Lyme Serology: DNA Probes Reveal a Phalanx of False Negatives

April 2016

It is widely agreed that Lyme Borreliosis serology can be falsely negative in the first few weeks after a tick-bite. Yet the phenomenon of late-stage false negatives has flown largely under the radar. 
The oft-repeated mantra that the CDC-recommended two-tier protocol easily detects all stages of Lyme after the immediate post-tick bite period is proving itself, more and more, to be a hollow incantation.
For those patients in whom chronic Borrelia infection remains undetected, the consequences can be dire. Outcomes ranging from chronic pain and disability to total incapacitation and death have been recorded.
At the Dr. Paul Duray Research Fellowship Endowment, the renowned Lyme pathologist Alan B. MacDonald MD, FCAP is seeking to change all that. 
He has been researching the phenomenon of antibody-negative, yet DNA-positive Borreliosis, utilising the FISH (fluorescence in situ DNA hybridization) technique with highly specific “molecular beacon” DNA probes which he has developed for this purpose.
This method, Dr MacDonald explains, has enabled “the identification of a large number of sero-negative chronic Borrelia infections caused by Borrelia species in the overall greater family of burgdorferi borrelia  (sl) strains...whose DNA differs significantly from the B31 strain to justify the assignment of a separate species and sub-species name. 
“These sub-strains are endowed with proteins that do not cross react with B31 proteins in ELISA and Western Blot test kits. 
Protein mismatches which are a consequence of DNA diversities tell us why a patient may harbour living Borrelia with negative antibody results in blood testing (ELISA/ Western Blots).
B31 is the prototype Borrelia burgdorferi strain on which the current CDC-recommended protocol is based. 
Yet B31, a lab strain cloned from a single tick-derived Borrelia cell, has never been detected in a human and can differ significantly from “wild-type” strains. 
Dr. MacDonald stresses the importance of the VBNC’s – the “viable but uncultivatable” strains that are circulating in the environment and which may be causing undetected disease. 
Once upon a time, he reminds us, all the named strains known today were “VBNCs”.
The molecular beacon DNA probes utilized by the Duray foundation researchers are extremely unlikely to throw up false positives, as they require a 100% nucleotide match before they will release their telltale fluorescent signal.
Dr. MacDonald has named the following Bb sensu lato strains as among those detectable using his probes:

“1. Burgdorferi: (sub-strains : CA283, N40, JD1,  ZS7)
2. Afzelii (sub strains : K78, Tom3107, HLJ01, Pko)
3. Valaisiana (sub-strain: Tom4006)
4. Garinii (sub-strains: PBi, BgVir, SZ)
5. Bissetti (DN127)”
This list includes strains detected in Europe and Asia.
As Dr. MacDonald points out:
“It is established that 60-70% patients infected with European Borrelia species and subspecies will fail to be detected with USA blood testing kits.  This failure to detect Borrelia and Lyme-like infections is entirely traced back to DNA differences.”
Because differing DNA profiles give rise to differing proteins, antigens in American Elisas and Western blots based on Bb sensu stricto B31 can fail to bind to the antibodies in a patient’s blood, rendering the test results – as Dr. MacDonald himself puts it – “meaningless”.
A separate yet equally significant issue is the dismal performance of current antibody tests in patients infected with Borrelia strains which are outside of the Borrelia burgdorferi sensu lato group (Bbsl) altogether.
These belong to species like Borrelia miyamotoi which resemble relapsing fever genetically, yet cause a clinical picture more compatible with Lyme borreliosis. Furthermore, unlike traditional relapsing fever borreliae, which are found in soft ticks, these inhabit hard ticks – often the same Ixodes ticks which transmit Bb.
As Dr. MacDonald points out:
“All Miyamotoi Borrelia are not detected in commercial testing.”
To address this problem, the Duray foundation has developed two DNA probes specific for Miyamotoi – one binding to its unique flagellin gene and the other to the gene glpQ
The latter, which is found in all relapsing fever borrelia, distinguishes Miyamotoi from all strains in the Borrelia burgdorferi (Bbsl) group, where it is absent.
The glpQ gene of B. miyamotoi shows some differences from traditional relapsing fever borrelia.
Dr. MacDonald explains:
“Some workers have placed Miyamotoi Borrelia in the Relapsing Fever Borrelia group, but detailed DNA studies fail to show 100% DNA matches (Miyamotoi vs Relapsing Fever) across the entire chromosome DNA pool and plasmid DNA pools of borrelia spirochetes.”
While European serological tests use some European strains, the Duray foundation have detected Borrelia in patients infected in Europe who were seronegative on European antibody tests too.
Some commentators, such as IDSA Lyme guidelines co-author Linda Bockenstedt, have alleged that evidence of persisting borrelia in antibiotic-treated patients must be simply due to “debris” of “dead” borrelia.
This flies in the face of all that is known about the normal course of events in infection. Normally, remnants of dead infective organism do not linger long-term in the body. They are cleared by phagocytic cells of the immune system.
But this is almost beside the point. What Dr. MacDonald and his colleagues are seeing under the microscope clearly illustrates the fallacy of the “dead bacteria” assumption. 
The fluorescent probes reveal Borrelia biofilms – highly structured and organised “villages” of bacteria with inbuilt water channels and waste removal outlets. 
The whole is enveloped in a protective layer resistant to the immune system and to many antibiotics. A far cry from “debris” indeed.
The recent announcement that Borrelia mayonii -a species new to science – was present in US patients’ blood constitutes the first admission by CDC that Lyme Borrelia species other than Bb sensu stricto are present on American soil. But this “awakening” is occurring at an agonizingly snail-like pace.
The new molecular beacon Borrelia probes developed by Dr. MacDonald are showing that much more than Borrelia mayonii has been missed. 
Shockingly, the probes have detected Borrelia in the aggressive brain tumour Glioblastoma multiforme, in Multiple Sclerosis, in psychiatric syndromes, and in the hallmark brain plaques in Alzheimer’s.
Many of the patients in whom these results were found had been seronegative on the recommended two-tier antibody testing protocol.
As Dr. MacDonald explains:
“DNA probe detection of Borrelia in blood by direct microscopic identification in the symptom-positive, but antibody-negative, patient cohort trumps the negative serology test results that such patients and their physicians have received.”
The probe used by Dr. MacDonald to detect the broad family of Bb sensu lato strains is based on a gene which encodes an inner cell membrane protein of borrelia – the gene bb 0740.
“I am very pleased that to date the gene 740 target represents the best possible DNA probe for European and for American Borrelia strains including B31.”
The Duray researchers continue to strive to maximize the sensitivity, evaluating other possible target DNA sequences.

“I am continually in search of a gene which is shared across Borrelia species  and sub-species,” Dr. MacDonald reveals. “Some day I may discover the universal DNA probe target.”
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Jargon-buster for Lay readers:

Bb sensu lato (Bbsl) – the “species-group” of Borrelia burgdorferi. Under this umbrella are many species such as Bb sensu stricto, Borrelia garinii, Borrelia americana etc..

Bb sensu stricto (Bbss) – a species within the wider Bbsl group, mainly associated with North America. The lab strain B31 on which US test-kits are based belongs to Bbss.

DNA Probes – unique stretches of DNA which will locate and bind to their complementary matching DNA only. DNA probes specific to a particular microbe can be used to uniquely identify the organism from a blood or tissue sample.

FISH (fluorescence in situ DNA hybridization) – a technique for detecting and locating a specific DNA sequence (for example of a micro-organism), using DNA probes attached to a fluorescent compound.
Nucleotide – one of the repeating units that make up DNA. There are four different types, and their order defines a DNA sequence.
Phagocytic cells – cells of the immune system which engulf and destroy invading microbes and cellular debris
Serology – identification of antibodies in a serum sample for diagnostic purposes.
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