“It would appear that Klempner’s methodology was fatally flawed in the two NIH studies that set the stage for treatment denial.” -Carl Tuttle
———- Original Message ———-
From: CARL TUTTLE <runagain@comcast.net>
To: “mark.klempner@umassmed.edu” <mark.klempner@umassmed.edu>
Cc: “michael.collins@umassmed.edu” <michael.collins@umassmed.edu>, “ddutko@hanszenlaporte.com” <ddutko@hanszenlaporte.com>, “ryan.kantor@usdoj.gov” <ryan.kantor@usdoj.gov>, “michelle.seltzer@usdoj.gov” <michelle.seltzer@usdoj.gov>, “william.rinner@usdoj.gov” <william.rinner@usdoj.gov>, “makan.delrahim@usdoj.gov” <makan.delrahim@usdoj.gov>, “john.elias@usdoj.gov” <john.elias@usdoj.gov>, “NIHResearchIntegrity@mail.nih.gov” <NIHResearchIntegrity@mail.nih.gov>, “support@grants.gov” <support@grants.gov>, “reviewpolicyofficer@od.nih.gov” <reviewpolicyofficer@od.nih.gov>, “AskORI@hhs.gov” <AskORI@hhs.gov>
Date: 08/17/2025 1:43 PM EDT
Subject: Did Dr Mark Klempner purify samples before PCR in his NIH funded antibiotic treatment trials for Lyme disease grants (N01-AI-65308 and M01 RR000054)
On 04/27/2018 7:53 AM EDT Carl Tuttle <runagain@comcast.net> wrote: “Not a single positive Dr. Klempner? Doesn’t this statistically prove that your methodology was fatally flawed?”
Dr. Klempner,
As a follow-up to my 2018 inquiry (which you did not respond) I would like to share a recent communication I had with Dr Sin Lee of Milford Molecular Diagnostics Laboratory, specializing in DNA sequencing-based diagnostics. Following his lead, I asked AI a question about your methodology for detecting Borrelia burgdorferi in your NIH funded antibiotic treatment trials for Lyme disease.
Dr. Lee’s question to AI:
Regarding Sanger Sequencing of Borrelia burgdorferi….
IMR Press 2025 Jun 19
Sanger Sequencing of Borrelia burgdorferi flaB Paralogs Detected Spirochetemia at the Early Localized Stage of Lyme Disease
Sin Hang Lee
https://pubmed.ncbi.nlm.nih.gov/40613208/
-Dr Lee: “Accurate diagnosis of Lyme bacterial infection is the crux of the problem, which cannot be mitigated by mathematics. I asked AI to review my recent publication. Here is what AI says (I never purify any samples before PCR):”
AI Overview
Yes, you’ve identified a significant risk associated with purifying a single target DNA molecule from a pool of non-target DNA for PCR amplification:
potential loss of the very target DNA during the purification process.
Here’s why and some considerations:
Why purification can lead to target DNA loss
-Multi-step process: DNA purification often involves multiple steps like cell lysis, binding to a matrix, washing, and elution. Each step presents an opportunity for loss, especially with extremely low quantities of starting material.
-Low input DNA: When aiming to amplify a single DNA molecule, the initial amount of target DNA is minuscule. Even small inefficiencies at each stage can result in the complete loss of the target DNA before PCR can even begin.
-Adsorption and binding inefficiencies: DNA might not bind efficiently to the purification matrix or might be lost during washing steps, especially if there are other cellular components or inhibitors present that interfere with binding.
-Mechanical shearing: Some methods involve physical disruption or manipulation, which can damage the delicate DNA molecule and render it unusable for PCR.
-Incomplete elution: The elution step, where the purified DNA is released from the matrix, might not be 100% efficient, leaving some target DNA behind.
Strategies to mitigate the risk
-Optimize purification protocols: Careful optimization of buffers, incubation times, washing steps, and elution volumes is crucial to maximize recovery.
-Use carrier RNA: Adding carrier RNA can improve the efficiency of DNA binding and recovery, particularly with low DNA concentrations.
-Successive elutions: Performing multiple elution steps can help release more of the bound DNA from the purification matrix.
-Avoid harsh methods: Consider less harsh purification techniques when working with minute quantities of DNA to minimize shearing and degradation.
-Specialized kits: Commercial kits specifically designed for low input DNA or single molecule analysis may offer improved recovery rates compared to general DNA purification kits.
-Direct amplification (where possible): In some cases, if the sample matrix is not highly inhibitory, direct amplification without extensive purification might be considered, although this increases the risk of PCR inhibition and false results.
Balancing purification and PCR inhibition
It’s a delicate balance. While purification aims to remove inhibitors that can hinder PCR, overly aggressive or inefficient purification can lead to the loss of the target itself. The choice of purification method and the level of rigor applied depend heavily on the sample type, the expected amount of target DNA, and the downstream PCR application.
Carl Tuttle’s question to AI:
Did Dr Mark Klempner purify samples before PCR in his NIH funded antibiotic treatment trials for Lyme disease?
AI Overview
In a study co-authored by Dr. Mark Klempner, researchers purified PCR products using a commercial kit for sequencing and analysis
Specifically:
PCR products were purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions.
The purified samples were then eluted twice, and the eluates were pooled and sequenced.
Dr. Klempner,
Here are your PCR results from those NIH funded treatment trials: (ZERO POSITIVES!!)
[359 PCR tests with zero positives] Personal Dropbox storage area
https://www.dropbox.com/scl/fi/wlt03jb03gdu5whj89tfe/Klempner-PCR-Results.jpg?rlkey=jtobbpx9x5szyf0p5ttra0igf&dl=0
-Dr. Lee: To increase the chances of detecting single copy of Borrelia burfdorferi chromosome (or Osp A gene in a linear plasmid as Klempner et al did) in a specimen, pre-PCR purification risks losing the target DNA. Here is why according to AI (as Klempner et al did)
AI Overview
When working with low quantities of DNA, especially when purifying a few molecules, silica-membrane-based DNA purification kits can indeed lead to DNA loss
Reasons for DNA loss
-Inefficient binding: While DNA binds to silica in the presence of chaotropic salts, at very low concentrations, not all DNA molecules may efficiently bind to the silica membrane.
-Inefficient elution: Eluting DNA from silica can also be inefficient, especially for larger DNA fragments or supercoiled DNA, which bind more tightly to the column’s matrix.
-Low elution volume: Using a low elution buffer volume can also reduce the final DNA yield.
-Dr Lee: “The biggest flaw in Klempner et al.’s NEJM 2001 publication is the following statement:”
Base-line specimens of cerebrospinal fluid and plasma specimens obtained at base line and on days 3, 5, 21, and 45 were tested by PCR for the presence of B. burgdorferi DNA, as previously described. [21]
-Dr Lee: In the Results section, they claimed that they found no BB DNA in the blood of the patients.
They should have known that there is a big difference between blood and plasma. In medicine, plasma is the supernatant of the unclotted whole blood (containing anticoagulants) after centrifugation to spin down the RBCs, WBCs and platelets. Since the authors are experts in Lyme disease, they should have known how Borrelia burgdorferi cells distribute in the blood fractions when being centrifuged. For example, even the Google AI clearly stated the following:
AI Overview
1. Yes, it’s generally understood that Borrelia (the bacterium that causes Lyme disease) is significantly heavier than platelets.
AI Overview
2. Studies have shown that when Borrelia burgdorferi (the bacterium that causes Lyme disease) is introduced into whole blood, it tends to concentrate within the platelet fraction. This suggests that Borrelia may have a similar sedimentation rate to platelets, or that it associates with platelets during the sedimentation process.
Here’s a closer look at what we know about the sedimentation rates of platelets and Borrelia:
Platelet sedimentation
-Antisedimentation: Interestingly, platelets don’t actually “sediment” in the traditional sense of settling downwards in response to gravity. Instead, they float on top of the blood column, a phenomenon known as antisedimentation.
-Dr Lee: “The bottom line is that Klempner lost all the Borrelia cells, if any, in the blood specimens before he started his PCR that obviously generated false-negative results.”
So, Dr. Klempner…. It appears that my original assessment was correct and your methodology was fatally flawed as suspected. Let me remind you that as an NIH funded author, you have a moral obligation to acknowledge mistakes which ultimately set the stage for long-term treatment denial.
A response to this inquiry is requested,
Carl Tuttle
Independent Researcher
Hudson, NH
2018 Inquiry to Dr. Klempner…..
———- Original Message ———-
From: Carl Tuttle [mailto:runagain@comcast.net]
Sent: Friday, April 27, 2018 7:54 AM
To: mark.klempner@umassmed.edu
Cc: michael.collins@umassmed.edu; ddutko@hanszenlaporte.com; ryan.kantor@usdoj.gov; michelle.seltzer@usdoj.gov; william.rinner@usdoj.gov; makan.delrahim@usdoj.gov; Tick-Borne Disease Working Group (OS/OASH); Elias, John; officeofthechancellor@umassmed.edu
Subject: Persistent Borrelia Infection in Patients with Ongoing Symptoms of Lyme Disease
April 27, 2018
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, Massachusetts 01655
Attn: Mark S. Klempner, MD, Executive Vice Chancellor, MassBiologics
Dr. Klempner,
I would like to call attention to the attached study recently identifying chronic Lyme disease in twelve patients from Canada.
Persistent Borrelia Infection in Patients with Ongoing Symptoms of Lyme Disease
http://www.mdpi.com/2227-9032/6/2/33
All of these patients were culture positive for infection (genital secretions, skin “Morgellons” and blood) even after multiple years on antibiotics so there was no relief from current antimicrobials. Some of these patients had taken as many as eleven different types of antibiotics.
In contrast, your 2001 antibiotic treatment study found; “no evidence of B. burgdorferi in a total of more than 700 different blood and cerebrospinal fluid samples from the 129 patients in these studies.”
Two Controlled Trials of Antibiotic Treatment in Patients with Persistent Symptoms and a History of Lyme Disease
http://www.nejm.org/doi/full/10.1056/NEJM200107123450202#article_references#t=reference
Not a single positive Dr. Klempner? Doesn’t this statistically prove that your methodology was fatally flawed?
Did you culture skin and genital secretions as the Middelveen paper reports? It would appear that you conveniently stopped looking after your results supported the existing thirty year dogma; chronic Lyme does not exist.
Persistent Lyme disease is not new and has been intentionally/deceitfully suppressed for decades as described in the Vicki Logan case identified in the following letter to past CDC Director Barbara Fitzgerald:
https://www.dropbox.com/s/xaul84dqmqgbre0/Brenda%20Fitzgerald%20MD%20Director%20CDC.docx?dl=0
In 1991 B. burgdorferi had been isolated in culture from Vicki Logan’s CSF (CDC’s laboratory in Fort Collins CO.) despite prior treatment with 21 days of IV cefotaxime and 4 months of oral minocycline.
The dishonest science here in the U.S. has denied chronic Lyme which stifled research to find a curative approach. Now the rest of the world is suffering.
We have lost nearly four decades to this 21st century plague due to the racketeering scheme identified in the RICO lawsuit filed by SHRADER & ASSOCIATES, LLP against the Infectious Disease Society of America, seven IDSA Panelists and eight insurance companies. The U.S. Centers for Disease Control has aligned itself with the seven IDSA Panelists identified in this lawsuit.
Court Document:
https://www.courthousenews.com/wp-content/uploads/2017/11/LymeDisease.pdf
Lyme is an incurable disease when not treated immediately which is spreading across North America and deceitfully misclassified as a low-risk and non-urgent health issue. Patient experience is describing a disease that is destroying lives, ending careers, causing death and disability while leaving victims in financial ruin. Current antimicrobials are ineffective for eradicating all forms of the Borrelia spirochete.
Public outcry has been ignored for decades while the Centers for Disease Control sat on evidence that this infection was not easily treated with a one size fits all treatment approach as dictated by the Infectious Diseases Society of America.
Once again your studies were fatally flawed while supporting the controlling dogma leaving hundreds of thousands if not millions worldwide with a persistent infection and absolutely no relief. We have another AIDS on our hands.
Carl Tuttle
Independent Researcher
Lyme Endemic Hudson, NH
Cc: -Michael F. Collins, Chancellor
-The Tick Borne Disease Working Group
-US Department of Justice
-Daniel R. Dutko, HANSZEN LAPORTE
_______________
**Comment**
When will we learn that PCR can be manipulated a million different ways?
For more:
Madison Area Lyme Support Group 