In this particular case, all other methods had failed to succeed. Neither were the lab physicians able to identify the pathogen with any of the classical procedures of microbiology nor by PCR or other established methods of DNA technology. The alleged killer of three organ recipients could only be convicted by a new technique of DNA sequencing. Automated “pyro-sequencing” enables the scientists at the New York Columbia University together with their colleagues at the Infectious Diseases Reference Laboratory in Victoria, Australia, to define a viral strain of Arena-Viruses not yet known to be the trigger of the deadly encephalitis.
Sequencing 100.000 DNA-pieces simultaneously
Normally, the course of infections with Arena Viruses proceeds asymptomatic or the disease causes light symptoms similar to flu if transmitted by rodents. Well-known members of the family are the Lymphocytic Choriomeningitis-Virus (LCMV) and the much more dangerous pathogen of Lassa-Fever. Gustavo Palacios and his colleagues describe the case in the New England Journal of Medicine: “There was absolutely no indication of any acute infection of the organ donor.” But the three Australian women who received the kidney and liver after the deadly cerebral hemorrhage of the donor died within four to six weeks. Those negative results did not let the Australian microbiologists rest. They approached Ian Lipkin, Professor of epidemiology, neurology and pathology at the Columbia University in New York.
From the RNA-isolates of kidney and liver as well as the liquor of two recipients, his team won about 100,000 DNA segments by PCR-technology which could be sequenced. He reduced the data by comparing them with the known human genome. What remained were the sequences of a potential microbial pathogen. The translation into amino-acid sequences then delivered the crucial evidence. The succession of the protein modules resembles those of the LCMV. Actually they found RNA sequences of such a novel Arena-Virus in 22 out of 30 samples. With this learning, the microbiologists succeeded in proving virus material in the cell cultures serologically.
Ideal for germs, viruses and Neanderthals
In a comment on the article, Richard Whitley, Professor of microbiology at the Alabama University, praises the new sequencing method: “It is perfect for applications with unknown germs, funguses, parasites or viruses”. Different from PCR or other methods applied so far, automated pyro-sequencing is able to analyze many short DNA segments very fast. Similar to the two classical sequencing techniques, a detector registers the integration of different nucleotides to the pattern of the DNA counter-strand. Pyrophosphate is released in the reaction which can be translated into flashes. 454 Life Sciences, a company belonging to the Roche group, finally automated the technology to “High throughput Sequencing” with a data output of 100 million nucleotides in a seven-hours run.
Since the method falters when it comes to DNA-snippets longer than 150 to 200 base pairs, the use for analysis of long genome segments for example in men is controversial – but not if the samples are about 40,000 years old. Because Svante Pääbo and his colleagues at the Max-Planck-Institut für evolutionäre Anthropologie in Leipzig were able to decode more than 1 million base pairs of the genotypes of our ancestors from small DNA pieces of Neanderthal bones thus making this technology known worldwide. The target of the project: Deciphering the genome completely and then comparing it to the one of today’s Homo sapiens. The method works particularly well for short DNA segments, i. e. also for analysis of microbes-DNA.
In four out of ten cases, the search for pathogens of encephalitis in American labs is without results. The doctors only discover about 30-60 percent in cases of acute respiratory disease in children, as Whitley writes in his article. “A rapid and exact identification of pathogens is becoming more and more important in a globalized world – especially when the treatment, the control of a pandemic or like in our case the safety of organ transplantations depend on it”, as Ian Lipkin considers the importance of the method. The technology to characterize yet unknown pathogens seems to be ready. But it is not standard yet in laboratory medicine. Andreas Nitsche of the Robert-Koch-Institut says: “Up to now there are only few of those machines in Germany – mainly for other applications. I really hope that will change soon.”