Guest guest Posted January 31, 2006 Report Share Posted January 31, 2006 On Tue, Jan 31, 2006 at 01:03:27PM -0000, Barb Peck wrote: >Hi Guys: > > Regarding PCR: >Before my initial lyme testing at MDL, I took about two months of >Doxycycline at maximum dose for my body weight. I know I had classic >herxheimer several hrs. after the first dose (and had to wait 3 days to >recover before taking another dose). After I finished the Doxy, I >waited about a month, then had blood sent to MDL to test for various >things. >Most of the tests were antibody tests, but I had QPCR for Lyme. That >was positive, with a high spirochete load. Western Blot for Lyme >showed only one band for IgG and IgM and that was the non descript >41 kDa. (I have since become seropositive over the insuing years, post >therapy - showing every band on the WB test except OspD at one time or >another,). > > Ive had a couple of questions about Lyme PCR that I'm curious about. >I've been told that PCR can't differentiate between Live and Dead >organisms (and a positive test indicates one or the other is found), >but your discussion below indicates that the organism must be destroyed >in order to find the DNA and be positive.... if I'm reading that right. > >QUESTION: Does PCR find living and dead organisms DNA? Yes. Destroying the organism is something they do in the process of PCR testing, not something you have to do in your body via antibiotics. >The other question has to do with the time lag between when I ended the >antibiotic and had the blood draw. Don'tya think the body should have >cleared the debris within a month? Or maybe there are antibodies, but >their complexed with the bits of dead organisms - and they aren't >cleared easily. I've heard of Lyme hiding inside red blood cells, which have lifetimes of about 120 days. It's conceivable that bacteria killed inside red blood cells could remain there until the cells' lifetimes were up. I don't know how likely this is. -- Norman Yarvin http://yarchive.net Quote Link to comment Share on other sites More sharing options...
Guest guest Posted January 31, 2006 Report Share Posted January 31, 2006 > That > was positive, with a high spirochete load. Western Blot for Lyme > showed only one band for IgG and IgM and that was the non descript > 41 kDa. (I have since become seropositive over the insuing years, post > therapy - showing every band on the WB test except OspD at one time or > another,). Does anyone have refereed lit on the induction of Bb seropositivity by abx? At any rate I know that experienced physicians assert it to be very common. I notice that a static infection, with no bacterial replication and no bacterial death, is a rather felicitous model for this. > QUESTION: Does PCR find living and dead organisms DNA? > > The other question has to do with the time lag between when I ended the > antibiotic and had the blood draw. Don'tya think the body should have > cleared the debris within a month? Or maybe there are antibodies, but > their complexed with the bits of dead organisms - and they aren't > cleared easily. From PMID 10834975 (free full text): Quantification of DNA from heat-killed B. burgdorferi organisms in skin punch biopsy samples by PCR. To determine how long the DNA of B. burgdorferi organisms remains in mammalian tissue, heat-killed borrelia organisms were injected into the skin of an uninfected dog. Low-passage-number B. burgdorferi organisms (strain N40; a gift from A. R. Pachner, Newark, N.J.) were grown in culture for 7 days to a density of 1.2 × 107 bacteria per ml. Spirochetes in the culture medium were killed at 65°C for 1 h. Twenty-five 5- & #956;l aliquots of this spirochete suspension (1.5 × 106 organisms total) were injected intradermally into the skin of an anesthetized dog. Injection sites were located on the left rib cage of the dog and were positioned 1 cm apart (area, 4 by 4 cm). Starting 1 day after inoculation, 4-mm skin punch biopsy samples were collected at weekly intervals under local anesthesia from the area where spirochetes had been injected previously. DNA extraction and PCR testing were done as outlined above. [...] Titration of DNA showed that more than 1 & #956;g of total DNA resulted in the inhibition of the PCR amplification. Five microliters of the DNA solution was used for subsequent PCR tests (approximately 30 to 300 ng of DNA per PCR tube). [...] 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 & #956;g of extracted DNA. No DNA was detected on days 8 and 15 after injection. > QUESTION: Can PCR find the DNA from bound antibody/antigen? Hmm... any DNA bound directly or indirectly to antibody would be marked for phagocytosis. The Fc ( " butt " ) of the Ab binds phagocyte receptors. This system is supposed to be pretty effective, but I'm not closely familiar with its study. Assuming phagocytosis, I would think DNA would be degraded in the phagolysosome. As for free DNA found in human humors, I am not sure how it would end up getting degraded. Quote Link to comment Share on other sites More sharing options...
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