'There are lone cemeteries, tombs full of soundless bones …"
So wrote Pablo Neruda in the opening lines of a poem about death. But in Canada and Denmark, parallel teams of scientists are now in the midst of a unique investigation to determine if the Chilean poet's bones can speak from beyond the grave.
Working with samples from Mr. Neruda's exhumed remains, the researchers are busy sifting through a microscopic haystack of DNA fragments, looking for signs of something that shouldn't be there. That something might be the traces of an infectious pathogen – a find that would add weight to the theory that Mr. Neruda was the victim of a bizarre assassination in the early days of Chile's 1973 military coup.
One way or another, investigators hope to shed light on the mysterious death of one of the 20th century's most celebrated poets. Their results could prove explosive if they show that Mr. Neruda, who had prostate cancer and was initially reported to have died of heart failure, was exposed to a deadly biological agent.
"I have no doubt that if it's there, we'll find it. But then the next question becomes 'Was it deliberately put there?'" said Hendrik Poinar, the molecular evolutionary geneticist at McMaster University whose lab is central to the effort.
Indeed, the definitive detection of such a sophisticated form of biological weapon would immediately point to sources beyond Chile's borders. It would also likely reignite long-standing questions about covert U.S. involvement in the overthrow of leftist president Salvador Allende, whom Mr. Neruda supported.
But scientists caution that the outcome of the investigation cannot be predicted and that their methods, while cutting edge, might well leave the matter unresolved.
The investigation represents the latest and perhaps final chapter in the quest to reveal what happened to Mr. Neruda. The Nobel Prize-winning poet gained world fame for his writings, particularly his love poems, and he remains Chile's best known literary figure.
"He was a person who could bring magic to language and in so doing bring magic to life and imagination … but at the same time, he did so by talking about things that were important in the daily lives of many people, particularly those less fortunate," said Cristian Orrego Benavente, a biochemist and senior fellow in forensic genetics at the Human Rights Center at University of California, Berkeley.
Mr. Neruda was denounced by right-wing opponents for his Communist ties and lived in exile for a time during the early years of the Cold War, returning in 1952. He campaigned for Mr. Allende during the 1970 election and served as Chile's ambassador to France until 1972. He was living in Chile again when the bloody coup that installed General Augusto Pinochet as dictator was launched.
Mr. Neruda was already diagnosed with cancer and in poor health as the coup began on Sept. 11, 1973. A few days later, he went to a clinic in Santiago, where he died on Sept. 23 at age 69.
A Chilean court opened an inquest into his death in 2011, prompted by accounts that Mr. Neruda was injected with something at the clinic that may have killed him. No medical records have survived from that time. In 2013, Mr. Neruda's remains were exhumed and a detailed forensic analysis was conducted. That analysis revealed the presence of proteins in Mr. Neruda's bones that are related to the staphylococcus aureus bacteria, also known as "golden staph," a potentially lethal organism. This set the stage for a second expert panel that would probe for genetic traces of the bacteria and other possible agents.
Dr. Orrego Benavente, a panel member, said McMaster's ancient DNA centre was a natural choice for the assignment because of Dr. Poinar's technical expertise in fishing genetic clues out of old bones, a technique he developed while working on the mammoth genome from fossils. Dr. Poinar's lab has since applied the same method to human bones and tissues, tracing the different strains of bubonic plague that first devastated Europe during the sixth century based on skeletons from medieval cemeteries. Working with preserved tissues from a medical museum in Philadelphia, the lab has also shed light on the evolution of the bacterium that causes cholera.
The work is challenging because DNA is a fragile molecule that typically breaks down within weeks of death. The successful extraction of DNA from bones that have been interred for just a few years can be nearly as challenging as from 10,000-year-old fossils. The lab's track record has meant the FBI and other agencies sometimes come knocking, Dr. Poinar said.
"When forensic teams come to the end of what they can do, they call us up," he said.
But the lab has never handled a case that is as high-profile and politically sensitive as the Neruda file, said Debi Poinar, a research associate at the centre (and Dr. Poinar's spouse) who has been conducting the DNA extraction and sourcing historical clues that could help the team interpret its results.
She said the special nature of the assignment hit home last fall, when the Poinars travelled to Chile to select samples for analysis from Mr. Neruda's remains, which are currently housed at a medical institute in Chile.
"This is a person with a face to us," she said. "I had to take a few deep breaths before I walked in."
The samples arrived in Hamilton by diplomatic courier three weeks ago and the lab set to work immediately. Samples have also been sent to the department of forensic medicine at the University of Copenhagen, where another team will conduct a parallel analysis. This will allow the labs to validate each other when their results are compared.
Exactly what the investigation will reveal remains a matter of speculation. If DNA from the golden staph turns up, the labs may be able to identify the strain, to see if it matches local strains that were present in Chile at the time. While a naturally occurring staph infection might be an unlikely coincidence, it cannot be ruled out, Dr. Poinar said. Alternatively, if the lab turns up a strain that doesn't match local varieties, it could point to a specially selected pathogen that originated elsewhere and may have been chosen specifically to trigger an aggressive infection.
The Chilean government has already stated in November that it is "highly probable" that Mr. Neruda was murdered. If something more conclusive turns up, panel members say the investigation would mark an important moment in the effort to come to terms with the human-rights abuses that were waged in Chile after the coup – and it may give those who aided those abuses some cause to worry.
"It would have reverberations [in the United States]," said Charles Brenner, a panel member and professor of forensic math and genetics at the University of California, Berkeley. "And it would be a general lesson to people of arrogance around the world that you can't hide."
Retrieving the past
Over the past decade, Hendrik Poinar's molecular genetics laboratory at McMaster University has become a world-leading centre known for its prowess in retrieving DNA from ancient and not-so-ancient bones and teeth.
The central part of a bone or the pulp within the root of the tooth tend to be places that are especially good at harbouring DNA fragments – including the DNA of infectious bacteria that may have been present at the time of death.
Many of the lab's techniques were developed and perfected in-house and they have helped shed light on a wide range of scientific questions concerning everything from ice-age mammals to medieval pandemics.
The basic process involves five steps that must be performed with exacting care:
1) Small quantities of bone or tooth are ground up and put into a chemical solution that absorbs calcium. That helps the mineral parts of the specimen gradually dissolve away, leaving behind everything else from cells that may be present, including short fragments of DNA.
2) The DNA fragments are separated from other components because under the right chemical conditions they can be coaxed to stick to glass. After everything else is washed away, the DNA is left behind. A simple change in PH will release the DNA from the glass and put it back into a purified solution.
3) Special sequences of DNA called adapters are added to the purified DNA from the sample. These act like ID tags for each piece of specimen DNA.
4) Next, the DNA fragments are added to a solution that contains RNA, a complementary molecule that can be engineered to attach only to particular DNA sequences of interest – for example, the DNA of a specific bacterium. The RNA acts like bait on a molecular fishing hook made of protein and metal. Researchers use a magnet to pull these microscopic hooks from the solution together with any DNA that's sticking to the RNA bait.
5) Researchers sequence the DNA they find and reassemble the genome of the target organism.
The process was first used to help retrieve DNA from ancient mammoth bones. Since then, the lab has turned its attention to other ancient beasts including, most recently, the glyptodon, a large and extinct relative of the armadillo. The same technique has proved ideal for isolating and comparing the DNA of pathogens whose genetic traces can linger for centuries in the bones of the people they infect.