Asymptomatic renal colonization of humans in the peruvian Amazon by Leptospira.

Abstract

Renal carriage and shedding of leptospires is characteristic of carrier or maintenance animal hosts. Sporadic reports indicate that after infection, humans may excrete leptospires for extended periods. We hypothesized that, like mammalian reservoir hosts, humans develop asymptomatic leptospiruria in settings of high disease transmission such as the Peruvian Amazon.
Using a cross-sectional study design, we used a combination of epidemiological data, serology and molecular detection of the leptospiral 16S rRNA gene to identify asymptomatic urinary shedders of Leptospira. Approximately one-third of the 314 asymptomatic participants had circulating anti-leptospiral antibodies. Among enrolled participants, 189/314 (59%) had evidence of recent infection (microscopic agglutination test (MAT0 >or=1:800 or ELISA IgM-positive or both). The proportion of MAT-positive and high MAT-titer (>or=1:800) persons was higher in men than women (p = 0.006). Among these people, 13/314 (4.1%) had Leptospira DNA-positive urine samples. Of these, the 16S rRNA gene from 10 samples was able to be sequenced. The urine-derived species clustered within both pathogenic (n = 6) and intermediate clades of Leptospira (n = 4). All of the thirteen participants with leptospiral DNA in urine were women. The median age of the DNA-positive group was older compared to the negative group (p<or=0.05). A group of asymptomatic participants ("long-term asymptomatic individuals," 102/341 (32.5%) of enrolled individuals) without serological evidence of recent infection was identified; within this group, 6/102 (5.9%) excreted pathogenic and intermediate-pathogenic Leptospira (75-229 bacteria/mL of urine).
Asymptomatic renal colonization of leptospires in a region of high disease transmission is common, including among people without serological or clinical evidence of recent infection. Both pathogenic and intermediate Leptospira can persist as renal colonization in humans. The pathogenic significance of this finding remains to be explored but is of fundamental biological significance.

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Asymptomatic Renal Colonization of Humans in the
Peruvian Amazon by Leptospira
Christian A. Ganoza1,2¤, Michael A. Matthias2, Mayuko Saito3,4,5, Manuel Cespedes6, Eduardo Gotuzzo1,
Joseph M. Vinetz2*
1Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru, 2Division of Infectious Diseases, Department of Medicine,
University of California San Diego School of Medicine, La Jolla, California, United States of America, 3Department of International Health, Johns Hopkins Bloomberg
School of Public Health, Baltimore, Maryland, United States of America, 4Department of Microbiology, Universidad Peruana Cayetano Heredia, Lima, Peru, 5Asociacion
Benefica PRISMA, Lima, Peru, 6Instituto Nacional de Salud, Lima, Peru
Abstract
Background: Renal carriage and shedding of leptospires is characteristic of carrier or maintenance animal hosts. Sporadic
reports indicate that after infection, humans may excrete leptospires for extended periods. We hypothesized that, like
mammalian reservoir hosts, humans develop asymptomatic leptospiruria in settings of high disease transmission such as
the Peruvian Amazon.
Methodology/Principal Findings: Using a cross-sectional study design, we used a combination of epidemiological data,
serology and molecular detection of the leptospiral 16S rRNA gene to identify asymptomatic urinary shedders of Leptospira.
Approximately one-third of the 314 asymptomatic participants had circulating anti-leptospiral antibodies. Among enrolled
participants, 189/314 (59%) had evidence of recent infection (microscopic agglutination test (MAT0 $1:800 or ELISA IgM-
positive or both). The proportion of MAT-positive and high MAT-titer ($1:800) persons was higher in men than women
(p=0.006). Among these people, 13/314 (4.1%) had Leptospira DNA-positive urine samples. Of these, the 16S rRNA gene
from 10 samples was able to be sequenced. The urine-derived species clustered within both pathogenic (n=6) and
intermediate clades of Leptospira (n=4). All of the thirteen participants with leptospiral DNA in urine were women. The
median age of the DNA-positive group was older compared to the negative group (p#0.05). A group of asymptomatic
participants (‘‘long-term asymptomatic individuals,’’ 102/341 (32.5%) of enrolled individuals) without serological evidence of
recent infection was identified; within this group, 6/102 (5.9%) excreted pathogenic and intermediate-pathogenic Leptospira
(75–229 bacteria/mL of urine).
Conclusions/Significance: Asymptomatic renal colonization of leptospires in a region of high disease transmission is
common, including among people without serological or clinical evidence of recent infection. Both pathogenic and
intermediate Leptospira can persist as renal colonization in humans. The pathogenic significance of this finding remains to
be explored but is of fundamental biological significance.
Citation: Ganoza CA, Matthias MA, Saito M, Cespedes M, Gotuzzo E, et al. (2010) Asymptomatic Renal Colonization of Humans in the Peruvian Amazon by
Leptospira. PLoS Negl Trop Dis 4(2): e612. doi:10.1371/journal.pntd.0000612
Editor: Sharon J. Peacock, Cambridge University, United Kingdom
Received October 30, 2009; Accepted December 31, 2009; Published February 23, 2010
Copyright: ? 2010 Ganoza et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by U.S. Public Health Service grants D43TW007120, R01TW005860, and K24AI068903. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jvinetz@ucsd.edu
¤ Current address: Department of Immunology, Max Planck Institute for Infection Biology, Charite ´platz 1, 10117 Berlin, Germany
Introduction
Leptospirosis is a zoonotic disease caused by spirochetes of the
genus Leptospira. Found worldwide, leptospirosis is more common
in tropical and sub-tropical areas where environmental and
socioeconomic conditions favor its transmission. It has been
identified in recent years as a global public health problem because
of its increased mortality and morbidity. The disease is principally
transmitted to humans indirectly by contact with water or soil
contaminated with the urine of domestic and wild animals with
persistent renal infection by Leptospira [1–3].
The tropical climate of the Peruvian Amazon region of Iquitos
is ideal for the maintenance and transmission of leptospirosis.
In developing countries, impoverished populations typically live
either in rural areas or under highly crowded conditions in urban
slums. These factors increase the risk of human exposure to the
urine of Leptospira-infected animals [4,5]. In the Iquitos region,
leptospirosis is common. Seropositivity as seen in cross-sectional
surveys is high [6]; more than half of patients presenting to urban
and rural community-based health posts with non-malarial acute
febrile illness have been observed to have diagnostic levels of anti-
leptospiral antibodies suggestive of acute leptospirosis [4]. The
majority of patients enrolled presented with a self-resolving
undifferentiated febrile illness with 70% of them having antibodies
against a newly described Leptospira species, L. licerasiae [7]. These
data suggest that exposure to Leptospira is common in daily life in
this tropical setting [5,8], and that, in general, Iquitos is accurately
classified as hyper-endemic for leptospirosis infection.
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Leptospirosis in humans is frequently misidentified because of
several factors: 1) variable and nonspecific clinical presentation; 2)
lack of awareness of the disease among clinicians; and 3) difficulty
in access to reliable and rapid diagnostic tests. Clinical
manifestations, when present, vary from a mild ‘flu-like’ febrile
illness to a severe disease variably including jaundice, renal failure,
pulmonary hemorrhage, refractory shock and other grave
manifestations. However, many if not most people infected by
Leptospira develop sub-clinical disease or have very mild symptoms,
and thus do not seek medical attention [1,2]. Asymptomatic
infection, common in endemic areas, has been reported in several
studies [8–12]. For example, in one study, 9–48% of healthy
subjects were diagnosed as having asymptomatic leptospiral
infection by serology (ELISA-IgM) and PCR [10]. However, in
this study, the identity of the infecting strains could not be
determined because of study design. We have observed in one
study that patients can have asymptomatic leptospiruria for
prolonged periods of time [4]. Hence an essential question about
the pathogenicity of Leptospira remains: are some serovars are more
likely than others to establish asymptomatic renal infection in
man?
Renal colonization and persistent shedding of leptospires is
characteristic of carrier or maintenance animal hosts [13–15].
Animals, especially rodents, are known reservoirs of pathogenic
Leptospira species, but rarely develop symptoms and are not
impaired by the infection of their kidneys. After infection, humans
can also excrete leptospires into the urine transiently for weeks or,
more rarely, months or more [1,2,16].
We hypothesized that like mammalian reservoir hosts, humans
develop asymptomatic leptospiruria, including pathogenic Leptospira
such as L. interrogans and intermediate pathogens such as the newly
discovered L. licerasiae [7]. To test this hypothesis, we carried out a
cross-sectional, population-based study in a rural village near the
city of Iquitos to identify the presence and species of infecting
Leptospira directly in the urine of healthy ambulatory people. If
found, we reasoned that the high prevalence of asymptomatic
urinaryinfectionmightprovidefundamentalinsightsintothenature
of Leptospira-human interactions, where humans are considered to
be accidental hosts. Such a finding would also provide the basis for
understanding mechanisms of naturally acquired immunity in
human leptospiral infection.
Materials and Methods
Ethics Statement
This study was approved by the Human Subjects Protection
Program, University of California San Diego, and the Ethical
Committees of Asociacion Benefica PRISMA, Lima, Peru, and
Universidad Peruana Cayetano Heredia, Lima, Peru. All human
subjects provided written informed consent before being enrolled
in the study.
Description of Study Area
This study was carried out in the village of Padrecocha, a rural
community near Iquitos, located north of the city along the Nanay
River, a tributary that branches from the Amazon River 15 km
downstream from Iquitos. The climate is tropical: rainfall averages
300 mm per year and temperatures range from 21.8uC to 31.6uC;
the village is surrounded by a vast expanse of humid tropical
rainforest. The population of this village is approximately 1,500.
Most inhabitants live in brick houses, and their water supply
comes from wells and local streams. These water sources harbor
pathogenic and intermediate-pathogenic Leptospira [5]. Residents
use water from wells or from the local streams for their daily needs
(cooking, bathing and washing clothes). There is no sewage system;
most households have pit latrines. Livestock (mostly chickens, pigs,
and cattle) roam free through the village and its streams; the
inhabitants observe rats frequently.
Enrollment
Using a whole-village canvassing strategy to develop a set of
candidate houses from which to randomly select asymptomatic
inhabitants of the rural village Padrecocha of age $5 years for
enrollment. Subjects were excluded if they had fever within the
previous 2 weeks or if they declined participation (Figure 1). All
participants were clinically evaluated and subjected to an
epidemiologic questionnaire. Whole blood (5 mL) and urine
samples (5—50 mL) were collected from each enrollee.
Serological Testing
Venous blood samples were drawn into tubes without
anticoagulant (Becton-Dickinson, USA) and transported to the
study laboratory within 4 hr at ambient temperature. Serum was
separated, frozen in 1 mL aliquots at 220uC, and transported on
dry ice to the National Leptospirosis Reference Laboratory at the
Instituto Nacional de Salud (INS) in Lima, where the presence of
anti-leptospiral antibodies was determined. An ELISA incorpo-
rating 6 pathogenic serovars (strains)–Icterohaemorrhagiae (RGA),
Australis (Ballico), Bratislava (Jez Bratislava), Ballum (MUS127),
Canicola (Hond Utretch IV), Cynopteri (3522 C), and Grippoty-
phosa (Moskva V)–was used to detect anti-leptospiral IgM
antibodies. An ELISA IgM result of 11.0 IU/mL or more was
considered to be positive [4,7]. Microscopic agglutination testing
(MAT) was performed using 25 leptospiral antigens, using the
Centers for Disease Control and Prevention (CDC) panel [17].
MAT titers were reported as the reciprocal of the number of
dilutions still agglutinating 50% of live bacterial antigen and a titer
of 1:100 or more was considered as positive.
Detection of Leptospiral DNA in Urine Samples
Sample collection and DNA extraction.
were collected in 50 mL sterile polypropylene centrifuge tubes and
Urine samples
Author Summary
Leptospirosis is a bacterial disease commonly transmitted
from animals to humans. The more than 200 types of
spiral-shaped bacteria (spirochetes) in the genus Leptospi-
ra are classified as pathogenic, intermediately pathogenic,
or saprophytic (meaning not causing infection in any
mammal) based on their ability to cause disease and on
genetic information. Unique among the spirochetes that
infect humans, Leptospira live both in the environment (in
surface waters and moist soils), and in mammals, where
they cause chronic infection by colonizing kidney tubules.
Infected animals are the source of human infection, but
humans have not been systematically studied as chronic
Leptospira carriers. In our study, we found that more than
5% of people (in fact, only women) in a rural Amazonian
village, without clinical evidence of infection by Leptospira,
were chronically colonized by the bacteria. Chronic
infection was not associated with a detectable immune
response against the spirochete. Pathogenic and interme-
diatelypathogenicLeptospira
chronic kidney infections. Future work is needed to
determine whether such chronic infection can lead to
human-to-human transmission
whether subtle measures of kidney disease are associated
with asymptomatic, long-term leptospiral infection.
caused asymptomatic,
ofleptospirosis, and
Leptospira Renal Persistence in Humans
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centrifuged at 3,000 g for 30 min at room temperature. DNA
from the pellet was extracted using the QIAampH DNA Mini Kit
(Qiagen, USA) following manufacturer’s directions. DNA was
extracted from samples on-site in the Iquitos laboratory the same
day of collection and stored at 220uC.
Quantitiative real time PCR (qPCR) screening.
samples collected were screened for the presence of pathogenic
and intermediate-pathogenic Leptospira using a published qPCR
TaqMan assay targeting the leptospiral 16S ribosomal gene [18];
this was performed on-site in our Iquitos laboratory using an
Opticon 2 real-time PCR machine (MJ Research, USA). The
assay protocol was modified from the published version by using
the fluorescent probe at a final concentration of 0.2 mM, primers
at a final concentration of 0.5 mM, and a 20 mL reaction volume
[5]. Standard curves for quantification were made using Leptospira
interrogans serovar Copenhageni strain M20. Standards were
prepared as follows. Leptospires were counted using a Petroff-
Hauser counting chamber (Hauser Scientific, USA) and serially
diluted with sterile double-distilled H2O to 108to 100leptospires/
ml. Genomic DNA was subsequently prepared using the DNeasy
All urine
Tissue Kit (Qiagen, USA). Standards were run in triplicate to
generate a standard curve with each run. A negative result was
assigned where no amplification occurred before 40 cycles.
Controls lacking template were extracted and added to qPCR
master mix to detect the presence of contaminating DNA.
Nested PCR amplification of leptospiral 16S rDNA gene
and sequencing of PCR products.
limitations of sensitivity, a nested PCR strategy was used using
the general eubacterial outer primers fD1 and rD1 [19] in a first
round of amplification followed by amplification with the specific
leptospiral 16S rDNA primers lepto16S11f and lepto16S1338r
[5]. Total genomic DNA from 63 qPCR-positive urine samples
was amplified using the universal bacterial 16S rDNA primers
fD1/rD1 as described previously. PCR products were purified
from 1.0% agarose gels in TAE buffer using the QIAquick Gel
Extraction Kit (Qiagen, USA) according to manufacturer’s
directions,diluted 1:100 in
subjected to a second round of amplification using the nested
primers lepto16S11f and lepto16S1338r with a previously
described protocol [5], and then cloned into the pCR2.1-TOPO
To address potential
steriledouble-distilled water,
Figure 1. Flow chart showing study subject enrollment and sample processing.
doi:10.1371/journal.pntd.0000612.g001
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vector (Invitrogen, USA). Recombinant plasmids containing
nested PCR products were then transformed into TOP10 cells
(Invitrogen, USA) and plated on LB agar containing 100 mg/mL
ampicillin. Individual clones were grown overnight in LB broth
containing 100 mg/mL ampicillin. Plasmid DNA from these
clones was purified using the QIAprep Spin Miniprep Kit
(Qiagen, USA) and cycle sequenced. Sequencing was performed
on an ABI 3100 automated sequencer (Applied Biosystems, USA)
using the M13 forward and reverse primers (Invitrogen, USA).
Reaction conditions were according to manufacturer’s directions.
Southern hybridization (dot blot).
luation of the DNA sequences of the first PCR-positive samples we
observed a high rate of false positivity for the nested PCR in urine
samples (75%, data not shown). The 16S ribosomal RNA gene
sequences of the organisms present in the urine of the false positive
PCR samples were frequently identical to the 16S ribosomal RNA
gene sequence of the bacterium Atopobium vaginae, an organism that
has been recently recognized in the microflora associated with
bacterial vaginosis [20]. The 16S ribosomal RNA gene inner nested
primers used to amplify Leptospira shared sufficient sequence
homology with the Atopobium 16S rDNA gene that under the
conditions described these primers generated amplicons for both
Leptospira (,1327 bp) and the Atopobium (,1345 bp) species, making
it difficult to distinguish this amplicon by agarose gel electro-
phoresis alone. To overcome this problem we developed a dot blot
hybridization assay using a digoxigenin (DIG)-labeled DNA probe.
Briefly, a 111bp DIG-labeled DNA probe (Lepto_DIG_probe) was
synthesized byPCR-labeling withthe PCR DIGProbeSynthesisKit
(Roche, USA). Leptospira interrogans serovar Copenhageni strain
Fiocruz L1-130 genomic DNA was used as template; primers
lepto16S620f(59-GAGTTTGGGAGAGGCAAGTGGAATTC-39)
and lepto16S730r (59-GTGCCTCAGCGTCAGTTTTAGGCC-
39) for the probe synthesis. These primers were selected from
conserved regions of the 16S rDNA genes of pathogenic and
intermediate-pathogenic leptospiral species, but were absent from
the saprophytes (Figure 2). Published 16S ribosomal RNA sequences
(n=178) retrieved from the Ribosomal Database Project II release 9
(http://rdp.cme.msu.edu) and aligned using clustalW 1.83 (http://
www.ebi.ac.uk/clustalw) were used. The PCR reaction mix was
prepared following the manufacturer’s directions with the primers at
a final concentration of 0.4 mM and a 1:12 ratio of DIG-
dUTP:dTTP nucleotides. The synthesis was conducted in a DNA
Engine PCT-200 Peltier Thermal Cycler (MJ Research). The
After the initial eva-
amplification protocol consisted of 95uC for 2 min, followed by 30
cycles of amplification, each cycle consisting of 95uC for 10 s, 64uC
for 30 s, and 72uC for 2 min.
Dot blotting of DNA samples.
samples and controls were PCR-amplified using the aforementioned
nested PCR assay, and the PCR products were separated by agar
electrophoresis and purified from the agarose gels. PCR products
were then denatured by boiling for 10 min in a denaturing solution
(0.4 M NaOH/10 mM EDTA, final concentration), then cooled on
ice and spotted into an Immobilon-Ny+ Charged Nylon Transfer
Membrane (Millipore, USA) using a 96-well vacuum manifold (Bio-
Dot Microfiltration Apparatus, Bio-Rad, USA). After spotting the
samples, the membrane was air-dried and the DNA was fixed
by UV cross-linking using the Stratalinker UV Crosslinker 2400
(Stratagene,USA).Thespotted membranewashybridizedovernight
at 56uC with 5 pmol of Lepto_DNA_probe in 3.5 mL of DIG Easy
Hyb hybridization buffer (Roche, USA) according to manufacturer’s
directions. After hybridization and stringency washes, the blot was
developed using a chemiluminescent assay with Anti-Digoxigenin-
AP and CPD-Star (Roche, USA) according to manufacturer’s
directions. For detection of the chemiluminescent signal, X-ray film
was exposed to the blot for 1 minute.
Colony hybridization assay.
contained a high proportion of contaminating Atopobium DNA (data
not shown). Thus to efficiently distinguish clones containing
leptospiral 16S rDNA amplicons for sequencing, we developed a
colony hybridization assay using the Lepto_DIG_probe whereby we
could screen all the transformants on a plate simultaneously. Briefly,
16S ribosomal RNA genes previously PCR-amplified using the
nested PCR assay from dot blot-positive samples (n=13) were
cloned using the PCR2.1-TOPO vector in E. coli as previously
described. Transformed bacteria were grown at 37uC in Petri dishes
(LB agar with 100 mg/mL ampicillin) overnight. Colonies bearing
the cloned 16S rDNA gene were adsorbed onto positively charged
circular nylon membranes (Immobilon-Ny+ membrane discs,
Millipore) and lifted from the agar plate. After adsorption, the
bacteria were denatured in the membranes and their DNA fixed to
them by UV cross-linking. A mean of 4 Petri dishes (or ,200
colonies) with transformed bacteria bearing the 16S ribosomal RNA
gene per urine sample were screened with this method. Membranes
were hybridized with the Lepto_DIG_probe and developed as
described above. Positive colonies in this assay were handpicked and
grown overnight. Plasmid DNA was purified and cycle sequenced
Genomic DNA from urine
The dot-blot positive samples
Figure 2. Sequence alignment of the Leptospira 16 s rRNA gene probe and target. The digoxigenin-labeled Leptospira 16 s rRNA gene
probe (111 bp) aligned with fragments of the 16 s rRNA genes of Leptospira (n=6), Leptonema (n=1) and Atopobium (n=2) species. Pathogenic and
intermediate Leptospira species are shaded in gray.
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as described previously to identify the present leptospires to the
species level.
Identification of infecting Leptospira by phylogenetic
analysis of 16S rRNA gene sequences from urine samples.
DNA sequences were assembled (using 4- to 6-times coverage per
nucleotide base) using the program CAP3 (http://pbil.univ-lyon1.
fr/cap3.php) then aligned using CLUSTALW v1.83 with default
parameters. Leptospira 16S ribosomal RNA gene sequences from
GenBank were used as controls in the tree (Table 1). Sequences
derived from urine samples were analyzed simultaneously and with
identical parameters for the nucleotide substitution model. Missing
(gaps) and ambiguous characters were excluded from the analysis.
However, a separate data partitionwas included whereby gaps were
coded as binary state data; with gap characters coded as 1 while all
others were coded as 0. This data partition was analyzed using the
restriction site model as implemented in MrBayes. A phylogenetic
dendrogram was generated using MrBayes v3.1.2 for Macintosh
(http://mrbayes.csit.fsu.edu/) running for0 3,000,000 generations.
The data were analyzed using the GTR+I+G nucleotide
substitution model with gamma-distributed rates and proportion
of invariant sites. The resulting data sets were then analyzed using
flat priors for the substitution rate parameters [5,7]. New leptospiral
16S sequences were deposited into GenBank (Table 1).
Statistical Analysis
The proportion of the seropositivity rate (at any MAT titer) and
distribution of the demographic variables were compared between
the subjects with and without leptospiruria using the chi-square test
and Mann-Whitney U tests using Stata v8 for Windows (StataCorp,
College Station, Texas) with a significance level (a) of 0.05.
Results
Demographic Description of the Study Population
In the pre-study census and sampling period, 1320 people in 225
houses were identified in the Peruvian Amazon village of Padre
Cocha near Iquitos. The study enrolled 354 participants of age $5
years from 175 households randomly picked from a census map. Of
those 354, 40 participants were excluded since 19 presented with
fever within 2 weeks of enrollment and 21 did not provide urine
samples (patient enrollment diagrammed in Figure 1). The study
included 314 participants with a median age of 27 (range 5—64).
More were female than male (212 vs. 102); 63 (20%) were children
younger than 15 years old (Table 2). Men (median =28.5 years,
range (25%–75%)=(16.5 – 37) were on average slightly older than
women (median =25, range =19–43) with borderline significance
(p=0.051).
Prevalence of Leptospiral Seropositivity
Blood samples were available from 282 of 314 participants
(89.8%). Of these, 97 were from males and 185 from females.
Circulating anti-leptospiral antibodies were found, by either IgM
ELISA or MAT or both, in 108 (38%) of the 281 subjects for whom
serological data were available (Table 2). The most frequently
observed serological reactivity (highest titer by MAT) was to
serogroup Australis (34/281, 12.1%); with serogroups Djasiman
(16/281, 5.7%), Icterohaemorrhagiae (13/281, 4.6%) and Cynopteri
(12/281, 4.3%) also represented. Of the 108 seropositive samples, 64
had serological evidence of recent sub-clinical infection: seven had
MAT titers ($1:800), 23 were IgM-positive but MAT-negative and
34 were IgM and MAT-positive, indicative of recent or current
leptospiral infection. The proportion of MAT-positive (reflecting any
previous exposure to Leptospira) and high MAT-titer ($800, reflecting
recent infection) persons was higher in men (40.6% and 9.4%,
respectively) than women (24.9% (p=0.006) and 2.2% (p=0.006)).
The difference stayed significant after adjusting for the age.
Detection and Molecular Identification of Leptospiral
Species from Urine Samples
The initial qPCR screening performed on-site in Iquitos
detected 63 (20%) positive samples. Further evaluation of these
Table 1. GenBank accession numbers of leptospiral 16S rRNA
gene sequences used in this study to construct phylogenetic
trees, including new sequences from patient urine samples.
Accession No.Strain designation or source
Leptonema
AY714984Leptonema illini serovar Illini strain 3055
Pathogenic Leptospira
AY631877 Leptospira weilii serovar Celledoni strain Celledoni
AY631880L. alexanderi serovar Manhao 3 strain L 60
AY631881 L. genomosp. 1 serovar Sichuan
AY631883L. santarosai serovar Shermani strain LT 821
AY631884L. borgpetersenii serovar Ballum strain Mus 127
AY631886 L. noguchii serovar Panama strain CZ 214
AY631894L. interrogans serovar Icterohaemorrhagiae strain RGA
AY631895L. kirschneri serovar Cynopteri strain 3522
FJ154542L. interrogans serovar Copenhageni strain M 20
Intermediately Pathogenic Leptospira
AY631885L. fainei serovar Hurstbridge strain BUT 6
AY631887L. inadai serovar Kaup strain LT 64–68
AY631891 L. inadai serovar Aguaruna strain MW 4
AY631896L. inadai serovar Lyme strain 10
AY792329 L. broomii strain L065
AY796065 L. broomii strain 5399
EF612284L. licerasiae serovar Varillal strain VAR010
Saprophytic ‘free-living’ Leptospira
AY631876L. biflexa serovar Patoc strain Patoc I
AY631878L. meyeri serovar Ranarum strain Iowa City Frog
AY631879L. wolbachii serovar Codice strain CDC
AY631882L. genomosp. 5 serovar Saopaulo strain Sao Paulo
AY631888 L. genomosp. 4 serovar Hualin strain LT 11–33
AY631889 L. meyeri serovar Hardjo strain Went5
AY631892L. meyeri serovar Semaranga strain Veldrat Samarang
AY631897L. genomosp. 3 serovar Holland strain WaZ Holland
Leptospiral Sequences from Patient Urine Samples in this Study
GU254499PAD062 L. interrogans
GU254500 PAD254 L. interrogans
GU254501PAD328 L. interrogans
GU254502 PAD115 L. interrogans
GU254503PAD061 L. interrogans
GU254504 PAD117 L. interrogans
GU254505 PAD216 L. fainei
GU254506PAD304 L. fainei
GU254507 PAD236 L. broomi
GU254508PAD274 L. licerasiae
doi:10.1371/journal.pntd.0000612.t001
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samples with the nested PCR assay confirmed their positivity.
Among these 63 PCR-positive samples, a newly designed dot-blot
assay, designed to exclude false-positive samples containing only
Atopobium DNA (Figure 3), identified 13/63 (21%) pCR-positive
samples as true positives. We successfully cloned and sequenced
the 16S rRNA gene from 10 of these dot-blot confirmed samples
(Table 1); sequence data were not obtained from 3 samples.
Species assignments were made by Bayesian phylogenetic analysis
of the cloned 16S rRNA gene.
Analysis of these 10 dot-blot-confirmed urine samples showed
that the 16S ribosomal RNA gene sequences clustered within both
the pathogenic (n=6) and intermediate clades of Leptospira (n=4)
(Figure 4). Although asymptomatic, one inhabitant (PAD304,
Table 3) had serological evidence of acute infection (IgM-positive)
indicating sub-clinical infection, and consequently excreted on
average one hundred-fold more Leptospira/ml compared to IgM-
and MAT-negative enrollees.
Serological Results and Characteristics of the Participants
with Leptospiral DNA in Urine
Serological results were available from 281 of the 314
participants including eight of the ten with Leptospira DNA positive
urine (Table 3). Ten of 13 people with DNA-positive urine had
negative results in both IgM and MAT.
All thirteen participants who had leptospiral DNA in their urine
were women and the proportion (100%, 13/13) of the women was
significantly higher compared to that in the leptospiral-DNA-
negative group (66%, 199/301, p=0.011). The median age of the
DNA positive group (43 years, range (min – max)=9258) was
older compared to the women in the negative group (median, 24;
range, 5–60; years, p=0.005). The difference stayed significant if
the men were included in the negative group (median, 27 (5–64)
years, p=0.011). Univariate analysis did not show significant
association between other epidemiological factors and leptospiral
DNA positivity in urine (data not shown).
Asymptomatic Urinary Shedding
Thirteen of the enrolled 314 asymptomatic inhabitants (4.1%)
were confirmed to excrete Leptospira by detection of leptospiral
DNA in their urine; of these, one participant may have had recent
but sub-clinical leptospiral infection, based on an ELISA finding of
IgM positive (Table 3). After clinical and epidemiological
assessment, a group of asymptomatic participants was identified
(n=102, 32.5% of enrolled individuals) that had no evidence of
recent infection (without febrile episodes in the previous year
before enrollment and without anti-Leptospira IgM antibodies
detected); we call them ‘‘long-term asymptomatic individuals.’’
Within this group, six (5.9%) excreted pathogenic and interme-
diate-pathogenic Leptospira (75–229 bacteria/mL of urine, Table 3).
Discussion
This study has several important findings. First, asymptomatic
individuals living in a region hyperendemic for leptospirosis had a
high rate of seropositivity (at any level) for leptospiral infection
(38% of 314 participants). Almost 60% of the seropositive
individuals had evidence of recent sub-clinical infection, as
indicated by MAT titer $1/800. Second, and of unique interest,
a novel 16S rDNA hybridization assay used to screen urine
samples for the presence of leptospiral DNA found that almost 5%
of healthy people living in a rural Amazonian community were
urinary shedders of Leptospira but did not have serological or
clinical evidence of recent infection. Third, we found that both
pathogenic and intermediately pathogenic Leptospira persistent
infected the renal tubules of humans. Such observations have not
been reported previously and are particularly notable because they
demonstrate that inapparent leptospiral infection is common and
frequently leads to shedding of organisms in urine. The long-term
clinical significance of this finding remains to be determined.
The occurrence of leptospirosis, and indeed many infectious
diseases, depends on several interacting variables. These include
favorable environmental conditions, the density of local reservoir
host populations, the type and frequency of exposure, exposure to
infectious doses of the etiologic agent, the virulence of the infecting
strain, and the lifestyle preferences and susceptibility of individuals
Table 2. Characteristics of 314 asymptomatic participants
from the Peruvian Amazon village of Padrecocha, Loreto
Department, Peru.
n=314 (%)
Age median (min – max)27 (5 – 64)
Gender (M:F)102:212
ELISA IgM positive 57/282(20.2)
MAT positive (.1:100)*85/281 (30.2)
ELISA IgM and/or MAT positive108/281(38.4)
MAT high titer (.1:800)*13/281 (4.6)
ELISA IgM and/or MAT high titer (.1:800)*64/281(22.8)
Fever within a year 171/314 (54.5)
Fever by malaria within a year 79/314 (25.2)
*Any serovar tested by MAT=Microscopic Agglutination Test. The ELISA
incorporated serovars Icterohaemorrhagiae (RGA), Australis (Ballico), Bratislava
(Jez Bratislava), Ballum (MUS127), Canicola (Hond Utretch IV), Cynopteri (3522
C), and Grippotyphosa (Moskva V).
doi:10.1371/journal.pntd.0000612.t002
Figure 3. Dot blot hybridization confirming leptospiral DNA in patient urine samples. A= L. interrogans ser. Icterohaemorrhagiae, B= L.
licerasiae ser. Var10, C= Atopobium vaginae, D= Negative urine, E= Water.
doi:10.1371/journal.pntd.0000612.g003
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within the exposed human population [21]. In the context of this
zoonotic infection, the density of local animal reservoir popula-
tions is likely an important determinant of the extent to which the
environment may become contaminated by leptospires through
urine from chronically infected carriers. When environmental
conditions are ideal and background contamination is prevalent,
social practices that predispose to infection, and the virulence of
local strains are significant factors that affect the incidence of the
disease [1]. To date, there is no evidence that humans contribute
to environmental contamination with Leptospira, but the data
presented here do not rule out this possibility.
Exposure to Leptospira in this rural Amazonian study population
was common (,39% were serologically positive at any MAT titer)
with many subjects having evidence of recent sub-clinical infection.
However, the serological data presented here need to be interpreted
with caution: in an endemic setting, a high individual MAT titer
($1:800) and/or IgM positivity are not reliable indicators of recent
or current infection as antibodies may persist for prolonged periods
[22]. The high background exposure rates and relative absence of
severe disease in this hyper-endemic region do suggest that long-
term urinary shedding may occur more frequently here than
elsewhere, where natural immunity may not be as common.
Figure 4. Phylogenetic relatedness of leptospiral 16 s rRNA gene sequences using a bayesian approach.
doi:10.1371/journal.pntd.0000612.g004
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It is generally accepted that humans can excrete leptospires
from weeks to months after infection [23,24]. However the data
presented here indicate that humans may excrete Leptospira for
periods exceeding a year; extending previous understandings of
the carrier state. Ten asymptomatic individuals without clinical (no
febrile episodes in more than a year) or serological evidence of
recent exposure were found to be shedding either pathogenic or
intermediately pathogenic Leptospira in their urine. Although these
persons may have been recently sub-clinically infected and either
failed to produce anti-leptospiral antibodies or all produced ‘false-
negative’ serology, these explanations seem unlikely. It is more
likely that they represent long-term renal asymptomatic shedders
of Leptospira, regardless of whether patients were subclinically
infected or had acute illness. However, a prospective study would
be needed to assess this possibility. Nonetheless, prospective
observational studies of such patients are required to confirm this
hypothesis
Our data also suggest that women (especially mature women)
are more likely to develop long-term renal carriage of Leptospira
than are men; with a significant increase in incidence with age in
women, possibly reflecting increased exposure with age or
alternatively increased susceptibility. It is possible that the
conclusion that women are more likely to be long-term
asymptomatic urinary shedders than men may reflect a bias in
the study, considering its relative underrepresentation of men.
However, this observation may also reflect increased susceptibility
of women to persistent leptospiral kidney infections; the reasons for
this are unclear. However in our study population, the MAT titer
was significantly lower in women than in men perhaps indicating
that men are able to mount a more effective immune response
than are women. Alternatively, men may have persistently higher
antibody titers as a result of more frequent exposure due to work
or recreational practices. Such possibilities require prospective
study to address.
The long-term consequences of human renal infection by
Leptospira need to be explored, in particularly the effect of persistent
infection on renal function and electrolyte balance. Moreover, the
nature of the infecting strains needs to be more carefully explored
as some strains may be more likely than others to result in
persistent renal infections in humans. Although we have identified
the species of the infecting strains in the present study, other
methods that are able to identify serovars, particularly isolation,
will be more informative.
While humans are considered to be exclusively incidental hosts,
animals can be maintenance and/or incidental hosts; maintenance
hosts are defined as species in which infection is endemic, of low or
no pathogenicity and (as a key factor) transmitted directly to the
same species [25–28]. Although human-to-human transmission
has been rarely documented, it is unlikely that asymptomatic
infected individuals have an important role in disease maintenance
and transmission [1,11,29,30]. An increased risk of having
leptospiral antibodies in households of leptospirosis index cases
compared to controls in an epidemic setting has been shown
recently [31], but this is most likely related to common
environmental exposure risks or genetic susceptibilities rather
than direct transmission. In light of these data, further studies
should address the possibility that long-term urinary shedders may
represent a source of Leptospira for their families and explore
human-human transmission more carefully. Infection in carrier
animals is usually acquired at an early age, and the prevalence of
chronic excretion in the urine increases with age; we observed a
similar trend in this population. Of note, none of the long-term
urinary shedders had circulating anti-leptospiral antibodies; this
is in accordance with early observations in Leptospira-carrier
mammals, where chronic urinary carriage was associated with
low seropositivity to urinary culture rates in asymptomatic well-
established serovar-specific carriers [16]. Taken together, these
observations make us speculate that in regions with high disease
transmission, humans can develop some clinical and serological
characteristics of asymptomatic urinary carriers, an attribute
classically restricted to animals. Further longitudinal studies should
address this possibility since the impact on disease transmission
and in renal function of the affected individuals are unknown.
The study design had several limitations. First, we relied only on
the recall of participants to define absence of fever in 1 year. Men
were underrepresented; fewer men were recruited because of a
lack of availability at the time of recruitment (most were away
working). Thus, no leptospiruric males were detected. This
observation suggests that we may have underestimated the overall
number of asymptomatic shedders, as men have been typically
Table 3. Characteristics of Leptospira dot blot-positive subjects (N=13).
ID GenderAge (years)Fever # #1 year IgMMATSpeciesBacteria/mL urine
PAD115*F9---L. interrogans 1.376102
PAD216*F58--- L. fainei2.296102
PAD232*F44---NSNS
PAD254*F29---L. interrogans0.796102
PAD274*F 31---L. licerasiae0.756102
PAD328*F49--- L. interrogans 2.266102
PAD062F 50- NA NAL. interrogans 5.626102
PAD061F 32
+
+
+
+
+
NANA L. interrogans2.276102
PAD117F 52--L. interrogans0.346102
PAD234F43-- NSNS
PAD236F 46-- L. broomii0.326102
PAD285F 40-- NSNS
PAD304F 15-
+
-L. fainei4.646104
*Subjects reported as long-term non-febrile ($1 year) and IgM-negative.
NA=Serum sample not available, NS=Not able to be sequenced.
doi:10.1371/journal.pntd.0000612.t003
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associated with a higher risk of exposure due to work-related
contact and behavioral practices. Another limitation was the initial
non-efficient PCR screening strategy. The presence of other
bacterial DNA hindered the identification of Leptospira-positive
clones; we detected both Leptospira and Atopobium DNA in multiple
samples, making the selection of colonies harboring the leptospiral
16S gene less efficient, in some instances, several hundred colonies
had to be screened; we were unable to sequence the infecting
strain in three enrollees due these technical limitations. Though
unlikely, it is also possible that in these three instances the dot-blot
gave false positive results. A third limitation of this study is that
culture isolation of leptospires from urine was not attempted.
Future work will be needed to further validate the molecular
results presented here, and will use the PCR method to screen
patients who then would have urine cultured for Leptospira.
Nonetheless, the deployment of a valid molecular tool to detect
leptospiruria represents a new approach to assessing chronic
asymptomatic infections in humans without the need for obtaining
isolates. Finally, because L. licerasiae serovar Varillal [7] had not
been fully characterized nor its epidemiological implications
known, this strain was not used as antigen in the MAT panel or
ELISA used to study patient sera, nor are these sera available for
retrospective analysis.
Few of the published Leptospira-specific PCR have beenapplied in
clinical or field settings[32]. Furthermore, detection of bacterial
DNA in urine is cumbersome because of the presence of PCR
inhibitors and samples are often contaminated by multiple bacterial
species whose DNA can interfere with the PCR assay [33].
Current understanding of host immune responses to Leptospira or
the pathogenesis of leptospirosis remains limited. Naturally
acquired immunity that protects against re-infection by Leptospira
does occur and has been shown in animal models. It has been
assumed that naturally acquired immunity is humorally-mediated
particularly by antibodies against oligosaccharides of leptospiral
LPS. Evidence also suggests that antibodies specific to Leptospira
membrane-associated proteins may play a role in host defense
[2,34]. We have documented that in this hyperendemic area, in
spite of the high levels of environmental exposure to Leptospira and
high prevalence of seropositivity, the prevalence of severe disease is
low [4,7]. These observations suggest the possibility that protective
immunity against severe disease from repeated infection may
develop in areas with high leptospirosis transmission, especially if
high frequency of infection leads to cross-serovar protection. Based
on the finding that asymptomatic infection and urinary carriage
are prevalent in this area where transmission is high and the
prevalence of severe disease is low, we suggest that repeated
exposure to Leptospira and asymptomatic infection could induce
protective acquired immunity. Longitudinal studies are needed to
test this hypothesis.
In conclusion, we have identified a long-term renal shedder
group among persons asymptomatically infected with pathogenic
and intermediately pathogenic Leptospira. The health implications
of long-term renal colonization and whether antibiotic treatment
of such patients is required remain to be determined.
Supporting Information
Alternative Language Abstract S1
abstract by MS.
Found at: doi:10.1371/journal.pntd.0000612.s001 (0.03 MB
DOC)
Japanese translation of the
Alternative Language Abstract S2
abstract by CAG.
Found at: doi:10.1371/journal.pntd.0000612.s002 (0.03 MB
DOC)
Spanish translation of the
Checklist S1
Found at: doi:10.1371/journal.pntd.0000612.s003 (0.09 MB
DOC)
STROBE checklist.
Acknowledgments
We thank the population of Padrecocha, Loreto, Peru ´ for their
understanding and collaboration. We thank Dr. Eddy Segura, Dr.
Katherine Remick, Asuncio ´n Gonzalez, biologists Flor Pacheco and Nahir
Chuquipiondo at the laboratory in Iquitos for helping to setting up the
study site and with sample collection in Padrecocha. Dr. Kailash Patra and
Dr. Fengwu Li at UCSD for technical advice and support. We thank the
laboratory staff of the Leptospirosis Reference Laboratory at the Instituto
Nacional de Salud in Lima for support with serological diagnosis. We
remain grateful to Dr. Carlos Vidal, Head, Directorate of Health, Loreto,
Peru for his continued support of these investigations. Paula Maguina of
the University of California San Diego made essential contributions in
terms of ethics management and international coordination and logistical
support for this project.
Author Contributions
Conceived and designed the experiments: CAG MAM EG JMV.
Performed the experiments: CAG MAM. Analyzed the data: CAG
MAM MS MC EG JMV. Contributed reagents/materials/analysis tools:
CAG MAM MS MC EG JMV. Wrote the paper: CAG MAM MS EG
JMV.
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