US Military Confirms Ebola PCR Tests Produce Contradictory Results
A U.S. military-linked paper published in Scientific Reports in 2023 reveals that Ebola RT-PCR test outcomes changed depending on how the assay’s synthetic primers and probes were engineered—with the same human samples testing negative under one Ebola PCR configuration and positive under another
PCR tests are currently being used to count Ebola cases, which governments in turn use to justify quarantines and other authoritarian outbreak response measures.
But if there’s reason to doubt the tests themselves, then there’s reason to doubt government response to “cases” determined by them.
The study, conducted by researchers from the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) and funded in part by the Defense Threat Reduction Agency (DTRA), directly undermines the idea that Ebola PCR testing is a fixed, stable, or independently self-validating diagnostic system.
The study was funded under:
“Defense Threat Reduction Agency (DTRA) and the Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Branch.”
According to the paper:
“Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.”
In other words, the same human samples produced contradictory Ebola test outcomes depending on which synthetic primer/probe configuration was used.
The paper repeatedly admits that tiny sequence mismatches between the assay’s primers/probes and the intended target sequence materially altered Ebola test results.
According to the study:
“mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results.”
The researchers found that just one or two nucleotide mismatches dramatically changed amplification behavior.
The paper states:
“one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction.”
The study further reported:
“A single mismatch between primer and template had a minor impact (<0.5 Cq change) on assay sensitivity while two mismatches resulted in a significant impact (> 4.5 Cq change).”
PCR amplification is exponential, meaning a 4.5-cycle shift is substantial.
The paper specifically found:
“the reverse primer in assay Ebo-TM reverse (G18A) had two mismatches for Kikwit and Makona, resulting in a significant 4.9 and 4.55 Cq decrease in sensitivity”
The study also admits that earlier Ebola PCR systems were built using incomplete genomic assumptions from the start.
According to the paper:
“assays developed at the time using the limited genomic information available”
The researchers acknowledged that later outbreak sequencing revealed additional Ebola sequence diversity capable of undermining those earlier assays.
The paper states:
“Rapid genomic sequencing used during the outbreak identified genomic diversity that could negatively impact previously developed assays.”
The study repeatedly warns that purportedly “undiscovered” sequence diversity can cause Ebola-positive samples to be missed entirely.
According to the paper:
“increasing the risk of false-negative test results due to undiscovered genetic diversity.”
The researchers then altered the assay’s molecular detection rules themselves by redesigning the primers and probes and introducing “degenerate nucleotides” to broaden sequence matching.
According to the study:
“We re-designed the Ebo-TM assay to account for all currently established EBOV variance within the assay target region by incorporating degenerate nucleotides to mitigate the mismatches impact on assay performance.”
The redesigned assay was then computationally validated against sequence databases.
According to the paper:
“An in silico analysis found this new assay showed 100% identity to 99.7% of all EBOV sequences in GenBank”
The paper also acknowledges that the redesigned assay was only built around “all known” Ebola variants.
According to the study:
“Inclusivity testing showed the new EBO-TM2 assay detected all known EBOV variants”
The wording “all known” is significant because it implicitly acknowledges that additional undiscovered sequence diversity could again undermine current assays in the future—just as newly discovered sequence diversity undermined earlier ones.
The study also suggests that mixed sequence populations within samples themselves may alter amplification behavior.
According to the paper:
“a small viral population within the EBOV Mayinga stock could have the variants captured within the G14A and G18A primers, resulting in a slight improvement in amplification efficiency”
Critically, while the paper assumes the redesigned assay was more accurate, the study never independently proved which assay outcome was actually correct for the disputed human samples.
That means the paper never independently established whether the original assay was wrong, the redesigned assay was wrong, or whether the contradictory positive and negative Ebola results were simply artifacts of changing PCR primer and probe assumptions.
What the paper directly demonstrated was:
- one assay configuration produced negative results,
- another assay configuration produced positive results,
- and the same human samples changed diagnostic classification depending on how the PCR system itself was engineered.
The paper ultimately portrays Ebola RT-PCR testing as a highly sequence-dependent molecular detection framework whose outputs are heavily shaped by:
- evolving sequence assumptions,
- incomplete genomic databases,
- primer/probe engineering,
- mismatch tolerance,
- and ongoing assay redesign decisions.
The Bottom line
The paper reveals that Ebola PCR outcomes changed when the assay’s primers and probes were altered, that the same human samples produced contradictory positive and negative Ebola classifications under different assay configurations, and that newly discovered sequence diversity undermined earlier Ebola PCR systems built using incomplete genomic assumptions.
Critically, the study never independently proved which assay outcome was actually correct for the disputed human samples. Instead, the paper demonstrated that Ebola diagnostic outcomes themselves changed depending on how the PCR system was engineered.
The study further admits that purportedly “undiscovered” sequence diversity can continue undermining current assays in the future just as newly discovered sequence diversity undermined earlier ones.
Meaning Ebola RT-PCR systems remain perpetually dependent on incomplete genomic databases, evolving sequence assumptions, changing primer/probe designs, mismatch tolerance decisions, and continual redesign of the assay itself.
If the same human samples can produce contradictory Ebola test outcomes under different PCR configurations—without independently establishing which result was actually correct—then the reliability of the tests being used to count Ebola “cases,” and justify quarantines and other outbreak response measures based on those case counts, is called into serious question.
See more here substack.com
Bold emphasis added
Header image: WebMD
very old white guy
| #
The PCR test cannot do what they are using it for.
Reply