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Journal of Clinical Microbiology, November 1999, p. 3764-3766, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transport and Storage of Fresh and Frozen Gastric
Biopsy Specimens for Optimal Recovery of Helicobacter
pylori
Markus
Heep,*
Karl
Scheibl,
Andrea
Degrell, and
Norbert
Lehn
Institute for Medical Microbiology, Uniklinik
Regensburg, Regensburg, Germany
Received 8 February 1999/Returned for modification 2 May
1999/Accepted 19 July 1999
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ABSTRACT |
The viability of Helicobacter pylori in vitro and in
gastric biopsy specimens was determined. Recovery rates were 94, 87, and 77% from biopsy specimens in Portagerm pylori in cooled containers after 1, 2, and 3 days of transport, respectively (n = 307), and 97% if stored and shipped in glycerol broth at 
70°C
(n = 232).
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TEXT |
The stability of Helicobacter
pylori in biopsy material during transport is a limiting factor
for culture and susceptibility testing.
For a multicenter therapy study in Southeast Asia, our institute in
Germany was requested to provide the necessary microbiological data.
The biopsy specimens were to be stored and shipped in a frozen state.
In preliminary investigations, we evaluated various transport media
with regard to optimal recovery of H. pylori either from
culture suspensions or from fresh and frozen gastric biopsy specimens,
and we analyzed our results with biopsy specimens transported from
distant national centers.
Reported data on stability and culture results, even from fresh biopsy
specimens in different media, vary considerably (4-6, 8, 10,
11). Few data are available on transport media and the conditions
for isolation of H. pylori from gastric biopsy specimens
when the distances they are being transported are long or
transportation is delayed (1, 7, 9). In one extensive evaluation, biopsy specimens were most successfully frozen in media
composed of skimmed milk or cysteine-Albimi broth supplemented with 17 to 20% glycerol (3). We used brucella broth (Becton Dickinson, Heidelberg, Germany) supplemented with 8% fetal calf serum, Dent Helicobacter selective supplement (Oxoid, Wesel, Germany), and 20% glycerol as one of the candidates for a transport medium (TM)
with 0.1% agar (TM1), 0.4% agar (TM4), 0.8% agar (TM8), and 1.2%
agar (TM12). We compared it to Portagerm pylori (BioMérieux, Nürtingen, Germany) and Stuart medium (Oxoid) with 0.5% agar (ST5) and 1% agar (ST10).
(Part of the results were presented at the XI International Workshop on
Gastroduodenal Pathology and Helicobacter pylori, Budapest,
Hungary, 1998.)
Stability of suspensions.
We used four fresh clinical isolates
either as a suspension or soaked in paper discs that resulted in
an inoculum of 105 to 106 CFU per U when
processed and cultured like gastric biopsy specimens. The samples
were stored at least in duplicate at room temperature (18 to
24°C) or other temperatures for up to 28 days. After each period,
every sample was used one time for quantitative culture on
Wilkins-Chalgren agar (with Dent [Oxoid] and 8% lysed horse blood
[Froschek, Mühlheim, Germany]). The density of CFUs per plate was graded as follows: no growth, 0; <10, 1; 100, 2; <1,000, 3;
and >1,000, 4. In TM, the recovery from suspensions stored at
70° and 25°C was very high for up to 28 days. In Portagerm pylori, recovery was acceptable at 70°C but not at 25°C.
Suspensions of H. pylori in saline did not survive at 70
and 25°C, so we avoided the evaluation of biopsy specimens under
these conditions. At room temperature and at 4°C, the performance of
Portagerm pylori in terms of storage of suspensions was slightly better
than that of TM on day 1. On day 2 or 3, there was no growth from any
suspension. The recovery from discs and biopsy specimens was higher
(Table 1).
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TABLE 1.
Mean density of CFUs (graded from 0 to 4) from paper
discs soaked with suspensions of H. pylori (n = 4) after storage for 0, 24, and 72 h in TM, Stuart
medium, or Portagerm pylori (PG) at different temperatures
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Stability in biopsy specimens.
Biopsy specimens were
streaked on Wilkins-Chalgren and chocolate agars with sterile
cotton swabs. From anaerobe jars (Schütt, Göttingen,
Germany), the air was evacuated to 0.4 bar and then substituted by a
gas mix resulting in an atmosphere of 9% CO2, 11%
O2, and 80% N2.
Multiple biopsy specimens were obtained from the antrums of 22 patients
with suspected
H. pylori infection and highly positive
(within 30 min) urease tests (Astra, Wedel, Germany). Patients
volunteered (informed consent) to provide up to eight additional
biopsy
specimens.
After endoscopy, the biopsy specimens were immediately placed in
transport media (saline, TM1 or TM4, and Portagerm pylori)
and then
put into transport containers at the respective
temperatures.
At room temperature, the recovery of
H. pylori from biopsy
specimens in TM was lower than that in Portagerm pylori or NaCl.
In the
worst-case scenario
sample delayed by 72 h without cooling
the
recovery from biopsy specimens stored in Portagerm pylori was
superior
to those in all other media (Fig.
1).
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FIG. 1.
Recovery of H. pylori from biopsy specimens
stored in NaCl, TM1 or TM4, and Portagerm pylori at room
temperature for 24 and 72 h (n = 14). The boxes
include all values from the 25th to the 75th percentile; the line in
the box shows the median values and the whisker extreme values.
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The highest recovery of CFU from biopsy specimens after 24 and 72 h in both TM and Portagerm pylori was observed at the constant
temperature of 4°C. From all biopsy specimens stored at
25°C,
including Portagerm pylori,
H. pylori could be cultured,
except
for one in TM. The results of storage for 0, 24, and 72 h
at constant
temperatures (
25 and 4°C) (
n = 8) were
compared to the recovery
after storage at room temperature with
different starting temperatures
(
n = 6) as a simulation
of the conditions during shipment (Fig.
2). With low starting temperatures, the
recovery was as high as
it was with the constantly cooled biopsy
specimens.
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FIG. 2.
Biopsy specimens stored for 72 h at a constant
temperature of 4° or 25°C (group A; n = 8) or at
room temperature in cooling containers with a starting temperature of
4° or 25°C (group B; n = 6).
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Clinical studies.
For the German study, two biopsy specimens
obtained from each of 307 patients who recently had a duodenal ulcer
and positive rapid urease tests (Jatrox; Procter & Gamble, Weiterstadt,
Germany) were placed in Portagerm pylori transport medium in cooled
transport containers (Sarstedt, Nümbrecht-Rommelsdorf,
Germany). The initial temperature was 25°C, and after transport for
1, 2, or 3 days, the recovery rates were 94, 87, and 77%,
respectively. After more than 2 days, the yields and densities of
H. pylori cultures were significantly decreased (Table
2).
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TABLE 2.
Recovery and density of H. pylori isolates
from biopsy specimens after transport for 1 to 3 days in
Portagerm pylori in cooled transport containers
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For the study in Southeast Asia (Singapore, Manila, Kuala Lumpur,
and Bangkok), our in-house transport medium (TM1) was used.
One
biopsy specimen each from the antrum and corpus were obtained
from 232 patients before and from 216 patients after triple therapy
(two
biopsies each from the antrum and corpus). The vials were
then
put in a prefrozen (
20°C) transport container (Sarstedt)
without
delay. The container was then put in a polystyrene transport
box
and sent to a central refrigeration facility within 24 h.
The specimens were stored at
70°C for 2 to 8 weeks. Finally,
a
carrier (World Courier) delivered them, on dry ice, to our laboratory
for
culture.
The recovery rate was 97% (Table
3) of
all primary samples (232 patients). We also obtained 16 isolates
(Table
3) after
failure of therapy (216 patients). Incubation exceeding
7 days
never resulted in additional isolates in either of the two
clinical
studies.
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TABLE 3.
Positive and negative H. pylori detection from
frozen biopsy specimens by culture and histology before and
after therapy
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In all of our experiments, recovery was optimal at a constant
temperature of 4°C after a few days. But most transport devices
attain room temperature in 12 to 24 h (data provided by Sarstedt).
The maintenance of cooling during long transports is barely possible
without dry ice, i.e., frozen samples. Freezing may result in
an
initial decrease in colony counts but allows for stable storage
over
several
weeks.
In contrast to the results obtained with suspensions, the recovery from
biopsy specimens stored at
25°C in Portagerm pylori
was very
good. It is possible that the presence of gastric tissue
and mucus or
the rapid immobilization of the organism within the
biopsy specimen
exerts a protective effect on
H. pylori during
freezing
in Portagerm pylori or that the bacterial load was much
higher in
the biopsy
specimens.
For transporting gastric biopsy specimens over longer distances, we
currently recommend cooled transport containers initially
cooled to
4°C or frozen at
25°C and the use of Portagerm pylori
because
of its superior performance at room temperature. For biopsy
specimens
that may arrive at our institute over the weekend, we
advocate storing
them in Portagerm pylori in a cooled transport
container at
25°C and shipping on a Monday. The possible loss
of amoxicillin
resistance after freezing requires further studies
(
2).
Our data affirm that freezing and the addition of glycerol permit the
storage of gastric biopsy specimens for a few weeks
at
25°C, and
probably for several months at temperatures below
70°C.
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ACKNOWLEDGMENTS |
We thank Karin Fenn, Brigitte Reisinger, and Regine Birngruber for
excellent technical assistance. We also thank E. Schütz, S. Weidenhiller, H. Worlicek, and R. Keller from Regensburg, Germany, for
providing the biopsy specimens for the preliminary studies. The
histopathological examinations for the Southeast Asia study were
done in the laboratory of Pentti Sipponen, Espoo, Finland.
The study in Germany was financed by Hoechst-Marrion-Roussel. The study
in Southeast Asia was financed by Astra Pharmaceuticals (Singapore).
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Medizinische Mikrobiologie, Uniklinik Regensburg, D-93053
Regensburg, Germany. Phone: 49 941 944-6414. Fax: 49 941 944-6402. E-mail: Markus.Heep{at}klinik.uni-regensburg.de.
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Journal of Clinical Microbiology, November 1999, p. 3764-3766, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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