Gastroenterology Unit,4 Massachusetts General Hospital, Boston, Massachusetts 02114. Received 19 October 1987/Accepted 9 February 1988. A study of 84 ...
Vol. 26, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, May 1988, p. 948-949
0095-1137/88/050948-02$02.00/0 Copyright © 1988, American Society for Microbiology
Simple Microbiologic Detection of Campylobacter pylori JULIE PARSONNET,' KAREN WELCH,2 CAROLYN COMPTON,3 ROBERT STRAUSS,4 TIMOTHY WANG,4 PETER KELSEY,4 AND MARY JANE FERRARO2* Diseases Infectious Unit,' Francis Blake Bacteriology Laboratories,2 Pathology Department,3 and Gastroenterology Unit,4 Massachusetts General Hospital, Boston, Massachusetts 02114 Received 19 October 1987/Accepted 9 February 1988
A study of 84 gastric biopsies taken from 42 adult patients revealed simple techniques for Gram stain and culture for Campylobacterpylori. In an initial study of 18 biopsies, Gram stains prepared from ground, diluted tissue were all negative for curved, gram-negative rods, whereas 13 of these specimens were positive for C. pylori by culture. The Gram stains for the remaining 66 biopsies were prepared by a rinse-imprint technique. In this group, there were 30 Gram stains positive for organisms resembling C. pylori and 32 positive cultures. By Gram-staining two sites, fundus and antrum, the sensitivity of the Gram stain for identifying a positive specimen increased from 91 to 100%. Gram stain may be the preferred technique for rapid diagnosis. When cultured, C. pylori was recovered most often on modified Thayer-Martin medium incubated microaerophilically at 35°C. The presence of antibiotics in modified Thayer-Martin medium limited upper respiratory flora overgrowth, which was often present on nonselective media.
London, England) or Bio-Bag type CF (Marion Scientific, Div. Marion Laboratories, Inc., Kansas City, Mo.). An additional BAP plate was inoculated and incubated at 35°C in 5% C02. All plates were examined for growth at 2 to 4 days and at 1 week. Small, clear colonies suggestive of C. pylori were Gram stained for typical C. pylori morphology. Positive catalase, oxidase, and rapid urease tests confirmed the identification. Endoscopic specimens were fixed and stained with silver by the Warthin-Starry method and were evaluated for the presence of both inflammation and typical, curved organisms.
Over the last several years, an association between Campylobacter pylori and gastritis has been recognized. Despite growing interest in C. pylori-related disease, diagnostic microbiologic techniques are not standardized. We present a simple protocol, using techniques and culture media available in all general microbiology laboratories, that has excellent diagnostic yield. MATERIALS AND METHODS Specimens were received from 42 adult patients referred to hospital gastroenterologists for elective endoscopy. All patients gave informed consent. Gastric biopsies were taken first from the fundus and then from the antrum in each patient. Biopsy forceps were washed thoroughly between biopsies. Histology specimens were placed in Formalin and routinely processed. Microbiology specimens were transported in sterile containers (without diluent or preservative) to the microbiology laboratory, where they were Gram stained and cultured within 1 h of biopsy. Two techniques were used for Gram stain. An initial 18 biopsy specimens (from 9 patients) were ground in a mortar and diluted with 0.5 ml of dextrose phosphate broth. A portion of this ground material was then smeared on a slide. The next 66 biopsy specimens (from 33 patients) were rinsed in dextrose phosphate broth and blotted on a sterile surface to remove excess broth, and an impression smear was made on a sterile slide. These specimens were later ground in 0.6 ml of dextrose phosphate broth for culture. All slides were heat fixed and Gram stained. They were then examined under oil for 5 min or until typical curved, gram-negative rods were observed. A sample (0.1 ml) of each ground specimen was inoculated and streaked for isolation on plates containing brucella agar with 5% horse blood (BAP) (GIBCO Diagnostics, Madison, Wis., or BBL Microbiology Systems, Cockeysville, Md.) and on modified Thayer-Martin medium (MTM) (GIBCO Diagnostics). These plates were incubated microaerophilically at 35°C in an atmosphere of 10% C02, 5% 02, and 85% H2, using a Campylobacter Gas Generating kit (Oxoid Ltd., *
RESULTS None of the initial 18 biopsy specimen Gram stains performed on ground, dilute tissue was positive. Of these 18 specimens, 13 grew C. pylori. The remaining 66 biopsies yielded 30 positive Gram stains using the rinse-imprint technique. In this group, there were 32 positive cultures (Table 1). There were three positive cultures from specimens with negative Gram stains. There was one positive Gram stain from a culture-negative specimen. The sensitivity of Gram stain compared with culture was 91%. Of those patients who had rinse-imprint Gram stains performed, there were a total of 17 patients with one or more culture-positive biopsies (Table 1). All of these patients had at least one positive Gram stain. Staining two sites, one fundal and one antral, increased the sensitivity of Gram stain to 100%. C. pylori was recovered most often on microaerophilically incubated MTM. Of 33 culture- or Gram-stain-positive specimens, 30 (91%) grew on MTM. C. pylori was recovered from microaerophilically incubated BAP in 61% of cases (20 of 33). BAP plates incubated in 5% C02 had poor yield (4 of 33). It was noted that there was often a region of poor growth surrounding the site of tissue inoculation on both media, suggesting "tissue inhibition." For this reason, quantitative culture may be unreliable. Table 1 summarizes the data for all 17 patients who had impression Gram stains as well as either a positive culture or a positive silver stain. Overall cultures correlated very well with histopathology. All patients who showed C. pylori on
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DETECTION OF C. PYLORI
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TABLE 1. Correlation of Gram stain, culture, and silver stain for 17 C. pylori-positive patients with specimens taken from two gastric sites Patient no.
Gram stain (13/16;
81%)" 1 5
+ -
7 9
+ -
il
+ + + +
16 17 18 19 20 21 27 28 29 30 31 32
Silver stain (13/15; 87%)
Gram stain (17/17; 100%)
Culture (16/17; 94%)
Silver stain
+
+
+
+
+
+ + +
+
+ + +
+ + +
+ + +
+
+ +
+ +
+ + + + + + +
+ + + + -
+ + +
Outcome of biopsy 2 (fundal)
Outcome of biopsy 1 (antral) Culture (16/16; 100%)
+ + +
-
+
(15/16; 93%)
+
+
NDb
+
+
+
+
+
+
+
+
+ + +
+ + + +
+ + + +
NDC
+ + + +
+ + + + +
+ + + + +
+
+ + + + + + +
a No. of true positives/no. tested; sensitivity. A true positive is defined as a positive silver stain or positive culture from the biopsy site. b Not determined; pathologic section showed fundal histology. C Not determined; pathologic section showed esophageal histology.
silver stain had at least one positive culture. One biopsy showed typical organisms histologically, but the culture was negative. Three biopsy specimens grew C. pylori but had negative silver stain results. Histologically, all three of these patients had chronic active gastritis. DISCUSSION In our experience, C. pylori is easy to isolate and identify. Imprint Gram stains of rinsed and blotted tissue, when performed by a trained technologist, were accurate as well as simple, quick, and economical. They can readily be done during the endoscopic procedure. Gram stains of ground tissue were unsuccessful. Urease tests of intact tissue have been shown to have similar sensitivity but require up to 24 h to react (5, 6). In addition, a recent study fails to confirm the high sensitivity of urease tests (2). Gram stain may be the preferred technique for rapid diagnosis. Because of the patchy nature of C. pylori gastritis, however, we would recommend stains from at least two separate sites. MTM proved to be an excellent medium for C. pylori growth. The presence of vancomycin, colistin, and trimethoprim in this medium limited the upper respiratory flora overgrowth often present on BAP cultures. The colistin concentration does not seem to be sufficient to inhibit C. pylori. Supplementation with starch (which is present in MTM) has recently been shown to facilitate C. pylori growth on blood agar (1). The additional glucose in MTM may have similarly improved C. pylori growth. We did not formally compare MTM with chocolate agar, brain heart infusion, or Skirrow medium, which are also considered to be acceptable media for culture of C. pylori (3, 4, 7). In a smaller series of patients tested before this study, however, we found growth on MTM and chocolate agar to be similar except in cases of bacterial overgrowth, for which MTM was superior. We have not evaluated other gono-
coccus-selective media such as New York or Martin-Lewis agar. In summary, the techniques for microbiologic diagnosis of gastric C. pylori are simple and easily performed by a general microbiology laboratory. Gram stain can provide rapid diagnosis. Culture on MTM has excellent sensitivity, equal to that of histopathology. Although the clinical significance of C. pylori is unclear, the ease with which microbiologic diagnosis can be made should readily facilitate further investigation.
1.
2. 3.
4.
5.
6.
7.
LITERATURE CITED Buck, G. E., and J. S. Smith. 1987. Medium supplementation for growth of Campylobacter pyloridis. J. Clin. Microbiol. 25:597599. Drumm, B., P. Sherman, E. Cutz, and M. Karmali. 1987. Association of Campylobacter pylori on the gastric mucosa with antral gastritis in children. N. Engl. J. Med. 316:1557-1561. Goodwin, C. S., E. D. Blincow, J. R. Warren, T. E. Waters, C. R. Sanderson, and L. Easton. 1985. Evaluation of cultural techniques for isolating Campylobacter pyloridis from endoscopic biopsies of gastric mucosa. J. Clin. Pathol. 38:1127-1131. Krajden, S., J. Bohnen, J. Anderson, J. Kempston, M. Fuksa, A. Matlow, N. Marcon, G. Haber, P. Kortan, M. Karmali, P. Corey, C. Petrea, C. Babida, and S. Hayman. 1987. Comparison of selective and nonselective media for recovery of Campylobacter pylori from antral biopsies. J. Clin. Microbiol. 25:1117-1118. Marshall, B. J., J. R. Warren, G. J. Francis, S. R. Langton, C. S. Goodwin, and E. D. Blincow. 1987. Rapid urease test in the management of Campylobacter pyloridis-associated gastritis. Am. J. Gastroenterol. 82:200-210. McNulty, C. A. M., and R. Wise. 1985. Rapid diagnosis of campylobacter-associated gastritis. Làncet i:1443-1444. Megraud, F., F. Bonnet, M. Garnier, and H. Lamouliatte. 1985. Characterization of "Campylobacter pyloridis" by culture, enzymatic profile, and protein content. J. Clin. Microbiol. 22:10071010.
