Antibiotic cream is NOT 100% effective in preventing Lyme disease

A topical antibiotic cream applied to tick bites did not perform any better than placebo in preventing Lyme disease, according to results of a randomized clinical trial conducted in Europe.  The study was published in Lancet Infectious Diseases.

I wasn't planning to blog about the study, but I changed my mind after a reader emailed me a link to a news article reporting that the antibiotic cream was 100% effective.  The lead investigator even claimed, "None of the test subjects went on to develop Lyme borreliosis."  As described by the news sources, seven subjects in the control group developed Lyme disease.  But the abstract of the paper states clearly that the antibiotic cream (azithromycin being the antibiotic) was not any better than the control cream; the investigators were even told to stop recruiting additional patients because the results was so clear with the patients who had already completed the study:

The trial was stopped early because an improvement in the primary endpoint in the group receiving azithromycin was not reached.  At 8 weeks, 11 (2%) of 505 in the azithromycin group and 11 (2%) of 490 in the placebo group had treatment failure.

So how is it possible for the lead author to claim that "none" of the subjects treated with azithromycin came down with Lyme disease?  The answer lies with the very last sentence of the abstract:

A subgroup analysis in this study suggested that topical azithromycin reduces erythema migrans after bites of infected ticks.

The subgroup analysis was done post-hoc (after looking at the data).  I won't dwell on why we shouldn't make definitive conclusions from any post-hoc analysis since the investigators themselves emphasized its exploratory nature in the Discussion of their paper.  However, even if you set that aside, you'll find another problem with the post-hoc analysis if you dig into the numbers.

Before I tell you what the problem is, let me first describe the study in greater detail so that you understand the issues that led to the post-hoc analysis.

The subjects were adults who had been bitten by a tick within the previous 72 hours and were able to save the tick.  The subjects were randomized to receive a topical azithromycin cream or placebo cream.  The cream was applied over the tick bite twice a day for three straight days.  The patients were followed for 8 weeks.  They were monitored for erythema migrans (EM), the characteristic rash of Lyme disease.  Blood was drawn for serological testing at the beginning and at the end of the 8 week study period.  "Treatment failure" was defined as the appearance of EM, seroconversion, or both by the end of 8 weeks.  The ticks were tested for the bacteria that cause Lyme disease (Borrelia garinii, B. afzelii, and B. burgdorferi) by PCR.

As I alluded to earlier, the independent committee monitoring the trial recommended that the investigators stop recruiting new subjects.  Among the patients who already completed the study, the group receiving azithromycin did not fare any better than the placebo group, and recruiting more patients to the study was unlikely to change the conclusion.  I provided the numbers above, but you can also find them in the table below ("ITT population," first row of data).

The researchers also did a pre-planned subanalysis with the per-protocol group, an idealized situation to directly test the question, "Does topical azithromycin prevent Lyme disease in those who are bitten by an infected tick?".  Patients bitten by a PCR-negative tick were excluded from the subanalysis.  The small number of patients who failed to follow or complete the study protocol were also excluded.

Again, azithromycin was not any better than placebo in preventing EM or seroconversion (see table, "Per-protocol population").  Treatment failure was observed in 5% (3/62) of the azithromycin group and 7% (5/72) of the placebo group (P = 0.34).

The researchers could have stopped the analysis there and write up the study, but the monitoring committee pointed out that none of the patients in the azithromycin group had erythema migrans by day 30 whereas five in the placebo group did.  The committee suggested that the investigators do a post-hoc subgroup analysis using a modified definition of treatment failure as EM by 30 days.  Seroconversion was removed from the modified definition.

Looking at the numbers in the table ("Reanalyzed ITT population"), we now see where the news media got their numbers.  No one in the azithromycin group (0/87, 0%) had EM by day 30, but seven in the placebo group (7/87, 8%) did.  The difference was statiscially significant (absolute risk reduction in those receiving azithromycin: 8.05%, 95% CI 1.18-14.91).  So, it's true that azithromycin prevented Lyme disease in all who were bitten by an infected tick - but only if you ignored the two patients who came down with EM after day 30 and a third patient who seroconverted.

This is why I'm so baffled by the lead author's quote, which I will repeat:  "None of the test subjects went on to develop Lyme borreiosis."  I'm guessing that the two patients with delayed EM would disagree.


Reference

Schwameis M, Kündig T, Huber G, von Bidder L, Meinel L, Weisser R, Aberer E, Härter G, Weinke T, Jelinek T, Fätkenheuer G, Wollina U, Burchard GD, Aschoff R, Nischik R, Sattler G, Popp G, Lotte W, Wiechert D, Eder G, Maus O, Staubach-Renz P, Gräfe A, Geigenberger V, Naudts I, Sebastian M, Reider N, Weber R, Heckmann M, Reisinger EC, Klein G, Wantzen J, & Jilma B (2016). Topical azithromycin for the prevention of Lyme borreliosis: a randomised, placebo-controlled, phase 3 efficacy trial. The Lancet. Infectious Diseases PMID: 28007428


Related posts

• A flawed study claiming prevention of Lyme spirochete infection with topical antibiotics
• A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease

The Lyme disease spirochete lives without thiamine

Thiamine, or vitamin B1, is vital for the survival of all living things.  One of the biologically functional forms of thiamine, thiamine pyrophosphate (TPP), is essential for the catalytic activity of several critical metabolic enzymes.  For this reason, we must get thiamine from the food that we eat (or the vitamin pills that we swallow).  Microbes obtain the vitamin from their surroundings, but many can also make their own thiamine if it's not available.

It turns out that the Lyme disease spirochete Borrelia burgdorferi does not need thiamine, as described by Zhang and colleagues in Nature Microbiology.  The B. burgdorferi genome lacks the genes encoding the dedicated transporters that bring thiamine into the cell.  The genes encoding the enzymes that produce thiamine are also absent.  Chemical analysis of B. burgdorferi by HPLC failed to detect thiamine or TPP.  Despite lacking the means to make or acquire thiamine, B. burgdorferi grew just fine in culture medium devoid of thiamine.

The researchers conducted stringent tests to verify that B. burgdorferi could live without thiamine.  To remove all traces of thiamine, they introduced the bcmE gene from Clostridium botulinum into the spirochete.  The bcmE gene encodes an enzyme that rapidly breaks down thiamine.  In culture, the spirochete grew at the same rate whether or not it had bcmE.  The bcmE gene did not affect B. burgdorferi's ability to infect mice or to survive in feeding Ixodes scapularis ticks.  The results of these experiments provided strong evidence that B. burgdorferi doesn't need thiamine to infect the tick vector or mouse.

How does B. burgdorferi manage to live without thiamine?  It can do without most of the enzymes that require the TPP coenzyme, but it's less obvious how B. burgdorferi copes without pyruvate dehydrogenase (PDH), a TPP-dependent enzyme that converts pyruvate to acetyl-CoA (see figure).  Acetyl-CoA is an essential precursor to the bacterial cell wall, something that B. burgdorferi obviously needs.  The researchers proposed that B. burgdorferi makes acetyl-CoA by an alternative pathway that starts with acetate.  B. burgdorferi possesses the enzymes acetate kinase (ACK) and phosphate acetyltransferase (PTA), which convert acetate to acetyl-CoA (see figure).

Figure 4 from Zhang et al., 2016.  Enzymes in red (PDC, PDH, and POX) require the TPP coenzyme.  Metabolic pathways found in other bacteria but missing in B. burgdorferi are shown with dashed lines.

B. burgdorferi may not be alone in living without thiamine.  The researchers also looked at the genomes of other bacterial pathogens that are transmitted by arthropods.  Borrelia hermsii (relapsing fever), Rickettsia prowazekii (epidemic typhus), and R. conorii (Mediterranean spotted fever) were missing the genes for thiamine biosynthesis and the enzymes that use thiamine pyrophosphate as a coenzyme.

The presence of the alternative pathway to acetyl-CoA synthesis assumes that acetate is available in the microenvironment surrounding the arthropod-borne pathogen.  According to measurements presented in a 2010 paper, acetate is present in the midgut of fed I. scapularis ticks and in mouse blood.  The B. burgdorferi protein BBA34 may be a transporter that brings acetate into the cell.

References

Zhang K, Bian J, Deng Y, Smith A, Nunez RE, Li MB, Pal U, Yu AM, Qiu W, Ealick SE, & Li C (2016). Lyme disease spirochaete Borrelia burgdorferi does not require thiamin. Nature Microbiology, 2 PMID: 27869793

Xu H, Caimano MJ, Lin T, He M, Radolf JD, Norris SJ, Gherardini F, Wolfe AJ, & Yang XF (2010). Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi. PLoS pathogens, 6 (9) PMID: 20862323

Subba Raju BV, Esteve-Gassent MD, Karna SL, Miller CL, Van Laar TA, & Seshu J (2011). Oligopeptide permease A5 modulates vertebrate host-specific adaptation of Borrelia burgdorferi. Infection and immunity, 79 (8), 3407-20 PMID: 21628523

Are NETs involved in fighting Leptospira interrogans infections?

Neutrophils are the most abundant white blood cells in the bloodstream.  As the first immune cells to be recruited to infected tissues, they play a key role in the fighting microbial intruders.  It's long been known that they engulf microbes by phagocytosis, which results in the microbes being imprisoned within phagosomes inside the neutrophil.  Deadly proteases, antimicrobial proteins, and reactive oxygen species are released into the phagosome to kill the microbes.

Another means used by neutrophils to kill microbes was discovered just a decade ago.  When mixed with bacteria, neutrophils cast nets of DNA impregnated with antimicrobial proteins to trap and kill the bacteria.  The web-like DNA goes by the name "neutrophil extracellular trap" (NET).  Several bacteria are known to trigger neutrophils to cast NETs, and NETs have even been observed by microscopy within infected tissues.

Fluorescence staining of a neutrophil exudate in an appendicitis case.  NETs are the fibrous material.  Figure 4H from Brinkmann et al., 2004.  Bar = 50 μm.

A study published last year in PLOS NTD showed that the spirochete Leptospira interrogans is also killed by NETs.  The image below shows the spirochetes trapped in a NET cast by a human neutrophil.

Human neutrophils were cultured with L. interrogans for 3 hours.  Figure 1A from Scharrig et al., 2015.  Bar = 50 μm.

The real question is whether NETs are involved in killing L. interrogans during infection.  To answer this question, the investigators turned to the mouse model of leptospirosis.  They found that the number of spirochetes in the bloodstream more than doubled when the neutrophils in the mice were depleted by injection of a monoclonal antibody targeting a antigen located on the neutrophil surface.  Later in the infection, there was 10-fold more spirochetes in the kidneys of mice whose neutrophils were depleted than in those with normal numbers of neutrophils.  This confirmed that neutrophils were involved in limiting infections by L. interrogans, but did the neutrophils fight the infection by casting NETs?

The investigators used an indirect method to measure the amount of NETs generated during infection.  Neutrophils often expel nuclear DNA in the form of nucleosomes to generate NETs.  (Nucleosomes are assembled by wrapping nuclear DNA around histones.)  For this reason, the investigators measured the levels of free nucleosomes in the bloodstream of infected mice by ELISA. They concluded that NETs were generated by neutrophils in the bloodstream because they detected free nucleosomes in blood drawn from infected mice.  Much less was detected when neutrophils were first depleted with the anti-neutrophil antibody, confirming that the main source of free nucleosomes was neutrophils.

These results don't convince me that NETs are generated by neutrophils during L. interrogans infection.  There could be other reasons for free nucleosomes being present in the bloodstream.  For example, nucleosomes could be released from neutrophils simply dying from their battle against L. interrogans.  More convincing evidence would be direct observation of NETs in infected animals, as done in this study of mice with E. coli blood infections.

References

Scharrig E, Carestia A, Ferrer MF, Cédola M, Pretre G, Drut R, Picardeau M, Schattner M, & Gómez RM (2015). Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira. PLoS Neglected Tropical Diseases, 9 (7) PMID: 26161745

Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, & Zychlinsky A (2004). Neutrophil extracellular traps kill bacteria. Science (New York, N.Y.), 303 (5663), 1532-5 PMID: 15001782

Xenodiagnosis to detect Borrelia burgdorferi in humans

We've seen that live Borrelia burgdorferi persists (in unculturable form) when infected mice are treated with antibiotics.  What we don't know is whether they persist in humans with post-treatment Lyme disease syndrome (PTLDS), which refers to the lingering long-term symptoms experienced by a minority of Lyme disease patients who have been treated with the standard course of antibiotics.

In theory, one could simply determine whether B. burgdorferi can be detected in bits of tissue or blood extracted from volunteers with post-treatment symptoms.  This is what was done in the mouse studies that I described in my previous post.  It's easy to culture B. burgdorferi from untreated mice that have been infected for a long time.  However, humans are not mice.  Except in those with Lyme arthritis, the spirochete is hard to detect by culture or PCR in patients at later stages of Lyme disease, even in those who haven't taken antibiotics.

In fact, three of the four randomized controlled retreatment trials that I keep on bringing up on this blog included attempts to detect B. burgdorferi in cerebral spinal fluid or blood of PTLDS patients by culture and PCR.  No specimen was culture positive except for one, and none were PCR positive.  The single positive culture turned out to be a contaminant.

The rest of the scientific literature is littered with claims that Lyme Borrelia can be detected by culture or PCR in blood, urine, or CSF of treated patients.  However, critics have raised several concerns about these studies.  For instance, alternative explanations for the findings such as contamination or reinfection weren't ruled out.

With all of this as background, Marques and colleagues decided to test a different approach – xenodiagnosis.  For this procedure, uninfected ticks are deliberately placed on the skin and left for several days to give them time to take a blood meal.  If there are any spirochetes in the skin nearby, they will move towards the feeding site because they are attracted to the tick's saliva.  The spirochetes then get drawn into the tick's feeding tube along with the blood meal.  The fed ticks are then removed and tested for the presence of B. burgdorferi.  The sensitivity of xenodiagnosis can be enhanced by placing multiple ticks to increase the chance that at least one tick will drink blood containing B. burgdorferi.  Xenodiagnosis is done routinely with mice in the research setting, and I mentioned in my previous post that B. burgdorferi can be detected in antibiotic-treated mice by xenodiagnosis.

The first thing to do was a pilot study to make sure that the procedure was safe for volunteers.  25 subjects who had been treated for Lyme disease took part in the study.  10 of the 25 had PTLDS.  Ten healthy volunteers and one subject with untreated erythema migrans (EM), the skin rash of early-stage Lyme disease, were included in the study.

As for the ticks, the investigators bred and maintained Ixodes scapularis in the laboratory.  The ticks were carefully screened to make sure they were free of known infectious agents.

25-30 ticks were placed on each volunteer and covered with a special dressing to keep them in place (see images below).  The ticks were left alone for a week so that they could consume a blood meal.  Some of the fed ticks were tested for the presence of B. burgdorferi DNA by standard PCR or by a more sensitive technique:  isothermal amplification followed by PCR and mass spectrometry (IA/PCR/ESI-MS).  The remaining ticks were cultured or were placed on immune-deficient mice to determine whether B. burgdorferi, if present, could be transmitted.

Figure 1 from Marques et al., 2014.  Left panel: ticks covered with a special dressing on forearm.  Right panel: feeding ticks attached to forearm, dressing removed.

So did anyone test positive by xenodiagnosis? Yes. B. burgdorferi DNA was detected in two subjects.  One was the subject with untreated EM.  This subject served as sort of a positive control.  I say "sort of" because antibiotic therapy was started at the same time that the ticks were placed on the EM lesion – it would not have been ethical to delay treatment while the ticks were feeding.  B. burgdorferi DNA was detected in two of the ten ticks tested.  The subject was tested by xenodiagnosis again seven months later, and all ten ticks that were tested were negative for B. burgdorferi DNA.

The other positive test came from one of the PTLDS subjects.  One of the five ticks that were tested was positive for B. burgdorferi DNA.  The same subject tested positive by xenodiagnosis again 8 months later:  one of three ticks tested positive for B. burgdorferi DNA by IA/PCR/ESI-MS.

Of course DNA doesn't equal viability.  The study didn't provide much evidence that the DNA detected in the single case of PTLDS came from spirochetes that were alive at the time that the ticks were placed.  A skin biopsy taken from where the xenodiagnostic ticks were feeding was culture negative, as were the fed ticks themselves.  The ticks also failed to transmit B. burgdorferi to immune-deficient mice, a process that probably requires live, motile spirochetes.  To be fair, this was just a pilot study with the primary goal to assess the safety of xenodiagnosis.  Nothing terrible happened to the volunteers, although half experienced mild itching at the feeding site.  The investigators are recruiting additional subjects for a larger study to determine whether positive test results by xenodiagnosis are associated with post-treatment symptoms.


References

Marques A, Telford SR 3rd, Turk SP, Chung E, Williams C, Dardick K, Krause PJ, Brandeburg C, Crowder CD, Carolan HE, Eshoo MW, Shaw PA, & Hu LT (2014). Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study. Clinical Infectious Diseases, 58 (7), 937-45 PMID: 24523212

Bockenstedt LK, & Radolf JD (2014). Xenodiagnosis for posttreatment Lyme disease syndrome: resolving the conundrum or adding to it? Clinical Infectious Diseases, 58 (7), 946-8 PMID: 24523213

Telford SR 3rd, Hu LT, & Marques A (2014). Is there a place for xenodiagnosis in the clinic? Expert Review of Anti-infective Therapy, 12 (11), 1307-10 PMID: 25301228


Related posts

• Video microscopy of ticks acquiring the Lyme disease spirochete from mice
• Resurgence of Borrelia burgdorferi in mice a year after antibiotic treatment

Resurgence of Borrelia burgdorferi in mice a year after antibiotic treatment

As a follow up to my previous post, I would like to say something about several mouse studies from Stephen Barthold's group.  These papers are often cited by those who believe that retreatment is needed in patients who continue to experience symptoms following treatment of Lyme disease with conventional antibiotic regimens.  The assumption is that post-treatment symptoms stem from spirochetes surviving the initial antibiotic therapy.

In the 2008 and 2010 studies (described in detail here and here), Barthold's group gave doxycycline, ceftriaxone, or tigecycline to mice with disseminated Borrelia burgdorferi infection.  As expected, all tissues were culture negative up to three months following antibiotic therapy.  Tissues from untreated mice were culture positive.  However, B. burgdorferi DNA and mRNA were detected by PCR in up to half the treated mice, and microscopy revealed a few intact spirochetes in collagen-rich tissues from these mice.  Ticks allowed to feed on the treated mice even transmitted the spirochetes to other mice (albeit immune deficient ones), where B. burgdorferi DNA was detected by PCR.  Clearly, the spirochetes that survived antibiotic treatment were alive despite being unculturable.

Although live spirochetes remained following antibiotic therapy, there was no evidence that they were capable of causing disease.  Lyme disease is driven by inflammation, but no inflammatory response in the form of infiltrating immune cells were seen in tissues harboring the spirochetes.  A critic of the work also pointed out that the number of spirochetes declined during the 3 months following treatment, implying that any lingering spirochetes would eventually disappear.  It seemed unlikely that a similar phenomenon was responsible for persisting symptoms following treatment of Lyme disease in human patients, who may suffer with disabling symptoms for years.

In 2014 Barthold's group came out with another paper, which I'm discussing here for the first time.  Again, mice with disseminated B. burgdorferi infections were treated with antibiotics, ceftriaxone in this case.  But this time, the mice were left for up to a year before their tissues were examined for the presence of B. burgdorferi.  Control mice were mock treated with saline and examined along with the treated mice.

There weren't any surprises when tissues were tested by culture.  Most of the control mice were culture positive at all time points (2, 4, 8, and 12 months) with both tissues tested, the urinary bladder and the skin where B. burgdorferi was inoculated to initiate infection.  None of the treated mice were culture positive at either site at any time point.

PCR testing for B. burgdorferi DNA was done with tissue obtained from six sites in the mice.  Ticks allowed to feed on the mice were also tested for the presence B. burgdorferi DNA by PCR in a method called xenodiagnosis.  All saline-treated mice were PCR positive in most tissues tested, and most tested positive by xenodiagnosis.

The results with the mice treated with ceftriaxone are shown in the table below.  Each row represents a single mouse.  Note that each tissue homogenate was tested three times.

Table 2 from Hodzic et al., 2014.  "Interval" = time after completion of treatment; "Inoc" = skin from inoculation site; "HB" = heart base; "VM" = ventricular muscle; "QM" = quadriceps muscle; "Tt" = tibiotarsus; "XenoDx" = xenodiagnostic ticks (# ticks testing positive/# ticks placed on mouse).

They saw something remarkable with the mice left for 12 months.  Although few tissues were positive at earlier time points, most tissues extracted from mice a year after treatment tested positive.  6 of the 8 mice also tested positive by xenodiagnosis.  So, instead of eventually disappearing, the spirochetes proliferated starting at some point after 8 months elapsed following treatment.  This resurgence occurred even though the spirochetes remained unculturable.

Barthold's group also looked for evidence of inflammation.  Despite the resurgence of spirochetes, they did not see much evidence of inflammation by microscopy of the tissues 12 months  following antibiotic treatment.  However, the researchers pointed out that no conclusions can be drawn about the ability of the persisting spirochetes to cause disease since inflammation was minimal even in saline-treated mice, which harbored culturable spirochetes.

The researchers next looked for molecular evidence of inflammation.  They measured transcript levels of 18 cytokines in the base of the heart, heart muscle, quadriceps muscle, and leg joint 12 months after treatment with ceftriaxone or saline.  The levels of cytokine transcripts in the two groups were compared to those in age-matched uninfected mice.  Not surprisingly, saline-treated mice had what the authors deemed a "proinflammatory" cytokine profile, most likely due to their ongoing infection.  Antibiotic-treated mice also had a proinflammatory cytokine profile, although it differed from that of the saline-treated mice.  This observation is the first to suggest that the mice were responding to persisting spirochetes that survived antibiotic treatment.

In conclusion, the evidence is convincing that B. burgdorferi persists in mice for a long time after antibiotic treatment.  They don't eventually disappear and may even proliferate.  Whether these unculturable spirochetes are capable of generating an inflammatory condition necessary for disease is less clear, though mice do appear to generate a unique cytokine profile in response to the persisting spirochetes.

Barthold's group caution readers from applying the findings too broadly:

Because of the controversial nature of these findings, they should not be over-interpreted and certainly not translated directly into clinical management of human Lyme borreliosis.

So is there any relevance of these findings to post-treatment symptoms in humans?  I will touch upon this issue in a future post.

Reference

Hodzic E, Imai D, Feng S, & Barthold SW (2014). Resurgence of persisting non-cultivable Borrelia burgdorferi following antibiotic treatment in mice. PLOS One, 9 (1) PMID: 24466286

Related posts

• Chronic Lyme disease in mice?
• Tigecycline fails to eradicate persisting Borrelia burgdorferi
• Long-term antibiotics for those with chronic symptoms that may or may not be related to Lyme disease

Long-term antibiotics for those with chronic symptoms that may or may not be related to Lyme disease

A Lyme disease study published a few weeks ago in the New England Journal of Medicine has received a lot of coverage in the press.  According to the abstract of the study, Berende and colleagues conducted a randomized placebo-controlled clinical trial to test the effectiveness of long-term "longer-term" antibiotics in treating "longer-term" chronic symptoms "attributed" to Lyme disease.

As many readers of this blog know, treatment of Lyme disease is a controversial topic.  Antibiotics are effective in treating Lyme disease, but 10-20% experience symptoms such as fatigue, muscular aches, and joint pain for at least 6 months following conventional treatment.  The cause of the persisting symptoms is not known.  They could be due to tissue damage caused by the infection, ongoing inflammation, or bacteria that survived antibiotic treatment.  Mainstream medical societies such as the IDSA do not believe that lingering infection is responsible for the persisting symptoms, and they do not recommend retreatment with antibiotics.  Four randomized controlled studies conducted in the U.S. showed little benefit of retreating these patients with antibiotics for up to 3 months.  On the other hand, not-so-mainstream groups such as ILADS dispute the interpretation of the data.  They insist that the treatment groups did show some improvement and that longer treatment regimens lasting longer than 3 months are needed for complete recovery of these patients.

There is another group of patients that also suffer from enduring fatigue, muscle aches, and joint pain.  They may or may not have had Lyme disease in the past, but their ongoing symptoms stem from some other condition.  Unfortunately, they may be misdiagnosed with "chronic Lyme disease" and end up being treated for a long time with antibiotics in an attempt to eradicate an infection that they don't have.

The new NEJM paper describes a randomized placebo-controlled trial that was conducted in the Netherlands.  281 subjects who had been experiencing chronic symptoms blamed on Lyme disease (fatigue, muscle aches, joint pain) were randomized into three groups.  All three groups were treated with ceftriaxone intravenously for two weeks.  The subjects were next given oral antibiotics or placebo for 12 weeks.  One group was treated with doxycycline, another group with both clarithromycin and hydroxychloroquine, and the third group was given placebo pills.

The graph below shows that the physical quality of life, the primary outcome measure, improved a little for all groups.  Because there was no difference in outcome among the three groups, the authors concluded that longer-term antibiotics were no better than short-term antibiotics in alleviating symptoms.  We don't know whether the initial two-week treatment with ceftriaxone had anything to do with the slight improvement since there was no true placebo (antibiotic-free) group.

Change in mean SF-36 physical component summary scores before and after treatment period.  Figure 2 from Berende et al., 2016.

Why wasn't a true placebo group?  The authors worried about withholding antibiotics from subjects who might have an infection that should be treated.  11% of the subjects hadn't been treated with antibiotics for their symptoms prior to their acceptance into the study.  This wasn't an issue with the earlier U.S. trials since previous treatment of Lyme disease with antibiotics was a requirement for acceptance into those studies, which included true placebo groups.

One baffling aspect of the study was the inclusion of subjects who might not have had Lyme disease prior to the appearance of their chronic symptoms. Only a third of the subjects had objective clinical features of Lyme disease (erythema migrans, meningoradiculitis, or acrodermatitis chronica atrophicans) immediately preceding their chronic symptoms, and a little more than a half recalled a tick bite.  Contrast this with the earlier U.S. studies, which only accepted patients with chronic symptoms that followed antibiotic treatment of a well-documented case of Lyme disease.

The remaining subjects did not have any objective features of Lyme disease before their chronic symptoms appeared.  The only evidence of a previous episode of Lyme disease was positive antibody testing by Western blot.  However, the antibodies may have been elicited by a Borrelia infection in the distant past.  Their past episode of Lyme disease may not be related to their chronic symptoms, which aren't specific for Lyme disease.  Another problem with relying solely on Western blots to diagnose Lyme disease is that false positives occur.  Without additional evidence, it is hard to be sure that their chronic symptoms were related to Lyme disease.

Nevertheless, the editorial accompanying the paper expressed support for the relaxed inclusion criteria used to select the subjects for the study:

Critics may rightly say that this trial does not truly capture with certainty the consequences of bona fide Lyme disease. However, studies with more stringent inclusion criteria have already been conducted, and the approach used by Berende and colleagues probably reflects the common practice in ambulatory care settings, in which patient presentations of fatigue or nonspecific pain prompt serologic checks for Lyme disease, despite evidence suggesting that these tests will not identify a probable cause or result in a treatment benefit.

The study population may reflect what's encountered by clinicians in the real world, but for a clinical trial it doesn't seem right to lump those whose chronic problems followed a real episode of Lyme disease with those whose issues had nothing to do with Lyme.  Any benefit of the antibiotics experienced by those who had genuine Lyme disease (assuming that there was any benefit) may have been obscured by the lack of benefit in those whose chronic symptoms aren't related to Lyme disease.

Berende and colleagues also defended the length of treatment, which is considered to be on the short end by those who support lengthy courses of antibiotic therapy:

...it may be argued that 14 weeks of treatment is insufficient to show a beneficial treatment effect. However, whereas prolonged antimicrobial treatment is not uncommon for various infectious diseases, the purpose of prolonged therapy for such diseases is for the prevention of microbiologic relapse rather than for a delayed onset of clinical alleviation of signs or symptoms. We are not aware of any infectious disease in which the initial effect on signs, symptoms, and laboratory findings is delayed beyond the first 3 months of effective therapy.

But the graph above clearly shows that the subjects felt better following the treatment period.  Unfortunately, as I mentioned earlier, the improvement in the quality of life can't be attributed to the antibiotics because there was no true placebo group.

With these issues, I'm not sure how this study got published in NEJM.  Regardless of my opinion, it will undoubtedly be cited as further proof that long-term antibiotics don't alleviate long-term symptoms that stem from Lyme disease.

Edit: Corrected quotes in first paragraph.

References

Berende A, ter Hofstede HJ, Vos FJ, van Middendorp H, Vogelaar ML, Tromp M, van den Hoogen FH, Donders AR, Evers AW, & Kullberg BJ (2016). Randomized Trial of Longer-Term Therapy for Symptoms Attributed to Lyme Disease. The New England Journal of Medicine, 374 (13), 1209-20 PMID: 27028911

Melia MT, & Auwaerter PG (2016). Time for a Different Approach to Lyme Disease and Long-Term Symptoms. The New England Journal of Medicine, 374 (13), 1277-8 PMID: 27028918

The genomes of 20 species of Leptospira

A massive study describing the genomes of 20 species of Leptospira was published a few weeks ago in PLOS Neglected Tropical Diseases.  The deluge of sequence information will be valuable to those in the leptospirosis field.  Scientists will be able to examine differences in genetic content between various categories of Leptospira species to generate hypotheses for experimental testing.  For example, genes present in species that cause infections but missing in species that don't may be important factors responsible for the pathogenesis of Leptospira.  The genome information will also aid in vaccine and serodiagnostics development by allowing researchers to identify protein antigens that are conserved among Leptospira species circulating within a region of interest.

The 20 Leptospira species are divided into 14 infectious and six noninfectous species.  (Actually, there are now 22 species known but only 20 when this study was launched.)  The infectious species are divided further into nine pathogenic and five "intermediate" species based on their genetic relatedness.

The Venn diagram below shows the number of genes that are shared among and within the three categories of Leptospira and Leptonema illini, a closely-related spirochete.  Looking at the relevant intersection (overlap) in the diagram, there are 255 genes that are carried by infectious Leptospira but not by saprophytic Leptospira.  (The other two figures in the overlap are the number of shared genes tabulated using looser criteria.  In these cases there are 302 genes found in all but one infectious Leptospira and 369 genes when those found in the majority of infectious species are counted.)  Similarly, there are 109 genes unique to the pathogenic species (or 161 or 416, if you want to use less stringent criteria).  The small circles at the periphery show the number of genes unique to each species.  So for example, L. interrogans, the species favored for study in molecular biology labs, has 672 genes that are not found in any other Leptospira species.

Figure 2A from Fouts et al., 2016.  Source.

Reference

Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL, Haake DA, Haft DH, Hartskeerl R, Ko AI, Levett PN, Matsunaga J, Mechaly AE, Monk JM, Nascimento AL, Nelson KE, Palsson B, Peacock SJ, Picardeau M, Ricaldi JN, Thaipandungpanit J, Wunder EA Jr, Yang XF, Zhang JJ, & Vinetz JM (2016). What makes a bacterial species pathogenic?: Comparative genomic analysis of the genus Leptospira. PLoS Neglected Tropical Diseases, 10 (2) PMID: 26890609

How a new species of Lyme disease bacteria was discovered

A new agent of the tick-borne illness known as Lyme disease has emerged in the upper Midwest.  The bacterium is genetically related to Borrelia burgdorferi, until now believed to be the only cause of Lyme disease in the United States.  The name proposed for the bacterium is Borrelia mayonii because the work was conducted at the Mayo Clinic.  B. mayonii has not been detected in patients outside of the Midwest (so far).  The findings are described in The Lancet Infectious Diseases.

The new species was discovered at the Mayo Clinic during routine testing of specimens (blood, cerebral spinal fluid, and joint fluid) received from all regions of the U.S.  Over 100,000 specimens collected from 2003 through 2014 were tested for Lyme disease bacteria by real-time PCR . The PCR probes were designed to detect the oppA1 gene from Borrelia species belonging to the Lyme disease group, known in the scientific literature as "B. burgdorferi sensu lato."  The Lyme disease group comprises 18 species that fall into the same genetic cluster within the genus Borrelia.  They include species known or suspected to cause Lyme disease (B. burgdorferi, B. garinii, B. afzelii, B. spielmanii, B. valaisiana, B bissettii, B. bavariensis, and B. lusitaniae) and another ten species that do not cause illness.  The PCR probes do not react with DNA from species belonging to the other cluster of Borrelia, the relapsing fever group.

The key to the discovery of the new species was the melting temperature analysis routinely programmed onto the end of real-time PCR runs.  The oppA1 PCR products amplified from B. burgdorferi strains have melting temperatures of 63.6 through 64.9°C.  For other Lyme disease species, the melting temperature ranges from 52.3°C (B. valaisiana) to 59.2°C (B. californiensis).  Therefore, the melting temperature of the oppA1 PCR product was used to distinguish B. burgdorferi from other Lyme disease Borrelia.

Over 9,000 specimens were collected from the states of Minnesota, Wisconsin, and North Dakota from January 2012 through September 2014.  102 were PCR positive, and most of the PCR products had the melting temperature profile of B. burgdorferi.  However, six had melting temperatures ranging from 60.4°C to 61.2°C, too low to be B. burgdorferi but too high to be any other member of the Lyme disease group.  The novel spirochetes were cultured from the blood of two of the patients.  The DNA sequence of several "housekeeping" genes of the new isolates differed enough from those of other Borrelia species to signify that a new Borrelia species has been found.  The investigators named the new spirochete Borrelia mayonii.  No specimen collected from other regions of the U.S. exhibited the atypical melting temperatures, and neither did any collected earlier than 2012 from the Midwest.  These findings led the authors to conclude that B. mayonii has recently emerged in the upper Midwest and that the six patients are the first known cases of Lyme disease to be caused by the new species.

The investigators also collected Ixodes scapularis ticks in Wisconsin.  PCR and melting temperature analysis showed that 19 of 658 ticks (2.9%) were positive for B. mayonii, 195 (29.6%) positive for B. burgdorferi, and two positive for both.

One striking feature of B. mayonii infections is the large number of spirochetes circulating within the patients.  The densities ranged from 420,000 to 6,400,000 bacterial cells per milliliter, at least a hundred times higher than observed in the blood of patients with B. burgdorferi infections.  The numbers were high enough that spirochetes could be seen in blood collected from one of the patients.

Fig. 1b from Pritt et al., 2016

The six patients had many of the typical Lyme disease symptoms:  headache, neck pain, muscle aches, joint pain, and fatigue.  Although mild fever is also common in Lyme disease, two of the six patients had severe fevers with temperature readings approaching 40°C (104°F).  Four had nausea or were vomiting, which are also uncommon Lyme disease symptoms.  Two patients were hospitalized because of the severity of their illness.  Lyme disease may be missed in those infected with B. mayonii because of the unusual symptoms.

The standard two-tier antibody test, which uses B. burgdorferi antigens to detect reactive antibody, may help with the diagnosis.  Blood specimens from five of the six patients were tested.  Four patients either tested positive or, if negative initially, tested positive with blood drawn weeks later.  The one patient who tested negative had blood drawn only on the first day of illness, so it's likely that the antibody response hadn't kicked in fully.  The test appears to help with the diagnosis of Lyme disease caused by B. mayonii, but the number of patients tested was too small to draw firm conclusions.

The authors conclude:

In view of the differing clinical manifestations for patients infected with the novel B burgdorferi sensu lato genospecies, it is likely that Lyme borreliosis is not being considered—and therefore not diagnosed—in some patients with this infection. The clinical range of illness must be better defined in additional patients to ensure that physicians can recognise the infection and distinguish it from other tick-borne infections. Many tick-borne pathogens have global distribution, therefore studies are needed to establish the geographic distribution of human beings and ticks infected with the novel B. burgdorferi sensu lato genopecies. Finally, clinicians should be aware of the potential role of oppA1 PCR for diagnosing infection with this novel pathogen.

Reference

Pritt BS, Mead PS, Johnson DK, Neitzel DF, Respicio-Kingry LB, Davis JP, Schiffman E, Sloan LM, Schriefer ME, Replogle AJ, Paskewitz SM, Ray JA, Bjork J, Steward CR, Deedon A, Lee X, Kingry LC, Miller TK, Feist MA, Theel ES, Patel R, Irish CL, & Petersen JM (2016). Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. The Lancet. Infectious diseases. PMID: 26856777

Lighting up Leptospira interrogans to test antibiotic treatment of chronically-infected mice

A group at the Pasteur Institute succeeded in generating a bioluminescent strain of Leptospira interrogans.  Their study was published last year in PLOS Neglected Tropical Diseases.

The benefit of having a bioluminescent strain is that infections of small laboratory rodents can be monitored without sacrificing the animals.  Instead, the animals are placed in a special whole-body imager that detects light emitted from the bodies.  The location of the infection in the animals can be determined from the images, and the light intensity measured by the imager gives an idea of the bacterial load in infected tissues.  After imaging, the animal can be returned to its cage, and additional images of the same animal can be taken later as the infection progresses.

Another advantage of using bioluminescent bacteria is that the luciferase reaction requires ATP, meaning that the bacteria must be metabolically active to light up.  Dead bacteria containing luciferase will not generate light, unlike green fluorescent protein (GFP), another reporter used to image bacteria (see this post that describes an experiment with Borrelia burgdorferi expressing GFP).

To generate bioluminescent L. interrogans, the researchers hooked the firefly luciferase gene up to a strong L. interrogans promoter and inserted the construct into a transposon carried on a suicide plasmid.  The plasmid was then introduced into L. interrogans by conjugation (see this blog post for details of the process).  Cultures of the engineered spirochetes lit up when luciferin, the luciferase substrate, was added.  The amount of light emitted depended on the culture density: more light was detected at higher culture densities.

Mice are ideal models to study persistent infection of the kidney since many rodents are chronic carriers of Leptospira out in nature.  These rodents don't get sick from the infection, but they contaminate the environment with the spirochete every time they urinate.

To see how chronic infection is established, the investigators injected a sublethal dose of 10⁷ bioluminescent L. interrogans cells into the abdominal cavity of C57BL6/J mice and took sequential images of the animals over the following months.  Albino mice were used because dark fur blocks the signal emitted by the bioluminescent bacteria.  (They later showed that standard C57BL6/J mice with black fur could be used as long as they shaved the fur off before placing them in the imager.)  The mice were injected with luciferin 10 minutes prior to imaging.

There turned out to be two phases of infection (see the "MFlum1" plot in the graph below).  In the acute phase, the bioluminescent signal rose to a peak by day 4 and quickly declined to background levels by day 7.  The signal then started increasing again slowly and plateaued after a month.

Figure 2A from Ratet et al., 2014.  Images of a single mouse taken sequentially are shown below the graph.  Click for larger image.  Source.

Images of a single mouse taken at different times after inoculation are shown below the graph.  Thirty minutes after inoculation, signal was detected in the abdominal cavity.  By day 3, the signal consumed the entire mouse.  At this point, the bacteria were probably circulating in the bloodstream.  By day 6, the signal was almost completely gone.  After day 6, the signal appeared again, but it was confined to the kidneys.  The intensity of the signal in the kidneys increased with time.  They did not detect signal anywhere else in the animals during the second phase.  They even sacrificed some of the infected mice 2 months into the infection to check the organs directly, but they failed to detect Leptospira by bioluminescence and qPCR in the brain, lungs, spleen, liver, or blood.  Not surprisingly, bioluminescence was detected in urine, confirming that the mice were shedding live L. interrogans.

Next, the investigators tested the effectiveness of antibiotics in treating mice infected with the bioluminescent L. interrogans.  Several antibiotics are used to treat acute leptospirosis in humans, including penicillin and azithromycin.  It is generally believed that antibiotics are more effective if provided early in acute illness.  Therefore, the investigators tested whether the timing of antibiotic treatment was important for effectiveness.

As expected, penicillin treatment was most effective when treatment was started at the beginning of the acute phase.  In mice treated with daily injections of penicillin for 5 days starting a day after infection, no bioluminescence was detected in the kidneys, and urine was free of L. interrogans as measured by qPCR.  However, if treatment was delayed until three days after inoculation, during the peak of acute infection, a low level of L. interrogans was detected in urine by qPCR even though no bioluminescence was detected in the kidneys.  It is likely L. interrogans was present in the kidneys but at levels too low to be detected by the imager. The bioluminescence approach clearly does not have the sensitivity of qPCR.  Additional experiments revealed that the limit of detection was 100 bioluminescent L. interrogans cells in 100 μl of buffer.

Penicillin was even less effective when administered after the spirochetes settled in the kidneys. When penicillin treatment was initiated at peak bacterial load in the kidneys, day 25 of infection, the signal diminished by over 90% but then bounced back to the level observed before treatment began (see figure below).  Ciprofloxacin also failed to eradicate the bacteria.

Figure 5A from Ratet et al., 2014.  Antibiotics were administered for 5 days started on day 25 of infection.  Cipr, ciprofloxacin; Pen, penicillin.  Source.

On the other hand, azithromycin managed to knock the signal in the kidneys down to background levels (see graph below).  However, the signal came back within a week, although not to the high levels seen in untreated mice.  A second course of antibiotics starting on day 112 knocked the signal back down to near background levels, but again, spirochete numbers rebounded, although not to the levels seen before retreatment.

Figure 5B from Ratet et al., 2014.  Azithromycin was administered for 5 days starting on day 25 and day 112 of infection.  Source.

Why are antibiotics ineffective in eradicating L. interrogans during the chronic phase?  Like other bacteria, L. interrogans can form biofilms in vitro.  Scientists who work with Leptospira believe that they also assemble into biofilms within the kidney tubules during chronic infection.  Biofilms are hard to eliminate in part because they harbor persister cells that tolerate antibiotics.  (See this post for some background on persister cells.)

I should caution readers from concluding that tolerance accounts for the poor effectiveness of antibiotics in treating human cases of acute leptospirosis.  As the authors point out, leptospirosis patients die because the infection severely injure vital organs.  By the time lethal damage occurs, it does not matter whether antibiotics kill all of the spirochetes.

So does the mouse model have any relevance to human leptospirosis?  The authors argue that asymptomatic carriage of Leptospira has been overlooked.  A 2013 study from the Netherlands revealed that 21% of patients who contracted  leptospirosis continued to suffer from headaches, muscle aches, and extreme fatigue two years later.  This may reflect unrepaired tissue damage inflicted during acute infection, but no one checked for the presence of Leptospira in these patients.  Another study from Peru (see this post) describes asymptomatic individuals who may have persistent Leptospira infection. Kidney function was not checked in the Peruvians, but there is reason to believe that chronic infection affects the kidneys despite the lack of symptoms.  Mice chronically infected with L. interrogans are not visibly sick, but they end up with scarred kidneys (fibrosis), as explained in this study.

If persistent asymptomatic infections really do occur in humans, it may be sensible to treat with antibiotics.  The chronically-infected mouse will serve as a nice model for testing antibacterial regimens that target Leptospira living in the kidneys.

References

Ratet G, Veyrier FJ, Fanton d'Andon M, Kammerscheit X, Nicola MA, Picardeau M, Boneca IG, & Werts C (2014). Live imaging of bioluminescent Leptospira interrogans in mice reveals renal colonization as a stealth escape from the blood defenses and antibiotics. PLoS Neglected Tropical Diseases, 8 (12) PMID: 25474719

Goris MG, Kikken V, Straetemans M, Alba S, Goeijenbier M, van Gorp EC, Boer KR, Wagenaar JF, & Hartskeerl RA (2013). Towards the burden of human leptospirosis: duration of acute illness and occurrence of post-leptospirosis symptoms of patients in the Netherlands. PloS One, 8 (10) PMID: 24098528

Fanton d'Andon M, Quellard N, Fernandez B, Ratet G, Lacroix-Lamandé S, Vandewalle A, Boneca IG, Goujon JM, & Werts C (2014). Leptospira Interrogans induces fibrosis in the mouse kidney through Inos-dependent, TLR- and NLR-independent signaling pathways. PLoS Neglected Tropical Diseases, 8 (1) PMID: 24498450


Related posts

• Life after leptospirosis, a pilot study
• Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon
• The magic of antibiotic tolerance

Do Lyme disease spirochetes produce a toxin?

According to the current view of Lyme disease pathogenesis, tissue damage is caused by the inflammatory response to the spirochetes.  Borrelia species do not produce toxins that injure the host directly.  A new study published in BMC Microbiology may force us to modify our view.

The study shows that some Borrelia strains carry a set of genes with the potential to generate a peptide resembling streptolysin S (SLS), a potent toxin produced by the pathogen Streptococcus pyogenes.  The enzymes that produce SLS in S. pyogenes are expressed from a cluster of genes surrounding sagA, a tiny gene encoding the SLS precursor.  The peptide produced from sagA is nontoxic; it has to undergo several alterations to its structure to become toxic.  A critical modification is carried out by the SagBCD protein complex, which converts the side chains of cysteine, serine, and threonine into ring structures.

Figure 2 from Molloy et al., 2011

Other genes surrounding sagA encode a peptidase that is thought to trim the leader peptide from the amino terminus of the SLS precursor and an ABC transporter that may be responsible for expelling SLS from the cytoplasm.

Figure 1A from Molloy et al., 2011

SLS targets neutrophils and possibly other immune cells during S. pyogenes infection.  SLS-like toxins are also produced by other Gram-positive pathogens, including Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum.

The investigators mined the genomes of other bacteria in search for genes encoding the machinery that generates SLS-like toxins.  They found SLS-like gene clusters in various Firmicutes and Actinobacteria, both Gram-positive groups of bacteria.

The researchers also found the gene cluster in the genomes of Borrelia afzelii strain PKo, Borrelia valaisiana strain VS116, and Borrelia spielmanii strain A14S.  B. afzelii is a major cause of Lyme disease in Europe and Asia.  B. valaisiana and B. spielmanii are responsible for occasional cases of Lyme disease.

Figure 4 from Molloy et al., 2015.  Top: organization of SLS-like gene cluster in S. pyogenes and three Borrelia strains. Bottom: sequence of the SLS precursor (SagA) and the borrelial SLS-like precursors.

They also used PCR to screen the DNA of 140 patient and tick isolates of Lyme Borrelia for the genes encoding the SLS-like biosynthetic machinery.  Most of the isolates were obtained from Europe and the U.S., with a few coming from Asia.  Design of the PCR primers was based on the sequence of the B. valaisiana bvalB, bvalC, and bvalD genes, which encode homologs of the S. pyogenes sagB, sagC, and sagD gene products.  Most of the B. garinii, B. afzelii, B. valaisiana, B. spielmanii, and B. lusitaniae isolates that were examined tested positive.  On the other hand, none of the 22 isolates of B. burgdorferi or 13 isolates of B. bavariensis were PCR positive.  These results indicate that SLS-like sequences are widespread among Lyme disease spirochetes (though not in B. burgdorferi).

The next step was to show that the SLS-like borrelial gene actually encoded a peptide that damages mammalian cells.  A simple assay based on the ability of many toxins to rupture (hemolyze) red blood cell in vitro is available.  Hemolysis is measured easily by mixing the toxin with sheep red blood cells.  Hemoglobin released from the ruptured cells is quantified with a spectrophotometer.

They decided to test the SLS-like peptide encoded by B. valaisiana, BvalA, for hemolytic activity.  The researchers succeeded in expressing and purifying a recombinant form of BvalA.  Not surprisingly,  BvalA was not hemolytic because its amino acid side chains had to be converted into ring structures necessary for the peptide to injure red blood cells.  They wanted to mix BvalA with the BvalBCD protein complex so that the peptide would be modified, but they could not generate the protein complex.  Instead, they used the SagBCD complex from S. pyogenes to modify the BvalA peptide.  When they did this, they finally observed hemolytic activity.

Red blood cells are unlikely to be a major target of borrelial SLS-like peptides during infection.  So what is the real target?  More studies are needed to answer this question, but we should consider the possibility that the toxin has nothing to do with Lyme disease.  Instead, it may help the spirochete to survive during its residence within the tick vector.  A number of nonpathogenic bacteria carry gene clusters distantly related to the ones that produce SLS.  Several peptide toxins produced by these bacteria are known to kill competing microbes.  Like us humans, ticks have a microbiome inhabiting their gut.  Some Lyme spirochetes may need to secrete the toxin to ward off their microbial neighbors.

References

Molloy EM, Casjens SR, Cox CL, Maxson T, Ethridge NA, Margos G, Fingerle V, & Mitchell DA (2015). Identification of the minimal cytolytic unit for streptolysin S and an expansion of the toxin family. BMC Microbiology, 15 PMID: 26204951

Molloy EM, Cotter PD, Hill C, Mitchell DA, & Ross RP (2011). Streptolysin S-like virulence factors: the continuing sagA. Nature Reviews Microbiology, 9 (9), 670-81 PMID: 21822292

A biosignature of early Lyme disease

Laboratory testing for Lyme disease involves two-tier antibody testing with sera from patients suspected of having the disease.  The first step is usually an ELISA with a cell lysate of Borrelia burgdorferi as antigen.  If the ELISA results are positive or borderline, a Western blot is done to confirm that the patient has Lyme disease.  Direct detection of Borrelia burgdorferi by culture would be the preferred laboratory test, but it takes too long for the spirochete to grow.  Culture of patient specimens is done only for research studies.

Source: CDC

In general, the problem with antibody testing for infectious diseases is that it takes time for the immune system to generate antibody against the pathogen.  Therefore, patients in the early stages of infection may test negative.  False-negative tests may delay appropriate treatment until the illness worsens.  For these reasons, scientists have been trying to come up with better laboratory tests for infections whose diagnosis relies on detecting antibody against the infectious agent.

One new approach being developed for a few pathogens involves measuring the amounts of each of the thousands of small molecules found in the sera of infected patients.  This is done by liquid chromatography/mass spectrometry (LC-MS), which accurately and precisely measures the size of small molecules, even in complex substances like serum.  The assumption is that the composition of small molecules (the so-called "metabolome") starts to change in a predictable manner as soon as someone is infected.  The metabolome changes because tissues react to the pathogen by generating inflammatory molecules that leak into the bloodstream.  Another critical assumption is that the changes that occur in the metabolome are unique to each pathogen.  Analysis of the patient's metabolome may therefore allow clinicians to quick diagnose any infectious disease whose metabolome has been characterized.

A recent CDC study revealed the metabolome of early Lyme disease.  The investigators obtained sera from 89 patients who had early-stage Lyme disease.  All had erythema migrans (EM), the rash characteristic of Lyme disease.  Most were also culture or PCR positive for Borrelia burgdorferi.  The patient sera were compared with sera from 50 healthy individuals by LC-MS.

After two runs of LC-MS with each sample, the researchers identified a set of 95 small molecules whose levels consistently differed between patients with early Lyme disease and healthy individuals.  Statistical modeling of the data allowed the investigators to refine the biosignature to a set of 44 molecules that identified Lyme disease in the 139 (89 + 50) subjects with the highest sensitivity and specificity.

To better gauge the performance of the biosignature in identifying those with early Lyme disease, the investigators conducted LC-MS on sera from another group of 91 patients shown to have early Lyme disease by the same criteria as the first set of patients.  Control sera came from 108 healthy individuals.  Another set of control sera was obtained from 101 patients with other diseases that could be confused with Lyme disease clinically, serologically, or microbiologically: syphilis, severe periodontitis, infectious mononucleosis, and fibromyalgia.  All patient and control sera were also tested by the standard two-tier antibody test.

The sensitivity of LC-MS testing turned out to be much higher than that of two-tier testing: 88% vs. 44%.  The specificity of LC-MS was 94% with healthy sera and 95% when sera from patients with other diseases were tested.  These values were not significantly different from the specificities of 100% and 95% achieved with two-tier testing.

These results show the promise of using the metabolic biosignature to help diagnose early Lyme disease.  However, note that all patient sera used to uncover the biosignature and assess its performance came from individuals with EM.  In practice, a clinical diagnosis involving the classic bulls-eye EM with a patient history suggestive of Lyme disease does not require confirmation by laboratory testing.  Patients without EM are more likely to need laboratory testing.  According to the CDC, 20-40% of Lyme disease patients do not have EM.

To get an idea of how well the biosignature performs on patients without EM, the investigators obtained sera drawn from 22 cases with early Lyme disease who tested positive with the C6 ELISA, a newer antibody test.  The antigen for the C6 ELISA is a highly-conserved peptide from the B. burgdorferi surface protein VlsE.  Eight of the 22 patients did not have EM.  The EM status was unknown in another eight patients.  The remaining six patients had EM.  Unfortunately, the results for each subgroup were not presented by the authors, so we don't have a firm answer about the performance of LC-MS testing on patients without EM.  What we can say is that even though more than a third of the 22 patients did not have EM, the sensitivity of LC-MS testing remained high at 86%.  In contrast, the sensitivity of two-tier testing with this group was only 41%, even though the investigators stacked the deck by using the C6 ELISA as the first tier with this group.  Future testing of the biosignature should include a larger number of sera from EM-negative patients in the early stages of Lyme disease.

As discussed in the paper, sera from patients with skin conditions that could be confused with EM (e.g., STARI, cellulitis) should be examined in future studies to make sure that the early Lyme biosignature can be used to rule out those conditions.  The authors recommend that sera from patients with neurologic, cardiac, and arthritic forms of Lyme disease also be examined to see if biosignatures specific for these more serious forms of Lyme disease could be identified.

Reference

Molins CR, Ashton LV, Wormser GP, Hess AM, Delorey MJ, Mahapatra S, Schriefer ME, & Belisle JT (2015). Development of a metabolic biosignature for detection of early Lyme disease. Clinical Infectious Diseases, 60 (12), 1767-1775 PMID: 25761869