AGBT 2010 - Thomas Briese - Columbia University

New Frontiers in Molecular Diagnosis of Infectious Diseases

The Zoonotic Pool (Morse 1993)

* Assuming that 20 viruses exist for each vertebrate, more than 99% of viruses remain to be found.

Recent Emerging /re-emerging diseases - threats to global health.

[Pictures of diseases, where they come from.... comic about king Tut being diagnosed with West Nile virus...]

* More serious discussion of history of West Nile Virus.

* Identified separately as both human encephalitis, and death in birds... eventually converged

Hong Kong & Sars...

* unfortunately add: "Visiting hong kong will take your breath away."

Why do we need new methods?

* 40-60% of samples will be unidentified or undiagnosed - acute respiratory infection

* 60% of encephalitis is never explained

* enteric infection is undiagnosed in 40% of cases

Introducing a staged sample for pathogen detection.

* this is faster

* more sensitive

* automate-able

* work with non-infectious nucleotides

* incoming samples screened (multiplexed PCR)

* if not known, move to array...

* if still not known, move to sequencing.

* Surveillance assays, QPCR, Serology, Koch's Postulates...

MassTag PCR.

* New technology for sensitive, highly multiplexed, rapid differential diagnose of common viral diseases.

* Has software, aligners, etc.

* primers are tagged, pcr purification on plate, elute to well, use mass spec, automated injection, identification of pathogen by signal analysis.

Has been applied - eg, New York State, 2004-2005.

* decrease in positive signals for infuenza, so a retrospective study was taken.

* 151 patients sample - found identical previously results, but also were able to diagnose 30% more samples. eg, tripple infections, etc.

* undiagnosed also included rhinoviruses (common cold)

* many rhinovirses turne out to be type C, which was unexpected. [not sure if it's because type C wasn't well known, or if it's just these types were not known.]

Not everything is resolved by multiplex PCR... Greene chips.

* named after Donor.

* highly multiplexed

Task 1: Select probes, devised selection mechanism that represents all sequence in genebank

* QUarterly results

Task 2: Amplify and label. Random amplification in sample, and lift to clinically relevant detection level.

Tested a sample on MARV, couldn't be found in standard pcr, etc...

ended up diagnosing as something else... plasmodia? [not sure how that fits with the gene chip.]

Pathogen discovery by NGS.

* used to sequence EVERYTHING in sample.

* if massTag PCR and GreeneChip fail, do HTS.

* Started with Colony Collapse Disorder

* All technologies were geared to vertebrate. but they did isolate a virus... however, not necessarily associated with causation.

Example: Transplant associated pathogenicity.

* Had a difficult time, as virus was very different from known viruses... only 14 tags observed

Forgotten Scripts and the puzzled librarian.

* How do you work with lost languages?

* Use patterns or word probabilities

* Did the same thing for viruses.

Unknown Disease of Parrots

* took tissue from brain of parrots

* discovered Bonavirus[?]

[This talk is incredibly difficult to follow - it's a lot or anecdotes on a theme. I'm sure that's reflected in my notes. Each item is interesting, but it's difficult to tie the common elements together to form a single picture. Mixing method anecdote and acknowledgements together...]

Nosocomial infection

* all died - except patient number 5.

* no disease was initially identified.

* eventually discovered new Hemorrhagic fever by sequencing.

* it has a very diverse biology, sequence, etc.

Hot spots for infectious diseases.

* Go to where the emerging diseases are likely to be..

* Look at the reservoirs

* work on preventative issues.

Projects include

* bats from bangledesh

Lastly: Unexplained encephalopathy

* hts revealed two sequences w 30% homology to mink astrovirus

* Not an efficient path.

* PCR gave more,

* Eventually identified new astrovirus.

* caused them to rethink pipeline, which is leading to new methods.

* better performance.

Prospective birth cohort samples with Norwegian govt.

* building a biobank from mothers, cord blood, fathers, etc

Quote from Einstein

Student: The questions are all the same from last year

Einstein: True, but this year the answers are all different

Question: how often are outbreaks followed up? And how many can't be solved?

Answer: depends on how many they are made aware of. The better the quality of the sample, the better the odds of getting the disease. Can even handle tissue samples.