectly despite the proclivity of the virus to mutate rapidly in each host body. Schmidt got 50 years in prison in this first use of phylogenetics in U.S. courts.

The method does have its limitations, however. Phylogenetic analyses can offer supporting evidence — that a virus found in person A is very likely to have come from person B, say — but can never prove direct transmission on their own. It is unlike DNA analysis which provides a very high likelihood of determining the donor.

Still phylogenetics was crucial in tracing the path of infection back to Maeso. The crime first came to light when a Spanish utility company began noticing a very high incidence of HCV among its workers. Charged with getting to the bottom of this outbreak, Dr.  Manuel Beltran poured over their medical  records and found that all had been in the same hospital for minor surgery procedures. Beltran contacted the local public health authority which embarked on a massive study of 66,000 patients to find the cause. Maeso name was linked almost immediately but police needed more to prove a crime had occurred.

Dr. Fernando González-Candelas, who led the scientific investigation at the University of Valencia,and his colleagues analyzed patterns of changes in a highly variable region of the HCV genome to sort the viruses into  branches of a genetic tree known as clades. The clades illustrated the evolutionary relationships of the genome changes.  González-Candelas analysed, on average, “11 such viral sequences per person from 321 people believed to have been infected by Maeso and 42 controls — local HCV-infected patients with no known connection to the case. When printed out, the tree that the researchers developed was 11 metres long.”

“Using all the data, the team determined for each infected individual a ‘likelihood ratio’ — that is, the probability that the infection was related to Maeso’s and others whom Maeso had presumably infected, versus the probability that it had come from a source unrelated to the outbreak. Because there were so many samples and a strong phylogenetic signal, the likelihood ratios the scientists got were high. Most were higher than 105, and the highest was 6.6 × 1095, exceptionally strong support for this type of analysis.”

“The Valencia work was also notable in that it attempted to pinpoint when individuals had contracted the virus, using a ‘molecular clock’ technique. To do this, the researchers sampled the genetic diversity of viruses in each person, and then used the mutation rate of HCV in the outbreak to estimate when they had been infected. Almost two-thirds of the estimated dates of infection lined up with when the patients had visited the Valencia hospitals, adding to the evidence that Maeso was the source.”

The science thus lined up almost perfectly with  the records review in pointing the finger at Maeso. Maeso denied the charges saying that he had contracted the HCV from a patient and that he was as much a victim as everyone else.  However, Maeso’s position in the clades at ground zero refuted that argument.

The technique is not without its critics. Many patient advocates complain that sequencing the genealogy of certain viruses like HIV will stigmatize the victims or lead to disclosure of embarrassing sources of the disease which could provide a disincentive to seeking treatment. Failing to get treatment could enlarge outbreaks and lead to public health problems, they argue.

But it seems unlikely these concerns will hinder the rapid growth of the field or delay its usefulness in both civil and criminal cases where infections serve as the weapon of choice.

Source:  Nature, Vol. 506, pp 424-6, Feb. 2014.

~Mark Esposito, Weekend Contributor

22 thoughts on “Phylogenetics: Finding The Smoking Genome

  1. Great piece. It is heartening to finally see HIV treated as an epidemic and not as a PC issue. Unfortunately, for the millions who died because of the PC approach, it’s much too later. Interestingly, it took criminal acts to have scientists act as scientists and not as puppets of PC politicians. One always much appreciate irony.

  2. As the the Ivins case, it is bunk:

    A former Army microbiologist who worked for years with Bruce E. Ivins, whom the F.B.I. has blamed for the anthrax letter attacks that killed five people in 2001, told a National Academy of Sciences panel on Thursday that he believed it was impossible that the deadly spores had been produced undetected in Dr. Ivins’s laboratory, as the F.B.I. asserts.

    Asked by reporters after his testimony whether he believed that there was any chance that Dr. Ivins, who committed suicide in 2008, had carried out the attacks, the microbiologist, Henry S. Heine, replied, “Absolutely not.” At the Army’s biodefense laboratory in Maryland, where Dr. Ivins and Dr. Heine worked, he said, “among the senior scientists, no one believes it.”

    Dr. Heine said he did not dispute that there was a genetic link between the spores in the letters and the anthrax in Dr. Ivins’s flask — a link that led the F.B.I. to conclude that Dr. Ivins had grown the spores from a sample taken from the flask. But samples from the flask were widely shared, Dr. Heine said. Accusing Dr. Ivins of the attacks, he said, was like tracing a murder to the clerk at the sporting goods shop who sold the bullets.

    “Whoever did this is still running around out there,” Dr. Heine said. “I truly believe that.”

    (Mysto Army Anthrax Lab Shut Down – 2). The lab was so loosy goosy they had to shut it down.

  3. The article linked to says:

    But the intersection of this science with the legal system makes many uneasy, says Anne-Mieke Vandamme, an evolutionary geneticist at the University of Leuven in Belgium, who has worked on 19 criminal cases since 2002, mostly for the defence. Unlike DNA evidence, which is routinely used in legal settings around the world, the results of phylogenetic forensics are rarely definitive. “You can never prove guilt,” she says.

    “What we are doing is a virus genealogy,” says Oliver Pybus, who studies evolution and infectious diseases at the University of Oxford, UK.

    Outside the courtroom scientists seem to be confident in their virus geneology work, even looking at it in the context that viruses evolved before cells did:

    The existence of several genes that are central to virus replication and structure, are shared by a broad variety of viruses but are missing from cellular genomes (virus hallmark genes) suggests the model of an ancient virus world, a flow of virus-specific genes that went uninterrupted from the precellular stage of life’s evolution to this day. This concept is tightly linked to two key conjectures on evolution of cells: existence of a complex, precellular, compartmentalized but extensively mixing and recombining pool of genes, and origin of the eukaryotic cell by archaeo-bacterial fusion. The virus world concept and these models of major transitions in the evolution of cells provide complementary pieces of an emerging coherent picture of life’s history.

    (The Uncertain Gene – 9). That may bode well for the forensic aspects Mark E reports on today.

  4. You believe that hogwash that Ivins was the culprit and then committed suicide when ALL his colleages said it was totally out of charicter and WTC7 proves that the government used explosives on 911 beyond any shadow of a doubt?
    You are not nearly as smart as I have been giving you credit for. That or you are just another MSM lying, stinking traitor continuing to give cover to the biggest act of treason in history. Either way I am done with you. Yesterday you had a video up mocking the Bible and today you are selling the completely debunked government version of 911.
    Good Ridence to you. Unsubscribing NOW.

  5. Good stuff Mark. This Maeso character belongs in a jail where he can’t hurt anyone else.
    Alain,
    Get a life.

  6. Another thing about the Ivins 9/11 anthrax case is that anthrax is not a virus, it is a single celled microbe, a bacterium.

    That raises some issues, for example: whether a virus is alive or instead is a conglomerate of molecular machines, like cells are:

    For about 100 years, the scientific community has repeatedly changed its collective mind over what viruses are. First seen as poisons, then as life-forms, then biological chemicals, viruses today are thought of as being in a gray area between living and nonliving: they cannot replicate on their own but can do so in truly living cells and can also affect the behavior of their hosts profoundly. The categorization of viruses as nonliving during much of the modern era of biological science has had an unintended consequence: it has led most researchers to ignore viruses in the study of evolution. Finally, however, scientists are beginning to appreciate viruses as fundamental players in the history of life.

    (The Uncertain Gene – 9). The anthrax is alive, in the context of it being a cell, a carbon-based life form, however the lowly virus is not always considered to be alive.

    That is big because the more complex the entity’s RNA or DNA genetic material being sequenced is, there tends to be more “junk” genetic material, now being called “dark matter.”

    That is a code phrase that means “we don’t have much of a clue about it” (it was considered “junk” after all).

    Viruses have less dark matter than cellular microbes have, and humans have far more dark matter than either of those two, and that material is packed with somewhat mysterious molecular machines:

    “We took this approach because so many RNAs are rapidly destroyed soon after they are made, and this makes them hard to detect,” Pugh said. “So rather than look for the RNA product of transcription we looked for the ‘initiation machine’ that makes the RNA. This machine assembles RNA polymerase, which goes on to make RNA, which goes on to make a protein.” Pugh added that he and Venters were stunned to find 160,000 of these “initiation machines,” because humans only have about 30,000 genes. “This finding is even more remarkable, given that fewer than 10,000 of these machines actually were found right at the site of genes. Since most genes are turned off in cells, it is understandable why they are typically devoid of the initiation machinery.”

    The remaining 150,000 initiation machines — those Pugh and Venters did not find right at genes — remained somewhat mysterious. “These initiation machines that were not associated with genes were clearly active since they were making RNA and aligned with fragments of RNA discovered by other scientists,” Pugh said. “In the early days, these fragments of RNA were generally dismissed as irrelevant [“junk”] since they did not code for proteins.” Pugh added that it was easy to dismiss these fragments because they lacked a feature called polyadenylation — a long string of genetic material, adenosine bases — that protect the RNA from being destroyed. Pugh and Venters further validated their surprising findings by determining that these non-coding initiation machines recognized the same DNA sequences as the ones at coding genes, indicating that they have a specific origin and that their production is regulated, just like it is at coding genes.

    (The Uncertain Gene – 8). In one sense, that makes this forensic technique applied to viruses, which Mark E reports on today, more acceptable to me than even traditional human DNA forensics.

  7. Dredd:

    what do you think about this? It seems reasonable or does virus DNA behave differently? As it goes from host to host does it keep some original DNA or does it just keep modifying based on the host?

    What exactly is it the scientist are looking for?

  8. Dredd:

    I think evolution is about changes in DNA brought about by viral mutations. That stuff about a wolf like creature changing to a whale because it needed to catch fish is bullsh*t.

  9. As this new documentary illistrates, lots of folks have been wrongfully convicted because of junk forensics. More so in this case, because the conviction is already eight years old. No lawyer who believes his client is everything, would want to be without this documentary or at the very least reading the transcript (link enclosed).

    Frontline
    The Real CSI
    CORRESPONDENT
    Lowell Bergman
    http://www.pbs.org/wgbh/pages/frontline/criminal-justice/real-csi/transcript-18/

  10. Bron

    Dredd:

    what do you think about this? It seems reasonable or does virus DNA behave differently? As it goes from host to host does it keep some original DNA or does it just keep modifying based on the host?

    What exactly is it the scientist are looking for?
    =================
    The virus research is quite hot now, because we now know that viruses outnumber single-celled microbes.

    And that most viruses are helpful, not harmful.

    The typically have RNA rather than DNA like living cells have.

    The molecular machinery behaves the same in principle as DNA in terms of storing, coding, and saving information.

    And both have the molecular machinery to do that.

    The anthrax case involved cells, the AIDS case involved viruses.

    There is a lot of horizantal gene transfer (virus to host) which in most cases helps the host.

    It is the rogue parasitic and pathogenic viruses that cause us problems.

    The scientists map out the RNA sequences at various points, then work backward or forward, to see patterns of change in the map or blueprint.

    Those patterns of change work like footprints or fingerprints which can be followed to a common point, a Y or fork in the road where mutations or gene transfer took place that changed the pattern or blueprint.

    In ultimate terms it is called LUCA (Last Universal Common Ancestor, or Last Universal Cellular Ancestor]) as the case may be.

  11. Bron

    Dredd:

    I think evolution is about changes in DNA brought about by viral mutations. That stuff about a wolf like creature changing to a whale because it needed to catch fish is bullsh*t.
    =============
    Yes, me too.

    It smacks of “Teleology” (a word that sorta means intense “scientific imagination”, like “The Selfish Gene”).

    A lot of scientists complain about that licentious use of nomenclature which still plagues biology more than some of the other scientific disciplines.

  12. samantha,

    That Frontline program you linked to is unsettling but instructive.

    It reminds me of the smug decades of Eugenics when people were castrated, lobotomized, or put in asylums based on pseudo-science which was upheld by the U.S. Supreme Court.

    Your link has this:

    LOWELL BERGMAN: For generations, the FBI and their fingerprint examiners have maintained that fingerprint identification is “infallible,” routinely testifying that they are “100 percent certain” and there’s “zero percent” chance they could be wrong.

    Judge DONALD SHELTON, Circuit Court, Michigan: Fingerprint examiners have been taught that there’s only one person in the world who could have left this fingerprint. There’s no scientific basis for that.

    LOWELL BERGMAN: Ken Moses is a veteran fingerprint examiner who has investigated more than 17,000 crime scenes in his 40-year career.

    …LOWELL BERGMAN: [on camera] When you’re looking at this partial print versus the known print, when do you decide that it’s a match? How do you decide it’s a match?

    KENNETH MOSES: At some point, you are examining this evidence, and based on your training and experience, you make a leap of faith.

    LOWELL BERGMAN: You make a leap of faith?

    KENNETH MOSES: It’s a point of decision making. It’s where you go from doubt to no doubt. That’s a leap of faith.

    I don’t think I have to tell the bloggers here about how smug geneticists were during the scientific dark age in the early decades of the 1900’s, regarding Eugenics.

    American Eugenics was a pseudo-science that was fully accepted in the U.S. Court System, but was fully wrong.

    On the heals of that came the same euphoric exuberance which manifested in the human genome project:

    There was nothing extraordinary about the Japanese canopy plant — that is, until Pellicer analyzed the stained cells using flow cytometry, a high-throughput technique to detect features of cells suspended in liquid. To Pellicer’s eye, the balls of DNA inside P. japonica’s nuclei looked “really, really big,” he recalls. Soon, he confirmed that P. japonica carries the largest known eukaryotic genome on the planet, with a whopping 150 billion base pairs — 50 times the size of the human genome. “We were astonished,” says Pellicer. Plants in general are known to have sizable genomes, often as a result of whole-genome duplications, so “we were expecting to find big genomes, but nothing that big.”

    The Human Genome Project’s most startling finding was that human genes, as currently defined, make up less than 2 percent of all the DNA on the genome, and that the total number of genes is relatively small. Scientists had predicted there might be 80,000 to 140,000 human genes, but the current tally is fewer than 25,000 — as one scientific paper put it, SOMEWHERE BETWEEN that of A CHICKEN AND A GRAPE. The remaining 98 percent of our DNA, once dismissed as “junk DNA,” is now taken more seriously.

    (Weekend Rebel Science Excursion – 26). The results of the human genome project were dumbfounding, emphasizing how wrong our “understanding” of genetics really was.

    It has been stated many times that microbes have less “junk DNA”, i.e. less “dark matter”, than humans have.

    In humans it is 98% junk DNA, in microbes only 2% junk DNA, and even less in viruses.

    Thus, since the “junk” DNA is not junk, now called “dark matter” to emphasize that reality, we should not be so smug about what we think we know (the real “dark matter” is in our smug, exceptionalist attitude).

    Especially in light of recent research which shows alarming results in that light:

    From biology class to “C.S.I.,” we are told again and again that our genome is at the heart of our identity. Read the sequences in the chromosomes of a single cell, and learn everything about a person’s genetic information …

    But scientists are discovering that — to a surprising degree — we contain genetic multitudes. Not long ago, researchers had thought it was rare for the cells in a single healthy person to differ genetically in a significant way. But scientists are finding that it’s quite common for an individual to have multiple genomes. Some people, for example, have groups of cells with mutations that are not found in the rest of the body. Some have genomes that came from other people.

    Medical researchers aren’t the only scientists interested in our multitudes of personal genomes. So are forensic scientists. When they attempt to identify criminals or murder victims by matching DNA, they want to avoid being misled by the variety of genomes inside a single person.

    Last year, for example, forensic scientists at the Washington State Patrol Crime Laboratory Division described how a saliva sample and a sperm sample from the same suspect in a sexual assault case didn’t match.

    (The “It’s In Your Genes” Myth – 2). Thus, two of our most cherished forensic techniques, fingerprinting and DNA, may have some problems.

    Virus genetic mapping is not likely to be as problematic as human genetic mapping however.

    The key is to develop methods and techniques that work with the differences, rather than ignore or deny them.

  13. I’m wondering what percentage of folks in the judicial system and law enforcement have viewed this documentary. much less know about it? I wouldn’t doubt corrupt officials suppress this film.

  14. Bron

    Dredd:

    that was interesting.
    ===============
    Samantha’s link about fingerprint forensics being upended was a show stopper.

    A lawyer who had never been to Spain was fingered as one of the bombers who bombed Madrid, Spain killing many innocents.

    His fingerprints from his military records matched those found at the bombing site.

    Three FBI forensic experts agreed.

    He was arrested out of the blue while working in his office, put in jail, and was facing the death penalty.

    Then the Spanish Police said they found a guy with the same prints who was actually in the area of the bombin at the time it took place.

    Can you imagine the impact on the FBI expert who had done tens of thousands of fingerprint cases over his career, who believed fingerprinting forensics was infallible?

    Nigtmares about sending innocent people to their deaths perhaps?

    Now we face a similar quandry with DNA forensics because we now know that people have multiple genetic material within them, not just one single unique set to us alone.

    In fact, our own DNA from one area of our body often does not match DNA from other areas of our body.

    In a significant number of cases.

    Yep, interesting for sure.

    Hopefully not as interesting as Eugenics.

    That was catastrophic.

  15. samantha

    I’m wondering what percentage of folks in the judicial system and law enforcement have viewed this documentary. much less know about it? I wouldn’t doubt corrupt officials suppress this film.
    ===============
    Good question.

    Time for the criminal defense bar to take notice.

    The prosecutors are not likely going to be the first to contemplate the implications.

  16. Mar 2, 2014 7:00:08 PM
    As if fuzzy science is not enough to land you in jail, there’s plenty of this:

    Unreliable or Improper Forensic Science
    Since the late 1980s, DNA analysis has helped identify the guilty and exonerate the innocent nationwide. While DNA testing was developed through extensive scientific research at top academic centers, many other forensic techniques � such as hair microscopy, bite mark comparisons, firearm tool mark analysis and shoe print comparisons � have never been subjected to rigorous scientific evaluation. Meanwhile, forensics techniques that have been properly validated � such as serology, commonly known as blood typing � are sometimes improperly conducted or inaccurately conveyed in trial testimony. In some cases, forensic analysts have fabricated results or engaged in other misconduct.

    Forensic Science Misconduct
    Because forensic science results can mean the difference between life and death in many cases, fraud and other types of misconduct in the field are particularly troubling. False testimony, exaggerated statistics and laboratory fraud have led to wrongful conviction in several states.

    Since forensic evidence is offered by “experts,” jurors routinely give it much more weight than other evidence. But when misconduct occurs, the weight is misplaced. In some instances, labs or their personnel have allied themselves with police and prosecutors, rather than prioritizing the search for truth. Other times, criminalists lacking the requisite knowledge have embellished findings and eluded detection because judges and juries lacked background in the relevant sciences, themselves.

    In some cases, critical evidence has been consumed or destroyed, so that re-testing to uncover misconduct has proven impossible. Evidence in these cases can never be tested again, preventing the truth from being revealed.

    One weak link
    The identification, collection, testing, storage, handling and reporting of any piece of forensic evidence involves a number of people. Evidence can be deliberately or accidentally mishandled at any stage of this process.

    The risk of misconduct starts at the crime scene, where evidence can be planted, destroyed or mishandled. Evidence is later sent to a forensic lab or independent contractor, where it can be contaminated, poorly tested, consumed unnecessarily or mislabeled. Then, in the reporting of test results, technicians and their superiors sometimes have misrepresented their findings. DNA exonerations have even revealed instances of “drylabbing” evidence – reporting results when no test was actually performed.

    Fraudulent crime labs all over the map
    more at:
    http://www.innocenceproject.org/understand/Forensic-Science-Misconduct.php

  17. Pure tobacco… Addictive sure… But easier to break the habit…. Rather than get chemically addicted…..

  18. Anonymously Yours

    Pure tobacco… Addictive sure… But easier to break the habit…. Rather than get chemically addicted…..
    =============
    And pure coal and oil … easy to quit … ;)

  19. Interesting case – a similar one in melbourne Australia where an anaesthetist, james peters, was found to have infected 50 patients with hep c at an abortion clinic. He was addicted to fentanyl and shared his infection with patients with similar forensic techniques used to establish his guilt – 14 years, non parole of 10. Tragic for all.

Comments are closed.