Biofilms and their Implications Regarding Treatment

A few weeks ago Joyce peeked my curiousity regarding biofilms, gallium and persistent infectionsi

It's an area that as I have delved further into over the past several weeks,  I have come to realize has very important implications for the treatment of stealth infections such as CPNi, mycoplasma and borrelia.

What follows is a synopsis from some of my personal research notes.  Please don't consider it scientific fact as I didn't take the time to footnote but I did want to throw this topic out to the group for pondering and possible discussion.  

Biofilms for those not familiar are the most common mode of bacterial growth in nature. 

An easy explanation is that they represent colonies of bacteria, often including several life forms of that bacteria in a gelatinous type matrix (think jello).  They also often include other types of bacteria, protozoa, fungi and algae that all band together to increase the odds of survival of the individual members.  Sort of like bacterial cities or rather fortresses. 

Some common examples that you might be familiar with would be the scum on a stagnant pond or the slimy feeling of rocks in streams, etc... 

That slimy gelatinous matrix is what holds the bacteria together in a biofilm formation.    It does so for many reasons. 

First it acts as a shield for the cells within the biofilm protecting them from hostile substances such as disinfectants, detergents and antibioticsi.   The gel matrix literally making it tougher for the antibiotics to get to the bacteria. 

Under certain conditions, the slimy matrix can even protect a biofilm/bacterial city enough that it can actually become fossilized.  A recent article that I read on the melting polar ice caps indicated that scientists have concerns about biofilms of previously eradicated bacteria such as small pox resurfacing from the melting ice.

Second, the matrix acts as a communication system allowing the bacteria to share information amongst themselves thereby increasing their likelihood of survival. 

Third,  the gelatinous matrix allows at least some bacteria to share food and other life giving substances. 

Interestingly enough, the same species of bacteria in a biofilm is often quiet different from free floating bacteria in the same species.  For example mutated CPN in a biofilm might be harder to eradicate than CPN that is a free floating bacteria.  This bacterial mutation is likely caused by the sharing of information, food and other life giving substances and it makes the bacteria much more harmful to human and animal health.

Biofilms, in addition to being found consistently throughout nature are also found in humans.  They are implicated in catheter infections as well as other nosocomially acquired infections.

Here< is a great slide presentation from Alan MacDonald, MD, a pathologist and researcher at a New York health system on the subject of biofilms.  I highly recommend taking a gander.  Several slides contain amazing photographs of bacteria and biofilms.  Slide 19 in Dr. MacDonald's presentation even graphically depicts a biofilm and the issues surrounding antibiotic penetration.  

For those with MS brain lesions, Dr. MacDonald's slide 52, shows an actual biofilm taken from a human brain . 

Amazingly, one of the centers of excellent in biofilms is Montana State University (MSU). 

Here's a quote from their website<, "The word biofilm was coined in 1978 by Bill Costerton, former director of MSU's Center for Biofilm Engineering. In the 29 years since, biofilms have been blamed for causing billions of dollars of damage to U.S. industry by fouling pipes, contaminating products and damaging equipment.

Recently, interest in medical biofilms has exploded. The National Institutes of Health now estimates that nearly 80 percent of all human infections -- everything from cystic fibrosis, to dental plaque, to chronic wounds -- are biofilm based. "

So I don't get caught in the spam filter, I will follow up with another attachment.

Biofilms and Chlamydia  

 The only mention of biofilms that I found in Dr. Strattons patent on chlamydia was the following:

"From Dr. Stratton’s patent “Vitamin C (2 gms bidi) has also been introduced based on the report thatVitamin C (ascorbic acid) at moderate intracellulari concentrations stimulates replication of C. trachomatis (Wang et al., J. Clin. Micro.
30:2551-2554 (1992)) as well as its potential effect on biofilm charge and infectivity of the bacterium and specifically the EBi (Hancock, R. E. W., Annual Review in Microbiology, 38:237-264 (1984))."

There is a good article in the Townsend Newsletter LINK< on CPNi and biofilms specifically.  Too many quotes from the article and I  recommend reading it if you haven't already.  Title is "New discoveries in the successful treatment of Chlamydia, parasites and biofilms"

more to follow in another post to avoid the spam catcher

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Daisy, Will read through this when able. But, here is a LINK< to a common mouthwash that treats Biofilm. Just bought some that I am using to treat osteomyelitis in my jaw. It returned after I had temporarily stopped CAPi for a few weeks to try to recover from secondary porphyriai caused by the IV ABXi. Had it right before starting CAP and it didn't come back until stopping the Doxyi and Roxy. I do think that this mouthwash is helping me to heal by treating the Biofilm. Thank you for your endeavours.  --Minai

 

 

RRMSi, diagnosed 2/04. NACi 4/06. Started Wheldon/Stratton regime 8/30/06. Doxycycline, 8/06, Azith, 10/06. Switched to Roxithromycin 11/06. Psuedo relapse/die-off with hospitalization 1/07. GAD-enhanced MRI of brain and spine shows NO NEW DISEASE ACTIVITY. LDNi 4/07. 1st Tinidazole Pulse, 8/11/07. Keflex 2/08. IV Rocephini 3/08. IV Clindamycin 5/08. USA

Hi Daisy,

Interresting stuff. Have you done a quick search through pubmed on NAC's apparent effect on biofilms by any chance?:

Pubmed search: N acetyl cysteine biofilms <

 

Treatment for Rosaceai<

  • CAPi:  01/06-07/07
  • High-Dose Vit D3, NACi:  07/07-11/08
  • Intermtnt CAP, HDose Vit D3:  11/08-01/09
  • HDose Vit D3, Mg, Zn: 01/09-
And, yet, another "Eureka" moment based on what you quoted from Dr. Stratton's patent...had also stopped drinking the grams of sodium abscorbate mixed with water, too! Thanks, Daisy! --Minai

Red -

I had only seen the tigecycline NAC link as I have been heavily researching tigecycline and it made sense to me as NAC is used as a mucosal thinning agent. 

Didn't think about it much beyond that.  Your links have given me more to go on  and more food for thought regarding increasing NAC dosage.

Thanks!

 

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Another good article on biofilms is in PLOS ( a personal favorite).

Looking for Chinks in the Armor of Bacterial Biofilms, Nov 2007LINK<

Great quote from the article " As the cells grow in these varying environments, they diverge genetically as well. In one study of biofilm growth, Singh says, “if we started with genetically identical population of cells, after five or ten days we'd find that population had actually diversified and was more like an old-growth forest than an monoculture of cells.” Like the forest, Singh suggests, the diversity of the biofilm could be a key element of its robust response to antibioticsi and other assaults.

The diversity clearly lets biofilms recover rapidly. Researchers speak of a small population of cells, called “persisters,” that for one reason or another survive an immunei or antibiotic attack. Afterwards, says Bill Costerton of the University of Southern California, “if you are a persister, you wake up in a puree of the guts of your neighbors that contains every molecule you ever needed.” These well-fed survivors can rapidly reestablish the film once the assault is over, he says. “Biofilms have a regrowth rate that is truly phenomenal.”"

'“If you look at a lot of other treatment regimens for other diseases—cancer, HIV—it's almost always a cocktail of drugs targeting different aspects of the disease process,” says Dartmouth's O'Toole. Combining antibiotics with other compounds that disrupt the formation or survival of the film might render the bacteria more susceptible to antibiotics—and could reduce antibiotic resistance, as well." 

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Great article! Has me wondering which of the recommended CAPi supplementsi contain or are phages? Vitamin C? NACi? Have even seen sea salt mentioned. Did stop drinking and irrigating my sinuses, with sea salt, too. Sorry to over simplify, have just been so, so ill. You've probably found this, too, Daisy...but it's a LINK< to a list of 11,000+ known bacteriophages. Too much, probably... downloads as a spreadsheet. --Minai

 

 

RRMSi, diagnosed 2/04. NAC 4/06. Started Wheldon/Stratton regime 8/30/06. Doxycycline, 8/06, Azith, 10/06. Switched to Roxithromycin 11/06. Psuedo relapse/die-off with hospitalization 1/07. GAD-enhanced MRI of brain and spine shows NO NEW DISEASE ACTIVITY. LDNi 4/07. 1st Tinidazole Pulse, 8/11/07. Keflex 2/08. IV Rocephini 3/08. IV Clindamycin 5/08. USA
One thing about an obligate intracellulari pathogen like Cpni is that we don't have to worry about it forming biofilms. That's for extracellular pathogens only. In fact, if a pathogen forms biofilms, it's almost by definition not a stealth pathogen, since biofilms are pretty obvious -- they're macroscopic slimy things, which pathologists and researchers often notice without even trying: "oh, yuck, that looks nasty".

That's not to say that some other pathogen couldn't be around and forming biofilms, at the same time as the Cpn is causing problems -- in fact, that probably happens in lots of cases, especially in places like the nose or the teeth where there are surfaces for biofilms to grow on. As for borreliae, they can live extracellularly, and so conceivably could form biofilms... but since McDonald doesn't explain his images, it's hard to tell whether that's really what he's showing.

Smallpox is a virus, which won't be forming biofilms any more than Cpn will. If a smallpox virus did get trapped inside a biofilm, it'd probably get eaten by the resident bacteria.

For an impressive case of treatment of a biofilm, see this abstract<.

It seems that there are a number of agents that have been known to affect biofilms - well - at least surface biofilms as well as some in vitro research.

Lactoferrin, green tea, NACi, Vitamin C, chitosan , digestive enzymes and probiotics, lumbrokinase, melittin from bee stings, GSE, etc...

 

 

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Biofilms and the chronic bladder infection folks

HERE< is quick link to Intracellulari< Bacterial Biofilm-Like Pods in Urinary Tract Infectionsi<

Another< link from PLOS - Detection of Intracellular Bacterial Communities in Human Urinary Tract Infection

Same type of information seems to exist regarding biofilms and bacteria for chronic prostatitis as well.

 

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Interesting editorial quote on biofilms as the potential originators of athrosclerotic plaque from Circulation.

Bacteria and Coronary Atheroma   More Fingerprints but No Smoking Gun              Link<

"Finally, the diversity of bacteria reported in atherectomy samples in the present study and their association with mature or advanced as opposed to early lesions raise the possibility that atherosclerotic plaques may secondarily form functional biofilms.

A biofilm is an assemblage of microbial cells that are associated with a surface in a matrix of polysaccharide material. Biofilm-associated organisms differ from their planktonic counterparts with respect to gene transcription, nutritional needs, secretory protein products, and reproductive rates. Biofilms develop attachments to specific surfaces based on properties of the surface and aqueous medium interaction.

Mature atheroma are just such surfaces because of their eccentricity and the perturbed flow characteristics in the microenvironment. These unique characteristics would explain the lack of efficacy of antibioticsi<2 in clinical trials to date but represent a persistent source of antigenemia fueling a chronic inflammatory state and leaving a plethora of bacterial fingerprints."

 

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Daisy, thanks for posting this info. I am also curious of iodine's effect on biofilms. I recently increased my dosage to 75 mgs a day and am aiming for 100mgs. Jorge Flechas, MD has some things to say about this level of Iodine as antiviral and antibacterial.( in conjunction with B2 and B3) Definite die off at 75 mgs so far. Here's a link on Candida biofilms and Iodine: http://iadr.confex.com/iadr/2007orleans/techprogram/abstract_90538.htm <

Lots of other links out there...

 

Raven

 

CAPi since 8-05 for Cpni and Mycoplasma P. for MS and/or CFSi

Feeling 98% well-going for 100. Very low test for Cpni. CAPi since 8-05 for Cpn/Mycoplasma P.,Lyme, Bartonella, Mold exposure,NACi,BHRT, MethyB12 FIRi Sauna. 1-18-11 begin new treatment plan with naturopath

Norman -

Thanks for catching the small pox.    I meant to say Bubonic Plague (Yersinia).  The article did mention a resurgence of small pox from the melting ice caps but not relative to biofilms.  It's what I get for multitasking and trying to do too many things at once yesterday.

Re MacDonald.  I am a big fan of his research.  He's given this talk several times, so I will see if I can find a transcript.  Believe he's giving it again this week at Univ of New Haven so perhaps the whole talk with his voice will pop up on their website.

It's interesting about the intracellulari bacteria, seems that some studies show many of them such as Legionella and Mycobacteria have two forms - biofilm forming and non biofilm forming.  I'd like to research this more when I get a chance.

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

An interesting subject, Daisy.

Biofilms can really protect bacteria against antibioticsi. I recall a young man (an IV drug user) with a Group B streptococcal endocarditis. The organism was exquisitely sensitive to penicillin, and this was given in massive doses, together with rifampicin and gentamicin. This treatment made not the slightest difference to his rapid decline. (He was not a candidate for operation.) One could postulate another organism, never cultured, breaking down the penicillin and protecting the sensitive streptococcus.

Chlamydiae, causing vast cellular damage, may pave the way for other organisms to form films, as Norman says.

Just as the blood stream isn't sterile, but rather an aquarium, so the disordered arterial plaque is a menagerie.

D W - [Myalgia and hypertension">i (typically 155/95.) Began (2003) taking doxycycline and macrolide and later adding metronidazolei. No medication now. Morning BP typically 110/75]

DW - Thanks for your comments!

All - This was more or less where I was headed with the biofilms relative to treatment implications. 

It seems many posters here at CPNhelp have multiple infectionsi which for some complicate treatment.  Some seem to improve quickly on various versions of CAPi while others struggle along.  I am not always sure this represents duration and severity of one infection or the other but perhaps instead the location or mixture of infections.

Theoretically mixed pathogen biofilm formation may further complicate the picture as well.  Might time prove that you need higher doses, longer pulses, more antibioticsi or more aggressive treatment in your cocktail as my husband has, to successfully eradicate bacteria if you have mixed infections?  At what point and under what circumstances should a more aggressive anti-infective treatment protocol be initiated?

Answers all unclear to me at this time. 

A study from Stamford indicated  "We invented a real-time method to visualize biofilm cells coming in contact with tetracycline, and showed that all the cells in the biofilm were almost instantaneously exposed to the antibiotic, and yet they were more resistant than their planktonic (free-living) counterparts"

Since even intracellulari pathogens can form biofilms on medical equipment surfaces or in public water supplies do they morph and form more resistant life forms as humans are infected with these pathogens?  What difference, if any, does this make in treatment?  Are there clinically proven methods to reduce biofilms invivo?  Again, all answers seem unclear.

I also often wonder about my husband's crainiotomy when they reported that what appeared on a high quality MRI machine to be a solid golf ball sized mass and likely a glioblastomia was really a large sac containing microcysts in a fine trabecular mesh resembling a honeycomb and filled with a preponderance of foamy macrophages and neutrophils.  Immunei system attacking a biofilm?  Unclear?

I think about biofilms being potentially implicated in coronary artery disease as the site of future plaques or biofilms relative to the formation of gad enhancing lesions in the brains of MS'ers. 

Loads of questions in my curious mind to which I find no clear answers.  Interesting subject though.

 

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

This article caught my eye

Clinical and Environmental Burkholderia Strains: Biofilm Production and Intracellulari Survival

Authors: Savoia, Dianella; Zucca, Mario

Source: Current Microbiology<, Volume 54, Number 6, June 2007 , pp. 440-444(5)

Abstract:

Bacteria belonging to the Burkholderia species are important pulmonary pathogens in cystic fibrosis (CF) patients. Their ability to establish chronic and sometimes fatal infectionsi seems linked to the quorum sensing-regulated expression of virulence factors.
We examined 23 Burkholderia isolates, 19 obtained from CF patients and 4 from the environment, to evaluate their ability to form biofilm and to penetrate and replicate inside J774 macrophagic cells. Our results indicate that biofilm formation and intracellular survival are behavioral traits frequently expressed by Burkholderia strains isolated from CF patients.
Successive isolates obtained from each of four chronically infected patients yielded bacteria consistently belonging to the same strain but showing increasing ability to replicate intracellularly and to produce biofilm, possibly due to in vivo bacterial microevolution driven by the selective lung environmental conditions. Protection against antimicrobials granted to burkholderiae by the expression of these two virulence factors might account for the frequent failures of antibiotic treatment in CF patients.

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Daisy, Is it still possible for someone else (alan macdonald?) to look at the specimen (or whatever it is called) of your husband's brain?
--------------- "Chance favors the prepared mind." --Louis Pasteur Husband treating MSi with CAPi

nope - they were suppose to get the 5 specimens in deep freeze for future testing, some grad student in the brain cancer program made a decision to throw the samples out since it wasn't brain cancer.  You can imagine what I had to say when I learned that...

Good thought though!

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Daisy, you are simply amazing along with all our science people here!.

Great information.

 

Thank you

 

CFIDSi/ME, FMSi, MCS, IBSi, EBVi, CMV, Cpni, H1, chronic insomnia, Chronic Lyme, HME, Babesia, Natural HRT-menopause, NAC 2.4 gm,Full CAP 6-2-07, all supplementsi+Iodorol, Inositol-depression, ultra Chitosan, L lysine Pulse#27 04-19-10 1gm Flagyli/day-5 days<

I found a comment made in this article< (link is to the PDF) interesting:
A disarmingly simple approach to sterilize an infection was first proposed by Bigger in 1944 (REF. 1). The proposal is to kill bacterial cells with a high dose of an antibiotic, then allow the antibiotic concentration to decrease, which will enable persisters to resuscitate and start to grow. If a second dose of antibiotic is administered shortly after persisters start to grow, a complete sterilization might be achieved. This approach is successful in vitro, and a P. aeruginosa biofilm can essentially be sterilized with 2 consecutive applications of a fluoroquinolone (K. L., unpublished observations). Perhaps understandably, this approach has not been received with enthusiasm by specialists in clinical microbiology. The goal of established therapies is to maintain the plasma level of an antibiotic at a maximum concentration, in order to discourage the development of resistance. Most importantly, an optimal pulse-dosing regimen would probably vary from patient to patient. However, it seems that some patients might have inadvertently taken solving the problem of intractable persistent infectionsi into their own hands. Individuals who suffer from persistent infections that require a lengthy therapy are often cured, but why a year-long regimen is better than a month-long one is unclear. An efficacious fluctuating dose of antibioticsi administered serendipitously by the patient might be responsible for persister eradication in these cases. The patients might adjust drug dosing simply through being absent-minded, which sooner or later could produce the perfect drug-administration regimen. Curing persistent infections might therefore result from patient non-compliance. Analysing how persistent infections are cured might shed light on the likelihood of developing a rational regimen for the pulse-dosing sterilization of infection.
(In case that link breaks, the article is Persister cells, dormancy and infectious disease, by K. Lewis, Nat Rev Microbiol. 2007 Jan;5(1):48-56.)

Norman - Great article !

I can just imagine some CAPper going into a doctor's office one day and dealing with a doctor's reluctance to prescribe long term multi drug antibiotic cocktails by dazzling them with information on biofilms.

The majority of practicing doctor's haven't a clue what they are.

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Good girl Daisy,  I was hoping you would dive more deeply into this subject.  So, did you see any gallium when you were swimming around down there?

Joyce~caregiver-advocate in Dallas for Steve J (SPMSi).  CAPi since August 06, Cpni, Mpn, B. burgdorferi, systemic candidiasis, EBVi, CMV & other herpes family viral infectionsi, elevated heavy metals, gluten+casein sensitivity. 

Joyce -

I did look at a good deal of information on gallium but was hoping to find something a little more human friendly to experiment with.  Been digging around to see if I can separate fact from fiction on some of the natural alts mentioned in post above.  No plans yet... just interested. 

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Norman and Daisy, you might be interested in seeing Alan McDonald's discussion of the importance of biofilms in the treatment of Lyme disease in the new film "Under Our Skin".

The film is being shown on a limited basis around the country but you can purchase the dvd for $41 on the website, www.underourskin.com<. There are links to youtube clips on the main page, though I don't think they include McDonald's discussion

Thanks for your research on this subject.

Marysia

And I thought a biofilm was one of those movies they showed us in Health class, and that a gallium was the collective plural of a gal, you know, one gal, many gallium?

 

CAPi for Cpni 11/04. Dxi: 25+yrs CFSi & FMSi. Currently: 250 aithromycin mwf, doxycycline 100mg BIDi, restarted Tinii pulses; Vit D2000 units, T4 & T3, 6mg Iodoral

Hi Daisy,

Just ran across this and thought you might be interested if you hadn't seen it already:

The human host defence peptide LL-37 prevents bacterial biofilm formation<

Potentially one more reason for higher vitamin di levels... 

 

Treatment for Rosaceai<

  • CAPi:  01/06-07/07
  • High-Dose Vit D3, NACi:  07/07-11/08
  • Intermtnt CAP, HDose Vit D3:  11/08-01/09
  • HDose Vit D3, Mg, Zn: 01/09-
Here< is link to a great recent article on Biofilms.  It's the second link down on the webpage "Biofilms and Chronic Infection"

Daisy - Husband on CAPi 5/07.  Husband died from Acute Myelogenous Leukemia Secondary to the Infusion of Novantrone.  Ie - the treatment with the conventional MSi drugs killed him.

Daisy on her own CAP 11/2012. 

Hello Daisy ... I tried your link and it didn't work for me.   MM

2002:CFSi. (2008-09:CPNi - CAPi/5 pulses)  3/2010: Restart CAP: 200Doxy/250Zith-MWF/Tinii pulses. 6/2010: HighBP/Benicar, 7/2010: EBVi, HHV6. 2012: 6,000 IU daily vit. D., Citioline CDP choline = sleep improvement dramatic. 

Try this.  http://home.swipnet.se/isop/biofilms.htm<

The difference between what we do and what we are capable of doing would suffice to solve most of the world’s problems. Mohandas Gandhi

Thank you. Got it, but had to copy & paste it. Interesting article. Finding agents to break down the biofilm so the antibiotic works is fascinating stuff. Thanks to you folks I've got DH using biotene PBF toothpaste & mouthwash. Will soon see if his next check up shows progress. Hoping for it. MM

2002:CFSi. (2008-09:CPNi - CAPi/5 pulses)  3/2010: Restart CAP: 200Doxy/250Zith-MWF/Tinii pulses. 6/2010: HighBP/Benicar, 7/2010: EBVi, HHV6. 2012: 6,000 IU daily vit. D., Citioline CDP choline = sleep improvement dramatic. 

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