By Mark Esposito, Weekend Contributor

Bespectacled 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