Darkfield microscope mayhem— Intraerythrocytic spirochetes... theories??

Referring to Alan MacDonald fluorescence images (seen in his YouTube presentations) and Dr Laane (norway) microscopy research.

No doubt spirochetes can become intracellular within red blood cells, but how/where does this happen in the body?? Can’t find any literature on this, most texts wrongly assert borrelia is purely extracellular.

I’ve been playing with darkfield microscopy using a 50x-oil lens and good LED, reliably seeing spirochetes. Still chasing better image quality, slowly perfecting a 3d-printed filter stop for 100x.

METHOD I’m using is based on Dr Laane’s published paper, using ~1.4mM sodium citrate solution. Dissolving similar amount of table salt and white sugar into the solution seems to be beneficial (pure glucose might be better). Sodium citrate is the key to making magic happen (nobody understands why?).. Higher concentrations exceeding 2mM citrate will cause spirochetes to quickly wake up and go bonkers, but they die very rapidly (long before they can swim off and split.). Approx 1.4mM citrate is the sweet spot.

I pipette a thick line of solution onto a glass slide, add a drop of blood and mix, then pipette tiny drops of diluted blood onto several fresh slides. After adding cover slips, I let slides sit for several minutes to air dry the edges, then seal the cover slip edges with thick immersion oil (type B), based on the accidental discovery by thatdudefromkansas/“borrelia tamer”(YouTube), then place them in a small plastic container with tight lid at room temp (cool, winter/spring)

After 24-30 hours has been the best time to observe textbook tight spirals, corkscrew ketes. From 24-72 hours, it’s fairly easy to find wiggling live ketes dangling from their “holsters”(cysts) and from a few red blood cells. Sometimes 6 or more per cell wiggling out in every direction, maybe up to 1% of erythrocytes do this. about a week later, I can find crazy numbers of exhausted daughter spirochetes as straight rods scattered across the slide (doesn’t happen without using sodium citrate.). Extracellular spirochetes are hard to follow until they straighten out and die, because they randomly move in and out of focus so quickly; some are more vigorous than others (it’s not from brownian motion.).

Hopefully this method helps someone!

Very confident of what I’ve seen, but totally confused how/why/where the erythrocytes get infected... thoughts?

Re—Garzie, regarding the negative PCR conundrum, the dna primers/amplicons in use by labs are too specific and the dna extraction methods for blood are insufficient to reliably capture the dna from borrelia, especially round/cyst forms.

Also curious if Sodium Citrate might have some future potential with combo antimicrobial treatment, it’s plausible to drink enough to reach a relevant concentration in blood. Kinda terrifying to contemplate deliberately waking up billions of dormant spirochetes at the same time... probably need to see about this in an animal model, the herx could be devastating/lethal after a few days.

(haven’t been on the forum in a few months, upgraded my scope to a Cree LED that gets crazy bright at up to 12w. 100% confirmed I have babesia, might get to send samples to CDC parasite lab, hopefully start antimalarial treatment in a few weeks, will post a PDF of my imagery somewhere, eventually )

TLDR ? =]

thanks for posting

yea-ok said...

Re—Garzie, regarding the negative PCR conundrum, the dna primers/amplicons in use by labs are too specific and the dna extraction methods for blood are insufficient to reliably capture the dna from borrelia, especially round/cyst forms.

I'm not sure about that - after all the same PCR tests have no difficulty detecting borrelia in tissues in multiple studies.....

yea-ok said...
will post a PDF of my imagery somewhere, eventually )

would be interested to see your images - please do post

video of borrelia / spirochetes buzzing about at speed would be pretty definitive

The limerick page has some good excerpts, very interesting. Google isnt returning anything helpful.

For PCR, from my reading the various research studies doing animal tissues don’t appear to be using the fda-approved commercial PCR testing, they carefully select a dna primer that precisely suits them. I suspect the issue of extraction from blood is a bigger issue.

Garzie, i sent you a 5mb email.

I recently pulsed 750mg disulfiram for two days and had herx symptoms for 3 weeks, samples since then still have ketes, but not nearly as many as I was finding before upping the dosage of DSF. Possibly similar story for babesia, but I haven’t spent enough time looking to make a fair assessment on whether babesia parasitemia is actually any lower.

yea-ok: any updates? How are you doing?

I'm also starting to wonder if I have Babesia. I feel kind of bad about not thinking about it any sooner after all these years.., but it is what it is. Live and learn.

Anyway, I wanted to share this study with you, hoping you get something out of it that might help you. It's basically claiming that elevated procalcitonin levels (a simple blood test), correlate with Babesia levels.

A few notes:

- Wormser reviewed the study so take it with a grain of salt, the guy has been nothing but trouble for us.
- Procalcitonin can be elevated in other bacterial infections as well. Babesia of course not being bacterial.

I've been trying to figure out how we could use this finding to our advantage but nothing hit me so far. The issue is that it wouldn't really narrow it down to Babesia if we also have other infections.

However, I haven't looked into whether Lyme and Bart specifically can elevate procalcitonin. If they don't, then this is huge news. Anyway, maybe you get more out of it than me:

https://www.ncbi.nlm.nih.gov/pmc/articles/pmc9698394/

"Our study demonstrated that the log-transformed procalcitonin levels had a linear correlation with log-transformed maximum parasitemia, which suggests that procalcitonin directly correlates with parasitemia values."

"Furthermore [...] suggesting that procalcitonin may assist in determining the severity of disease."

"We demonstrate that procalcitonin may directly correlate with the parasitemia percentage and have prognostic capabilities, which suggests that procalcitonin may have biomarker potential in human babesiosis."

“whereas i think parasitaemia levels are well under 1% in most people with chronic babesiosis”

Perhaps also why bilirubin levels are often slightly out of range but not ER alarming.

the graph makes it clearer to me rainy

that the dots are in the real world all over the place - and the correlation was found in a data set of cases with mostly very high parasitaemia - so i'm not convinced that it can be used predictively in people with low levels of parasitaemia - as those of us with chronic disease would be interested in

of put another way - how much predictive value would there be if all the cases they looked at had around 0.1% parasitaemia - as is typically the case in chronic babs

I'm not trying to poo poo your ideas Rainy - or start an argument - just trying to explain the pitfalls of applying this study's findings to the real world - without getting too far into the weeds on the statistics

the statistical analysis just doesn't work as you are indicating

in simple terms - even if 1/3 of the data points are under 1% parasitaemia ( which is still 10x more than typical in chronic Babesia in humans )
the P number stated - (which is used to convey the statistical significance of the findings) is still dependent on and driven mainly by the majority of data points that are way way outside of the range seen in chronic babesia - so their results are describing a very different use-case than we in the chronic disease population would be interested in

to try to illustrate - imagine a straight line drawn through a cluster of points at the bottom end of the % parasitaemia graph ( all under 1%) - and a group of points at the top end of the % graph so you have a slope rising from left to right

if you now take away the cluster of points at the top end of the graph and try to draw a line of best fit through only those at the bottom - what is your p value now?

i don't plan on doing the math - but i hope you can see that - if you took out 2/3rds of the data points the result would be very different

even if this were not the case and say only 1/3 of the data points were outside the range of % parasitaemia we are interested in - the distortion effect would still invalidate the P value

they also do not show a control population which would be particularly important at these low levels - as there could well be significant overlap between levels in people with low levels of parasitaemia and uninfected people. as in pretty much all other standard blood tests

maybe there are other studies which show this marker is well correlated at v low levels of parasitaemia seen in chronic human babesiosis - i am just stating that the conclusions to this one cannot be safely extended to such a different use case / population