Friday, April 30, 2010

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.

ResearchBlogging.orgIn 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