------------------------------------------------- Int J Mol Sci. 2014 Mar 11;15(3):4284-98. doi: 10.3390/ijms15034284.
Detection of borreliae in archived sera from patients with clinically suspect lyme disease.
Lee SH, Vigliotti JS, Vigliotti VS, Jones W, Shearer DM.
Abstract
The diagnoses of Lyme disease based on clinical manifestations, serological findings and detection of infectious agents often contradict each other.
We tested 52 blind-coded serum samples, including 20 pre-treatment and 12 post-treatment sera from clinically suspect Lyme disease patients, for the presence of residual Lyme disease infectious agents,
using nested PCR amplification of a signature segment of the borrelial 16S ribosomal RNA gene for detection and direct DNA sequencing of the PCR amplicon for molecular validation.
These archived sera were split from the samples drawn for the 2-tier serology tests performed by a CDC-approved laboratory, and are used as reference materials for evaluating new diagnostic reagents.
Of the 12 post-treatment serum samples, we found DNA evidence of a novel borrelia of uncertain significance in one, which was also positive for the 2-tier serology test. The rest of the post-treatment sera and all 20 control sera were PCR-negative.
Of the 20 pre-treatment sera from clinically suspect early Lyme disease patients, we found Borrelia miyamotoi in one which was 2-tier serology-negative, and a Borrelia burgdorferi in two-one negative and one positive for 2-tier serology.
We conclude that a sensitive and reliable DNA-based test is needed to support the diagnosis of Lyme disease and Lyme disease-like borreliosis.
Free Article
PMID: 24619223 [PubMed - in process]
-------------------------------------------------- The study tried to do PCR tests to confirm the Western Blot results.
It should be noted that the IDSA guidelines specifically disallow a diagnosis of lyme disease based on a positive PCR test result.
One of the amazing statements found in the article -- Quote -- It is also reasonable to expect that the blood of a Lyme disease patient may be positive for the diagnostic antibody bands, but negative for the causative agent, or vice versa, at any point in time.-- End Quote
Basically what the study proves is that the antibody bands are frequently absent in early disease (13/20 untreated patients with EM rash were negative using 2 tier band results). Of those 13 negative patients 1 tested positive by PCR for lyme and 1 tested positive by PCR for b. miyamotoi. Only one of the 7 patients that were positive using 2 tier band results was positive by PCR for lyme.
Of course the missing piece of the puzzle is the patients without a rash who had known tickbites. The control patients do not fit that definition. Also only 2 control patients had fibromyalgia and 2 more had MS -- those were all negative by 2 tier band results.
12 treated patients (8 with EM rashes, 2 with neurological lyme and 2 with lyme arthritis) were tested by PCR. 11 were negative but one of the neurological lyme patients was found to have a novel borrelia species -- b. coriaceae is a species known to infect the soft tick Ornithodoros coriaceus, classified in the relapsing fever group, and not known to be a human pathogen.
According to the article the number of borrelial bacteria or bacterial fragments in the serum samples that tested positive by PCR were less than 30. This means that the test may just not be sensitive enough to detect very low levels of borrelia in some samples.
One of the big problems with the study is the low number of patients -- and also the overabundance of patients with an EM rash -- 28 of the 32 lyme patients.
posted
This study which showed inconsistency between CDC sample serological testing and PCR results plus finding an unknown spirochete and a B miyamotoi spirochete in a CDC provided sample, was done by the same people who provide the $150 16S PCR + sequencing when positive testing. Its interesting the CDC never did nested PCR testing on samples they provide for validation studies. If they had, they would have found the new spirochete and B miyamotoi.
The test they offer now is more sensitive to the number of spirochetes than the test in the study of CDC provided samples. I believe the purpose of this study is to validate the PCR + sequencing test they would like to provide to other labs as a confirmative or alternative to the 2 tier serology. $150 for 3 nested PCR runs plus sequencing is quite inexpensive.
Posts: 51 | From California | Registered: Aug 2013
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posted
Obviously PCR testing is not a valid alternative to serological testing -- it would miss even more patients than the serological testing does.
As an additional testing method it would diagnose a few more patients in the eyes of non LLMD's -- I think the vast majority of LLMD's would have considered the positive PCR test patients as being positive by serology as well.
I have posted many times that hubby never ever had anywhere close to a positive serological test. He only had band 41 show up once on probably 15 Western Blots from at least 4 or 5 different labs. All other bands were always negative -- never even had any indeterminates.
But he did luck out and get a positive PCR test from IGeneX once as well as 2 positive recombinant antigen tests from MDL (test is no longer done).
So according to the IDSA and CDC who do not recognize PCR lyme tests hubby never had lyme.
I am still working on getting his brain specimans from his autopsy samples tested for lyme.
Bea Seibert
Posts: 7306 | From Martinsville,VA,USA | Registered: Oct 2004
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lpkayak
Honored Contributor (10K+ posts)
Member # 5230
posted
What is nested pcr? I know what pcr is
-------------------- Lyme? Its complicated. Educate yourself. Posts: 13712 | From new england | Registered: Feb 2004
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posted
Nested PCR is a variant on PCR but is more sensitive and better able to discriminate and less likely to encounter a false sequence.
PCR uses a set of primers which are strings of nucleotide sequences that bracket the section of DNA you are looking for. In this study, they found a 358 nucleotide ( A, C, G or T ) sequence that ALL known Borrelia contain inside their 16S gene.
Nested PCR uses two sets of primers. It fist uses the outside primers ( OutSidePr )to amplify the section of interest plus the initial inside primers. Then it uses the second set of primers ( InSidePr ) that bracket the actual sequence of 358 in this case. So they call it nested because the sequence is inside a set of primers and they both are inside the outer primers i.e. they are nested.
It would look like:
OutSidePr1 InSidePr1 358 InSidePr2 OutSidePr2
So the first reaction detects and cuts out leaving
InSidePr1 358 InSidePr2
Then the second reaction amplifies only the 358
Then they sequence the 358 to see if it matches known Borrelia.
In this case, they found 2 B burgdorferi, 1 B miyamotoi and 1 unknown that most closely matches Borrelia coriaceae.
Posts: 51 | From California | Registered: Aug 2013
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