Monday, November 26, 2012

A post-Thanksgiving story of leptospirosis

I'm about half way through 1491, a book that gives readers a view of the Americas before Columbus showed up.  It also describes the devastating impact that foreign infectious diseases had on the native population as Europeans explored the New World.

One chapter tells the story of Tisquantum (Squanto), who lived in the village of Patuxet, one of the many Indian communities thriving along the coast of New England at the time.  In 1614 Thomas Hunt, a British slave trader, kidnapped Tisquantum and other Indians and shipped them to Spain.  Fortunately, Tisquantum was rescued by Spanish priests before he could be sold.  After convincing the priests to let him return home, he left for London, where he learned English while staying at a shipbuilder's home, and eventually made his way back to North America.  As he sailed down the New England shoreline in 1619 on a British ship, he realized that the world familiar to him had vanished.  A mysterious disease had wiped out 90% of the population of coastal New England.  When he arrived at his home village of Patuxet, he found it deserted.  Tisquantum was soon captured and sent to Massasoit, the leader of the Wampanoag confederacy, which encompassed Patuxet.  Massasoit did not trust Tisquantum because of his recent association with the British, yet he would later use him as a translator in a negotiation that turned out to be a pivotal event in American history.

The epidemic had been blamed at one time or another on smallpox, the plague, yellow fever, typhus, and hepatitis.  As I've mentioned before, a recent analysis has added leptospirosis to the list of suspects.  The symptoms and signs of leptospirosis match those reported from first-hand accounts of the mystery ailment.  Here's a post on the Slate website about the epidemic.  I'm glad to see that the story is getting attention from popular news sites.

Leptospirosis can be deadly, but could it account for the devastating 90% motality rate of the 1616-1619 epidemic?  A hypervirulent strain of Leptospira or genetic susceptibility of the Indians could be an explanation.  However, the authors of the study thought that the most critical factor was the Indian lifestyle, which brought them into repeated contact with Leptospira in the environment.  The Europeans who fished nearby were spared because they did not engage in activities that exposed them to Leptospira.  Therefore, only the Indians contracted the illness, according to the hypothesis.

Figure 3 from Marr and Cathey, 2010.

Whatever its cause, it's hard to overstate the significance of the epidemic.  Prior to 1616, the New England native communities traded with the Europeans and even welcomed them for brief stays.  However, all attempts by the foreigners to establish permanent settlements were fiercely resisted.  Coastal New England was well defended by the large native population.  The Wampanoag confederacy became especially hostile towards the Europeans after having their citizens abducted.

By the time the Mayflower landed in Patuxet (Plymouth) in December of 1620, the thinking of the Wampanoag had changed.  Their depleted population was vulnerable to attack by their longtime enemies to the west, the Narragansett, who remained untouched by the epidemic.  To forestall an attack, Massasoit felt that the best course of action was to form an alliance with the Pilgrims rather than expel them.  In the spring of 1621, with Tisquantum serving as the translator, Massasoit arranged a peace treaty with the Pilgrims.

Reference

Marr J.S. & Cathey J.T. (2010). New hypothesis for cause of epidemic among Native Americans, New England, 1616–1619, Emerging Infectious Diseases, 16 (2) 281-286. DOI:

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Thursday, November 8, 2012

Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease

According to the CDC, 10-20% of Lyme disease patients who have completed antibiotic therapy continue to suffer from symptoms such as joint, muscle, and neurological pain.  The following hypotheses are often presented as possible reasons for the lingering symptoms:  autoimmunity triggered by the infection, tissue damage inflicted by the spirochetes, and (depending on whom you ask) failure of antibiotics to kill all the spirochetes.  A new paper from Linda Bockenstedt's group at Yale proposes that antibiotic treatment of disseminated Borrelia burgdorferi infection leaves behind inflammatory pieces of dead spirochetes that are responsible for the persisting symptoms.

Bockenstedt's group used the mouse model of Lyme disease for the study.  To ensure that the tissues harbored enough B. burgdorferi spirochetes to be visible by intravital microscopy, the mice were genetically deficient in the intracellular signaling protein MyD88.  MyD88 links the recognition of microbial parts by most Toll-like receptors to activation of certain nuclear genes whose products are involved in the inflammatory process.  Mice lacking MyD88 are unable to control the proliferation of a number of bacterial pathogens, including B. burgdorferi.  The load of B. burgdorferi in tissues is about 100-fold higher in MyD88-deficient mice than in mice with a complete immune system.

The spirochetes were genetically altered to express green fluorescent protein (GFP).  The GFP+ B. burgdorferi was introduced into the MyD88-deficient mice by tick inoculation.  21 days later some of the mice were treated for one month with doxycycline, one of the antibiotics used to treat Lyme disease in humans.

The researchers next peered into the thin layer of skin covering the ear by intravital microscopy.  In the mice that were left untreated, they saw lots of spirochetes scurrying about in the dermis.

video

At the deepest depths of the dermis, they noticed immobile specks and patches of green material deposited near the cartilage.  They also saw the deposits in the doxycycline-treated mice.  The material was detected by immunofluorescence of ear sections with antibody against B. burgdorferi up to 10 weeks after antibiotic treatment was completed, indicating that the immune system was unable to clear the deposits.

There was no evidence that any spirochetes survived antibiotic treatment.  The researchers did not see any motile spirochetes in the skin by intravital microscopy.  In addition, tissues were culture negative, ticks that fed on the treated mice were culture negative (xenodiagnosis), and transplantation of skin from the treated mice failed to transmit the infection to recipient mice.  Based on these results, the authors concluded that the deposits were remnants of dead spirochetes.  As expected, untreated mice tested positive by these assays.

Since chronic infection can lead to Lyme arthritis, the investigators also examined the joints.  In another set of mice, the infection was allowed to proceed for four months.  The mice were then treated with the antibiotic ceftriaxone for 18 days.  When the researchers looked in the joints by intravital microscopy, they again saw the green material (see figure below).



Fig. 5 from Bockenstedt et al. showing the surface of the patella where it meets the tendon (enthesis).   Panel A, from mouse infected for 4 months, untreated.  Panel B, from mouse infected for 4 months and then treated with ceftriaxone for 18 days. Scale bar, 30 µm.

A critical issue to address is whether the amorphous material left behind following antibiotic treatment inflames the joints.   The authors could not answer this question directly because of the limitations of the mouse model. Histopathology is unlikely reveal joint inflammation, even in the untreated animals, because laboratory mice do not reliably exhibit joint inflammation so late (4-5 months) during B. burgdorferi infection.  Instead, the authors conducted a test tube experiment to see whether the deposits had inflammatory potential.  They ground up joint tissue from antibiotic-treated mice in buffer and applied the homogenate to cultured mouse macrophages.  The macrophages responded by producing TNF, a key cytokine that promotes inflammation.  The more tissue that was added, the more TNF that was produced by the macrophages.  In contrast, joint tissue from uninfected mice did not promote TNF production by the macrophages.  Therefore, the deposits had the potential to spark inflammation, even after motile spirochetes were eliminated by antibiotics.  The debris would continue to inflame the tissues even after antibiotics killed all live spirochetes, explaining why symptoms persist in ~10% of Lyme arthritis cases even after antibiotic treament.

The relevance of the deposits to Lyme disease in humans could be questioned because the MyD88-deficient mice did not have a complete immune system.  The authors addressed this concern in the Discussion by mentioning a recent study that described a TLR1 variant linked to severe inflammation and treatment failure in Lyme arthritis patients.  Although the gene encoding MyD88 has never been examined in Lyme disease patients, it is conceivable that the TLR1 variant or different forms of other immune genes lead to deposits of Borrelia antigen in the joint and other host tissues.

The authors also addressed the possibility that the deposits are really biofilms, which generally resist killing by antibiotics.  Biofilms are believed to be populated by persister cells, which are in a nondividing state that allows bacteria to tolerate antibiotics.  According to the authors, if the deposits had harbored persister cells, those cells should have resumed growing when conditions became favorable for growth again.  Because the skin and joints from the treated mice were culture negative and because the skin also tested negative by xenodiagnosis and transplantation assays, the authors quickly dismissed the biofilm hypothesis.

Stricly speaking, the authors are correct.  Persister cells should start multiplying again in fresh culture medium.  However, it's hard to dismiss the biofilm hypothesis completely given the known examples of culture-negative chronic infections associated with biofilms (see this review for one example).  Electron microscopy of the joint tissue could reveal whether these deposits are intact spirochetes or debris.

Regardless of their exact nature, deposits of antigen have never been detected within the joints of Lyme arthritis patients.  Allen Steere's group failed to find such deposits in pieces of synovial membrane removed from 26 patients with antibiotic-refractory Lyme arthritis.   The findings of Bockenstedt and colleagues, who detected the deposits in a location outside of the synovial membrane, suggest that Steere's group was looking in the wrong place.


Featured paper

Bockenstedt, L., Gonzalez, D., Haberman, A., & Belperron, A. (2012). Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy Journal of Clinical Investigation, 122 (7), 2652-2660 DOI: 10.1172/JCI58813
 
Helpful references

Bolz DD, Sundsbak RS, Ma Y, Akira S, Kirschning CJ, Zachary JF, Weis JH, and Weis JJ (August 1, 2004).  MyD88 plays a unique role in host defense but not arthritis development in Lyme disease.  The Journal of Immunology 173(3):2003-2010.  Link

Strle K, Shin JJ, Glickstein LJ, and Steere AC (May 2012).  Association of a Toll-like Receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis.  Arthritis and Rheumatism 64(5):1497-1507.  DOI: 10.1002/art.34383

Bakaletz LO (October 2007).  Bacterial biofilms in otitis media, evidence and relevance.  The Pediatric Infectious Disease Journal 26(10):S17-S19.  Link

Carlson D, Hernandez J, Bloom BJ, Coburn J, Aversa JM, Steere AC (December 1999).  Lack of Borrelia burgdorferi DNA in synovial samples from patients with antibiotic treatment-resistant Lyme arthritis.  Arthritis and Rheumatism 42(12):2705-2709.  DOI: 10.1002/1529-0131(199912)42:12<2705::aid-anr29>3.0.CO;2-H


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