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

Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon

The spirochete Leptospira is the agent of leptospirosis, a zoonosis that primarily burdens tropical regions of the world.  Moist conditions promote the survival of Leptospira in soil and fresh water. Although Leptospira could survive out in wet environments if they had to, they thrive in the kidneys of rats and other maintenance hosts, where they form dense masses lining the inner surface of the kidney tubules.  The spirochetes spill into the urine that forms in the tubules, which drain into the bladder.  Animals colonized by their "preferred" serovar (immune type) shed Leptospira throughout their lives without ever showing signs of illness.  The tainted urine ends up contaminating soil and water with infectious Leptospira.

Humans aren't regarded as long-term carriers of Leptospira.  Rather, they are deemed "accidental" (incidental) hosts who may suffer serious complications of acute disease, including kidney failure and lung hemorrhage.  Humans get infected when they come into contact with contaminated water or soil or following direct exposure to infectious animal urine or tissue.  Leptospira enters through cuts in the skin or mucous membranes.  From there the motile spirochete spreads via the bloodstream and invades internal organs, including the kidneys, where they remain for the duration of the disease.  Patients typically stop releasing Leptospira into their urine after they recover from the illness, presumably because the spirochetes have been eliminated from their kidneys.  However, there have been a few reports of Leptospira excreted in urine months or even years following recovery from leptospirosis.  The truth is that no one has ever done a systematic study to determine how common the chronic carrier state is in humans.

A team of investigators from the United States and Peru set out to find long-term carriers of Leptospira.  Their study is described in the February issue of PLoS Neglected Tropical Diseases.  The authors examined the inhabitants of a rural Amazon village of Padrecocha near the city of Iquitos, Peru, where leptospirosis is endemic. The tropical climate is ideal for the survival of Leptospira in the moist environment favored by the spirochete.  Indeed, in an earlier study the authors detected infectious strains of Leptospira in the streams and wells serving the village.  Cattle, pigs, dogs, and rats, all potential carriers, freely roam the area.

Ganoza and colleagues wanted to determine what percentage of the villagers were chronic carriers of Leptospira.  They first identified villagers who were not recently infected with Leptospira.  Out of the 314 healthy villagers enrolled in the study, 102 (32.5%) had no clinical or serological evidence of recent infection; they did not recall experiencing a fever during the previous year (fever is a typical symptom of leptospirosis), and they tested negative for newly-acquired Leptospira infection by IgM ELISA.

The investigators next identified those whose kidney were colonized by Leptospira among the 102 who were not newly infected.  Since Leptospira living in the kidney tubules are shed into urine, they screened urine samples by nested PCR using primers targeting the 16S rRNA gene of Leptospira.  To exclude false-positive signals, the investigators screened the PCR-generated DNA (amplicon) by dot blot analysis with a Leptospira 16S rRNA probe.  Many false positive signals occurred because their Leptospira PCR primers also hybridized to the 16S rRNA gene from Atopobium vaginae, a bacterium recently found to be associated with vaginosis. 

When urine from the 102 "long-term" healthy individuals was screened, Leptospira DNA was found in 6 (5.9%).  Sequencing of the 16S rRNA gene revealed that the carriers were colonized with L. interrogans, L. fainei, and L. licerasiae.  So it turns out that the asymptomatic carrier state is not as rare as initially believed.  More than 1 in 20 individuals who had been healthy for at least a year were colonized with Leptospira in their kidneys.

The investigators found seven additional individuals colonized with Leptospira by screening urine from the other 212 individuals in the study.  Overall the percentage of shedders of Leptospira among all healthy individuals, irrespective of when they were infected, was 4.1% (13/314).  The concentration of Leptospira in the urine of shedders, as measured by quantitative PCR, was low, in the 10²-10⁴/ml range.  In contrast, rats may shed up to 10⁸ spirochetes/ml!

The study unearthed another surprise.  All 13 individuals who were shedding Leptospira at the time of the study (including the 6 chronic carriers) were women.  The proportion of women with Leptospira DNA in their urine (13/13, 100%) was significantly higher than the proportion of women in the group lacking detectable DNA in their urine (199/301, 66%, p = 0.011).  This result raises the possibility that women are more likely to become persistent carriers than men.  However, the authors pointed out that men were underrepresented in the study sample.  Less than one third of the villagers enrolled in the study were men.  Most of the other men were away at work when the authors were recruiting people for the study.  Agricultural occupations, which bring workers into contact with environmental sources of Leptospira, are well-known risk factors for infection by the spirochete in endemic areas.  Hence, male shedders of Leptospira may have been inadvertently excluded from the study.

Another surprising result was that sera from all six chronic carriers failed to agglutinate Leptospira by MAT (microscopic agglutination test), a standard serological test used to check for Leptospira infection whether it occurred recently or years ago.  The authors mentioned that this was entirely consistent with old studies failing to detect agglutinating antibodies in the sera of some maintenance host animals excreting Leptospira. However, another possibility is that the "chronic human carriers" may have actually acquired asymptomatic infections very recently.  They could have been enrolled in the study before the anti-Leptospira IgM and agglutinating antibodies had enough time the accumulate to the cut-off values selected for the IgM ELISA and MAT, respectively.  Although the authors discounted the possibility of newly acquired asymptomatic infections accounting for the seronegativity of the shedders, they recommended a longitudinal study to clarify the issue.

The authors posed several questions raised by their study:

• Does persistent Leptospira infection of human kidneys have any subtle effect on their function? If so, is antibiotic treatment warranted?
• Are some strains of Leptospira more likely than others to persistently infect the kidneys of humans?  .
• Can persistent human shedders be a source of transmission of Leptospira to other humans (and animals)?

Future studies will need to include urine cultures to demonstrate that Leptospira shed by human carriers are alive.

In conclusion, this is an important study that challenges the simplistic notion that humans are incidental hosts of Leptospira.  The reality appears to be more complicated.

Featured paper

Ganoza, C.A., Matthias, M.A., Saito, M., Cespedes, M., Gotuzzo, E., & Vinetz, J.M. (2010). Asymptomatic renal colonization of humans in the Peruvian Amazon by Leptospira. PLoS Neglected Tropical Diseases, 4 (2) DOI: 10.1371/journal.pntd.0000612

Related paper

Ganoza, C.A., Matthias, M.A., Collins-Richards, D., Brouwer, K.C., Cunningham, C.B., Segura, E.R., Gilman, R.H., Gotuzzo, E., & Vinetz, J.M. (2006). Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira. PLoS Medicine, 3 (8) DOI: 10.1371/journal.pmed.0030308