There is a pathologist named Sin Hang Lee at MIlford Hospital in Conn. that is offering a combination nested PCR combined with sequencing that should be far more sensitive than standard PCR. The nested PCR can be up to a thousand fold more sensitive than standard 2 primer PCR if done carefully. Its the first time I'm aware of that nested PCR and sequencing is being offered for the detection and identification ( of strain and species) to mainstream doctors.
http://www.dnalymetest.com/lymediseasediagnostics.html
This is more interesting than usual because Sin Hang Lee has published papers giving his lab more credibility and a level of validation. The technique itself does not need validation since its common used and widely accepted. The question would be in whether a particular lab was sloppy. Nested DNA PCR must be done very carefully and the primer pairs chosen very carefully. The choice of the 16S ribosomal RNA gene is also a common approach and by Next-Gen sequencing the PCR results and then using the NCBI BLAST, it can be shown its really Borrelia DNA and its strain and species identified. If the sensitivity is as good as shown in the paper, we might finally have a PCR test for Lyme that has good sensitivity but also shows real human infection strains in the general population rather than a small human study.
Its also well known that nested PCR vastly outperforms standard PCR in detecting small amounts of DNA. So there is no magic here. They are simply using the best tools commonly used and validated. So this might be the "real deal" - University level tools available to regular doctors. They are almost certainly capable of doing the mRNA PCR so maybe that's next as was used in the Barthold mice study. Since the mRNA PCR requires the removal of all normal DNA followed by reverse transcription of the mRNA back into cDNA, it may not have the sensitivity to pick up small amounts of mRNA. Every cell has a complete set of DNA genes and plasmids and they are very stable. mRNA is only transcribed when a gene is expressed and then its quickly degraded intentionally after its used to build a protein or control the expression of another gene. So there is a lower volume to find and the choice of which mRNA is important. But when mRNA is reverse transcribed, its converted into cDNA which is just the complementary set of base pairs. So it then becomes identical to normal DNA.
http://ajcp.ascpjournals.org/content/133/4/569.abstract
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2984391/
Just like any lab test, if Borrelia contamination gets in the sample being analyzed, it will happily show the strain and species but be wrong. The good news is if they find strains and species that properly match what has been found in the past, its good evidence for the lack of contamination. For example, here in CA we have strains of Bb that are quite different from those more commonly found elsewhere. There has been enough tick Bb strain testing within each endemic state for there to be fairly good statistical idea of what strains are common. So if the labs detection of strains even roughly matches the known statistics of strain distribution, its strong evidence its not contamination. If they contaminate their samples somehow, the test results will cluster around the contamination source. Most Next-Gen Sequencing errors rates can be as high as the 1/2% range ( depending on how its done) so for every 200 base pairs of DNA, it would be possible to find 1 base pair error - very very roughly. I'm assuming they can't afford doing the most accurate techniques for a few hundred $ lab blood test. But even a single base pair error won't typically interfere with the strain ID.
http://www.dnalymetest.com/dnasequencetesting.html
This offering is also important because nested PCR if done correctly and without contamination is far more accurate for diagnosing Lyme disease. It will finally be able to provide data on which strains and species like B miyamotoi are really infecting humans as is being done by Kerry Clark. The main difference between this lab and Kerry Clark's search with nested PCR was he looked for the flaB gene. The nested PCR testing approach used by Kerry Clark and that described by Sin Hang Lee this are similar. Kerry Clark's results have been important because he has shown that Borrelia burgdorferi is not the only species found in the US along with the recent finding of B miyamotoi in humans. The California Health Department's Dr Padget has found about 1/2 the ticks tested positive for Bb also were infected with B miyamotoi. But to date, there have been no California studies testing humans for B miyamotoi using nested PCR and the antibody response to B miyamotoi and other distant strains or species is missed on the antibody tests.
http://www.medsci.org/v10p0915.htm
http://www.cdph.ca.gov/services/boards/ ... -15-13.pdf
Everybody here is aware of the numerous problems with the antibody based tests and the low sensitivity of standard PCR. I'm guessing this approach will become the replacement for the 2 tiered antibody test. Unfortunately in late disseminated lyme disease, the number of spirochetes in the blood could be very low to zero at times. But say someone was infected 10 years ago and treated 7 years ago. Even though the Borrelia DNA can persist in humans for a long time, its unlikely it will persist long in blood. Its probably going to persist in tissues or immune privileged places and rarely escape into the blood unless its replicating. So if someone treated 7 years ago is found positive for Lyme by PCR, its not proof of replication persistence but suspicious evidence. If one finds Borrelia DNA commonly in people treated many years ago and its suspected of being non-cultivable, it will become obvious to microbiologists to try mRNA PCR of multiple genes.
But its ALWAYS critical to avoid contamination so this should be interesting. It should be easier to avoid contamination with PCR over a culture given all else equal because the PCR test is done overnight while a culture needs to sit nearby other cultures for as long as a month. So the PCR test contamination exposure should be easier since it arrives and is tested versus the culture hanging around hundreds of high volume growing spirochetes from other cultures and in a commonly made culture medium. There is just more opportunity in the culturing environment but it obviously can and has been done without contamination successfully. I bet this is just the tip of the iceberg as other labs decide to follow the lead of Sin Hang Lee. It could be the beginning of the end of the testing nightmare.
The greater the ignorance, the greater the dogmatism.
Attributed to William Osler, 1902
