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Thursday, November 13, 2008

Bartonella- Babesia- Bugs- Testing

Clongen has a species PCR runnning for Babesia. It includes 15 species. This is the first test of its kind available. It still won't cover untyped strains. PCR methods are not fool proof.

Here is some evidence for Bartonella proponents. I got back a positive Bartonella species PCR with a negative Bartonella henselae PCR. This suggests that atypical strains of Bartonella in TBD patients do in fact exist. Side bar: On the meds that are supposed to work for atypical Bartonella- Levaquin and Rifampin, she is getting worse. Now that I know what this patient has, I will have to figure out what actually works for this non-typed Bartonella species.

Lyme disease patients have small highly motile gram negative bacteria in their blood. These bacteria are coccobacilli and appear uniform in morphology. They do not adhere to red blood cells. The are present in patients treated with Rocephin, Zithromax and Doxycycline. The DNA sequencing should be done next week. I think they may be GI anerobes from the tick gut. But that is just a wild guess.

Finding out what this bug is may represent a new turn our understanding of this complex poly-microbial disease, and hopefully will offer a new direction regarding possible therapy.

Dr. Kilani mentioned that many physicians are spending a tremendous amount of money ordering tests which are rarely helpful. Here is a suggestion of cost effective screening for patients: Lyme WB. Babesia species PCR. Bartonella species PCR. Ribosomal DNA 16S sequencing for other bacteria. The Bartonella test is optional since it may show up with the RDNA 16S screen. He does not do serology. I would add antibody studies for Ehrlichia and Anaplasma if available. PCR testing for Lyme is rarely positive and should not be done routinely. Other basic information including CBC,Chems,Sed rate, CRP, vit D 25 and 1,25, and B12/folate are essential in my experience. CD57, C3a, C4a: I like to get them out of habit. I have not found that they alter my clinical judgement regarding diagnosis or therapy.


cehansen said...

This new testing is exciting news. Will we be able to get an ID on what has previously been a BLO/bartonella? If so, that is groundbreaking!!

Seibertneurolyme said...

For your info, Fry Labs already has a Babesia PCR test available. This is a genus test (not species specific). They also have similar tests for Borrelia and Mycoplasma.

Unfortunately they do not offer the DNA tests that Clongen does.

I have a question for the doc. Did you also test your patient for mycoplasma?

If not, then I don't think we can definitively say that BLO is a bartonella species instead of a mycoplasma species. Both are gram negative bacteria which would show up on a bloodslide from either Fry Labs or Clongen as coccobacilli.

Lyme report: Montgomery County, MD said...

I AM SORRY: THERE IS ABSOLUTELY NO WAY TO MAKE A GENUS IDENTIFICATION BASED ON THE MORPHOLOGY ON A SLIDE. PERIOD. At the recent ILADS conference Fry labs had not yet developed an PCR tests. Perhaps this is something new.

There are hundreds of bacteria that are gram negative.


The only way to identify the organisms seen on a slide is to do a DNA sequencing. (Or by cuture).

They do not look anything like Bartonella.

Myclplasm can be Identified after an antbiotic challenge is given and a Mycoplasm species PCR is performed. There is a low yield for M. fermentans with this method.

Mycoplasm cannot be identified on a slide or blood smear.

It is not gram negative because it lacks a cell wall. Gram stains only work with bacteria which have a cell wall.

Seibertneurolyme said...

Ok, I am not an M.D. -- I am an accountant. Just got off the phone with Clongen Labs. Montgomery County, M.D. and I were both partly wrong.

Dr K at Clongen says that Bartonella are gram negative coccobacilli and they can be seen on a bloodsmear.

Dr K also says that mycoplasma do not have a cell wall so they are not gram negative bacteria and they can't be seen on a bloodsmear.

So it looks like Fry Labs is seeing Bartonella, but they have not yet identified the exact species.

Will be interesting to see what Clongen finds when hubby gets his bloodsmear and DNA 16 sequencing done on 12/16/08 at Clongen.

Maybe after 7 1/2 years we will actually have some answers.

Hubby had already been on numerous antibiotics when he got the 2 positive bloodslides from Fry last year and has done 6 additional antibiotics since then.

If the mystery bug is really a species of bartonella then it sure is tough to kill.

dogdoc said...

Everyones a bit right and a bit wrong. I've been studying all different bartonellas for days.

Barts include both gram neg rods AND coccobacilli.

Mycoplasma of all types don't stain

BLO is a set of clinical signs of what we don't know

Fry labs doesn't do PCR's- he does IFA's. Igenex and Clongen do PCR's. Igenex has B. henselae PCR only, not Bartonella species. They have babesia species and babesia fish.

What is unique about these coccobaccili is that they are motile. I looked my own under the microscope today and they are very active. My stained smears look a lot like what Fry labs calls Bart. The different Barts look different to me in different species and between the different types. However, what everyone thinks we are treating as Bart are the nonmotile ones. Will be interesting to see what they are. Lots of motile gram negative coccobacilli and short small rods (look the same really) to choose from. Can't assume they are Bart. If they are Bart, then they are unlike anyone we know of. They should be long dead with the types of drugs and lengths of treatments we use.

Seibertneurolyme said...

Thanks dogdoc for chiming in.

Do want to clarify that Fry Labs does do PCR testing -- very new and not on the website yet.

Prices I was quoted by the lab are as follows -- PCR for 70 Mycoplasma and Bartonella species combined -- test for genus only is $265. PCR test for I think 15 babesia species -- test for genus only is $265. Borrelia PCR test for 4 species -- test for genus only is also $265.

Discounts for multiple tests -- 2 PCR tests for $395 or 3 PCR tests for $495.

Unfortunately Fry Labs does not yet have the capability of doing the DNA 16 sequencing to identify the exact species of all these pathogens.

gale said...
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Lyme report: Montgomery County, MD said...

Dogdoc knows more that I do about microbiolgy and molecular biology.

My understanding is that classic Bartonella are bacilli. This means they are rod shaped. Cocci are spherically shaped. Coccobacilli have an appearance which is between rod and sphere. Ultimately it is the DNA which is important. We don't need to fuss about this distinction. Bartonella are known to be intracellular parasites and should be seen inside red blood cells, not swimming freely in blood.

Hemobartonella have been reclassified as Mycoplasms. They do not have a cell wall; so I don't understand how they can gram stain negative. From what I read, they adhere to the outside of red cells and penetrate into red cells.
They are associated with anemia in cats(correct?).Their microscopic morphology is variable- rods and cocci. Again, they are only seen in approximation to red cells. Mycoplasma species are not supposed to be visible under light microscopy, so I am confused by this phenomenon.

16S Ribosomal DNA typing has been around for a decade or more. In the past it was only used for research. Clongen is making this well established technology available for commercial diagnositic purposes. Front line doctors such as myself had never heard of the technology. Researchers can't believe this. This technology is far from cutting edge. This seems to point to a larger issue. Why are clinicians not informed about newer and better diagnostic procedures? Certainly, researchers at major medical schools are familiar with all of these hi tech approaches. IDSA types must know that newer technology can clarify any controversy, if it truly exists, regarding the chronic nature of Lyme and TBD. LLMDS use FRY labs, naively believing this is the best available technology.
This is a little like living in a world in which you think that a horse and buggy is the best from of transportation while folks buzz buy you in cars.

I know many of us are anxiously awaiting an answer. Sometimes we get more questions instead. Hang in there.

Lyme report: Montgomery County, MD said...

If Fry has newer technology great. I was referring to the interpretation of smears which has been used extensively by LLMDS over the past couple of years.
A multi-species PCR test may loose its validiy- Mycoplama and Bartonella?. The tests are only as good as the internal validation developed in the labs performing these tests. None of these new diagnostic tests will be FDA approved. They will be considered experimental. (AND IN FACT, THEY ARE EXPERIMENTAL). The DNA sequencing tests have well established credibility. I don't know the FDA approval status- but this is not a novel test which was put together in the last few weeks.
These words have little meaning to the IDSA. But we have to do better.

I live in a world were positive results from a state of the art lab are discounted by experts because the mill labs do not produce the same results.

Microsporidia are intracellular parasites. These are motile bacteria. Not in the running.

dogdoc said...

Sorry about the Fry labs one- I had just looked them up and nothing showed yet.

At least in my OWN blood, these are definately NOT microsporidia! They look entirely different- microsporidia stain gram positive and have entirely different morphological characteristics.

We need to be careful when describing morphological characteristics and cellular locations to describe the species and not just the genus. I believe this is the source of confusion. For example some Bartonella are capable of invading rbc's and are found inside and some are not and are found free floating and adherent to rbc's exteriors.

In this case, I think the main issue is to get an ID of exactly what the organisms are. There are many methods of ID- the only completely specific is to DNA fingerprint. Since this technology has become widely available (in research I mean) and large numbers of bacteria and other organisms have been catalogued, this is the only method standardly used in research. It has superceded all the other methods- immunostains, pcrs, cultures, ect. - because of its specificity. Out in clinical practice, we have not had the technology available or at least within a reasonable price range.

"Can't" culture is a misnomer- very few things cannot be cultured in some way. There are many special media, cell cultures, egg passage, anaerobic systems, ect. Researchers culture things very well in study of them. However, our standard labs have very low yield or completely miss many organisms. Its like asking Labquest to DNA fingerprint. You should see the miss rates of the cultures done thru our bioattack preparedness system - these were on easy to culture and ID organisms designed to key down as brucella species. Commercial labs performed horribly.
Once we get a fingerprint, we can work on finding some one who can culture it and see if we can get sensitivites. They are even using real time pcr methods for antibiotic sensitvities with some hard to culture organisms life Coxiella (agent of Q fever). Technology is complex but fascinating.
I would like to point out that the reason why this stuff is bleak looking sometimes is because we haven't figured out all of what is causing it and how to treat it yet. Look at AIDS in the 80's and now- big difference already and that disease is really horrible. This one is better. It is just not being worked on at any major level. We can start doing some of that by allying with research oriented folks like at Clongen and publishing. Usually that is the role of teaching hospitals, but they aren't doing their job here. Best would be to find small scale funding and copublish studies. And series of clinical cases reports of course. This is a good place to start. IF we can figure out what organisms and immune functions are involved- then we can get companies with real resources and money to take over. There is big money in developing new drugs and immune system altering products for common diseases in which no good treatment exists. Pharm R&D is faster and more effective than anything else we have to call on right now. But they can't be looking for "BLO's" and phantoms. We need to give them real organisms, real data of immune and cytokine dysfunctions, real incidence in subpopulations. Conjecture on what something might be won't help. We need DNA fingerprints, we need data. This disease complex is common enough that if we could get a handle on it, pharmaceutical companies would pick it up in a heart beat. They don't care about insurance companies or anything but making a buck. Look at the money made from MS immunomodulators. That is a fairly small population segment disease.

dogdoc said...

OOps- forgot to answer Haemobart ? Were 3 species I beleive transferred to mycoplasma- felis, canis and muris. Felis is supposed to stain gram neg (causes anemia in cats yes). We never gram stain it- turns purple in standard blood smears and you can see it just fine. Gram stains don't stain intracellular or cell wall less organisms - some others stain poorly as well. I beleive most Mycoplasmas and ureaplasmas stain pretty well with diff quick and other romanoski types stains. I think that is the source of confusion. Gram stains are designed to stain and differentiate cell wall structure. Our standard diff quik and similar blood stains stain intracellular components like chromatin and other structures as well as all different things- think of the variation of staining in just a blood smear of the different blood cells, platelets, ect. Really in practice, we diff quik everything first. We can see it all then- cells, bacteria, yeasts, parasites. If we need to, we go back and gram stain bacteria to see if they are gram positive or negative. Usually we get lazy and assume- that big blunt ended rod in the urine is assumed gram negative.

atomicdoc said...


Have you used zoologix, they do animal pcr with primers for

Bartonella henselae, B. bacilliformis, B. claridgeiae, B. elizabethae, B. quintana and B. vinsonii subsp. berkhoffii.

If so how was the service?

Also on the bartonella pathology, I always understood it to be a extracellular organism. The electron micrographs that I have seen show it adhereing to the RBC walls but pitting them so that it looks like they are inside. This was however many years ago???

atomicdoc said...
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atomicdoc said...

Forgot to mention, a vet friend of mine told me the pcr panels where pretty inexpensive ~$70 or so for a panel of 5 tests. He has not used them yet.

gale said...
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dogdoc said...

It's so refreshing having knowledgable people to blog with.

I have not used Zoologix. I know one LLMD that sends similar panel to NC states vet lab. Gets positives but also know of negative that is Fry lab positive.

Fry lab does blood smears with blood stains. Perhaps we should contact him and see if he can do wet mounts on his samples for a week or two and see if what he is seeing is motile also? That would be nice to know.

Microsporidia- yes, the spore form in lifecycle is what you see. Intracellularly in clusters usually. Did the pathologist look at stained blood smear only or gram, silver, and wet mount smears? I'm not a pathologist or an expert, but I have never heard of anything with a mobile spore! Spores don't swim by definition. Also- that is interesting. Must mean the pathologist was looking at intracellular inclusions to be calling them microsporridia. On the Fry slides I've seen posted on line, the "Bartonella" labled organisms were mostly extracellular and adherent to rbc margains. These looked like my own smears on my microscope. The Fry labs organisms labled "Babesia" again on the internet posted slides I've seen were variable intercellular organisms- I thought some looked like different babesias and there were some I might have called microsporidia. As far as I know, Clongen has found no intracellular organisms on the smears he has done. I certainly looked thru quite a number of my own and found none.

A note on microsporidia, do you have GI signs- chronic severe diarrhea and wasting/ weight loss? That is primarily a gi organism that can become systemic. Nasty hard to treat stuff. Should be very obvious clinically.

None of the Bartonella that I know of are obligately intracellular. All that I have seen in human pathogens and opportunists have been found in intracellular locations as well as the less pathogenic ones adhering to the surface of rbc's.

All of this cytological discussion is why we need DNA fingerprints- too much could be and looks like. We need IS.

Anybody who has a friend with EM- I volunteer blood.

On titers and PCR's cross species and specific. Titers for Bartonellas cross react between some and not others. This is a source of confusion. Recent study comparing rodent prepared and human Bartonellas found some of the rodent species showed serologic evidence in sick humans (real evidence- seroconversion and high titers forming over course of disease). Some cross reacted with human species serology, and some did not. Bartonella serology is also know to cross react with Coxiella (Q fever) and Chlamydias.
These are chronic intracellular bacteria that could be involved in this syndrome also.

On PCR's, all depends on if species you are testing has the exact sequence- one little mutation and you are negative. Our multi species PCR's may not be as multispecies as we think when all organisms are found and sequenced. Thats when we get to start renaming genus designations. The one thing that you can count on in microbiology is that organisms will be restudied and reclassified on an ongoing basis as we were previously wrong!

dogdoc said...

Tyge- I am very interested in your immune values. I don't find much on this and think it may be a key peice of the puzzle we are not testing in different peoples reactions. You said you are running high TNF's and low CD 20's with weak I am assuming fuction tests. Exactly what did you have tested and where? Multiple times? Any other immune parameters done- ie IgG or IgA profiles? Do you have markers of systemic inflammation or autoimmune phenomena- ie elevated CRP, sed rate, C3a/C4a, RA factor, ect. ?

dogdoc said...

Last bit of curiousity- why are we using a veterinary lab in the Philipines? My ex father in law (also a vet) went to UP. Small world.

lymie said...

What do you think of this:

Scanning electron micrographs of sheep (A) and chicken (B) erythrocytes after in vitro infection with a clonal derivative of M. gallisepticum strain Rlow. The arrows indicate mycoplasmas or imprints of mycoplasmas appearing to sink into the erythrocyte surface.

gale said...
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dogdoc said...

Remember- I'm not a microbiologist or a pathologist and I really don't know that much. I never heard of it not showing up in gut biopsies and being in the bloodstream. Your immune changes are interesting -they are consistant with what I've been putting together in bits and pieces from here and there. I'll mail for rest.

atomicdoc said...

Don't know where this mesage went so I'll try again.


Take a look at whipple's disease if you have not done so already.

dogdoc said...

Lymie- missed your comment, sorry. I am no expert on mycoplasmas. From what I know, most of the ones considered human pathogens adhere to respiratory and urogenital epithelium as well as lymphocytes and invade from there. Some of the vet ones attach to and invade rbcs (those reclassified as mycoplasmas esp). Other vet pathogens are primarily respiratory pathogens that can become systemic as well. I've only done a little research in this area.

gale said...
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dogdoc said...

Which vit d is low? Different groups seem to check different things.

loup said...


I also have Fry labs rare cocobacilli adherent to erythrocytes suggestive of Hemobart/Mycoplasma spp.

A pathologist (same one as tyge?)said it looks like microsporidia, we cannot identify it with PCR, it is untreatable.

I have been chronically ill for 6 years with urethral pain syndrome. I know from my various symptoms I definately have an infection. Doctors can't find anything. I recently had the Panel C test from neuroimmunologylabs. Came back Equivocal for Borrelia garinii and C2+C6 antibodies. Lots of Abnormal neurologic antibodies. Low Lymphocytes. Thats it! So I don't know whether this new organism is causing the infection in my urethral glands or if I have Lyme or Mycoplasma or Chlamydia etc. somehow hiding in my urethra. Is this new organism a red herring that I should give up pursuing? I think many people I am in contact with who have chronic prostatitis and IC will benefit from knowing the answer to this mystery.

dogdoc said...

Which abnormal neuro antibodies? I don't think these bacteria could be a complete red herring- they may not be the primary pathogen, but you just don't walk around bacteremic. Somethings got to be wrong immune wise. People that have bacteremia usually get septic and die. I'm no doc of people, but perhaps he will chime in here. What about Ureaplasma/ genital species mycoplasma secondary to immunosupression as possible cause of urogenital signs? E medicine describes this well in people. I would think a course of doxycyline combined with levoquin would cover the standard antibiotic resistances of ureaplasma/m. hom. as well as get a start on mystery organism if it is either a bart or a mycoplasma. That would also get started on that possible Borrelia. Seems to me a faster answer in a couple of months than more tests if that hasn't already been done. I'm not a doc- ask yours. I was just musing out loud on how one could cover those organisms and use drugs with decent prostate penetration (at least in dogs which I do treat).

gale said...
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gale said...
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loup said...

Thanks for the response. Your suggestion is more helpful than anything else I've been offered in the last 6 years. I've seen many docs/specialists. Either they say I can't possibly have an infection after 2 weeks doxycycline etc. or they believe me but shake me off as they don't know what to suggest. Saw new doc yesterday. He offered me macrodantin again (as I said I'd felt some improvement on that before!) I declined as I feel I need a better plan of action than that. The alternative he said was to continue doing what I'm doing which is managing the illness myself and searching for help elsewhere.

Oh - I had chronic tonsillitis as a child until removed and chronic sinusitis as adult so suggestion that this organism is suppressing my immune system makes sense. Do you think these illnesses can be caused by ureaplasma/mycoplasma? I currently get intermittent PID.

The abnormal neurologic antibodies I have are: BBB Protein IgM, Myelin Basic Protein IgA and IgM and Neurofilament IgM. I also have smooth muscle antibodies. I have a low grade fever.

tyge, yes it is the same pathologist I am in Europe too. I contacted you on lymeneteurope. Yes my symptoms are worse in the last 12 months and also new ones. Don't know whether it is due to chronic infection or mystery organism. I haven't tried treating it - still hoping for more info from Fry - I think you have better access to labs/docs than me. They have re-instated the thread on Lymenet about Fry's NEW bacteria.

dogdoc said...

Hi guys. Haven't had much time this pm to get back to anybody.

On mystery organism and possible ID- Dr K at Clongen has started first run of samples so hopefully that won't be far off. If mystery report is correct, off the cuff guess is we are looking for a Burkholderi possibly B cepacia, perhaps with increased pathogenicity by gaining B psuedomallei genes. Lots of antibiotic resistance possible, but we can kill effectively with appropriate drug combos with longterm I use believe. Studied that group in past- will need to go thru research with fine tooth comb when I get a chance.

On microsporidia- looked up a bit more in humans. Vet pathogen and it seems to be similar in humans. Did you guys get PCR's done on your intestinal biopsies? I could see pathologists and docs trying to bark up that for the longterm intestinal disease. A motile organism seen under light microscopy doesn't make sense for that- that form is too small. Did they know it was motile and not the cysts? I bet no one looked at a wet mount. By the way, if it is burkholderi, I think there is a gliche in ID I remember- have to use the interspacer sequence instead or something like that. Faint memory- I'll go back and look up when I get a chance. Didn't use the standard dna primers or something.
If cellular immunity is affected by this disease group (which I believe there are good signs of) then we should be susceptible to many intracellular pathogens- whether bacteria like mycoplasma, or blood parasites like babesia, or a whole host of things including those like microsporidia. Half of our immune system is immunosuppressed (like half AIDS so to speak) and the other half can be over active making antibodies against all sorts of things like our myelin. Nih published an extensive guideline for the diagnosis and treatment of opportunistic infections in AIDS. Some of these may be quite applicable to us. I'll pull mine out when I get a chance and review microsporridias and ureaplasmas. I beleive mepron has some efficacy against microsporidia as well as toxoplasmosis and babesia. Urogenital mycoplasmas and ureaplasmas are well described as causing problems in urogenital tracts of immunosupressed folks (not just AIDS but diabetes and the like also). You guys don't have LLMD's there- perhaps you have a good HIV/AIDS specialist that might be willing to help you look for these types of infections? They would be used to trying to diagnose them.
A long time ago, docs didn't have all the fancy tests. Vets still don't have use of them financially for many patients. We do as the country docs used to- treat the treatable. So you don't know exactly what you killed we that antibiotic- you got response to therapy and that is more important than a name or a test anyway. Ideal to have all of those- doesn't always happen.
If the mystery organism is a Burkholderi, it might be better to use minocycline with the levaquin and perhaps with bactrim or suprax. I don't understand what the big deal is trying antibiotics- they'll try immunosuppressives and shell out hefty pysch drugs and painkillers like they were going out of style. But oh know- god forbid, not the levaquin. That might hurt something! Sorry- its late here and I'm tired and punchy. Antibiotics can have serious side effects and need to be used and monitored carefully. The side effect of leaving someone to fall apart in pain by leaving someone unteated before you've exhausted everything that can be done is a big one. Patients shouldn't have to have some thing with a specific test and protocol before some one tries to help them. You should exhaust all that first- but if diagnosis is not forthcoming, you should try to help your patient. Who knows- you might find something that treats it and end up helping the next 15 patients that have the same thing with that knowledge. Why is that wrong? We are supposed to become docs to help alleviate suffering. Or was that just my oath? Better get some sleep -night.

loup said...

Could I contact you by private email? I have a couple of questions.

I can be contacted on


lymie said...

I was not suggesting that the mycoplasma species depicting at the website I posted was found in humans also. The purpose was to show the behavior and appearance, and note that it rather resembles what is being found in the Fry blood smear results.

However, I don't think that lab has yet decided on a positive ID. The lymenet thread someone else mentioned here says a Dept of Defense grant is going to fund further work. Hope this is correct as it might get farther than with NIH or CDC, who are apparently trying not to find new things.

dogdoc said...

Lymie- sorry, didn't mean to make it sound that way. Some of the vet mycoplasmas I know about which were renamed look like this stuff as far as adhering to rbcs- I agree. The human mycoplasma pathogens I have been able to find pics of don't seem to. These are just what has been described for these- we are always finding new things and reclassifying new things in all different species.
If they are- I have something different because they swim. If the thread is correct and DOD is involved, we may get somewhere faster. Unusual- they have all their own labs and such in place. Big operations. Wouldn't need to outsource to such a little operation. Something sounds fishy.

Seibertneurolyme said...


Interesting combo of antibiotics -- levaquin, bactrim and minocycline. Unfortunately, that didn't work for hubby. Next he stopped the Levaquin and phased in Zithromax, Rifampin and Alinia.

Pretty sure the mystery bug survived that 5 drug combo as well. Will know more in a week or so.

Unfortunately I think hubby has Bartonella, Mycoplasma and the mystery bug all 3 so that complicates matters. Add in Lyme and Babesia and a few viruses such as Borna virus and you have a big mess.

I do know which herbs put hubby in the hospital last year -- they worked too well and he broke out with lots of new petachia. If his Clongen tests show the mystery bug then I think I will ramp up the herbs as they seem just as effective or maybe more so than the antibiotics.

gale said...

when the rumours about proteobacteria as the ID of the "dots" began to appear I contacted the Lab doc who had made a negative "PAN bacteria" on my blood.
his reaction was that such a finding was the expression for contamination.he certainly knows what he is talking about.But I got the feeling that he was thinking,"I found it too, but maybe I made the wrong decision not to pursue the finding?"

However for the people here that have to find medical assistance far away from "germantown" be prepared for this kind of reasoning,when/if dr K comes up with an ID.

gale said...
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