The Difficulty of Culturing Spirochetes
by Tom Grier
Why is the culturing of spirochetes important? The obvious answer is that culturing offers the strongest of all proofs that an infection is present. But let me give you a historical example of why culturing spirochetes is so important. As far back as 1918 physicians related the lesions in the brains of MS patients that died to the presence of spirochetal bacteria. Using the techniques of the day, they were able to fix and stain the tissue and see the bacteria, but they could not culture it. Later, in the 1920's, microbiologists such as the late Gabriel Steiner (as in Steiner silver stain) were able to transfer this infectious agent to animal models and demonstrate that the bacteria had a tropism for the animals brain - and that the organism could then be isolated from the animal model and reinfect a second set of animals. This strongly suggested a possible spirochetal etiology for MS, but the work fell into obscurity. After 1957, almost nothing more was written on the subject until 1981, when Lyme Disease - which can mimic MS - was discovered to be a spirochete.
If Dr. Steiner had been able to culture the bacteria that he and others referred to as the "MS organism", wonderful things could have taken place. First of all, research on MS patients could have been easier, since a blood culture is always preferable to a brain biopsy! Also, once the bacteria is grown and purified in culture, other tests can be developed, such as fluorescent antibody stains or using the bacteria to create ELISA tests and Western Blots. Without a stable lab culture to use as source material, these antibody-based tests are impractical.
If an "MS spirochete" could be cultured, it would mean a PCR test - which are inexpensive and highly sensitive - could be developed . But most importantly, if Dr. Steiner could have cultured his spirochete, his work probably would never had died out in obscurity! If we can learn to cultivate spirochetes with greater proficiency, perhaps that work can be resurrected.
In the beginning : Ever since the great German physician, Robert Koch (1843-1910), first found that the cause of syphilis was a spirochete, scientists have sought to culture it. Unfortunately, in the past 100 years, the syphilis spirochete Treponema pallidum has never been successfully cultivated in vitro. The syphilis organism simply cannot be grown in any known culture media. The only way T. pallidum is kept alive in the laboratory is to transfer the live organism directly from one infected host to another, such as taking the infected exudate from a patient with a syphilitic lesion and transferring it to a living animal like the rabbit. Unfortunately, such animal systems are cumbersome and expensive, and do not reflect the exact pathogenic nature of the organism as when it is in the human host.
Fact: The only Treponemes that have ever been cultured are nonpathogenic. (Non disease causing.) Simply stated, the culturing of the syphilis spirochete is impossible in the absence of a living host. Why? We don't know why!
Other disease causing spirochetes, such as the genus Borrelia which causes Lyme disease and relapsing fever, have also proven difficult to culture. When they are cultured successfully, they require a very complicated culture medium and long incubation times - usually 12-48 hours or more, compared to 1-2 hours for other types of bacteria. (Some pathogenic Borrelia strains have taken months to culture.) The cultured bacteria usually exhibit a change in their surface proteins, indicating that the organisms have adapted to the culture media differently than they would in the human host. In other words, most disease causing spirochetes are very difficult, if not impossible, to culture! While we don't know why, we do have some clues as to what may be missing.
Spirochetes are in many ways more evolved than other bacteria. At times they almost exhibit animal-like tendencies. Borrelia bacteria have an internal propulsion system that allows them to be highly motile. This is an evolutionary advantage over nonmotile bacteria that allows Borrelia to seek out those areas in a host that are best suited to its needs. The spirochete can swim as easily forward as it can backward, and it now appears that there are receptors for host tissue in both tips of the spirochete. This allows each end of the bacteria to seek out sites of attachment as well as change direction when a better target is located.
The most important adaptation, however, may be the recently observed phenomena that Borrelia burgdorferi may have many morphogenic states and can change their appearance both in shape and chemical structure. Morphogenic changes is another way of saying that the bacteria can adapt itself to the host that it happens to be in, such as a tick, a mouse, or a man. These morphogenic changes may be as subtle as the absence of one surface protein from one division to the next, or, as some have suggested, the spirochete may shed its cell wall entirely and become a spherical-shaped blob, devoid of many of its proteins.
Borrelia bacteria appear to have several layers of protection not commonly seen in other bacteria. One such layer is a glyco-protein gel coat (the S-layer is not commonly seen in-vitro), which has been described as a kind of jelly which can encapsulate the bacteria. Beneath this lies a cell wall, and beneath that is a three-layer membrane. Embedded within the cell wall and membrane are a variety of proteins. Some of these proteins are now known to be receptors to attach to certain host tissues and cells. More insidious in nature are those proteins on the outer cell membrane that seem to be present to purposely fool the immune systems of mammalian hosts.
At least one OSP (outer surface protein) appears to trick the host immune system into producing antibodies which are ineffective and harmlessly coat the bacteria, but do not initiate the body's compliment system to destroy the pathogen. (Blocking antibodies in the IgG4 subclass has been reported for almost ten years.) Further, the Borrelia bacteria can turn off several surface proteins, causing the immune system to fall behind in its ability to track and kill the bacteria.
Relapses in relapsing fever are caused by the Borrelia bacteria changing its OSP proteins every few weeks, thus causing the body to develop a new febrile state of fevers and sweats when the immune system again starts its attack against the new set of bacterial proteins. This gives the bacteria a chance to use its motility to escape detection, and ultimately destruction, by sequestering themselves in immune privileged sites. Sites such as the brain and tendons are places where the immune system has a difficult time penetrating. What does this have to do with culturing?
The most common method of culturing is to use a blood sample to look for the organism, but Borrelia bacteria can quickly leave the blood stream. If there are fewer bacteria in the blood, then culturing becomes more difficult. So, the bacteria's affinity for certain tissues outside the blood stream, combined with its motility, makes the bacteria less likely to be in the blood. This means less successful blood cultures. Usually less than ten milliliters (10 cc) are used for a blood culture. A more successful culture rate might be achieved by taking an entire unit of blood (500 cc), and then using special lab techniques to concentrate the organisms before culturing is attempted. Although using larger blood samples have worked experimentally, it has obvious commercial limitations. Therefore, to maintain patient compliance, ease of shipping, and to reduce costs and potential side effects, 10 ml, inadequate or not, remains the standard sample size that is taken from a patient.
The most successful way to culture the Lyme spirochete from a patient has always been to take a skin biopsy from the edge of the bull's-eye rash and culture it in a commercially available culture media, such as BSK-II T (Barbour, Stoener, Kelly media). The success rate of this technique seems to vary from about 10-80 % from lab to lab. (In an abstract by D. Jesse Goodman from the U of MN comparing a new PCR test to culturing, they reported a dismal 4 % success rate at culturing EM rashes from early Lyme cases.) Restricting culturing to the EM rash is more successful, but what does it prove? The rash is already proof of the illness. Unfortunately, the success rate of culturing Bb from the spinal fluid, blood, and tissue, using commercial media, has been less successful than that of culturing the rash.
One of the things that makes spirochetes more difficult to culture from other bacteria is the fact that they cannot tolerate large quantities of oxygen. They are nearly completely anaerobic. (Technically B. burgdorferi is microaerophillic - meaning it can tolerate small amounts of oxygen.) Anaerobic bacteria generally require some special consideration in the laboratory so as not to mix large quantities of air into the media, and incubating the culture in a manner that reduces its exposure to circulating air, such as adding pressurized C02, nitrogen, and hydrogen to displace oxygenated air. Most labs that culture spirochetes do not use full anaerobic techniques, mostly due to the long incubation times. Anaerobic procedures are cumbersome and expensive, and techniques and results vary tremendously from lab to lab. I have not seen a study published comparing the use of full anaerobic culturing methods to standard culturing practice, so the effect that oxygen has on the process is not well documented.
This inability of Borrelia species to tolerate oxygen, combined with its slow reproduction rate, also causes another problem for microbiologists - the inability to easily test for antibiotic sensitivities. If you get an infection, such as an ear, bladder, or throat infection, the doctor will quite commonly order an antibiotic sensitivity profile. This is done because some bacteria have developed mechanisms to resist certain antibiotics. While one patient's strep throat may respond to amoxicillin, another patient's strep infection may be completely resistant to the drug. So, researchers developed a relatively quick technique to test for antibiotic effectiveness. It is called the Kirby-Bauer method of antibiotic sensitivity testing.
In the Kirby-Bauer method of testing, a large petri dish is filled with a special media, called nutrient agar, that hardens into a clear gel. In most cases, such as with a throat culture, it is possible to smear this plate with a sample of the bacteria from a previous patient culture and, within 24 hours, the plate will be covered with bacterial colonies. However, before the infected plate is incubated, several tiny paper disks saturated with several classes of different antibiotics are added to the plate. If the bacteria is sensitive to an antibiotic, the bacteria will not grow in and around that particular paper disk. This way, a doctor can know if a class of antibiotics will be effective, such as the teyracycline class verses the penicillin class, etc. Without this method of testing , the doctor must make a guess with each and every Lyme patient as to which antibiotic to use, with no way of knowing if it will be effective.
Why doesn't the Kirby-Bauer method of sensitivity testing work on spirochetes? This is probably due to several factors. First, there is too much exposure to oxygen, over a period of several weeks, to grow surface colonies of anaerobic Borrelia species. Although this technique has been tried even under anaerobic conditions, I am not aware of anyone growing spirochetes in semi solid agar. Second, Borrelia species require a very complex growth media which has never been successfully combined with a semi-solid nutrient agar. All spirochete cultures are done in solution. Third, even if spirochetes grow, they would not become surface colonies, and it is doubtful that you could visually observe zones of inhibition from the very small number of spirochetes that might survive deep within the agar. In short, anyone who can make this method of antibiotic sensitivity testing practical for Borrelia burgdorferi, or any other spirochete, would be destined to win major recognition and awards in microbiology, not to mention its obvious financial rewards!
I have never seen the Kirby-Bauer method of antibiotic sensitivity testing successfully applied to spirochetes, but other methods have been used. Ever since Borrelia species have been cultured in the lab, researchers have sought to find the most effective antibiotic. This was usually done by using a laboratory strain of Borrelia, such as B-31, and culturing the bacteria in several tubes of liquid media. Each tube would be incubated with its own separate antibiotic, and would later be compared to the control tube to determine the degree of bacterial growth inhibition. This is a time consuming and relatively labor intensive process that has never been standardized. This makes it expensive and generally impractical to market commercially. Growing lab strains of B. burgdorferi is hard enough, but trying to culture every patient successfully and then do sensitivities is a monumental task. Although one lab has claimed they can do it, their work has yet to be verified or confirmed.
In today's hospitals, doctors are used to ordering sensitivities today and having their results tomorrow. Spirochete sensitivities could take weeks to months. Using a lab strain of Borrelia burgdorferi, like B-31, can give you a generalized overview of antibiotics that are effective in the treatment of Lyme disease, but it does not reflect the exact strain the patient may actually have. For example, a lab director could never tell a doctor to treat an infant with an earache with penicillin simply because the on hand stock of laboratory bacteria happened to be sensitive to penicillin. The doctor would fully expect the patient to be cultured and then that organism, and no other, to be tested. Yet, in the treatment of Lyme disease, the medical community has made huge assumptions about the effectiveness of antibiotic treatments based solely on a few generalized laboratory observations that, in most cases, were made over ten years ago!
Lyme patient's personal accounts with antibiotic therapy have consistently been in conflict with what has been observed in the test tube. In the test tube, Erythromycin is one of the most effective drugs against the Lyme spirochete, yet its use in Lyme patients has been disappointing. Yes, we know Rocephin (ceftriaxone) is effective in Lyme disease, but does it work in every patient? We don't have enough data to really say for certainty that all patients respond equally well.
Speciation: How do you tell one Borrelia species from another? With other types of bacteria, the species was often determined by the nutrient requirements of the bacteria. If two identical-looking bacteria were grown on two slightly different nutrient agars, but you couldn't grow them both on either media, then the bacteria were a different species and had different growth requirements. A very complicated system of trial and elimination based on nutrient requirements ensued and, finally, the exact species was determined. Today, we have better and faster methods to identify bacteria species.
With the Borrelia bacteria, it was determined early on that there was a tremendous variation within the genus, resulting in dozens of species of Borrelia that cause "relapsing fevers". (Oscar Felsenfeld's book, Borrelia, 1971, predates Lyme by almost ten years, but stresses that variations of relapsing fevers due to regional vectors could lead to new, as yet unseen forms of the disease.) Each species was specific to its own tick or louse, and its own secondary host, such as rats and mice. Unfortunately, determining the species of Borrelia has been much more difficult than with other faster and easier to grow bacteria. In the last ten years, however, major advances have occurred in bacteriology. One of the most important is the use of the bacteria's genetic material to determine species.
Early DNA/RNA sequencing work to determine bacterial species concentrated on the bacteria's ribosomal RNA. If the RNA within the ribosomes of the Borrelia varied too much, it was determined to be a new species. Using similarities in rRNA, a phylogenic tree could be made that showed related species of Borrelia could be grouped together in families. Since the discovery and development of mechanized DNA sequencing and PCR, and DNA amplification technology, determining species has become faster and cheaper than ever before.
Ribosomes are organelles inside cells that convert the DNA code into actual proteins to be used by the cell. This occurs in our own cells every second we are alive! Bacteria can slow this process down and become metabolically inactive or dormant.)
Once a new species of any bacteria has been determined based on its genetic code, all one has to do to manufacture a test for determining that species in the future is find a short DNA sequence that is absolutely unique to that species and use it to make a PCR-DNA primer. Then, when you have a sample that contains your suspected bacteria species, you add the PCR-primer and run a PCR test. Within hours, it will - with a great deal of specificity and sensitivity - ascertain if the DNA you are looking for is present in the sample. If the DNA in the sample (culture) is amplified, then you know that you have, in fact, cultured the species you were looking for. If, however, the sample had bacteria of a different species, the PCR test would be negative.
If you had two or more species of bacteria in the same sample, the PCR test could only ascertain the single species you were probing for. For example, lets say you have a culture of spirochetes that you are certain are Borrelia, but you don't know what species - perhaps it is Borrelia burgdorferi or perhaps a European species that causes Lyme, such as Borrelia garinii. If you had specific DNA-PCR primers to these two species, you could tell if you had either of those. However, you could not determine if you had a third Lyme spirochete species, Borrelia azellii - or, for that matter, any other Borrelia species - unless you had specific primers for those species. In a second example, let's say you have a blood culture from a patient that reveals spirochetes, but the PCR tests are negative for B. burgdorferi - what about a relapsing fever spirochete? Unless you have an idea of the species to begin with and already have a PCR-primer for that species at your disposal, there would be no way to determine the exact species without further tests - or the use of different tests, such as an ELISA antibody profile of the patient for relapsing fever.
The point I'm trying to make is that isolating new bacterial pathogens is very difficult. I often hear patients say things like, "Oh, they tested me for everything and I'm fine." First of all, if they tested you for everything, they would have had to run about a thousand separate little tests - and insurance companies don't like that. Second, without a general idea of what to look for, a doctor could easily miss a diagnosis. You can't just throw blood in a test tube and expect everything to suddenly appear under a microscope. You have to start somewhere.
If your doctor orders a Lyme test, you get a Lyme test, not a syphilis test or a relapsing fever test. You get just what your doctor ordered. If you have a brand new disease, you might have to wait your whole life for a diagnosis. Where did all the Lyme patients from the 1940's, 50's, and 60's end up? Some of them probably died before they even heard of the disease! So what other new diseases are out there?
If you happen to culture an entirely new species of spirochete from a patient, you have a lot more work to do to determine what genus and species it is. In today's microbiology lab, this would almost certainly be done by DNA sequencing and by comparing it to known data bases (such as at the NIH) of other similar species. If there were slight variations in the genetic profile, it is considered a new strain. If the DNA sequences are even more varied, it may indeed be a new species. A few years ago, only so-called super computers had the storage capacity and speed to compare such huge data bases, but today it is relatively routine. DNA sequencing is as much a computer based science as it is a lab science.
While isolating a new bacteria is prestigious work that can lead to having a new species named after you, the real advantage that DNA sequencing and PCR technology has brought to medicine is the ability to instantly patent your discoveries. In the past, it was "publish or perish", but today it is "patent or perish", because patents mean money. Today, owning a good PCR primer can mean owning the test that diagnoses the disease. That process almost always depends upon the ability to culture the organism, so that other tests can then be developed, tested, and patented.
What has PCR got to do with the latest controversies in culturing Borrelia burgdorferi? First, despite what some have claimed, you cannot determine a spirochete's genus or species by looking at the bacteria under a light microscope. It certainly would not be evidence that stood a chance in court. Even under an electron microscope, ultrastructural variations between species can be so subtle that trained professionals cannot determine species this way. This is why culture samples are sent away to PCR labs for confirmation. Some researchers have claimed to have cultured a spherical form of Borrelia burgdorferi, known as an L-form. If it is B. burgdorferi, it can be confirmed by the use of the PCR-primer for B. burgdorferi. Just because the spirochetes now appear to be spherical instead of helical, the bacteria's DNA is unchanged. That means you can amplify it using PCR technology.
Nutrient requirements of the Lyme bacterium: It is generally accepted that the nutrient requirements of Borrelia burgdorferi species are the most complicated of any known bacteria. It needs an organic source of iron; it requires NAG; it requires several salts and sugars. Many microbiologists have concocted special liquid soups that are so complicated and difficult to mix, they are commercially nonviable. Those media mixes, which are currently commercially available, have often been accused of lacking all the nutrients necessary to grow fully pathogenic bacteria. In other words, they are designed to cultivate and maintain laboratory strains, but have a poor success rate at culturing pathogens from actual patient's samples of blood, spinal fluid, and tissues such as skin, brain, muscle, and collagen.
It was observed by a European researcher in 1991, that, if you added hydrolyzed mammalian collagen to modified Kelly media T, the Borrelia burgdorferi appeared to become more pathogenic in animal models. Why? It was surmised that the tissue offered some component that was lacking in the culture media. Prior to this observation, other researchers did an interesting experiment. They added cultured Borrelia burgdorferi to a mixture of mammalian tissues, including brain, heart, collagen, muscle, and whole blood vessel. It was found that the Bb bacteria seemed to have a preference for certain tissues.
With equal exposure times, the Lyme spirochete seemed to prefer tendons and brain cells, and the endothelial cells of blood vessels, to the other tissues. Since then, we have learned that there is a probable receptor site on the surface for Bb to attach to - N-acetyl glucasamine (NAG), a component of connective tissue. Although the rat brain model of neuro-Lyme suggests strongly that there is a receptor on neurons for B. burgdorferi, a receptor has yet to be fully described. Further, it now appears, through gene studies and observation, that the Lyme pathogen uses NAG as a food source. This might be how it survives in the tick, as the tick's shell is a very probable source of NAG.
What does this all mean? It means the current culture media that is used may be insufficient in some basic nutrients. More importantly, it may also be lacking the complicated receptor sites found on mammalian tissues that may somehow activate metabolic changes in the bacteria. This is supported by the observation that the Bb bacteria can regulate which surface proteins it will express, depending upon what host it is in - the tick, the mouse, or human. The numerous Borrelia species that cause relapsing fever have been observed to change their surface antigens as they are attacked by the host's immune cells. Is this regulated by chemicals; temperature; chance - or is it a receptor-regulated phenomena?
In one study, Dr. Andrew Pachner, M.D., experimentally infected a mouse with one strain of the Lyme bacteria. After a few weeks, he found the bacteria that was isolated from the brain had somehow become different from the spirochetes in the blood. How? Why? Once the bacteria was exposed to the brain, the bacteria appeared to change its surface proteins so completely that the antibodies from the bloodstream of the same mouse could not recognize the bacteria isolated from the brain of that mouse. Once the Lyme spirochete was isolated within the brain of the mouse, it quickly began to change its surface proteins. This as yet undelineated mechanism of receptor regulated change in antigenic morphology may account for why simple nutrient broths are inadequate to effectively culture spirochetes.
It has been hypothesized by some that, in order to make a more effective spirochetal culture media, it may require the addition of whole tissues to introduce complex three dimensional tissue receptors that trigger specific responses within the bacteria. In other words, just like a three dimensional key fits into a lock and causes the lock to turn, a tissue receptor might trigger the Lyme bacteria to react internally - perhaps to turn on or express a new gene, or turn off a gene currently being expressed.
Also, just like tiny pieces of the key are inadequate to fit in and turn the lock, pieces of the tissue receptors may not be enough to trigger the kind of bacterial growth that occurs within the host. In other words, a simple mixture of nutrients might allow the bacteria to survive, but not thrive! An analogy may be: A human can live on bread and water for a long time, but we require vitamins, minerals, proteins and fats to thrive. A Lyme spirochete might require NAG to survive, but would it do better on a diet of whole cell isolates of human tissue?
Are there different forms of spirochetes? Recently, there has been an ongoing debate as to whether Borrelia burgdorferi can have a nonspiral shape? It has been suggested by Dr. Lida Mattman, Ph.D., and others, that some spirochetes can lose their outer cell walls. When a spirochete loses its cell wall, it loses its' ability to maintain its' spiral shape. It is then hypothesized that it will take on a spherical shape held together by a thin membrane. Such cell wall deficient organisms are known as L-forms, as described by the Lister Institute in London. Since a cell wall deficient spirochete is more difficult to culture, and consequently study, than the classical form, few scientists have considered this unfamiliar form as being pathogenic. The commercially available media appears to be completely ineffective at cultivating this new form of the spirochete, but Lida Mattman and others have claimed success at culturing these new forms using a more complete culture media.
We know that the classic form of Borrelia burgdorferi does not particularly like the bloodstream, but a cell wall deficient form which would lack many of the surface antigens of its' spiral counterpart may well find the bloodstream more likable environment An L-form may be better at avoiding the human immune system, because it has fewer antigens to be targeted by antibodies and the host's immune cells. In other words, different forms of the Lyme spirochete may prefer different tissues.
How and where the L-forms reside in the blood stream remains controversial, but the recent article by Mattman and Phillips, in the Journal of Infection, January, 1999, suggests they are commonly present in the blood, and can be cultured even after a Lyme patient has received aggressive antibiotic therapy. This would indicate that the standard method of culturing classical forms from the blood may be flawed. If, in fact, the preferred form of the spirochete in the blood is a cell wall deficient form, then standard culturing methods would be only be expected to be effective when there is a high concentration of classical forms. Does that ever occur? Spirochatemias in Lyme disease are reportedly rare.
It is extremely important to prove the existence or nonexistence of L-forms of B. burgdorferi, because the therapy chosen depends on which form is surviving in the human host. An antibiotic like Rocephin (ceftriaxone) would be worthless against a cell wall deficient bacteria, because Rocephin kills only during cell division by disrupting new cell wall synthesis. So, if L-forms are prevalent in late-Lyme patients, it would require an antibiotic regimen that disrupts bacterial metabolism such as macrolides. Unfortunately, macrolides don't work well with cell wall agents like Rocephin, because they tend to slow down cell division. So, drugs like doxycycline inhibit the effectiveness of drugs like amoxicillin.
At this time, the scientific community is skeptical of accepting the existence of cell wall deficient spirochetes, and proof of the existence of these cultured L-forms is going to require repeated confirmations. How is this done?
Whether or not a bacteria has a cell wall is inconsequential to its DNA content. If you know the DNA sequence of a specific species of bacteria, you can use DNA amplification (PCR) to determine the presence of that bacteria in cultures. So, if you culture blood and you get CWD forms of Borrelia burgdorferi growing, then, using PCR testing, you should be able to detect DNA specific for Bb in those cultures. If DNA specific for Bb is not present, then whatever it is that you cultured cannot be Bb. Using this technique it appears that Lida Mattman's lab has been able to isolate, culture, and confirm the presence of a new form of Borrelia burgdorferi - a spherical cell wall deficient form. Samples of the Mattman/Phillips cultures were sent to an independent lab that used OSP-A PCR primers to confirm the presence of Borrelia burgdorferi DNA within the cultures.
Using past methods and techniques to culture spirochetes has proven difficult and inconsistent. If this new method of culturing L-forms can be duplicated and repeated by others, it will mean a change in our paradigm of how we view this disease. It will change our understanding of diagnosis, treatment, and the disease process. If this technique proves successful, it may even culture as yet undiscovered species of bacteria that may prove to be pathogens in common diseases that as yet have no known cause, such as MS, Lupus, Crohn's disease, ALS, sarcoidosis, or scleroderma.
We can only hope this technique will live up to its full potential and be fully developed by the medical community. We have the methods of fully testing this new technology, but diverting research time, money, and attention to this controversial new theory of culturing L-forms will be as great a political effort as it is a scientific one! Don't expect the nay sayers of chronic active infection to suddenly have a change of heart without some further proof - and don't expect them to be quick or cooperative in diverting research funds from current Lyme research into this new area. There are many researchers and institutions with economic interests to protect that may be threatened by this new technique.
While it is the fantasy of many chronic Lyme patients that their doctor will call them up and say, "Sorry! I was wrong! You were right! Despite treatment, you really are still infected", I somehow have doubts that this will happen any time soon, and it certainly won't occur before the work of Phillips/Mattman is confirmed by others within the medical research community.