Re: holy crap! qPCR of EM!

[Guest guest]

I don' know but if you read Lynn Margulis on spirochetes in mudflats,

you can't find anything at all until you put a bit of mud in the

right medium in a petri dish and out swim spirochetes.

>

> I sooo looked for this before, but didnt find it cause I used the

> alternative term qtPCR.

>

> This is of great importance for understanding lyme.

>

> " For those specimens in which B. burgdorferi was detectable by

qPCR,

> the mean ± standard deviation number of spirochetes present in a 2-

> mm skin biopsy specimen was 2,462 ± 2,942, the median number of

> organisms was 1,450, and the number of spirochetes in positive

> specimens varied over 3 orders of magnitude (Fig. 1). "

>

> Why are 1/2 of such biopsies negative for Bb by microscopy, the

rest

> showing only a few organisms (see Hulinska, see Aberer)? This may

be

> hefty evidence that most borrelia are suboptical in size and do not

> express OspA, the common antigen used in IEM. There are other

> possible interpretations, such as lots of dead organisms with

intact

> DNA... which seems unlikely to me.

>

> http://www.pubmedcentral.gov/articlerender.fcgi?

> tool=pubmed & pubmedid=11923340

>

[Guest guest]

> There are other

> possible interpretations, such as lots of dead organisms with intact

> DNA... which seems unlikely to me.

Maybe not so unlikely. Straubinger:

" Detection of heat-killed organisms by PCR. A total of 1.5 ~ 106 heat-

killed, low-passage B. burgdorferi organisms (strain N40) were

injected into the skin of an uninfected beagle. Ten sequential skin

punch biopsy samples were taken at weekly intervals starting 1 day

after injection. PCR analysis revealed that B. burgdorferi DNA was

detectable up to 3 weeks after injection. During that time the amount

of detected DNA equaled 242 spirochetes (day 1 after injection) and 78

spirochetes (22 days after injection) per 100 ƒÊg of extracted DNA. No

DNA was detected on days 8 and 15 after injection. "

http://www.pubmedcentral.gov/articlerender.fcgi?

tool=pubmed & pubmedid=10834975

[Guest guest]

But even the qPCR detected borrelia in only 80% of EM cases. The other

detection methods (the ones that can be used in normal diagnostic labs)

were even less sensitive. And number of organisms detected didn't

correlate with severity or number of symptoms. What if it's all just

not accurate enough to prove anything?

- Kate

On Sunday, October 16, 2005, at 08:38 PM, wrote:

> " For those specimens in which B. burgdorferi was detectable by qPCR,

> the mean ± standard deviation number of spirochetes present in a 2-

> mm skin biopsy specimen was 2,462 ± 2,942, the median number of

> organisms was 1,450, and the number of spirochetes in positive

> specimens varied over 3 orders of magnitude (Fig. 1). "

>

> Why are 1/2 of such biopsies negative for Bb by microscopy, the rest

> showing only a few organisms (see Hulinska, see Aberer)? This may be

> hefty evidence that most borrelia are suboptical in size and do not

> express OspA, the common antigen used in IEM. There are other

> possible interpretations, such as lots of dead organisms with intact

> DNA... which seems unlikely to me.

>

> http://www.pubmedcentral.gov/articlerender.fcgi?

> tool=pubmed & pubmedid=11923340

[Guest guest]

THey do note that theyre at a loss to explain the negatives. I dont

know why they didnt just run multiple aliquots (subsamples) from each

sample, since they only used a 50th of each sample for the PCR.

Alot of things just dont work every time. The grad students in my lab

joke that its all witchcraft. Basically with molecular bio techniques

you are always troubleshooting something, its virtually a big deal

when something actually works right.

I do wish they had commented on the consistency of their control

experiments as Straubinger did - I'm assuming (almost) that they did

do some.

I dono how this spread of organism density (3 mags) compares to other

infectious diseases... but the much-disconfirmed alz group found a 6

mag variation in the number of chlamydiae in alz.

Another possibility that has to be taken seriously is that some morphs

of Bb are less amenable to PCR than others - whether while living, or

after death, or both.

The Straubinger finding that Bb DNA can last so long in a dog is

really a big surprise to me, and sure complicates reading this finding.

>

> But even the qPCR detected borrelia in only 80% of EM cases. The

other

> detection methods (the ones that can be used in normal diagnostic

labs)

> were even less sensitive. And number of organisms detected didn't

> correlate with severity or number of symptoms. What if it's all just

> not accurate enough to prove anything?

>

> - Kate

[Guest guest]

Speaking of PCR results, does anyone know anything about the PCR

regents that Klepner used in his research study that was the demise

of long term antibiotic treatment of Lyme. What kind of positive

and negative controls did he use. Kind of makes you wonder if his

PCR reagents were even relevant to the disease.

-- In infections , " "

<usenethod@y...> wrote:

>

> THey do note that theyre at a loss to explain the negatives. I

dont

> know why they didnt just run multiple aliquots (subsamples) from

each

> sample, since they only used a 50th of each sample for the PCR.

>

> Alot of things just dont work every time. The grad students in my

lab

> joke that its all witchcraft. Basically with molecular bio

techniques

> you are always troubleshooting something, its virtually a big deal

> when something actually works right.

>

> I do wish they had commented on the consistency of their control

> experiments as Straubinger did - I'm assuming (almost) that they

did

> do some.

>

> I dono how this spread of organism density (3 mags) compares to

other

> infectious diseases... but the much-disconfirmed alz group found a

6

> mag variation in the number of chlamydiae in alz.

>

> Another possibility that has to be taken seriously is that some

morphs

> of Bb are less amenable to PCR than others - whether while living,

or

> after death, or both.

>

> The Straubinger finding that Bb DNA can last so long in a dog is

> really a big surprise to me, and sure complicates reading this

finding.

>

>

>

> >

> > But even the qPCR detected borrelia in only 80% of EM cases. The

> other

> > detection methods (the ones that can be used in normal

diagnostic

> labs)

> > were even less sensitive. And number of organisms detected

didn't

> > correlate with severity or number of symptoms. What if it's all

just

> > not accurate enough to prove anything?

> >

> > - Kate

>

[Guest guest]

Re blood PCR, check the Straubinger article upthread. Blood PCR was

almost always negative in infected dogs.

>

> Speaking of PCR results, does anyone know anything about the PCR

> regents that Klepner used in his research study that was the demise

> of long term antibiotic treatment of Lyme. What kind of positive

> and negative controls did he use. Kind of makes you wonder if his

> PCR reagents were even relevant to the disease.

[Guest guest]

I'm not following this thread. : What specificially is your

question?

I THINK you're asking why there are negatives in PCR testing, when

the host is known to be infected??? Right.

If that's the case, then the answer may lie in the fact that a

person may be infected with a strain thats not being tested for.

PCR can't distinguish between DNA thats from a live organism, or DNA

thats from dead organism. And if PCR capture dead bits of DNA - the

whole genome isn't going to be there (obviously).

And as far as borrelia DNA goes, Although Bb strains all share DNA -

there are differences.

PCR (like Igenex) try to set up their tests to capture all strains,

i.e. although, some PCR tests will be more senstivie to one strain

and less sensitive to others - so in a group, some may just be missed.

see this:

So yah- when you read these published articles, you have to read

their materials and methods to understand how they're setting the

experiment up in the first place.

http://jcm.asm.org/cgi/content/full/36/9/2658

> > >

> > > But even the qPCR detected borrelia in only 80% of EM cases.

The

> > other

> > > detection methods (the ones that can be used in normal

> diagnostic

> > labs)

> > > were even less sensitive. And number of organisms detected

> didn't

> > > correlate with severity or number of symptoms. What if it's all

> just

> > > not accurate enough to prove anything?

> > >

> > > - Kate

> >

>