Rebuttal published to CDC vs Advanced in J Clin Microbiology

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inmacdonald
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Rebuttal published to CDC vs Advanced in J Clin Microbiology

Post by inmacdonald » Wed 19 Nov 2014 15:40

Full text of published rebuttal to Johnson and CDC et al re: alleged contamination in methods of Advanced Labs

Link :

https://www.dropbox.com/s/7owth2aa6xdb0 ... 3.pdf?dl=0


Respectfully
Alan B . MacDonald MD FCAP
November 19, 2014

PS : We have recently witnessed in Network television coverage and admissions by the Director of The CDC, that CDC mistakes and egregious releases of SmallPox and other pathogens occurred on more than one occasion,
Due to poor techniques in CDC laboratories.
The CDC laboratorians are not models of ideal laboratory techniques with microbial pathogens.

A formal report from the CDC as to where the breaks in security occurred with the small pox releases,
And follow up news media reports of the outcomes of the 85 CDC EMPLOYEES WHO WERE EXPOSED TO THE SMALLPOX VIRUSES IN CDC LABORATORY BLUNDERS.

duncan
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by duncan » Wed 19 Nov 2014 16:19

Dr. MacDonald, congratulations on having your rebuttal published.

Would you have any insight on where ALS stands in having its culture test validated by independent sources? If I remember correctly, two universities were engaged in the validation process, but I think that was some time ago.

hv808ct
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by hv808ct » Wed 19 Nov 2014 16:30

Reply to “No Evidence for Contamination of Borrelia Blood Cultures: a Review of Facts”
Barbara J. B. Johnson, Mark A. Pilgard and Theresa M. Russell
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA

A basic obligation of a commercial clinical laboratory is to validate a test before offering it for sale to diagnose patients. Advanced Laboratory Services offers a culture method to diagnose patients with a Borrelia species infection (1), but we do not find the evidence provided in support of this method by Sapi et al. adequate to establish that the new culture test has the clinical sensitivity (94%) and specificity (100%) claimed (2). We disagree, therefore, with the opinion expressed by Dr. MacDonald in his letter to the editor (3) stating that the analytical methods of Advanced Laboratory Services are sound.

Sapi et al. sequenced a portion of a single gene after nested-PCR amplification and concluded that they had ruled out laboratory contamination. Our analysis demonstrated that the vast majority of the patient-related DNA sequences were identical to those of the laboratory strains used to develop this culture method. These identities can readily be seen in GenBank using BLASTn and support our claims (2). (One way among many to see these relationships is to enter the accession number of each patient-associated pyrG sequence into the search box of the BLASTn program.) The published data are insufficient to determine the source(s) of the DNA used by Sapi et al. to produce patient-related gene sequences. Possibilities include borrelial cells, DNA, and/or PCR amplicons. Because 80% of the patient-associated sequences matched the controls over the region sequenced, the possibility of contamination cannot be excluded. To show that purported Borrelia isolates from patients are different from control strains, more-robust analyses, such as multilocus sequence typing, would be required.

We also noted inconsistencies between the DNA sequences and the immunofluorescence results from patient-related material. According to Sapi et al., an internal validation study demonstrated that monoclonal antibody MA1-7006 recognized Borrelia burgdorferi strains B31 and 297 but did not recognize the B. garinii or B. afzelii reference strains. All patient-related cultures were reported to be positive for MA1-7006 staining, indicating the presence of B. burgdorferi cells. However, patient-related DNA sequences matched all three Borrelia species used as reference strains.

The majority of patient-related sequences matched the pyrG gene of the Japanese control strain of Borrelia garinii (Fuji P1). If well validated, these data would constitute the first evidence of human infection by B. garinii acquired in the Western Hemisphere. However, it is unwarranted to conclude based on the work of Sapi et al. (1) that B. garinii is endemic in the United States. The assertions made by Dr. MacDonald (3) are not supported by the literature, and none of the references cited established B. garinii as the infecting agent in the patients studied.

To properly validate a Lyme disease diagnostic test, investigators must examine blindly by the same procedures samples from well-characterized patients, healthy controls, and patients with unrelated conditions that have similar symptoms. Sapi et al. did not do this in their report. We are not aware of any published evaluation that appropriately characterizes the sensitivity and specificity of this culture method.

Finally, we recall the reason that culture is considered the “gold standard” for laboratory diagnosis in microbiology. Cultures can be archived and shared. Advanced Laboratory Services can send their isolates of Borrelia from clinical material to independent laboratories for analysis. However, despite commercial use for more than 2 years, this test has not been corroborated in an independent report. For all of these reasons, we stand by our critique and strongly recommend that patients and clinicians wait for independent verification of these findings before relying on results of this culture method to diagnose and treat patients.

duncan
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by duncan » Wed 19 Nov 2014 16:59

Does Barbara Johnson have a patent for a competitive diagnostic? Am I remembering that correctly? If she does, should she be recusing herself from commentary on the ALS product as a disinterested CDC representative?

I'm not sure how this sort of thing works in medical science communities.

nnecker
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by nnecker » Wed 19 Nov 2014 18:24

I didn't realize all of this was being posted so I quess this belongs here:

From "Why is the CDC Trying to Block an Accurate Lyme Disease Test?"
With 2 universities already testing the culture...
LHCTom said almost 2 years ago:

My understanding is that the ALS culture is being validated by 2 University studies right now. I was told this by 2 people close to the company.

I sure would like to know what 2 Universities they are talking about?I emailed Beth Daley about this matter and she emailed me back stating that she asked ALS this question many times.Every time the response was the same,"no comment."
I find this post from member Claudia interesting as well:

but as much as I was hoping for straight responses to my questions of Advanced Laboratory Services, I was also looking to see if there would be at the very least the courtesy of an acknowledgement to my contacting and inquiry, even if it was to direct me to information on their website and press releases already distributed. There was zero response from this lab, and I do find that questionable. THE TWO OTHER TIMES THAT I HAVE CONTACTED OTHER DIAGNOSTIC LABS IN THE PAST,MY CALL WAS TAKEN AND MY EMAIL ANSWERED
So there it is Dr MacDonald,can you please enlighten us?

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LHCTom
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by LHCTom » Wed 19 Nov 2014 19:01

Serious Concerns with CDC Lyme Culture Assessment

http://puurelyrandom.wordpress.com/
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

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LHCTom
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by LHCTom » Wed 19 Nov 2014 19:22

Another rebuttal to the Johnson analysis.

http://www.puurelyrandom.wordpress.com

If the current serological test performed anywhere near where claimed with 98% sensitivity and 97% specificity, than why are the "experts" patenting like its a goldrush? And no they are not all trying to achieve a test that only performs sooner after infection as is always the party line. These patents are not cheap and the names are a who's who of IDSA folks. The patent rush is not in agreement with party line.

The culture is considered the Gold Standard for obvious reasons. The experts claim it doesn't work well after treatment so have given up. Barbara Johnson's "laboratory Testing for Bb Infection" and almost everyone simply points to the 2005 Maria E. Aguero-Rosenfeld "Diagnosis of Lyme Disease" as the usual reference.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1195970/

But it makes reference to the culture technique going through various modifications over time citing studies from the 1980's and 1990's with no recent attempts at improvements in over a decade. It sounds like improving the culture was essentially given up over a decade ago with practical issues such as the long culture time, blood requirements exceed children and cost.

Most of the effort on Lyme testing seems focused on patentable approaches and lean toward higher and higher tech because its easy to patent successfully. Even though ALS and Sapi patented their recent attempt at an improved culture, its not easy to protect culturing variations. Of course it makes sense to find a test that is 100% sensitive and 100% specificity that is fast, cheap and works with all patients, at time during an infection and children in a clinical setting.

But given the controversy, why wouldn't everyone want to find a culture as the last line of diagnosis since it has the ability to be 100% specific. The culture when positive either by immunochemical and or DNA PCR verification is the only technique that can give a positive unequivocal result of an active infection. Redundant DNA testing can be used to minimize any risk of contamination. Of course its not a high volume first line test since it would only be used when all other testing is equivocal. It would decide the validity of persistence or not quite easily. It would help allow the differentiation of successfully treated or not when no other test can. If the sensitivity is only 50%, then redundant cultures can help push the sensitivity higher. Once persistence is shown real or not, treatment can be based on facts rather guessing.

With all the effort and patenting of endless testing techniques, why wouldn't the NIH and CDC put some resources into designing a culture that performs better. Because they gave up in the late 90's. A great deal has been learned in microbiology in the last decade which could be applied to the believed unculturability. Giving up when its known Borrelia does grow in a culture environment is foolish. Its because a handful of people in power in the research community have developed a dogmatic attitude and lost their imagination. That's bad science and they complain about pseudoscience - how about dead end imagination science? Just as bad.

Its rather obvious that Lyme culture design was retarded in the late 90's. The last great improvement was taking more blood. Duh, more blood more spirochetes = rocket science . The Marques xenodiagnosis is essentially a culture technique that is trying leverage mother natures strategy. The tick gut is the culture media and their saliva is a chemo-attractant and they feed for weeks. That alone says a few things about why the retarded culture media of the 90's failed. If ticks really have a chemo-attractant as Marques believes and seems a reasonable hypothesis, why not try and understand how it works and apply it in a culture environment. Wormser found doubling the blood volume increased sensitivity due to more spirochetes and letting ticks feed for weeks gives them a longer window and better blood exposure. An adult can give a pint of blood with no problems. People on dialysis for Kidney disease allow a machine to filter their entire blood supply many times. There are many obvious strategies to follow and why a spirochete becomes difficult to culture can be explored with transcriptome analysis or metabolomic analysis. There are many untapped roads of thought if only the imaginations can be turned on.

And now we here Dr Eshoo has developed a sufficiently sensitive molecular strategy that may approach "the culture" in its ability to detect extremely small numbers of spirochetes in PTLS when used redundantly.

I cant help but wonder if making a better culture simply isn't going to be profitable since it should only be used for a small percentage of Lyme cases where persistence is suspected and antibiotic treatment possibly failed. The scientists who claim persistence isn't real seem to block paths that they know could expose them to being wrong. That's worse than pseudoscience. Many ill people would happily sit in a dialysis like setting with chemo-attracttants collecting spirochetes on a newly designed medium and wait 12 weeks and pay for DNA analysis. That test could save them years of suffering and expensive treatments or prove the truth one way or another. I'm just pointing out the culture used in research today is only marginally better and more imaginative than the one used in 1990.

Just look at what happened with the ALS/Sapi culture. Instead of getting together and cooperating with Sapi et al, the CDC performed a paper analysis that was incomplete leading to pinching off interest in a better culture and FDA regulation. The Johnson Assessment chose to find a problem they could leverage but failed to analyze what they actually found. They also didn't even examine 16S gene sequences in NCBI that contradict what they found and provide evidence the contamination occurred in the nested PCR testing and not in the cultures. The Sapi team ran the afzelii and garinii test strains through 16S nested PCR and sequencing alongside the patient samples. The 16S sequences in NCBI show this. They also show the 16S sequences for the 20 patients the CDC found garinii were burgdorferi by 16S gene BLAST by 7 nucleotides. Its not possible for the spirochetes to be both garinii and burgdorferi. That alone suggests contamination occurred in the nested PCR or possibly the sequencing steps - But not in the cultures themselves.

That's because the 16S nested PCR testing or sequencing in Alabama became contaminated with the ATCC test afzelii and garinii so appeared to be in patient cultures. Wrong. The CDC team failed to even examine where the contamination entered the process and failed to mention any 16S sequence data in NCBI. That's questionable science. You cannot pick and choose your data and decide when to not look any deeper once you have the answer you want. So yes their was contamination in the PCR testing but not in the cultures. Once you realize that, then the Mab and Pab and controls testing plus 16S data all are consistent. It really looks bad when a journal like the JCM and all the peer reviews just overlooked the 16S data sitting in NCBI and to ask where the contamination entered and what does that mean?

Marques thought she found contamination in 2 subject PCR's and didn't throw out the whole study. She threw out what she thought was bad data. That's what scientists are supposed to do. Then if the remaining data s sufficient, its reasonable to draw conclusions about validity or not. That doesn't mean additional independent validations are not needed. They are. But the CDC would like to truncate this potential advance before they are shown wrong. That's worse than pseudoscience. Its manipulated bad science.

It really looks like the dogma is so strong its polluting the quality of the science. Oh well. I suggest we get Stanford to use both the Eshoo approach and ALS/Sapi culture side by side on a group of PTLS patients with likely persistence and put an end to the debate. I'll pay for it.

hv808ct - Why don't you put your money where your mouth is....


http://www.puurelyrandom.wordpress.com
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

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inmacdonald
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by inmacdonald » Wed 19 Nov 2014 20:19

Dear Duncan,

I am not a privy to any information concerning the validation of Dr Sapi's
Novel method for the isolation of borrelia species in pure culture from
Human blood specimens.

I offer the possibility of a new and very precise analytic method for VALIDATION OF DR SAPI's BLOOD CULTURE METHOD
Which I believe is currently still in use routinely at
Advanced Laboratories, Sharon,Pa. For primary isolation of Borrelia Spirochetes from human blood .

The method is the technique perfected br Dr Mark Eschoo which is described in detail
In another thread on this forum.
Dr. Eschoo has untilzed ISOTHERMAL DNA AMPLIFICATION . THIS METHOD UTILIZES 50 sets of DNA primers
To amplify borrelia species . The products of isothermal DNA amplification are then speciated by their melting curves.
In addition the DNA of any new borrelia isolate is subjected to the Mass Spect / high resolution analysis which Dr Eschoo has perfected.
From these methods, Dr. Eschoo now possesses 100 separate and unique GENOVARS of Borrelia species in the burgdorferi group Bbss.

If Advanced laboratories agrees , specimens from a large number of their patient borrelia isolates, to Dr Eschoo for his analysis. This validation, while not a word for word recapitulation of the Sapi blood culture method, would provid inconvertrobertible evidence of authenticity of the blood isolates.
The option for ANY OTHER LABORATORY IN THE WORLD, to repeat the Sapi method for blood cultures, is still on the table
For side but side blood culture method validation.

As of today, the CDC has Never attempted to follow Dr SAPI's blood culture recipe,
A recipe which is fully diet ailed and published in her MANUSCRIPT, WITH NO DETAILS WITH HELD OR REDACTED.

Laboratorians at the Ft Collins CDC lab know very well how to mix the reagents, obtain patient specimens, and repeat the Sapi blood culture method.
In place of this very basic procedure which is inured in microbiology professionals,
The Ft Collins CDC personnel opted to Vote with their " gut feelings". In place of following the scientific method
Which Dr Sapi clearly described in her paper.

Let the Final act of this DRAMA written by Dr. Barbara Johnson, be finalized by a rigorous repetition of the Dr Sapi
Method in a BIOSAFETY Level 2 clinical Microbiology Laboratory inside of a building owned by the CDC
At Ft. Collins

Respectfully
Alan B. MacDonald , MD FCAP
November 18' 2014

duncan
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by duncan » Wed 19 Nov 2014 20:41

Dr. MacDonald, although the Eschoo check would be intriguing and arguably supportive, it would not imo necessarily validate the ALS culture per se. To do that, I think an independent and reputable source has to replicate the conditions and the processes that characterize the ALS culture. Preferably more than one independent source.

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inmacdonald
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by inmacdonald » Thu 20 Nov 2014 0:19

Dear Duncan

Ideally, any VALIDATION of ANY LAB METHOD OR TEST PROTOCOL I
Is:
1. Completed by persons who are politically Neutral ( ie persons with no pre-analytical bias)
2. Completed By persons or labs who do not know the originating author or Laboratory
3. Completed with sufficient analytical cases ( analyses) to reach a statistically significant result ( p value)
4. Completed by following the identical protocol of the index laboratory, with no deviations from that protocol.
5. Completed by persons whose lifetime practice experience in diagnostic laboratory methods qualifies them as peer labs or referees.
6. Completed by persons who have been repeatedly been challenged by clinical laboratory CERTIFICATION
EXAMINATIONS ( UN announced spontaneous examinations supervised by national and state regulatory authorities)

The metrics in 1 - 6 above Exclude the laboratorians at Ft Collins CDC Laboratories.
The CDC is. NOTsubject to oversight by the rules of accreditation of The Joint Commission of Hospitals and Health Care Facilities ( JCAH)
Or
By Competency examinations in Laboratory Practices by the College of American Pathologists (CAP)

Validation is the obligation of the Director of each Diagnostic LAboratory.

Laboratories voluntarily participate in such certification exercises in the spirit of Quality Control
And Laboratory Credibility.

The CDC labs do not participate in such Quality Control / Certification activities,
Therefore we shall never know how the CDC Laboratories " measure up" when compared with
Hospital labs, Medical school labs, and private Labs who elect to obtain national certification.

Respectfully
Alan B. MacDonald MD FCAP
November 18, 2014

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