Robin Huiras (00:04):

Hello, and welcome to today's episode of Diagnostics in Practice. I'm Robin Huiras, a senior marketing specialist with Mayo Clinic Laboratories, and a firm believer in the power of advanced testing to change patients' lives. I'm really excited to be here today speaking with Dr. Ellie Theel, director of the Infectious Diseases Serology Laboratory at Mayo Clinic, about Mayo Clinic Lab's newest test to diagnose neuro invasive syphilis or neurosyphilis. This test, which is an IgG antibody index, is a significant advancement from the previous testing approach. Good morning, Dr. Theel. Thanks so much for joining us.

Elitza Theel, Ph.D. (00:41):

Good morning, Robin. I'm very happy to be here.

Robin Huiras (00:44):

Now, before we dig into today's topic, Dr. Theel, can you please share with us a bit about yourself and your experience at Mayo Clinic?

Elitza Theel, Ph.D. (00:51):

Sure, I'd be happy to. So I am a clinical microbiologist, and currently I'm the laboratory director for the Infectious Diseases Serology Laboratory, and I've been in this position since 2012. Then also for the last two years, I've overseen our testing for sexually transmitted infections as well here at Mayo Clinic and for Mayo Clinic Laboratories. And one of my areas of interest is in advancing some of the diagnostic tests that we offer for sexually transmitted infections, including outreach and increased access to some of this testing. But in particular for today's discussion, I've really spent quite a bit of time on attempting to improve some of our assays for detection of invasive syphilis. So a lot to talk about today.

Robin Huiras (01:41):

Now, you mentioned that one of your areas of focus is around sexually transmitted diseases like syphilis, and I'm hoping you can really kick off our conversation today by telling us about this particular infection.

Elitza Theel, Ph.D. (01:53):

Sure. So syphilis is a bacterial infection that's primarily transmitted through sexual contact. Syphilis in general has multiple different stages that patients progress through, starting with primary syphilis, which typically presents with one or more lesions or sores that are referred to as chancre. Patients can then go on to develop secondary syphilis, which typically has more systemic symptoms, including classically, this non itchy rash that can be on the torso, but also very characteristically occurs on the palms and soles of the feet. Patients during this stage can also go on to develop fever, malaise, myalgia, lymphadenopathy and sore throat amongst other things. And again, even with these symptoms, some patients, well, the symptoms will resolve without treatment and patients again will still continue to have syphilis even though they don't have those symptoms anymore. And at that point, patients enter what's called the latent stage of disease where they're still infected, but entirely asymptomatic, and that can last for decades, years to decades in some cases.

And then the last stage I want to talk about that's more relevant for our conversation today is that syphilis can spread to the brain and the nervous system leading to neurosyphilis. And this can occur at any stage of disease after that primary syphilitic stage. Patients typically present with severe headache muscle weakness, they have difficulty moving changes in their mental state, like difficulty focusing, confusion, memory problems. And unlike other stages of disease without treatment, neurosyphilis can lead to significant morbidity for patients. So as you can see, syphilis can present in a variety of different ways, which is why this disease has been referred to as the great imitator by physicians throughout time. So it's a tricky one.

Robin Huiras (03:51):

Yeah, it really does sound like it. Thank you so much for walking us through the different stages of the disease. It really does seem like it can be elusive in many cases.

Elitza Theel, Ph.D. (04:02):

So neurosyphilis may not be the first thing that physicians really jump to. It's really on the clinician to consider it as part of their differential diagnosis based on the patient's clinical history, including their sexual history, importantly, and also if they're seropositive for syphilis antibodies in blood. So those are kind of the key components that are necessary before a clinician goes down to the neurosyphilis differential diagnosis.

Robin Huiras (04:32):

Yeah, I see. And that leads me to ask what percentage of patients affected by syphilis actually do develop neurosyphilis?

Elitza Theel, Ph.D. (04:41):

Yeah, another great question. Unfortunately, neurosyphilis is not a nationally notifiable disease at this point, so rates are not captured by our national data. That being said, our best estimates is that anywhere between one to 2% of untreated patients will go on to develop neurosyphilis. But again, this is likely an underestimate because it's not tracked routinely, and our current diagnostic testing for neurosyphilis is unfortunately not ideal.

Robin Huiras (05:11):

So in the cases when the differential does point to syphilis or neurosyphilis, would confirming diagnosis be the next step?

Elitza Theel, Ph.D. (05:20):

Yes, exactly. So confirming a diagnosis of neurosyphilis using laboratory testing really does help to definitively establish the cause of the patient's symptoms and helps to really optimize their care and their treatment.

Robin Huiras (05:35):

It sounds like proper care and treatment are key to identifying the infection before it can be harmful to patients, but syphilis has been affecting humans for ages and testing has been available for decades. And I'm hoping you can tell us a little bit about why a change is needed in how individuals are tested at this point in time.

Elitza Theel, Ph.D. (05:55):

So the first for syphilis was developed and put into use over a hundred years ago in 1906, 1907. It's referred to as the Wasserman test, and that was used at Mayo Clinic in 1906, I believe. That is what's called a non-treponemal test, meaning that it detects antibodies to lipoidal products that are released from host cells. So patient cells that are damaged by Treponema pallidum, which is the causative agent of syphilis around 1945. The Wasserman test, which is a complement fixation based test, which is very challenging to run. That was replaced with the Venereal Diseases Research Laboratory Test or the VDRL assay, which also detects these non-treponemal antibodies, but it's an agglutination assay and is much easier to perform. And we as well as other laboratories throughout the country and worldwide have continued to use this VDRL assay for diagnosis of neurosyphilis really ever since. With all of our advancements in diagnostics though over the last few decades, I really thought that it is time to, and we have the opportunity to update our testing methods for neurosyphilis in particular.

Robin Huiras (07:17):

Yeah, I agree. And thank you so much for that really nice description of the non-treponemal assays. Can you tell me and our listeners if there are other syphilis assays that detect antibodies specific to the organism?

Elitza Theel, Ph.D. (07:30):

Yeah, so the diagnostic spectrum for syphilis is somewhat broad, and if we start up at the top, if you will, there's two different I guess categories of tests for syphilis. There's molecular testing and there's serologic testing. Molecular methods, I think we are all familiar with. They're used to detect nucleic acids from organisms. These are direct detection methods and are used for diagnosis of many infectious diseases. The problem for syphilis though is that this organism treponema pallidum doesn't really lead to or get to a high enough bacterial load in blood for it to be detectable routinely and effectively by molecular methods. And studies have shown that PCR or molecular detection, irrespective of what specimen source one uses, is fairly low ranging anywhere from 12 to about 50% in blood, for example. So not great. And then the highest sensitivity for molecular testing is actually observed from swabs of the lesion.

So those chancres that we talked about, sensitivity there is not bad. It ranges anywhere from 75 to 95% for PCR assays across studies. So unless you have a lesion of some sort to test a syphilis PCR just isn't going to give you an accurate answer most of the time. So as a result, we continue to rely on serologic methods to detect patient developed antibodies to the organism and with syphilis. There's two types of serologic tests that we use, the non-treponemal type that I already talked about, and the other type of syphilis serologic tests are the treponemal assays which detect antibodies specifically developed against treponema pallidum itself.

Robin Huiras (09:16):

Thanks so much for telling us about the difference between these two tests, which are both quite old. And it seems like based on the lifespan that these tests have been used, they were doing a good job. Am I correct in that assumption?

Elitza Theel, Ph.D. (09:30):

So the old tests we're talking about are the non-treponemal VDRL assays in particular, and it's been an okay test for neurosyphilis. The sensitivity of this test in cerebral spinal fluid or CSF from patients with neurosyphilis ranges anywhere from 49 to about 88% across studies. And then the specificity ranges anywhere from 74 to 95%. So it's not been great.

Robin Huiras (09:57):

Clearly there's been some room for improvements.

Elitza Theel, Ph.D. (09:59):

Yes. And the new tests we've just brought up, which is the only one of its kind available in the US currently, does give physicians a much more definitive answer, we feel.

Robin Huiras (10:10):

Tell us a little bit about those answers. Can you elaborate?

Elitza Theel, Ph.D. (10:13):

Yeah, so the new test, which is called the Neurosyphilis antibody Index assay, has a number of benefits over the VDRL test. First and foremost, this test is looking for antibodies that are specific to treponema pallidum, so it's a treponemal based antibody test versus the VDRL, which is a non-treponemal test. So we're looking for IgG antibodies developed against treponema pallidum. So right off the bat, because we are using a more specific targeted antibody test, we have improved specificity over the current standard test.

Robin Huiras (10:51):

Nice. Increased specificity is always a great advantage. Can you tell me, are there others to this antibody index test?

Elitza Theel, Ph.D. (10:58):

Yeah, the other key advantage of the neurosyphilis antibody index assay is that we're looking for what are called intrathecal synthesized antibodies. So intrathecal antibody synthesis means that the antibodies that we're detecting were developed and released by B cells in the central nervous system following recognition of the pathogen in that space. And so their presence would indicate true invasive disease. This approach is preferred and in a sense, better than the VDRL assay, which does not differentiate between intrathecally synthesized antibodies versus antibodies that are present in the spinal fluid simply due to passive diffusion of those antibodies from the serum into the CSF across the blood-brain barrier. Given the small size of IgG, passive diffusion occurs normally, and so the antibody index assay corrects for that diffusion and is able to differentiate between those two scenarios. So if the index is positive, that indicates the presence of true neuro invasive disease and not passive diffusion of antibodies into the spinal fluid.

Robin Huiras (12:11):

Now I'd like to go back to the specificity of the antibody index. Can you share with our listeners how much more specific this test is than the VDRL assay?

Elitza Theel, Ph.D. (12:21):

Yeah, so our validation studies show that about 30% of VDRL results are not confirmed by the antibody index, which would suggest that the VDL is positive in spinal fluid due to that passive diffusion of antibodies, and that could potentially lead to a misdiagnosis of neurosyphilis in patients that have something else entirely. And then additionally, other studies looking at antibody indices for syphilis have shown that the specificity of this approach ranges from 85 to a 100% percent amongst patients with neurosyphilis, which is quite a bit higher again than what we see for the VDRL assay, which can be as low as 75%. So definitely a bit more specific.

Robin Huiras (13:03):

Can you share a little bit about what misdiagnosis means for patients affected by syphilis or neurosyphilis?

Elitza Theel, Ph.D. (13:10):

Yeah, so misdiagnosis really means that the true causative agent of the patient's neurologic symptoms is entirely missed and patients end up receiving treatment for invasive syphilis, which has limited activity in most cases if it's not syphilis. And so the actual cause of their symptoms has not been identified. It prolongs in some cases inpatient hospital care, receiving antibiotics unnecessarily is a problem. So there's a lot of damage that can be done in patients that are misdiagnosed due to a false positive VDRL, for example.

Robin Huiras (13:44):

You talked already a little bit about the sensitivity of the antibody index, but can you compare and contrast between how the sensitivity is different between this new assay and the VDRL assay?

Elitza Theel, Ph.D. (13:57):

Yeah, so the sensitivity overall is higher for the antibody index assay ranging anywhere from 86 to 90% across studies. So I think we have a much tighter sensitivity range and definitely a bit higher than what we see for the VDRL assay. So a definite improvement there as well.

Robin Huiras (14:18):

Are there other differences with the new antibody index as compared to the current and standard VDRL assay?

Elitza Theel, Ph.D. (14:24):

Yeah, so there's a couple of key differences in the specimen requirements for the antibody index test specifically for this assay. In order to compare antibody levels between the CSF and the serum, we need to have both CSF and serum samples submitted for testing. And ideally, both of those samples are collected within about 24 hours of each other. The reason we need both sources is that the antibody index workflow testing process, we start by screening the spinal fluid first to see if there's any antibodies to syphilis in there. If there's no antibodies detected in the spinal fluid, the result goes out as negative. If, however, we do see some sort of reactivity in the spinal fluid, we then serially dilute and titer out the serum and the spinal fluid looking for and essentially quantifying the level of syphilis specific antibodies in both sources. Both of those sources are also tested for total IgG as well as total albumin, and we use total IgG and albumin in both sources to normalize the level of antibody specific to syphilis in CSF and serum.

And we specifically use the Reiber formula to calculate that intrathecal antibody index. That calculation takes into account things like molecular size, normal diffusion rates, normal concentrations of these analytes in CSF and so on, to ultimately, again, get this index and we report out the index value with a qualitative interpretation as well. Additionally, since the VDRL assay continues to be included in all guidelines for neurosyphilis diagnostics for any positive or equivocal antibody index result, we will also perform the VDRL assay on that and report a titer. Importantly though, as we just talked about, the antibody index assay is more sensitive than the VDRL assay, so there could be scenarios where you have a positive antibody index and a negative VDRL result. So that would indicate again, true intrathecal antibody synthesis and that the patient has invasive disease that would potentially have been missed by just doing a VDRL test by itself.

Robin Huiras (16:42):

I'd like to talk a little bit more about additional limitations for this test. Besides requiring both specimen types, can you share other items that our listeners ought to know about in terms of limitations?

Elitza Theel, Ph.D. (16:55):

Yeah, so the main limitation to be cognizant of with respect to the antibody index is that intrathecal antibody synthesis can remain detectable for quite some time. So patients that have been diagnosed with neurosyphilis in the past using an antibody index test or a VDRL test will be positive by the antibody index for potentially months after resolution of their disease and their symptoms. So very importantly, this test should be used as an acute diagnostic in symptomatic patients who are also seropositive for antibodies to syphilis in blood. This test should not be used to monitor response to therapy.

Robin Huiras (17:35):

And speaking of therapy, what is the treatment used for neurosyphilis infections?

Elitza Theel, Ph.D. (17:40):

So treatment for neurosyphilis remains penicillin based, although it is a bit more intensive than a non-neurosyphilis treatment regimen. Patients with neurosyphilis typically receive penicillin IV infusions for 10 to 14 days. So it's not a simple treatment, but it is very effective.

Robin Huiras (17:59):

I'm curious to know this particular type of antibody index approach. Is it used for other infections?

Elitza Theel, Ph.D. (18:06):

Great question. So at Mayo Clinic, we use this type of antibody index method for the diagnosis of invasive Lyme disease, and this approach has been and is I guess what is recommended in both the European and US guidelines for diagnosis of neuroborreliosis. So there is precedent for using an antibody index test for detecting intrathecal antibody synthesis, I guess a different pathogen. Additionally, there's other invasive diseases for which molecular testing is insensitive that require a similar antibody index approach. With both of those things, I think that there's an inability for clinicians to be really confident in the results that they're getting from the current diagnostic options.

Robin Huiras (18:55):

Right, and those options such as the VDRL are those that are in the clinical guidelines, but what about this new invasive antibody index? Is that included in the clinical guidelines?

Elitza Theel, Ph.D. (19:09):

Unfortunately not yet, and that's largely due to the novelty of this approach. A syphilis antibody index testing is used in Europe, however, the accepted use of this sort of index testing for invasive Lyme, which shares some similar features with the pathology similar to syphilis. I think the fact that it's in guidelines for Lyme disease, for example, really reinforces that this can be used effectively for other diseases like neurosyphilis. So I would hope and foresee that as more studies come out, this is considered and implemented in future guidelines.

Robin Huiras (19:49):

Yeah, I hope so too, because this test really does seem to offer the best of both worlds. It's a new assay that uses an optimized approach for both improved sensitivity and specificity.

Elitza Theel, Ph.D. (20:00):

Yeah, and we've been doing the neuro invasive Lyme disease antibody index test since 2018. We've had a longstanding experience with how to do these sorts of assays at this point. So I'd say we're really well poised to bring in antibody index testing for any disease where molecular testing is not ideal or where it's important to differentiate between whether antibodies are intrathecally synthesized or not.

Elitza Theel, Ph.D. (20:28):

Can you talk a little bit more about why our answers can equip physicians with more confidence about their decision making?

Elitza Theel, Ph.D. (20:35):

Yeah. I think once clinicians start using and getting comfortable with this test, I think their confidence will grow. If it is positive, it means that intrathecal synthesis is present. It means that there's an organism in the central nervous system that attracted B cells there. They reacted and created antibodies. We don't really have to ask the how confident are we questioned? I think we're decreasing the risk of misdiagnosis and mistreatment and hopefully providing more accurate diagnoses of what the patient really has. I think we're really moving the needle in the positive direction with this one for testing for syphilis. Finally, I would say it's a big advancement over where we've been in the past.

Robin Huiras (21:20):

Thank you so much for your time today and such an engaging conversation. I really appreciate all that you've said and hope our listeners agree.

Elitza Theel, Ph.D. (21:30):

Yeah, thank you. Happy to be on with you today.

Robin Huiras (21:33):

And to our listeners, thanks for joining us. We really hope you enjoy today's conversation, and we'll tune in again for the next installment of answers from the lab.