PLoS ONEplosplosonePLoS ONE1932-6203Public Library of ScienceSan Francisco, USAPONE-D-10-0378510.1371/journal.pone.0017366Research ArticleBiologyImmunologyMedicineClinical immunologyAutoimmune diseasesClinical research designClinical trialsGastroenterology and hepatologyCeliac diseaseSmall intestineInfectious diseasesParasitic diseasesHelminth infectionHookworm infectionHookwormSoil-transmitted helminthsImmunologyInfectious DiseasesGastroenterology and HepatologyEffect of Hookworm Infection on Wheat Challenge in Celiac Disease
– A Randomised Double-Blinded Placebo Controlled TrialTrialling Hookworms in Celiac
DiseaseDavesonA. James1JonesDianne M.1GazeSoraya23McSorleyHenry23CloustonAndrew4PascoeAndrew1CookeSharon5SpeareRichard3MacdonaldGraeme A.12AndersonRobert6McCarthyJames S.27LoukasAlex23CroeseJohn235*Princess Alexandra Hospital, Brisbane,
AustraliaQueensland Institute of Medical Research,
Brisbane, AustraliaJames Cook University, Townsville,
AustraliaEnvoi Specialist Pathologists, Brisbane,
AustraliaThe Townsville Hospital, Townsville,
AustraliaWalter and Eliza Hall Institute, Melbourne,
AustraliaUniversity of Queensland, Brisbane,
AustraliaGluudLise LotteEditorCopenhagen University Hospital Gentofte, Denmark* E-mail: jcroese@bigpond.com
Conceived and designed the experiments: JC AL JM. Performed the experiments:
JD DJ SG HM AC AP SC RS JM. Analyzed the data: JD DJ SG HM AC RS RA JM AL
JC. Wrote the manuscript: JD DJ SG HM RS RA JM AL JC. Clinical site study
supervision: GM.
The authors have declared that no competing interests exist. Envoi Specialist
Pathologists, Brisbane, QLD, Australia contributed to the processing and the
interpretation (Prof. Clouston) of the histological material, but neither the
organisation nor any employee has any commercial interest beyond this support
nor a competing interest. Envoi's involvement does not affect or alter the
authors' adherence to all the PLoS ONE policies on sharing data and
materials.
201183201163e17366110201031120112011Daveson et alThis 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.Background and Aims
The association between hygiene and prevalence of autoimmune disease has been
attributed in part to enteric helminth infection. A pilot study of
experimental infection with the hookworm Necator americanus
was undertaken among a group of otherwise healthy people with celiac disease
to test the potential of the helminth to suppress the immunopathology
induced by gluten.
Methods
In a 21-week, double-blinded, placebo-controlled study, we explored the
effects of N. americanus infection in 20 healthy,
helminth-naïve adults with celiac disease well controlled by diet.
Staged cutaneous inoculations with 10 and 5 infective 3rd stage
hookworm larvae or placebo were performed at week-0 and -12 respectively. At
week-20, a five day oral wheat challenge equivalent to 16 grams of gluten
per day was undertaken. Primary outcomes included duodenal Marsh score and
quantification of the immunodominant α-gliadin peptide (QE65)-specific
systemic interferon-γ-producing cells by ELISpot pre- and post-wheat
challenge.
Results
Enteric colonisation with hookworm established in all 10 cases, resulting in
transiently painful enteritis in 5. Chronic infection was asymptomatic, with
no effect on hemoglobin levels. Although some duodenal eosinophilia was
apparent, hookworm-infected mucosa retained a healthy appearance. In both
groups, wheat challenge caused deterioration in both primary and several
secondary outcomes.
Conclusions
Experimental N. americanus infection proved to be safe and
enabled testing its effect on a range of measures of the human autoimmune
response. Infection imposed no obvious benefit on pathology.
Trial Registration
ClinicalTrials.gov NCT00671138
The study was funded by the Broad Medical Research Program of The Broad
Foundation. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. Prof. Andrew
Clouston is a principal of and clinical pathologist at Envoi Specialist
Pathologists, Brisbane, QLD, Australia, which processed tissue sections for
histological grading. Prof. Clouston contributed to the interpretation and
grading of the histology, and the preparation of the manuscript. Neither Prof.
Clouston nor Envoi Specialist Pathologists has any patent or financial interest
in the product of this clinical trial.Introduction
The “hygiene hypothesis” proposes that the increasing prevalence of
allergic and autoimmune diseases in developed countries may be due to the reduction
in the incidence of infectious diseases [1]; epidemiologic and genetic studies
suggest that the decline in the prevalence of helminth infections is a major factor
[2]–[5]. Observational studies supporting this hypothesis include
a report that infection with the nematode Strongyloides stercoralis
is associated with protection against autoimmune liver disease [6], and another showing that
unspecified helminth infections with peripheral eosinophilia are associated with
reduced autoantigen-specific responses and disease progression in multiple sclerosis
[7].
Co-infection with helminths is also known to attenuate murine models of autoimmunity
and inflammatory bowel disease [8], [9].
Despite being classed as pathogens [10], helminth parasites are being proposed as treatments for
allergic and autoimmune diseases [11], [12]. Preliminary observations suggest that intentional
infection with the zoophilic Trichuris suis is safe [13], and reduces
the activity of inflammatory bowel disease [13], [14]. However, dosing with
T. suis every three weeks is required to maintain ongoing
infection because the human host does not sustain development of T.
suis to maturity [14].
Necator americanus (NA) is a long-lived hematophagous,
human-specific (anthropophilic) gastrointestinal nematode that infects over 500
million people in developing countries where heavy infection causes iron deficiency
anemia, and is associated with reduced physical and intellectual development [15]. Experimental
infection of healthy volunteers with NA infective third-stage larvae (L3)
may cause an acute, painful enteropathy [16]–[18]. Recent published data indicate
that low-dose inocula of NA are better tolerated [19], and because they do not
proliferate in humans, a defined dose can be administered and later fully eliminated
with anthelmintic therapy [19]. A further advantage is infected individuals pose no risk
to others because hookworms are soil-transmitted (geohelminths) and cannot be
propagated in modern sanitary environments.
We chose NA and celiac disease (CD) to explore the relationship between helminth
infection and intestinal inflammation due to a well characterised dietary antigen,
gluten. Our previous studies with this hookworm have provided us with a pure source
of infective larvae [18], [20]. Individuals carrying chronic infection with hookworms in
endemic settings demonstrate parasite-specific TH2 responses, but TH1 and TH2 immune
responses to other antigens are diminished [21].
Celiac disease is uniquely suited to explore the effects of helminth infection; it is
common [22] and
remission is achieved with elimination of dietary gluten allowing host-parasite
interaction to be studied free of potential artefacts caused by medications.
Clinical studies in volunteers with CD also have the advantage that the effects of
deliberate gluten exposure can be measured by symptom response, in blood and
intestinal tissue [23]–[26]. Over 90% of people with CD possess genes encoding
the major histocompatability (MHC) Class II molecule, HLA-DQ2, and HLA
DQ2-restricted CD4+ T cells specific for deamidated gluten peptides
can be isolated from intestinal tissue [26]. HLA DQ2-restricted
CD4+ TH1 cells specific for deamidated gluten including the
immunodominant α-gliadin 17-mer p57-73 (QE65) peptide are also present in blood
after oral wheat challenge [27].
In this study, we undertook a clinical trial to test whether NA infection reduces the
immunotoxic effects of gluten in CD.
Methods
The protocol for this trial and supporting CONSORT checklist are available as
supporting information; see Checklist S1 and Protocol
S1.
Ethics Statement
The Princess Alexandra Hospital, Queensland Institute of Medical Research and
Townsville Hospital Human Research Ethics Committees approved the study. Written
informed consent was obtained from all subjects.
Patients
Healthy people with CD aged 18 years or older were invited to participate through
the Queensland Coeliac Society. Inclusion criteria included: 1) pre-treatment
histological diagnosis of Marsh grade 3 celiac disease [25], 2) positive immunoglobulin
(Ig) A anti-tissue transglutaminase (tTG) or anti-endomysial antibody, 3)
HLA-DQ2 phenotype, and 4) adherence to a gluten-free diet (GFD) for at least 6
months pre-enrolment [28]. Subjects were excluded if they had 1) insulin
dependent diabetes mellitus or Addison's disease; 2) life threatening
allergy; 3) treatment with immunomodulatory therapies within the six months
prior to enrolment (including aspirin, non-steroidal anti-inflammatory drugs,
celocoxib inhibitors, statins or intramuscular and intravenous steroids); 4) an
unmanaged risk of pregnancy; 5) historical, fecal or serological evidence of a
prior helminth infection; 6) iron deficiency anemia, 7) any vaccination within
the 30-day period prior to study commencement, or 8) an elevated level of tTG
IgA.
Study Design
This was a prospective, randomized, double-blinded, placebo-controlled trial
evaluating the safety, tolerability and immunological effects of NA infection in
subjects with CD in remission on gluten free diet and during wheat challenge.
Participants were matched by age and gender (Table 1). Using a random number generation
sequence, the researcher preparing inocula independently of the clinical
support, assigned participants to the “hookworm” and
“control” groups. Primary end-points included duodenal histology
Marsh scores and systemic interferon-γ measured by QE65-ELISpot pre- and
post-wheat challenge. Secondary end-points included a) the clinical response to
inoculation with hookworm, and b) subsequent response to the in
vivo wheat challenge, as measured by symptom and laboratory
indices, intraepithelial duodenal CD4+, CD8+ and
CD3+ lymphocyte counts (IEL), and duodenal villus
height/crypt depth (Vh/Cd) ratios. Because this was a Phase 1b/2a clinical trial
assessing safety, the hookworm and control sample sizes were deliberately
limited to ten volunteers each. Subjects and investigators remained blinded
throughout the trial.
10.1371/journal.pone.0017366.t001
Participant demographics.
Control Subjects (n = 10)
Hookworm Subjects
(n = 10)
Age (years)1,2
44 (25–58)
47 (25–62)
Gender (M/F)
2/8
2/8
BMI (kg/m2)3,2
27 (22–32)
26 (18–31)
IgA tTG3 (U/ml)4,2
9 (5–15)
8 (5–19)
Duration (months)5,2
70 (10–325)
77 (15–143
Age at enrolment,
mean value (range),
Body Mass Index,
Pre-enrolment IgA anti-tissue transglutaminase (normal range below 20
U/ml),
duration of gluten free diet.
Inocula and inoculation
Inoculations were performed at wk 0 (either 10 L3 or placebo) and
wk 12 (either 5 L3 or placebo). Inocula of five or ten NA
L3 were prepared as previously described [18].
Placebo consisted of an identically presented inoculum containing 0.2 ml
McIlhenny Co Tabasco Pepper Sauce® to mimic the pruritus that
accompanies skin penetration by L3. A control sample of
L3 was submitted with every batch of inoculum including
placebo, and was inspected microscopically for larval motility to verify
viability. All vials and pipettes were examined following inoculation to
ensure that there were no residual hookworm larvae. Evidence of hookworm
infection was provided by peripheral blood eosinophil count
(×109/L), and documenting hookworm eggs in feces by
microscopy and/or by sighting a hookworm at endoscopy.
A strict GFD was maintained from weeks 0 to 20. Endoscopy was performed at
week 20 using an Olympus high resolution digital endoscope; five single-bite
biopsies were collected from the third part of the duodenum (D3), and seven
from the second part (D2). Volunteers consumed two 50 g slices of white
bread twice a day (equivalent to 16 g of gluten daily) for five days. On the
sixth day, blood was collected and a second endoscopy was performed
(referred to as the week 21 timepoint).
Monitoring, assessments and wheat challenge
Clinical reviews occurred at weeks 0, 4, 12, 20 and 21, and symptom diaries
were recorded weekly. From weeks 0–20 and daily during the wheat
challenge, participants were asked to complete a symptom score rating from 0
(none) to 3 (severe) bloating, constipation, flatulence, headaches,
lethargy, mouth ulcers, nausea, skin rash, skin itch, and vomiting, and to
record their number of bowel motions, loose bowel motions and urgent bowel
motions, and the number of episodes of pain per day. Also recorded were pain
intensity and general wellbeing on a visual analogue scale from “no
pain at all” to “very severe” and “very well”
to “severely ill” respectively.
Histopathological Analysis
Biopsies were fixed in neutral buffered formalin, processed and carefully
orientated and embedded in paraffin wax. Sections (3 µm) were stained with
hematoxylin and eosin (H&E) and immunostained with anti-CD8, anti-CD4 and
anti-CD3 antibodies (all from Novocastra Laboratories Ltd). The Marsh scores
were graded by two independent researchers [25]. The IELs per 100 nucleated
enterocytes (100NE) were counted at 24 randomly selected sites between the
villus tip to the base of the crypt in each biopsy. The Vh/Cd ratios were
measured independently of the Marsh grading [24] and were performed on 10
randomly selected well-orientated sites.
Peripheral blood interferon-γ ELISpot responses to the immunodominant
α-gliadin peptide p57–73 QE65
Peripheral blood mononuclear cells (PBMCs) were incubated with p57–73 QE65
(50 µg/ml) in overnight IFN-γ ELISpot assays, as previously described
[27].
Briefly, ELISpot plates were coated with anti-IFN-γ (clone 1-D1K, Mabtech)
and then blocked with 5% fetal calf serum (FCS). Freshly isolated PBMCs
were added to wells containing medium alone (RPMI 1640, Invitrogen, 10%
fetal bovine serum, 100 U/µl penicillin, 100 µg/ml streptomycin and
2 mM L-glutamine) or p57–73 QE65 in medium, and cultured for 24 h at
37°C in 5% CO2. Cells were removed and the plates were
incubated with anti-IFN-γ-biotin (clone 7-B6-1, Mabtech), then
streptavidin-alkaline phosphatase, and developed with BCIP/NBT substrate. Spots
were counted on an iSpot ELISpot reader (Autoimmun Diagnostika GmbH).
Statistical Analyses
Statistical analyses were performed using Prism5 (GraphPad). When comparing
non-continuous variables (eg. symptom scores), area under the curve analysis
from week 1 to week 20 followed by Mann-Whitney U test was used to compare
between groups. For continuous variables (eg. duodenal IEL counts), two-way
ANOVA was used to analyse between time-points and groups. In the figures,
* = p<0.05,
** = p<0.01,
*** = p<0.001.
Results
Between October 2007 and March 2008, 131 potential subjects indicated interest, 61
were screened and twenty were enrolled and completed the study without protocol
violations (Figure 1). Reasons
for exclusion of subjects are summarised in Figure 2.
10.1371/journal.pone.0017366.g001
CONSORT flowchart.
10.1371/journal.pone.0017366.g002
Recruitment and Protocol.
Recruitment and summation of those excluded post screening (A), and trial
outline (B).
Hookworm Tolerance and SafetyInoculation efficacy
Mature hookworm infection was confirmed in each of the hookworm group by the
identification of eggs in feces (n = 5), or by
endoscopic visualisation of at least one adult worm
(n = 8; Figure 3A), or by both (n = 3). In five
subjects with proven infection, patency could not be confirmed by fecal
microscopy despite the testing of 17 of 18 scheduled collections from wk
12–21. No control subject had evidence of hookworm infection and no
subject had evidence of any other helminth infection.
Symptom scores: No serious adverse event relating to
hookworm infection occurred. All subjects in the hookworm group developed
multiple tiny papules shortly after the application of L3 (Figure 3B). Symptoms were
scored by questionnaire over the 20 weeks of hookworm infection (Figure 4A–F). The
hookworm group experienced pain during the initial colonisation of the
intestine, but this resolved completely by week 16 (Figure 4A). There were trends towards the
hookworm group having less frequent bowel actions after colonisation was
established (Figure 4B),
and more flatulence (4C), nausea and bloating (data not shown) at earlier timepoints. The
inoculation site remained itchy until week 4 (Figure 4D). Unexpectedly, the hookworm
group experienced less lethargy than the controls (Figure 4E), and wellbeing was unaffected
(Figure 4F). At week
21, 5 of 10 subjects in the control group incorrectly guessed that they were
in the active hookworm-infected group, while 8 of the 10 in the hookworm
group confidently and correctly identified his or her infection status. At
the end of the study all infected subjects were offered anthelminthic
treatment but declined.
10.1371/journal.pone.0017366.g004
Symptom responses to hookworm infection.
Control group is indicated by black circles, hookworm group by grey
squares, values are mean +/− SEM. (A) pain events, (B)
bowel motions/day, (C) flatulence, (D) skin rash, (E) lethargy, (F)
wellbeing. Area under the curve analysis followed by Mann-Whitney U
test to compare between groups showed that pain events (A) was
significantly different between the groups.
Laboratory Indices: A modest leukocytosis with an
eosinophilia was noticeable from week 4 after hookworm inoculation (Figure 5A–B).
Hemoglobin levels were stable from week 0 to 20, but dropped at week 21 in
both groups after wheat challenge (Figure 5C).
10.1371/journal.pone.0017366.g005
Blood cell and marker levels.
(A) white cell, (B) eosinophil (C) hemoglobin. Control group is
indicated by black circles, hookworm group by grey squares, values
are mean +/− SEM. Data was analyzed by two-way ANOVA:
significant effects of time and interaction was shown in (B), and
post-hoc one-way ANOVA on each group showed the differences between
timepoints as indicated.
Oral wheat challengePrimary outcomes
Mucosal damage measured by Marsh score: In both the hookworm
and control groups, mucosal damage deteriorated after the 5-day wheat
challenge (Figure 6A).
Although this deterioration was pronounced in the control group
(p = 0.02), there was no statistical difference in
Marsh score between hookworm and control groups.
10.1371/journal.pone.0017366.g006
Histological and immunological results.
(A) Marsh score and (B) IFN-γ ELISpot. Data was analyzed by
two-way ANOVA: significant effects of time only were shown as
indicated, no significant effect of group or interaction was found
in (A) or (B).
Systemic inflammatory immune responses: Gluten-specific
IFN-γ-producing PBMCs were measured by ELISpot before and after wheat
challenge (Figure 6B).
As expected, T cells secreting IFN-γ in response to p57–73 QE65
were absent prior to wheat challenge (data not shown), but were mobilised
into blood following the challenge. The frequency of IFN-γ secreting T
cells specific for p57–73 QE65 increased significantly only in the
control group (p = 0.01), but the difference between
the groups was not significant.
Secondary Outcomes
Clinical response to wheat challenge — Symptom scores:
Nine subjects (4 with hookworm and 5 controls) developed abdominal
discomfort, bloating and vomiting 1–2 hours after the oral wheat
challenge. These symptoms resolved within 2–4 hours (data not shown).
These symptoms did not recur despite continuation and completion of the five
day wheat challenge. There was no significant difference between the groups
during week 20 in level of pain intensity or episodes of pain, number or
urgency of bowel motions, vomiting, nausea bloating, headaches, lethargy or
well-being.
Mucosal inflammatory response — Duodenal histological
measurements: After the 5-day in vivo wheat
challenge, the total, CD3+ and CD8+ duodenal
IEL counts increased in both groups (Table 2). The CD4+ IEL
counts were not significantly affected by either in vivo
wheat challenge or hookworm infection. A robust but almost identical
decrease in the Vh∶Cd ratio after gluten was measured in the hookworm
and control groups (Table
2).
10.1371/journal.pone.0017366.t002
Duodenal cellular responses following in vivo
gluten challenge.
Control
Hookworm
p values
Week 20
Week 21
Week 20
Week 21
Time
Group
Interaction
Total IEL
30.5(14.8–46.2)
52.55(32.5–72.6)
26.4(19.4–33.4)
43.5(32.7–54.3)
0.0037
0.30
0.70
CD8+ IEL
32.95(19.5–46.4)
45.75(33.6–57.9)
38.55(31.8–45.3)
54.05(36.6–71.5)
0.019
0.24
0.82
CD4+ IEL
0.85(−0.043–1.74)
2.75(−0.43–5.93)
0.55(0.090–1.01)
0.7(0.087–1.31)
0.18
0.13
0.25
CD3+ IEL
37.65(23.4–51.9)
58.9(44.4–73.4)
42.45(34.0–50.9)
60(42–78)
0.0039
0.64
0.77
Villus Height
40.44(32.9–48.0)
28.23(21.8–34.6)
38.27(34.6–41.9)
28.43(19.6–37.3)
0.0009
0.75
0.70
Crypt Depth
15.9(14.1–17.8)
18.08(15.8–20.3)
15.38(13.5–17.3)
18.54(15.5–21.6)
0.014
0.98
0.64
Vh/Cd ratio
2.669(2.0–3.3)
1.644(1.2–2.1)
2.575(2.1–3.0)
1.683(1.0–2.3)
0.0006
0.91
0.79
Levels were analyzed by two-way ANOVA. p values considered
significant are indicated in bold.
Discussion
The “hygiene hypothesis” is a plausible explanation for the increasing
incidence of autoimmune and allergic diseases in affluent societies, but it has been
difficult to directly elucidate the mechanism by which infectious agents such as
nematodes alter disease-causing immune responses in humans. In common with many
other immune diseases, the prevalence of CD has risen dramatically. We propose that
chronic helminthiasis, such as hookworm infection, may be immunomodulatory and alter
pathogenic immune responses in vivo.
In this Phase 1b/2a trial, we have established an experimental model allowing us to
explore how hookworm infection alters the effects of gluten in CD. Chronic hookworm
infection can be reliably and safely established in subjects with well controlled
disease. Lacking precedent, the size of this demanding study was small. We observed
at best weak trends towards reduced numbers of gluten peptide-specific T cells in
blood and histological damage following wheat challenge in CD.
As has been reported in another trial, standard fecal microscopy is relatively
insensitive in light infection; a negative test does not exclude colonization [29]. All patients
inoculated acquired adult hookworms in the intestine. The pathognomic findings of a
transient papular rash at the site of inoculation, together with the common
development of mild abdominal pain in the period soon after the initial inoculation
hampered attempts to blind the hookworm-infected participants and investigators. In
each control participant however, genuine confusion as to status was usual. It is
inherently difficult to mask the hallmark features of hookworm inoculation, just as
it is to mimic them. Rather than inoculating every participant and subsequently
treating the controls with an anthelminthic, we felt obliged to accept this
compromise. The occurrence of abdominal pain has implications when evaluating
hookworms as therapy in clinical trials, especially during the establishment phase
where symptom scores to measure outcomes may be confounded [20]. Because infection with NA
typically persists for years [30], [31], the morbidity occurring during early infection should be
accounted for by undertaking studies after chronic infection is established.
The refusal of all participants in the active arm to take anthelminthic therapy after
completion of the trial was not unique to this study [20], [32] and supports the contention that
chronic light hookworm infection does not compromise wellbeing, an argument
congruous with the trend in the improved lethargy score. Although it is well
recognized that heavy hookworm infection causes clinically significant blood loss
[15], the
legitimate concern that experimental infection would cause anemia in patients
already predisposed with CD did not eventuate. As anticipated from a previous
experimental inoculation study utilising capsule endoscopy [18], the hookworm group acquired
peripheral and mucosal eosinophilia but the mucosa at week 20 was not obviously
damaged.
Following the epidemiological evidence of a causal association between the
disappearance of helminths from societies with advanced sanitary infrastructure and
the apparent rise in incidence of autoimmune and allergic diseases [3], a number of
interventional clinical trials have been undertaken [12], [20], [32], [33], with inconsistent results.
The porcine whipworm, T. suis, has been reported as beneficial in
Crohn's disease and ulcerative colitis [33], both conditions which share
genetic traits with CD [34]. However, a recently reported controlled trial using this
helminth in patients with allergic rhinitis demonstrated that while an immunological
response to whipworm was elicited, no therapeutic benefit was apparent [12]. Similarly, in a
trial where NA infection was tested for an effect among patients with asthma, no
significant benefit from helminth infection was reported [32]. While our experience from a
proof of concept study where patients with active Crohn's disease were infected
with hookworm suggested an early benefit, their wellbeing was reliant on
continuation of immunosuppressive therapies [20].
Our study establishes that hookworm infection on its own will not obviate the
necessity for a restricted diet in CD. However, this experimental human challenge
system appears to be a safe way of investigating the effect helminth parasites might
impact on immune pathology. The advantages are that it directly addresses the human
response, the disease process is not affected by the clinical imperative to use
immune modulating therapy, intestinal tissue as well as blood is available for
analyses and antigen stimulation testing can be effected both in
vivo and in vitro.
Supporting Information
CONSORT Checklist.
(PDF)
Study Protocol.
(PDF)
The authors wish to gratefully acknowledge the valued contribution made by Dr Owen
Harris who agreed to be the clinical monitor for this trial. They also wish to
acknowledge the contribution made by Dr Linda Fletcher for assistance with specimen
handling.
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