Download Prevalence Study of Antibody to Ratborne Pathogens and Other

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CONCISE COMMUNICATION
Prevalence Study of Antibody to Ratborne Pathogens and Other Agents
among Patients Using a Free Clinic in Downtown Los Angeles
Heather M. Smith,1 Roshan Reporter,1
Michael P. Rood,2 Andrea J. Linscott,3,a
Laurene M. Mascola,1 Wayne Hogrefe,4
and Robert H. Purcell 5
1
Acute Communicable Disease Control Unit, 2Vector Management
Program, and 3Public Health Laboratory, Los Angeles County
Department of Health Services, Los Angeles, and 4Focus Technologies,
Cypress, California; 5Hepatitis Viruses Section, National Institute
of Allergy and Infectious Diseases, National Institutes
of Health, Bethesda, Maryland
Norway rats (Rattus norvegicus) are hosts for various microbes. Homeless people who have
contact with rats may be at risk of infection by them. The Los Angeles County Department
of Health Services initiated a seroepidemiologic study among patients who used a free clinic
in downtown Los Angeles; 200 serum specimens obtained for other routine assays were tested
for antibodies to ratborne pathogens and other agents. The seroprevalence of antibody to
hepatitis E virus in this population was 13.6%; to Bartonella elizabethae, 12.5%; to B. quintana,
9.5%; to B. henselae, 3.5%; to Seoul virus, 0.5%; and to Rickettsia typhi, 0.0%. This study
found that patients and locally trapped rats had antibodies to some of the same agents.
Homeless people may be at risk for certain infectious and
chronic diseases as a result of their lifestyle and of other social
factors [1]. There have been few studies involving the homeless
and their exposure to ratborne and other infectious diseases.
Surveys conducted from 1996 through 1998 of Norway rats
(Rattus norvegicus) in downtown Los Angeles revealed a 6.7%
seroprevalence (8 seropositive rats of 120 rats tested) to Seoul
virus (SEOV), a 25.9% seroprevalence (67 of 259) to Rickettsia
typhi (RT), and a 73.1% seroprevalence (98 of 134) to hepatitis
E virus (HEV) (M.P.R., unpublished data). SEOV and RE are
known ratborne pathogens. In a recent study, 2 novel genotypes
of Bartonella that were similar to Bartonella elizabethae (BE)
were isolated in culture and identified in 45.2% of Norway rats
trapped in downtown Los Angeles (19 of 42 rats) [2]. In response to these findings, the Los Angeles County Department
of Health Services, Acute Communicable Disease Control Unit,
initiated a study to determine the seroprevalence of antibody
Received 27 June 2002; revised 12 August 2002; electronically published
1 November 2002.
Presented in part: 39th annual meeting of the Infectious Diseases Society
of America, San Francisco, 25–28 October 2001 (abstract 909).
This study was approved by the Institutional Review Board of the Los
Angeles County Department of Health Services. Informed consent was not
required, because the serum that was used had been drawn from patients
for other tests ordered by the clinic. In addition, the study was unlinked;
follow-up with patients was not possible, because their identities were
unknown.
a
Present affiliation: Louisiana State University Health Sciences Center,
Department of Pathology, Shreveport.
Reprints or correspondence: Ms. Heather Smith, Los Angeles County
Dept. of Health Services, Acute Communicable Disease Control Unit, 313
N. Figuroa St., Rm. 212, Los Angeles, CA 90012 ([email protected]).
The Journal of Infectious Diseases 2002; 186:1673–6
䉷 2002 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2002/18611-0019$15.00
to ratborne pathogens and other agents among patients being
treated at a free clinic in downtown Los Angeles. The clinic
serves an area where many homeless people reside and where
many of the rats were trapped.
Methods
Specimen collection and submission. In June and August 2000,
serum specimens obtained for other routine tests were obtained
from patients who visited a free clinic in the “skid row” district of
downtown Los Angeles. Most of these patients had been homeless
during the previous year.
The serum samples were sent from the free clinic to the Los Angeles County Public Health Laboratory (LACPHL). At LACPHL,
3 aliquots were drawn from each specimen and stored at ⫺80⬚C
pending testing. An aliquot of each serum sample was sent to the
Hepatitis Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH; Bethesda, MD)
and to Focus Technologies (Cypress, CA) for HEV and hantavirus
antibody testing, respectively. Focus Technologies used a pan-hantavirus screening test and sent serum samples that were positive
for the virus to the Special Pathogens Branch, Division of Viral
and Rickettsial Diseases, Centers for Disease Control and Prevention (CDC; Atlanta) for SEOV-specific testing. The age and sex of
the subjects were the only demographic data available. No clinical
data were collected.
Laboratory methods. LACPHL tested the serum samples for
BE, B. quintana (BQ), B. henselae (BH), and RT antibodies, using
reagents provided by the Viral and Rickettsial Zoonoses Branch
of the CDC. An indirect fluorescent antibody method [3] was used
to screen for antibody production to these organisms. In brief, 4
antigens, RT, BE, BQ, and BH, were fixed to each slide, and serial
dilutions of serum samples were applied to the antigens. The slides
were incubated with the test samples, washed to remove unbound
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Smith et al.
immunoglobulins, and allowed to dry. The slides were then overlaid
with fluorescein isothiocyanate–labeled goat anti–human IgG, incubated, washed, and examined for fluorescence under UV illumination. The highest dilution at which distinct and specific fluorescence was seen was scored as the end-point titer for that serum
sample. The serologic cutoff point for RT, BQ, and BH was established at 64, and the serologic cutoff point for a positive result
for BE was set at 128.
The NIH tested the serum samples for HEV antibody. The putative capsid gene (open-reading frame 2) of HEV strain Sar-55
was cloned into a baculovirus expression vector, expressed in insect
cells, and purified by ion-exchange and gel-filtration chromatography, as described elsewhere [4]. The resultant protein is a 56-kDa
truncated form of the capsid protein that consists of aa 112–607
of the full-length protein. The protein was applied to the wells of
microtiter plates, and specific antibodies were detected with horseradish peroxidase–labeled anti–human IgG, as described elsewhere
[5]. The sensitivity and specificity of the assay were monitored with
positive and negative controls that, in turn, were calibrated against
a World Health Organization anti-HEV standard obtained from
the National Institute for Biological Standards and Control (Hertfordshire, England). Serum samples were tested at a dilution of
100. All samples were tested in 2 separate tests, and serum samples
with borderline results were tested at least once more.
The serum samples were screened at Focus Technologies for the
presence of IgG and IgM antibodies to hantavirus nucleoproteins,
using 2 commercial hantavirus ELISA kits, one for IgM and the
other for IgG, developed by Focus Technologies. The serum samples were diluted to 100 and incubated in a microtiter plate containing a cocktail of recombinant hantavirus nucleocapsid proteins.
After the addition of goat anti–human IgG Fc or goat anti–human
IgM Fc, an index value was obtained. Samples with IgG or IgM
values 11.1 were considered to be positive, those with values !0.9
were considered to be negative, and those with values of 0.91–1.09
were considered to be equivocal. Because of the high cross-reactivity of antibody to hantaviral nucleoproteins, IgG or IgM antibodies to hantaviruses pathogenic for humans are typically detected.
These ELISA tests do not discriminate among species-specific hantavirus antibodies. Therefore, all positive and equivocal specimens
were sent to the Special Pathogens Branch, Division of Viral and
Rickettsial Zoonoses, CDC, for confirmation, using methods described by Ksiazek et al. [6] and LeDuc et al. [7].
Statistical analysis. Epi Info version 6.0 software (CDC) was
used to analyze the frequency of and association between variables.
Mantel-Haenszel and Fisher’s exact tests (2-tailed) were used to
assess the strength of association (P ! .05 was considered to be
significant) between variables. SAS software (SAS Institute) was
used to compare, by t test, the mean age of the patient with respect
to presence of antibody.
Results
Samples from 200 patients were collected and tested. The ratio
of men to women in the study was 3:1, and the median age was
41 years (range, 21–66 years). Most participants (85%) were between the ages of 25 and 54 years. Univariate analysis showed
that sex, as a demographic factor, was not significantly associated
JID 2002;186 (1 December)
with any agent (P 1 .10). Patients who had antibody to HEV
were significantly older (mean age, 48 years; P p .0005) than
those who did not (mean age, 40 years). This association did not
reach significance for the other agents (P 1 .10).
Overall, 31.5% of all specimens (63 of 200) were positive for
antibody to at least 1 antigen. Seroprevalence for ⭓1 Bartonella
species was 17.5% (35 of 200 specimens) (table 1). Twenty-seven
specimens (13.5%) had antibody to HEV. Two of the specimens
tested moderately positive against the hantavirus panel of antigens, and 2 specimens had equivocal results of testing. However, only 1 specimen (0.5%) was confirmed to have antibody
to SEOV. None of the specimens had antibody to RT.
The seroprevalence was different for each Bartonella species.
The rates of antibody were 3.5% (7 of 200 specimens), 9.5%
(19 of 200), and 12.5% (25 of 200) for BH, BQ, and BE, respectively. Of those specimens seropositive for BE, 44% (11 of
25) were positive for BQ, and 20% (5 of 25) were positive for
BH. Of the specimens that were positive for BQ, 58% (11 of
19) were positive for BE, and 21% (4 of 19) were positive for
BH (table 1). In general, as measured by immunofluorescence,
titers were low: 35 (68.6%) of the 51 samples that tested positive
for any Bartonella species had titers that just exceeded the cutoff
value, and only 4 samples with antibody to BQ had a titer that
was ⭓4-fold greater than the cutoff value
The only significant association among different agents was
found in specimens with Bartonella antibody. Antibody to BE
antigen was significantly associated with antibody to BQ and
BH antigen (P ! .001). Specimens with HEV antibody were not
significantly more likely to have antibody to any other antigens
(P 1 .10), nor was the specimen with SEOV antibody (P 1 .10).
Discussion
In this study, we found that people using a free clinic had
antibodies to organisms similar to those found in rats trapped
in the same area and in a similar relative proportion, although
the absolute prevalence in the rats was higher. Studies suggest
that R. norvegicus is a reservoir host for BE and BE-like organisms [2]. How patients in our study acquired antibody to
Table 1. Prevalence of antibody to Bartonella species among 200
patients who were treated at a free clinic in downtown Los Angeles.
Bartonella species
No. (%) of patients
with antibody to
indicated species
Any
a
B. elizabethae only
b
B. quintana only
b
B. henselae only
B. elizabethae and B. quintana
B. elizabethae and B. henselae
B. quintana and B. henselae
B. elizabethae, B. quintana, and B. henselae
None
a
b
The serologic cutoff point was a titer of 1:128.
The serologic cutoff point was a titer of 1:64.
35
13
8
2
7
1
0
4
165
(17.5)
(6.5)
(4.0)
(1.0)
(3.5)
(0.5)
(0.0)
(2.0)
(82.5)
JID 2002;186 (1 December)
Prevalence of Antibody to Ratborne Pathogens
BE is not known, but if rats are reservoir hosts of BE, exposure
through living or foraging in rat-infested areas may be a risk
factor for humans.
Serum samples from the subjects in our study population
were also tested for Bartonella species that are not associated
with R. norvegicus. BQ is transmitted by the human body louse
and is the etiologic agent of trench fever. Studies conducted in
intravenous drug user and homeless populations have found
that the seroprevalence to this agent is higher than that in blood
donor populations (10%–20% vs. 0%–2%) [8, 9]. Although our
study lacked a control group, the seroprevalence of 9.5% to BQ
is consistent with the results of these other studies. Our data
also showed a seroprevalence to BH, the agent of cat scratch
disease, similar to that in nonindigent populations [10].
HEV is transmitted through the fecal-oral route and has been
implicated in epidemics in developing countries. The HEV seroprevalence we found was similar to the HEV seroprevalence in
healthy blood donor populations in Sacramento (CA) and Baltimore, which ranged from 13.7% to 21.3% [11]. In a recent
study, HEV-like viruses were recovered from wild rats trapped
in Los Angeles and were transmitted via serum and feces to
laboratory rats [12]. The zoonotic potential of HEV is under
investigation.
Seroprevalence studies of SEOV have been conducted in Baltimore. Two of these studies found that 0.16% of intravenous
drug users (1 of 635) and 0.25% of people visiting a sexually
transmitted disease clinic (3 of 1180) had antibody to SEOV
[13, 14]. The Los Angeles population had a seroprevalence that
was slightly higher; however, the sample size of the present
study was smaller than that of the Baltimore studies.
None of the patient specimens tested positive for antibody
to RT. Cases of murine typhus associated with residence in
downtown Los Angeles have been documented (M.P.R., unpublished data); however, the negative findings for RT in our
study population suggest that, although evidence of infection
in rodent reservoirs exists, the homeless human population is
at low risk of exposure to infected fleas.
Sex was not a risk factor for seroprevalence in this population,
which suggests that men and women are equally likely to be
exposed to these antigens. Increasing age was found to be significantly associated with antibody to HEV antigen, a finding
that has been documented in other studies [15, 16]. Neither HEV
nor SEOV antibody was significantly associated with BE, BH,
or BQ antibody, which may indicate that modes of transmission
of these organisms differ. However, BE, BH, and BQ antibody
were all significantly associated with each other, particularly BE
with BQ and BH. Significant associations between BQ- and BHpositive specimens appeared to be confounded by BE antigen,
because there were no specimens that tested positive for BQ and
BH only. These associations could be the result of cross-reactivity
of antibody to antigen or other factors.
The data presented here raise some questions about health
among the homeless population. For example, does antibody
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to these agents represent clinical disease? Are these infections
related to exposure to rats, or are they spread by other means?
Do other populations in Los Angeles County have serologic
evidence of exposure to these same agents? Additional studies
may lead to effective public health interventions. These studies
could include a seroprevalence survey of these agents among
the nonhomeless population of Los Angeles, a prospective clinical study to look for disease in the homeless caused by these
organisms, and interviews with homeless patients to ascertain
risk factors for infection.
Understanding the dynamics of disease among homeless people is important. The homeless in an urban setting represent a
subset of a larger population that may be less affected by certain
diseases. However, the homeless population may serve as a
predictor of disease in the general public, should the infrastructure of public health be undermined. Clinicians who treat homeless populations should be aware of the possibility of infection
with atypical agents of disease.
Acknowledgments
We thank Ellen Goudlock, Roweena Gler, and the Weingart Clinic,
for their cooperation in this study; Russ Regnery and the laboratory
at the Division of Viral and Rickettsial Diseases, Centers for Disease
Control and Prevention (CDC; Atlanta), for supplying us with Bartonella species and Rickettsia typhi reagents; Joni Young and the Special
Pathogens Branch laboratory, CDC, for Seoul virus testing; Caroline
Bautista and Mary Ledbetter at the Los Angeles County Public Health
Laboratory, for laboratory assistance; Grace Run, for helping with the
analysis of the data; and Douglas Frye and Esther Parker, for reviewing
the manuscript.
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