Mulige årsager til at (anti-flagel) antistof testen
for Borrelia burgdorferi (Bb)
kan udkomme negativ til trods for at pt. bevisligt ER inficeret
med Borrelia burgdorferi bakterien:

Seronegativ – antistof er IKKE TILSTEDE
Falsk seronegativ – antistof ER TILSTEDE, men kan ikke påvises med den anvendte test
 
Forskellen er stor, men desværre skelnes der ofte ikke imellem de to situationer i litteraturen !

  1. Tidlig infektion, før immun respons er blevet rejst. Nylig antibiotisk behandling har fjernet / hurtigt reduceret antigen-stimulationen i blodet, så produktionen af antistof falder, men har måske ikke formået at fjerne samtlige bakterier i dybere væv, hvorfra bakterierne kan vende tilbage senere, fordi infektionen ikke blev udraderet ~ ”eradikeret”.
    Næsten halvdelen af pt. med erythema migrans, hvor Borreliose verificeredes med dyrkning, udviklede aldrig antistof imod flagella ...

  2. Antistoffer bliver straks bundet i immunkomplekser (dvs. Antigen >> Antistof) og antistoftesten måler KUN FRIT ANTISTOF!
  3. Bakterierne skjuler sig for immunsystemet i dårligt vaskulariserede områder / væv (indre øje, senevæv, diskus …); spirokæterne kan vandre hurtigt i forhold til størrelsen selv i tæt senevæv uden at ødelægge vævets struktur

  4. Bakterierne dækker sine overflade antigener med værtens proteiner (ex. Fibronektin, “S-layer”), hindrer derved immunstimulation og dannede antistoffer preller af (S-layer kan binde antistof uden at skade bakterien)

  5. Bakterierne skjuler sig intracellulært, hvor de er beskyttet mod antibiotika der ikke trænger godt over cellemembraner og kun tidvis stimulerer immunsystemet når de tidvist (cykliske symptomer !) bryder ud af værtens celler i kort tidsrum

  6. Genetisk meget heterogen bakterie familie, hvor lokale stammers antigener måske kan afvige genetisk fra test-stammernes antigener (300 Borrelia strains).
    Antigen variabilitet, dvs. at samme bakterie-stamme fra tid til anden udskifter sine overflade antigener (Borrelia har for et antigen VlsE ligefrem udviklet et ”kassette system”, hvor kun den ydre del af antigenet udskiftes), så allerede dannede antistoffer mod det fjernede antigen efterlades uvirksomme og immunsystemet skal til at rejse reaktion forfra mod det nye antigen i stedet
    Antigen variabilitet kan være afhængig af omgivelsernes temperatur (ospA i flåter -> ospC i pattedyr og i lab. ved vækst ved kropsnær temp.)

  7. Flagella-fri "mutant" har reduceret bevægelighed og mister evnen til at trænge over karvæg (endothel), og stimulerer naturligvis ikke værtens immunsystem til dannelse af antistof mod flagella, når så længe den ikke har sådanne strukturer på sin overflade. Sådanne former kunne imidlertid spontant vende tilbage til vild-typen og kunne derved gendanne sin bevægelighed og evnen til at penetrere karvæg.

  8. Bruger værtens egne enzymer (proteazer) / proteiner istedet for mikrobielle stoffer; værtens egne stoffer virker ikke antigent på værten

  9. Bakterierne findes i alternative former (L-form) som er uden den normale cellevæg med de forventede antigener; ”cysteformen” af Borrelia har f.eks. ingen flagella, stimulerer derfor ikke til dannelse af antistof mod flagella, det eneste antistof den danske Borrelia test leder efter!
  10. Anti-inflammatorisk behandling (prednison; NSAID (gigtmidler) kan reducere antistofdannelsen med op til 50%)
     
  11. Blandingsinfektion med Monocytær Ehrlichia, Anaplasma phagocytophilum (tidligere Ehrlichia p.) og antagelig Babesia kan virke immunhæmmende og reducere antistof produktion.

  12. Andre årsager til immun hæmning (f.eks. HIV … EBV ? CMV ?)

  13. Borrelia kan ødelægge immunceller rettet mod den, både ved direkte invasion af immunceller og via celledræbende cytokiner 

  14. Nedregulering af immunforsvar via cytokiner (flåtspyt indeholder f.eks. immunhæmmende cytokiner)

  15. Dårlig antistof test; cut-off værdien sat så højt, så den ikke finder alle de inficerede og syge med relativt lav immunreaktion; testresultat er ikke reproducerbart fra laboratorie til laboratorie eller i samme laboratorie når undersøgelse af samme materiale gentages senere. 


REFERENCER:

1.  Long-term serological follow-up of patients treated for chronic cutaneous borreliosis or culture-positive erythema migrans.   
Lomholt H, Lebech AM, Hansen K, Brandrup F, Halkier-Sørensen L. Acta Derm Venereol 2000 Sep-Oct;80(5):362-6    PMID: 11200835

23 pt. med dyrkningsverificeret erythema migrans fulgtes i 23 +/- 14 mdr.: 41% forblev seronegative; 35% udviste isoleret IgM
[dvs. 76% havde enten slet intet eller kun umoden IgM antistof reaktion der ikke efterlader nogen immun-hukommelses-celler!],
8% havde et isoleret IgG og 16% et kombineret IgM+IgG respons.
Antistof niveauet toppede indenfor 3 måneder, hvorefter der sås gradvis fald indenfor et år.

22 pt. med kronisk hudaffektion (ACA) fulgtes i 23 +/- 11 mdr.: alle pt. forblev IgG-positive.
Næsten 3/4 udvist fald i IgG niveau over årene, mens resten ikke faldt.
Efter 9 +/- 1 år var 88% af pt. stadig IgG positive.

Konklusion: behandling for erythema migrans bør straks startes på baggrund af UDSLETTET, idet et stort antal pt. forbliver seronegative!"

2. Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease. Schutzer SE et al. Lancet 1990 Feb 10; 335(8685): 312-5 PMID: 1967770   

To find out whether apparent seronegativity in patients strongly suspected of having Lyme disease can be due to sequestration of antibodies in immune complexes, such complexes were isolated and tested for antibody to Borrelia burgdorferi. In a blinded analysis the antibody was detected in all 10 seronegative Lyme disease patients with erythema chronicum migrans (ECM), in none of 19 patients with other diseases, and in 4 of 12 seronegative patients who probably had Lyme disease but had no ECM. These findings were confirmed by western blot, which also showed that immune complex dissociation liberated mainly antibody reactive to the 41 kD antigen and sometimes antibody to an approximate 30 kD antigen. Complexed B burgdorferi antibody was also found in 21 of 22 (95%) of seropositive patients with active disease, 3 additional seronegative but cell mediated immune reactive patients, and 3 other seronegative patients who eventually became seropositive. Apparent B burgdorferi seronegativity in serum immune complexes may thus be due to sequestration of antibody in immune complexes.    In a blinded analysis the antibody was detected in all 10 seronegative patients with ECM, in non of 19 with other disease and in 4/12 seronegative patients who probably had LD, but no ECM. These findings were confirmed by WB. Complexed Bb antibody was also found in 21 of 22 (95%) of seropositive patients with active disease, 3 additional seronegative, but cell mediated immune reactive patients and 3 other patients who eventually became seropositive.   

Excerpt:
In a blinded analysis the antibody was detected in all 10 seronegative patients with ECM, in non of 19 with other disease and in 4/12 seronegative patients who probably had LD, but no ECM. These findings were confirmed by WB. Complexed Bb antibody was also found in 21 of 22 (95%) of seropositive patients with active disease, 3 additional seronegative, but cell mediated immune reactive patients and 3 other patients who eventually became seropositive.

8.  A flagella-less mutant of Borrelia burgdorferi. Structural, molecular, and in vitro functional characterization. Sadziene A, Thomas DD, Bundoc VG, Holt SC, Barbour.  J Clin Invest 1991 Jul; 88(1): 82-92   PMID:  2056133   PDF    
 
A nonmotile mutant of Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated and characterized. The mutant was compared with the wild-type predecessor as well as with a motile back-revertant of the same genetic background. The mutant lacked, by morphologic, biochemical, and immunologic criteria, the major structural protein of flagella, flagellin. This mutation was not associated with major DNA rearrangements or with failure of transcription. An apparent consequence of a loss of flagella was reduced ability to penetrate human endothelial cell layers in vitro. In another assessment of functional significance, the flagella-less mutant was equal if not superior to flagella-bearing, isogenic isolates when examined in an enzyme-linked immunosorbent assay for anti-B. burgdorferi antibodies in the sera of Lyme disease patients. These studies of a mutant, the first among pathogenic Borrelia spp. to be characterized, indicate that the flagellum and motility it confers play a role in B. burgdorferi's invasion of human tissues. A flagella-less B. burgdorferi may be useful as the basis of a more specific immunoassay and a vaccine for protection against Lyme disease.    

"From Results:
A sample of the variant cell population was plated on BSK agar for a second round of cloning. Four well-isolated colonies were picked and grown in broth medium. These other clonal populations had the same nonmotile phenotype when examined by phase contrast microscopy. One ofthe second group of clones was arbitrarily selected for use in subsequent experiments and was designated ""M"" for ""mutant."" When M cells were passed in medium without Yeastolate, at least 99% cells in each tube's population remained nonmotile after 10-15 passages, or 80-120 generations. However, when the M mutant was passed in complete BSK II, which contains Yeastolate, motile bacteria with a morphology like W cells constituted at least 0.1% of the cell population in the culture tubes by three to six passages, or 24-48 generations. When these mixed cultures were subsequently continuously passed in BSK II medium the motile cells consistently came to predominate. Thereafter, the population retained the wild-type phenotype even when recultivated in BSK I medium. Cultures of these revertant motile cells, unlike Mcells, changed the color of the phenol red in the medium at the same cell concentration as W cells. This revertant motile population, designated ""R."" was cloned by limiting dilution for subsequent experiments.

From discussion:
These nonmotile ""spirochetes"" are the first mutants ofa pathogenic Borrelia sp. to be characterized structurally and functionally. The distinctive morphologic and behavioral phenotype of M cells is attributable to lack of the major structural protein, flagellin. We concluded this because the revertant R regained flagella, helical morphology, and motility at the same time.
… .... A reasonable explanation for the significantly poorer penetration ofthe endothelial monolayer byMcells is that the motility conferred by flagella is an important factor for spirochetal invasion. It follows from this that we will find that a mutation in a gene concerned with flagella production is the proximate cause for a major loss of invasiveness. Alternatively, the poor invasiveness ofthe M isolate may be an effect ofan undetected and unrelated second mutation in another gene. This latter explanation is unlikely, because the revertant isolate regained at the same time flagella and, to a large extent, its invasive properties. A third model specifies that the loss ofinvasiveness is the consequence of single but pleiotropic mutation in the M mutant. In other words, one or more other borrelia genes are silenced in expression by a putative ""M"" regulatory mutation. This third explanation cannot at this time be excluded, but even if it holds, an association between flagella and invasion still exists."      
... Finally, we consider the biological significance of the flagella variation phenomenon described here. From the spirochete's viewpoint the switching on-and-off of flagella synthesis - in our less teleologic terminology a "mutation" and "backmutation" - provides possible advantage in its vertebrate or arthropod host. For instance, a nonmotile variant need expend less energy than its flagella-bearing counterpart to survive. Once a borrelia has gained access via motility to certain niches in the host, such as the nervous system, the pressure for further migration in its environment may diminish, and consequently nonmotile variants in the population may fare better over the long term.