The Spectrum of Gastrointestinal Manifestations in Children and
Adolescents with Lyme Disease
Adolescents with Lyme Disease
Martin D. Fried, MD, Departments of Pediatric Gastroenterology; Matthew
Abel, MD, Departments of Pediatric Gastroenterology; Dorothy Pietrucha,
MD, Pediatric Neurology; Yen-Hong Kuo, MS, Academic Affairs; and Aswine
Bal, MD, Pediatric Infectious Disease, Jersey Shore Medical Center,
Neptune, New Jersey.
[Journal of Spirochetal and Tick-Borne Diseases 6(4):89-93, 1999. ©
1999 Lyme Disease Foundation, Inc.]
Abstract
A clinical diagnosis of Lyme disease was made in 15 consecutive
patients between the ages of 8 and 20 years who presented with a
history of an erythema migrans rash followed by chronic
gastrointestinal symptoms and multiple organ system complaints.
Endoscopic evaluation was performed to assess the gastrointestinal
mucosa and to obtain biopsies for polymerase chain reaction (PCR) to
the outer surface protein A (Osp A) of Borrelia burgdorferi. As age
matched controls, 10 patients with biopsy-proven Crohn's disease were
also tested by PCR. The laboratories assessing the histopathology and
performing the PCR were blinded to the diagnosis of all specimens.
The presence of B burgdorferi DNA in the gastrointestinal tract was
confirmed by PCR in all of the patients with the clinical diagnosis of
Lyme disease who had chronic gastrointestinal symptoms and in two
control subjects with Crohn's disease. Biopsy evidence of chronic
gastritis, chronic duodenitis, and chronic colitis was found in
patients with Lyme disease who had chronic gastrointestinal symptoms
and was associated with the presence of B burgdorferi.
The chronic gastrointestinal symptoms that occurred within 6 months of
an erythema migrans rash and Lyme disease may be attributed to a direct
effect or immune mediated response to B burgdorferi.
Introduction
Lyme disease affects a wide range of organ systems, producing
dermatologic, musculoskeletal, neurologic, genitourinary, lymphatic,
hepatic, renal, respiratory, cardiovascular, and ocular manifestations.
[1,2] One report to date describes the presence of Borrelia burgdorferi
in the stomach, intestines, and colon of children.[3] To further
address the clinical manifestations of Lyme disease and the possibility
of direct involvement of the gastrointestinal (GI) tract, a prospective
study was made of 15 consecutive patients who had a physician
documented erythema migrans (EM) rash followed by chronic
gastrointestinal symptoms and multiple organ system complaints of Lyme
disease.
Methods
All patients included in our study had a physician documented EM rash
with no prior history of gastrointestinal complaints. They were
referred to the pediatric gastroenterology and nutrition service of
Jersey Shore Medical Center for evaluation of chronic abdominal pain,
chronic diarrhea, acid reflux, or blood in the stool that occurred
within 6 months after the onset of the EM rash. >From January 1998
through April 1999, 15 consecutive patients satisfying the above
clinical criteria[4] were evaluated prospectively. There were 6 boys
and 9 girls evaluated (mean age 1463.6 years, range 8-20). Each case
included a history, physical examination, complete blood cell count,
liver function tests, sedimentation rate, antinuclear antigen (ANA),
HLA B27, esophagogastroduodenoscopy (EGD), and/or colonoscopy. A Lyme
Western blot was performed for confirmation of an acute (immunoglobulin
M) or past (immunoglobulin G) B burgdorferi infection. A positive IgM
Western blot was interpreted as 2 of 3 bands (23, 39, 41 kd). A
positive IgG Western blot was interpreted as 5 or more of the following
B burgdorferi-specific bands: 18, 23, 28, 31, 34, 39, 41, 45, 58, 66,
93 kd. A diet history was taken to assess the dietary fat intake.
Ultrasonography of the abdomen was performed when the history suggested
a diagnosis of gallstones or pancreatitis. Stool samples were examined
for occult blood, Salmonella, Shigella, Yersinia, Campylobacter, ova
and parasites, and Clostridium difficile toxin. Gastrointestinal (GI)
biopsies were reviewed to assess the mucosa by microscopy and whether
Helicobacter pylori (on EGD only) or eosinophilia was present.
Biopsy specimens were taken from areas of the GI tract that looked
inflamed during EGD or colonoscopy. The biopsies were assigned randomly
to three histopathologists who were blinded to the diagnosis of the
specimens they received. The histopathologists did not perform a silver
stain for the detection of spirochetes because it is not routinely
done. Biopsies were reported as acutely inflamed when polymorphonuclear
cells were present in the mucosa and chronically inflamed if 6 or more
plasma cells and lymphocytes were present in the gastric mucosa without
polymorphonuclear cells. Chronic duodenitis or chronic colitis was
diagnosed when more than 6 intraepithelial lymphocytes per 100 surface
absorptive cells were present in tissue biopsies in conjunction with a
distortion in glandular architecture.
Polymerase chain reaction (PCR) for DNA to B burgdorferi[5] was
performed on all biopsies by Medical Diagnostic Laboratories in Mount
Laurel, New Jersey. In all patients in which B burgdorferi DNA was
detected, PCR for B burgdorferi RNA polymerase was performed and
results are reported in the Table. As a target for DNA amplification,
the gene coding for the outer surface protein A (OspA) of B burgdorferi
was selected and analyzed as described below.
DNA Isolation from Biopsy Specimens
Total DNA was extracted from duodenal, gastric, and colonic biopsies as
described by Maniaties et al.[6] The samples were centrifuged (5
minutes, 4°C, 14K rpm) and the pelleted biopsy was subjected to 500 µL
of cell lysis buffer [0.5% SDS, 470 µL TE buffer, 5 µL of proteinase K
(20 µg/µL)]. The samples were incubated for 24 hours at 50°C.
Proteinase K (5 µL) was added to the mixture every 6 hours. DNA was
extracted by phenol chloroform, followed by ethanol precipitation. DNA
concentrations were determined spectrophotometrically by measuring the
A260.
DNA Amplification
The SL primers (SLA 59-AAT AGG TCT AAT AAT AGC CTT AAT AGC-39 SLB 59
CTA GTG TTT TGC CAT CTT CTT TGA AAA-39) are suitable for amplification
of all B burgdorferi sensu lato isolates. The SL primers amplify a
region (nucleotide 21-328) of the B burgdorferi senso stricto B31 OspA
sequence. One µg of isolated DNA was used as a template DNA in the
presence of a 20 pmol sample of each primer in a 50 µL reaction
mixture. The samples were subjected to 35 amplification cycles in a
Perkin Elmer 2400 thermocycler (Foster City, CA) under the following
conditions: 93°C, 1 minute; 65°C, 1 minute; and 72°C, 1 minute. PCR
amplification products were resolved onto 1.5% agarose electrophoresis
gels and visualized under ultraviolet light with ethidium bromide.
To test the presence of inhibitory substances and to provide a positive
control in the PCR assay, amplifications were also performed with
primers targeting the histone gene. A positive control was performed
with every biopsy specimen. It included a PCR in the presence of 100% B
burgdorferi DNA that was purchased from the American Type Culture
Collection (Rockville, MD). This B burgdorferi DNA was isolated from
Ixodes scapularis tick, New York Type strain, and shipped frozen to the
laboratory.[7] The negative control performed with each biopsy specimen
included the PCR in the absence of DNA. A second genomic DNA control is
done weekly at the laboratory as part of their quality control.
Physical containment measures ensured the absence of DNA contamination
in the PCR procedure.
As age-matched controls, 10 adolescents with biopsy proven Crohn's
disease (5 boys, 5 girls, 13.562.5 years, range 10-17), who had not
been on antibiotics one year prior to endoscopy, were also tested by
PCR. The laboratory performing the PCR analysis was blinded to the
diagnosis of all specimens they received.
Statistical Analysis
The sensitivity and specificity of PCR for the detection of B
burgdorferi in the GI tract was calculated. The confidence intervals
(CI) were calculated by using the Fischer's Exact test method. A
Fischer's exact test was used to determine the association between
inflammation and PCR positivity in each of the biopsied sites.
Results
Patients with Lyme disease presented with chronic abdominal pain (n=10,
67%), chronic diarrhea (n=1, 7%), visibly evident blood in the stool
(n=2, 13%), and acid reflux with heartburn (n=2, 13%). In all 4
patients whose biopsies revealed evidence of colitis, the abdominal
pain was characterized as a crampy, periumbilical pain that started at
the right middle quadrant of the abdomen and spread to the left middle
quadrant of the abdomen or vice versa. The pain was unrelated to meals
and occurred throughout the day. In the remaining 6 patients with
abdominal pain whose biopsies revealed gastritis, duodenitis, or both,
the abdominal pain was characterized as periumbilical, burning, and
improved by avoiding fried foods and foods high in fat content.
Ultrasonography of the abdomen did not reveal any gallstones or
evidence of pancreatitis. In 2 patients who complained of acid reflux,
their pain was a burning midepigastric pain that radiated to the
esophagus. The pain occurred within the first postprandial hour and was
relieved by antacids. Ten of the 15 patients with Lyme disease had
evidence of inflammation at a biopsy site with detection of B
burgdorferi DNA at that site. Patients 2 and 11 had blood in their
stool and presented with the clinical features of Crohn's disease (ie,
15 pound weight loss in a year, arthritis of the knee, protein losing
enteropathy) and ulcerative colitis (6 bloody bowel movements a day for
a week), respectively. The biopsies of all the patients with Lyme
disease revealed no evidence of granulomas or terminal ileitis. In
patient 6, the IgM Western blot was positive and showed the 23, 31, 34,
39, 41, 58, and 66 kd bands. The IgG Western blot was negative (no
bands present). No other patient had a positive Western blot. All
control patients with Crohn's disease had biopsy proven terminal
ileitis and granulomatous colitis. Lyme disease was diagnosed in 15
patients and 2 with Crohn's disease had a positive PCR to B burgdorferi
DNA in biopsy specimens from the gastrointestinal tract (Table). In 6
patients with Lyme disease, B burgdorferi DNA was detected in the GI
tract and B burgdorferi RNA polymerase was detected by PCR.
A positive B burgdorferi PCR occurred with chronic inflammation in the
GI tract of 11 of 15 patients with Lyme disease. In patients 8 and 9,
inflammation occurred in the stomach; however, B burgdorferi DNA was
detected in the colon. B burgdorferi DNA was detected in the GI tract
in the absence of inflammation in 4 patients (27%), 3 of whom had
received at least 2 months of antibiotics prior to endoscopy. There was
no statistically significant association between PCR positivity in the
GI tract and chronic inflammation.
In 10 of 15 patients (67%), antibiotic therapy for Lyme disease had
been prescribed within 1 to 5 months prior to endoscopy (n=4, 1-2
months; n=3, 3-4 months, and n=3, 5 months). Despite prior antibiotic
use, all 4 patients with colitis were PCR positive for B burgdorferi
DNA in the colon while 5 of 9 with gastric inflammation were PCR
positive in gastric biopsies. Helicobacter pylori was not detected in
any of the gastric biopsies. Salmonella, Shigella, Yersinia,
Campylobacter, and Clostridium difficile toxin was not detected in any
of the stool samples. HLA B27 was positive in patients 1 and 11 and in
none of the controls. ANA was positive and had a speckled pattern in
patients 2, 5, and 6. An elevated sedimentation rate of 85 and 28 were
found in patients 4 and 13, respectively.
The lab performing the PCR had a false positive rate of 1 in 500 by
analyzing 6550 specimens from January 1998 through April 1999. The
sensitivity of GI B burgdorferi DNA detection was 100% (15/15) with a
95% CI (81.9%, 100%). The specificity was 80% (8/10) with a 95% CI
(44.4%, 97.5%). The positive predictive value was 88.2% (15/17) with a
95% CI (63.6%, 98.5%).
Discussion
Abdominal pain and the associated GI pathology in children with Lyme
disease whose biopsies are PCR positive for B burgdorferi has not been
reported previously. The presence of an EM rash in the past or a
positive Western blot and chronic GI symptoms in the past does not mean
that the two are related. However, positive detection of the OspA gene
in biopsies confirmed the presence of B burgdorferi DNA in the biopsied
tissue samples while offering the advantage of no cross reaction with
other spirochete species that have been previously detected in the GI
tract.[8-10] It is possible that PCR, a highly sensitive method, could
lead to false positive results because of the amplification of similar
sequences of related microorganisms. However, a false positive rate of
1 in 500 biopsy specimens suggests that this occurs infrequently.
B burgdorferi may contribute to GI symptoms by its presence directly in
the GI tract or by eliciting an inflammatory or immune response.[11,12]
In 11 patients (73%), inflammation in the GI tract was accompanied by a
positive PCR to B burgdorferi suggesting an association between the
infection and inflammation in these patients. In 2 patients the
detection of the DNA occurred at a site distant from the inflammation.
In the absence of inflammation, the presence of B burgdorferi may have
contributed to abdominal pain and acid reflux (patients 3, 6, and 13).
Most available evidence suggests that appropriate antimicrobial
treatment is highly efficacious to cure Lyme disease. As previously
reported,[13,14] we found that B burgdorferi persisted even after 1 to
5 months of antibiotic therapy. Despite prior antibiotic therapy, we
were still able to detect B burgdorferi DNA and RNA polymerase in these
patients. The detection of RNA polymerase in 5 patients suggests that
the infection was actively replicating. In two cases (patients 1 and
6), this active replication occurred despite 5 months of antibiotic
therapy for Lyme disease. Previous work has demonstrated that B
burgdorferi can invade human fibroblasts and be protected from
antimicrobial action.[15] The ability of the organism to survive in
this intracellular environment is one mechanism by which it may evade
the immune response of the host and thus persist. Antibiotic resistance
is another method that could explain the persistence of the organism
despite prior antibiotic therapy. While the detection of B burgdorferi
may represent evidence of prior or ongoing Lyme disease, it may not be
the only etiology of the patients' abdominal symptoms.
Duray and Steere[16] reported that B burgdorferi elicits interleukin-1,
collagenase, prostaglandin E2, and circulating immune complexes. Some
of these immune complexes may exert their effect at a distant site from
the infection. Two patients (8 and 9) illustrate this possibility. In
each case, B burgdorferi was detected in the colon but the inflammation
was found in the stomach.
An inflammatory or immune reaction as a result of Lyme disease could
affect the colon and may persist because of circulating immune
complexes.[16] Inflammatory and immune etiologies have also been
proposed in Crohn's disease[17,18]; however, this is the first report
to describe the detection of B burgdorferi in patients with Crohn's
disease. The role of B burgdorferi in patients with Crohn's disease
needs further investigation.
Conclusion
Children and adolescents with a history of Lyme disease and chronic GI
symptoms occurring within 6 months of an EM rash, had evidence of
inflammation in the stomach, duodenum, and colon. We found B
burgdorferi by PCR of GI biopsies to be associated with chronic
inflammation. The inflammatory reaction we describe may have been
caused by spirochetes or by immune system products elicited in response
to the spirochete presence.
Table. The gastrointestinal manifestations and biopsy results of
patients with Lyme (1-15) and Crohn's disease (A-J).
Patient Antibiotics (months)* Chief complaint Gastric biopsy Duodenal
biopsy Colon biopsy Gastric PCR? Duodenal PCR Colon Patient PCR
1 5 abdominal pain gastritis duodenitis colitis DNA DNA DNA/RNA
2 4 blood in stool gastritis (-) colitis DNA (-) DNA
3 4 acid reflux (-)[?] (-) NB DNA (-) NB
4 3 abdominal pain gastritis (-) (-) DNA DNA (-)
5 5 abdominal pain gastritis duodenitis (-) (-) DNA (-)
6 5 abdominal pain (-) (-) NB DNA/RNA (-) NB
7 2 diarrhea (-) (-) colitis DNA (-) DNA
8 0 abdominal pain gastritis (-) (-) (-) (-) DNA/RNA
9 2 abdominal pain gastritis (-) (-) (-) (-) DNA/RNA
10 0 abdominal pain gastritis (-) NB DNA (-) NB
11 0 blood in stool NB[§] NB colitis NB NB DNA/RNA
12 1 abdominal pain gastritis duodenitis NB (-) DNA NB
13 2 abdominal pain (-) (-) NB DNA (-) NB
14 0 abdominal pain (-) (-) NB (-) DNA/RNA NB
15 0 acid reflux gastritis (-) NB DNA (-) NB
A 0 blood in stool (-) (-) granuloma (-) (-) (-)
B 0 abdominal pain (-) (-) granuloma (-) (-) DNA
C 0 abdominal pain gastritis duodenitis granuloma (-) (-) (-)
D 0 abdominal pain (-) (-) granuloma (-) (-) (-)
E 0 abdominal pain gastritis duodenitis granuloma (-) (-) (-)
F 0 abdominal pain granuloma (-) granuloma (-) (-) (-)
G 0 blood in stool NB NB granuloma NB NB DNA
H 0 blood in stool NB NB granuloma NB NB (-)
I 0 abdominal pain gastritis duodenitis granuloma (-) (-) (-)
J 0 abdominal pain (-) (-) granuloma (-) (-) (-)
* Number of months of antibiotic treatment for Lyme disease prior to GI
biopsy.
? PCR-DNA denotes the detection of B burgdorferi DNA (the outer surface
protein A) by PCR of biopsy specimens. DNA/RNA denotes the detection of
B burgdorferi DNA and RNA polymerase by PCR of biopsy specimens.
? (-) denotes no histological pathology detected on biopsy or no
detection of B burgdorferi by PCR.
§ NB denotes an area of the gastrointestinal tract that was not
biopsied and a PCR test that was not performed due to the absence of a
specimen from that site.
Abbreviations: DNA = deoxyribonucleic acid; NB = not biopsied; PCR =
polymerase chain reaction; RNA = ribonucleic acid.
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