Tags: antibody, Borrelia, Borrelia burgdorferi, Borrelia hermsii, diagnosis, IFA, IgG, IgM, laboratory testing, Lyme Disease, medicine, PCR, Relapsing Fever, Western blot
Two weeks ago, my infectious disease specialist, Dr. David C. Wright, who treated both of my tick-borne infections, was kind enough to grant me an interview during our Borrelia patient support group meeting, as well as a follow-up interview last week to clarify some points. The Q&A below is the result of those two interviews.
Em: Can you briefly explain 1) the way an immune response to a Borrelia infection works and 2) how well currently available tests detect this response.
Dr. Wright: The real problem we have detecting Borrelia infections is that humans don’t usually amount much of a response acutely to these infections. People can have millions of organisms in their blood and not have much of a response. Let me explain how this response works. One type of antibody we make is called IgM, which is for an acute response (to a new infection). Shortly after we make IgM, organisms clear from the blood stream. It’s been shown that IgM antibody can kill organisms in the absence of any other factor (you don’t need complement) and this may be the only infection where this occurs.
B cells make IgM antibody, and with time, switch to another class, IgG, of which there are 4 types, IgG 1, IgG 2, IgG 3, and IgG 4. The body eventually switches from making IgM to making IgG. Sometimes it happens, and sometimes it doesn’t. We don’t know why. Other people make an IgM response, then switch, and all we see is IgG.
For B. burgdorferi, we have a 2 tier system with ELISA and Western Blot. The ELISA is not very sensitive, and many doctors do not like to use it, relying more on the Western Blot. The way a Western Blot works is that proteins that are present in the organism are run on a gel, paper is put up against the gel, they run currents though it, proteins are transferred to the paper, and they are separated by weight. Kilodalton (kDa) is a measure of molecular weight, so a reactive 39 kDa band means the patient is making antibody to the protein that has a molecular weight of 39,000 daltons. For the IgM Western Blot, you need 2 of 3 bands positive for the test to be considered positive.
For the IgG Western Blot, the government (CDC) defines positive as 5 bands (out of 10 or 11). The sticky wicket of all of this is that the organism that is used is called a B31 isolate (from New York in 1982). Only one organism, one isolate, is used to detect antibodies. That would be all well and good if the world were a nice clean place and there was only one strain of B. burgdorferi and it never changed. Worldwide, there are probably about 14 or 15 isolates in the Lyme-like Borrelia group (and there are at least 10 different species in the Relapsing Fever group), so it’s silly to think that you could detect antibodies to all the Borrelia with an ELISA and a Western Blot, and that’s what the government and most doctors would have you believe.
Just to show you how stupid this is, I’ll tell you about an article published by some doctors in Scotland, “Local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen improves western blot sensitivity.” The authors said it doesn’t make any sense to use the B31 isolate of B. burgdorferi when they have different isolates in Europe. They have B. afzelii there, so they took that and added it to B31, and they picked up 14 more positive patients (who would have been told they didn’t have anything). The Scots are not radical; they’re smart. What’s important to ask is, what are the isolates in your neighborhood or state? We need to make Western Blots with those organisms.
We also need more tests available for the other Borrelia in the Relapsing Fever group. For example, we know there’s an unusual European-like isolate in northern California. Now there’s B. hermsii, B. miyamotoi, B. parkeri, and we don’t have Western Blots for those species. Fortunately, Borrelia do have common proteins. For example,there is a BDR gene sequence that codes for relatively conserved proteins across all Borrelia species. In many cases, there is up to 60% homology. Some people may make antibodies that cross-react with several different strains based on this common protein sequence. Another way to think about the Western Blot is, if we had other isolates (ideally 6 or 7), because of these cross-reactivities, we wouldn’t be able to tell people which organism they’re infected with, but we’d stop missing Borrelia infections, which is the real point of the discussion, and it’s not discussed, because they’re just focused on one organism. It’s like we have scientific blinders on.
Another problem is that many strains haven’t been grown, so we don’t have tests for them. For example, B. persica, which causes Relapsing Fever and is common is Israel, has never been isolated and grown.
Em: What should patients know about the interpretation of IFAs and Western Blots? If a patient’s B. burgdorferi Western Blot has only one or two reactive bands, does that mean he/she doesn’t have a tick-borne illness?
Dr. W: Interpretation is pretty much a mess, but we’re kind of stuck with the current definition of what a positive is, even though we have inadequate tests. The official opinion for the IgG Western Blot is that anything less than 5 bands is a negative Western Blot. That’s interesting, but arbitrary. This was all decided at a conference in 1994. To my knowledge, none of this was ever published in a peer-reviewed journal. It’s based on this conference and data that was presented there, which most doctors have never been able to review. Over time, blots change. Banding patterns change. Organisms mutate. The reading of these assays is supposed to be standardized, but some labs use a densitometry reading and others use just a visual inspection. So it appears not to be standardized.
An article published in 2010 by Dr. Gary Wormser and his colleagues at New York Medical College, Harvard, and the CDC admits that we could increase the sensitivity of the IgG Western Blot for B. burgdorferi by changing the definition of a positive from 5 bands to 3 bands. ”With a requirement of ⩾99% specificity, the greatest sensitivity was achieved using a cutoff of 3 of 11 specific IgG bands (18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kD plus VlsE).” Doing this allowed them to detect 33% more infections.
When I see someone with one, two, or three bands on a Western Blot, it makes me really nervous, especially if they have been partially treated. That’s when we have a problem, because we have interrupted the immune response that would occur if nothing had been done. So then really the patient is in limbo. Insurance companies won’t pay for treatment unless a patient has five or more bands. What we do here in clinic is if I think a patient has symptoms and signs of a Borrelia infection and he or she wants to be treated, we offer to treat them as a cash patient. If they want to try Doxycycline, we do that, but if they’ve been sick for many years, it probably won’t work, so we offer daily Ceftriaxone therapy for 4 to 6 weeks. Even after a patient finishes this treatment, I like to follow him or her for an extended period of time and order labs every three months.
Em: Why do some patients have only positive IgM and others have only positive IgG? Can you explain your theory about the difference between these two groups?
Dr. W: I don’t know for sure why this happens. Dr. Gary Wormser and his fellow researchers say that if you have a positive IgM response, it means nothing. Others have published a paper that says other bacterial infections are associated with persistent elevated IgM. IgM is made within the first 3-5 days of infection. In a serious infection like Borrelia hermsii, if you don’t make that response, you might die, because the doctors aren’t going to recognize the infection and aren’t going to treat you. That’s why people die of Borrelia hermsii. If patients make it through the first cycle of organisms, maybe doctors will pick up on it and they will survive because they are making IgM antibodies. Because we have sophisticated tests for B. burgdorferi, we find a lot of people who have persistent IgM and no IgG. We also find people with IgG and no IgM, and with antibody to the C6 peptide. I’m not sure all these people are infected with the same organism. I worry about a persistent IgM response; those people should be followed because that means you have a clone of lymphocytes that are making antibody consistently. This occasionally happens in lymphoma. The opinion of a lot of B. burgdorferi experts is that a persistent IgM response is normal. They see it, ignore it, and don’t order additional tests. I don’t agree with this approach, and I think these patients need to be followed (with periodic lab tests).
Em: Can you explain the concept of cross-reactivity and how it relates to the detection of tick-borne infections?
Dr. W: Borrelia have internal flagella that allow them to be motile. These are inside the membrane, and motility structures are preserved across species. In other words, Borrelia flagella have a lot in common. Motility is key to survival, so you can’t change it much evolutionarily. The proteins are similar, so you would expect if you’re going to have cross-reactivity, you would see antibodies to the 41 kDa flagellar antigen [on the Western Blot]. If I see that, it makes me wonder whether someone has another Borrelia infection, like B. hermsii, because they don’t have enough bands. In fact, that’s actually what you had, Em.* Other doctors say it’s a false positive caused by some other organism that has flagella, but that doesn’t make a lot of sense because it’s more likely that you’d have a cross-reactive antibody response with another Borrelia strain than with something like E. coli.
*I had a reactive 41 kDa band on my Western Blot, and my antibody test was positive for B. hermsii.
Em: What’s a C6 peptide assay?
Dr. W: C6 is a 26 amino acid peptide from the sixth invariable region of Borrelia burgdorferi. A positive C6 peptide assay means a person is making antibody to a protein (C6) found in just one Borrelia—B. burgdorferi. This assay was developed for use during the B. burgdorferi vaccine trial. The researchers knew cultures were frequently (false) negative, so this test helped distinguish between people who were infected and not infected. Doctors have forgotten about this. It’s a nice thing to follow because you get a titer (as opposed to reactive/nonreactive bands), and you can see whether that titer goes up or down as you treat and follow a patient.
Em: When should a PCR be used to detect Borrelia infections?
Dr. W: It’s very useful to test on serum, cerebrospinal fluid (CSF), and biopsies of skin lesions (like Erythema chronicum migrans). The problem is that even in the most capable hands, culture and PCR are only positive in 94% of people with Erythema chronicum migrans. (A paper published by Dr. Wormser and colleagues found that 6% of patients with Erythema chronicum migrans tested negative on five different PCR tests for B. burgdorferi.) It’s possible that the other 6% have another type of Borrelia infection. The problem in general with PCR is that most assays can’t detect less than 200 copies of an organism in a milliliter of blood, so a person can have an infection but a negative PCR. It is useful to do if you can get access to one. ARUP Labs has a PCR for B. burgdorferi that they can run on serum and cerebrospinal fluid (CSF). It’s useful to do a PCR if you think someone might have an acute infection and their blood smear is negative. Currently we don’t have a commercially-available PCR assay for B. hermsii in the U.S.
Em: What does it mean to “re-isolate the organism” following antibiotic treatment?
Dr. W: If we were to isolate an organism from a blood culture after treatment, it means the infection was either not adequately treated or the patient has been re-infected. We rarely isolate Borrelia organisms in a rural health setting because of delays with delivering samples to the appropriate laboratories.
Em: Are you familiar with the CD-57 + NK panel used by Dr. Stricker and others? Is it useful?
Dr. W: NK stands for natural killer cells. Dr. Stricker and other LLMDs have published papers saying that if you follow the CD57 level and it is low, it means a person has a B. burgdorferi infection. It’s an interesting concept; however, my guess is that the CD57 count might be low in other infections as well. I don’t usually order an isolated CD-57 test. If a patient is lymphopenic (has an abnormal lymphocyte count on his or her CBC), I will order a lymphocyte subset panel (CD4-helper cell, CD8-suppressor cell, B-cells, and NK cells). If I think the patient may be hypogammaglobulinemic, I’ll test their immunoglobulin levels to see if they can make adequate amounts of antibody. If the antibody levels are really low, it might interfere with our ability to detect a Borrelia infection or other infection using antibody-based assays (like Western Blot and IFA).
Em: What new tests are needed?
Dr. W: We don’t necessarily need new types of tests; we need additional tests for other organisms. There are approximately 30 species of Borrelia worldwide. We can’t grow them all, but the ones we can grow, we should have a Western Blot for. For B. hermsii, the BipA antigen, which was discovered in a federal laboratory, has not become commercially available, despite the fact that the antigen is specific for B. hermsii and antibodies generated to BipA would only be found in a B. hermsii infection. It also would be nice to have a PCR for every one of the Borrelia types. In addition, more time needs to be spent developing media to grow Borrelia that have not yet been cultured.
Many thanks to Dr. Wright for donating his time. If you’d like to learn more about his practice in Monterey, CA, you can visit his website: http://davidcwrightmd.com.
If you have questions for me or Dr. Wright about any of the information in this post, please leave them in the comments or drop me an e-mail.
Well, Babs, you’re trickier than I thought 05/01/2012Posted by thetickthatbitme in Diagnosis, Peer-Reviewed, TBI Facts, Tick-Lit.
Tags: Babesia, Blood donation, Blood transfusion, health, IFA, labs, Lyme, medicine, PCR, smear, tick
1 comment so far
Welcome to the second installment of Tick-Lit Tuesday, where I comb through PubMed so you don’t have to. Today’s topic: Babesia and Blood Transfusions. Now, I know I posted about Babesia in the blood supply just a few days ago, but an interesting study has since come to my attention (thanks, Dr. W), and the implications are a bit scary. Okay, get your popcorn and let’s begin.
It has been well-documented that the tick-borne protozoan parasite Babesia can be contracted through blood transfusions. Blood centers aren’t required to test donated blood for Babesia, but this may change in the future, as Babesia infections contracted through transfusions are on the rise. So if we were to test all donors for Babesia prior to donation, which tests should we rely on to detect this pesky parasite? Let’s look at the candidates.
IFA: IFA is an abbreviation for indirect fluorescent antibody test. This type of test can also be referred to as serologic (as in blood serum) testing. If you’ve had one of these tests for Babesia, it’s probably titled something like “WA1 IGG ANTIBODY IFA” (for B. duncani) or “BABESIA MICROTI ABS IGG/IGM” on your lab results. If you’ve had Babesia in the past and been treated for it, your antibody test might still read positive because your body is still making antibodies to the parasite. This is one of the reasons why most insurance companies refuse to pay for treatment for Babesia if your only positive test is the IFA. They think maybe you had a past infection that you got over, so you don’t need treatment. (The other reason they refuse to pay is that they’re jerks, to put it nicely.) I’ll talk more about why this is such a problem later in this post.
Smear: When we talk about a smear for Babesia, we mean a Giemsa-stained thin blood smear. This test involves looking at blood samples under a microscope to see if there are any parasites hanging around. The problem with this test is that Babesia can infect fewer than 1% of your circulating red blood cells, so it could take many, many smears before any Babesia show up under the microscope. For more information about that phenomenon, read this.
PCR: This stands for polymerase chain reaction. It’s basically a DNA test that tries to identify whether a gene associated with Babesia is present in the blood. PCR has been found to be “as sensitive and specific” as blood smears for Babesia (see this study), which is not saying much, considering the tendency of Babesia to go undetected with smears.
Hmmm, for whom shall I cast my ballot, the antibody test insurance companies don’t trust, the inaccurate smear, or the inaccurate PCR? Choices, choices…
Can the donated blood of someone with a negative PCR and negative blood smear still be infected with Babesia and cause Babesia infection in transfusion recipients?
(Hint: This is a leading question.)
Let’s talk about a study published in the journal Transfusion in December of 2011 called “The third described case of transfusion-transmitted Babesia duncani.”
Here’s what happened:
In May 2008, a 59 year-old California resident (I’ll call him Cal) with sickle-cell disease had some red blood cell transfusions. Cal’s only risk factor for Babesia was the transfusions; he didn’t have any tick exposure. In September of 2008, Cal was diagnosed with a Babesia duncani (WA-1) infection. The parasites were visible on a blood smear, the indirect fluorescent antibody (IFA) test was positive, and the PCR was positive for the Babesia gene. This launched a transfusion investigation in which doctors tracked down 34 of the 38 blood donors whose blood could have infected Cal with Babesia. One donor, a 67-year-old California resident (who I’ll call Don) had a B. duncani titer of 1:4096 (on the IFA test). What does a titer of 1:4096 mean? Well, if the antibody test for B. duncani is negative, the titer will be < 1:256. That means that Don’s antibody test was positive.
What the article abstract doesn’t tell you, which the full article does, is that both Don’s PCR and blood smear were negative for Babesia. How did the researchers prove definitively that Don had Babesia in his blood? They injected the blood into Mongolian gerbils, and were later able to isolate the parasite from the gerbils. Conclusion: Even though Don showed no symptoms of Babesia and both his PCR and smear were negative, his donated blood caused Babesiosis in both Cal and the gerbils.
Here’s why the study’s findings are important:
1. Clearly, blood smears and PCRs are not good indicators of whether someone is infected with Babesia. Why insurance companies think these tests need to be positive before they’ll pay for treatment is a mystery to me. There are probably a lot of people out there who’ve had positive IFAs but negative smear and/or PCR who were then not treated for Babesia because either the doctor, the insurance company, or both said they didn’t have an infection.
2. As far as the blood donation goes, if we don’t start screening out donors with positive Babesia IFAs, we’re going to continue to contaminate the blood supply with Babesia. It should be as simple as that. Been bitten by a tick? No blood donation for you. Positive IFA? No blood donation for you.