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).
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).
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
| Temperature (°C) | Time (h) | Mean density of CFUs in:
|
||||||
|---|---|---|---|---|---|---|---|---|
| TM1 | TM4 | TM8 | TM12 | ST5 | ST10 | PG | ||
| 0 | 3.7 | 3.7 | 3.8 | 3.6 | 3.9 | 3.9 | 3.9 | |
| −25 | 24 | 3.5 | 3.5 | 3.6 | 3.6 | 2.3 | 2.3 | 2.5 |
| 72 | 3.0 | 3.0 | 3.0 | 3.3 | 2.5 | 2.6 | 1.6 | |
| 4 | 24 | 3.1 | 3.1 | 3.0 | 3.1 | 3.4 | 3.4 | 4.0 |
| 72 | 2.4 | 2.3 | 2.3 | 1.9 | 1.6 | 1.9 | 3.4 | |
| 10 | 24 | 2.8 | 2.6 | 2.5 | 2.5 | 2.6 | 2.6 | 3.6 |
| 72 | 1.6 | 1.8 | 1.4 | 1.5 | 1.0 | 1.4 | 2.8 | |
| 16 | 24 | 3.0 | 2.4 | 2.8 | 2.5 | 2.9 | 2.9 | 3.8 |
| 72 | 1.1 | 1.0 | 1.1 | 1.3 | 0.9 | 1.3 | 2.6 | |
| 20 | 24 | 1.5 | 1.4 | 1.5 | 1.8 | 2.0 | 2.0 | 4.0 |
| 72 | 0.0 | 0.1 | 0.2 | 0.2 | 0.6 | 0.7 | 3.1 | |
| 36 | 24 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| 72 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | |
| 4a | 24 | 3.5 | 3.5 | 3.5 | 3.0 | 2.5 | 4.0 | 4.0 |
| 72 | 0.0 | 0.5 | 0.5 | 0.5 | 1.0 | 1.0 | 4.0 | |
| −25a | 24 | 4.0 | 4.0 | 4.0 | 4.0 | 4.0 | 3.5 | 4.0 |
| 72 | 0.0 | 0.0 | 0.0 | 0.5 | 0.0 | 0.0 | 1.5 | |
Cooled container with starting temperature of 4 or −25°C that was then stored at room temperature.
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).
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.
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.
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).
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).
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
| Sample grade or description | Densitya (%) of isolates after transport for:
|
||
|---|---|---|---|
| 1 day (n = 270) | 2 days (n = 15) | 3 days (n = 22) | |
| 4 | 32 | 27 | 9 |
| 3 | 38 | 40 | 23 |
| 1–2 | 24 | 20 | 45 |
| Negativeb | 4 | 7 | 23 |
| Contaminated | 2 | 7 | 0 |
Significant difference of the distribution of density between days 1 and 3 (P < 0.001, Mann-Whitney test).
Significant difference of the positivity rate between days 1 and 3 (P < 0.001, Mann-Whitney test).
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.
TABLE 3.
Positive and negative H. pylori detection from frozen biopsy specimens by culture and histology before and after therapy
| Histology result | Culture result
|
Total | |
|---|---|---|---|
| Negative | Positive | ||
| Before therapy (n = 232) | |||
| Negative | 5 | 16 | 21 |
| Positive | 2 | 209 | 211 |
| Total | 7 | 225 | 232 |
| After therapy (n = 216) | |||
| Negative | 199 | 4 | 203 |
| Positive | 1 | 12 | 13 |
| Total | 200 | 16 | 216 |
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.
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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