Abstract
We evaluated stool specimens known to contain or be free of Campylobacter by traditional culture, using the ProSpecT Campylobacter microplate assay (Alexon-Trend, Ramsey, Minn.). This rapid enzyme immunoassay for the detection of Campylobacter-specific antigens demonstrated 96% sensitivity and 99% specificity and is an acceptable alternative method of Campylobacter detection.
Food- and water-borne bacteria cause gastroenteritis that affects millions of people each year in the United States, which costs billions of U.S. dollars and results in thousands of deaths (2, 15, 24). Campylobacter jejuni is the most common cause of bacterial gastroenteritis in the United States, surpassing disease caused by Salmonella and Shigella spp. combined (1, 8–13, 20, 21, 25, 33, 36, 42). The appropriate identification of the etiologic agent of infectious gastroenteritis is important, since there are differences in treatment; the possibilities of refractory disease and postinfectious sequelae also make identification of the etiologic agent important (3, 20, 32, 35, 38, 40, 43).
Campylobacter species are microaerophilic gram-negative, curved bacilli that may be detected in stool by direct microscopy, but more commonly are cultured using selective medium or stool filtration (5–7, 14, 17, 18, 22, 24, 28, 30, 33, 37, 39, 41). More recently, nucleic acid amplification methods and enzyme-linked immunoassays (EIA) have been used to detect these bacteria (16, 19, 26, 27, 31, 37, 44, 45; A. B. John and Y. A. Lue, Program Abstr. 98th Gen. Meet. Am. Soc. Microbiol. 1998, abstr. C-263).
We evaluated the ability of the ProSpecT Campylobacter microplate assay (Alexon-Trend, Ramsey, Minn.) to detect Campylobacter spp. in clinical stool specimens that were known to contain or be free of Campylobacter spp. by traditional culture. Clinical stool specimens were collected and frozen from three institutions; 50 Campylobacter culture-positive and 114 Campylobacter culture-negative stools were collected simultaneously.
Campylobacter species were detected and identified by standard methods (34). Fifteen of the 114 Campylobacter culture-negative stool specimens contained the following other bacterial enteric pathogens: six Salmonella spp., three Shigella spp., three Yersinia enterocolitica, and three Escherichia coli O157:H7 strains. All of the stool specimens in this study represented samples from individual patients; no duplicate specimens were tested. Seventy-seven of the stool specimens were received in transport medium. The remaining 87 stool samples were received fresh and frozen immediately after culture.
The 164 stool specimens were evaluated for the presence of Campylobacter using the the ProSpecT Campylobacter microplate assay (Alexon-Trend) according to the manufacturer's instructions. Stool specimens received in transport medium were not diluted. For the fresh-frozen stool specimens, 0.5 ml of stool was mixed with the diluent provided to obtain the four drops necessary for testing. A positive and negative control well were also prepared using the positive and negative control reagents, respectively. The reactions were read both visually and spectrophotometrically in a single-wavelength spectrophotometer at 450 nm. The validity of each test run was based on appropriate reactions in the positive and negative control wells. These interpretations were performed in accordance with the manufacturer's guidelines. Each stool specimen was tested in duplicate by different medical technologists, who were blinded to the culture results. Each medical technologist recorded a visual interpretation prior to recording the spectrophotometric interpretation. The agreement between the two independent visual interpretations and the two independent spectrophotometric interpretations was determined. The agreement was also determined between the manual and spectrophotometric interpretations. All indeterminate results were repeated. Repetitively indeterminate specimens were recorded as such and considered negatives in calculations, since the EIA was unable to generate a positive result. Upon completion of the study, specimens with discordant culture and EIA results were retested by EIA, and a review of the patient's medical record was performed.
The ProSpecT Campylobacter microplate assay correctly characterized 48 of 50 Campylobacter culture-positive stool specimens. Two culture-positive stools (one received in transport medium and one fresh-frozen) were characterized as negative by EIA. Repeat EIA testing of these two specimens demonstrated one negative result and one positive result. These specimens were both considered false-negatives, since repeat testing would not have been routinely performed. One hundred and twelve of the 114 Campylobacter culture-negative stool specimens were characterized by the ProSpecT Campylobacter microplate assay as negative on initial testing. Of the remaining two Campylobacter culture-negative stool specimens, one was repeatedly positive and one was initially indeterminate but negative upon repeat testing; these were characterized as false-positive and true-negative, respectively. In this analysis, the ProSpecT Campylobacter microplate assay demonstrated 96% sensitivity and 99% specificity. Review of the medical record, however, revealed that the one “false-positive” EIA was from a patient diagnosed with infectious enteritis that may have been campylobacteriosis. It is possible that this could represent a true-positive EIA and a false-negative stool culture, since viable organisms are not necessary for detection with the EIA. In this case, the sensitivity and specificity of the ProSpecT Campylobacter microplate assay would be increased to 96.1 and 100%, respectively. There was excellent interobserver agreement in both the visual and spectrophotometric test interpretations. Similarly, there was excellent agreement between the visual and spectrophotometric measurements.
The ProSpecT Campylobacter microplate assay is an EIA that recognizes a Campylobacter surface antigen which is shared by C. jejuni and Campylobacter coli (10, 42). This EIA demonstrated at least 96% sensitivity and 99% specificity using Campylobacter culture-positive and culture-negative specimens as the reference standard. It was rapid and easy to use, with excellent agreement between duplicate manual and duplicate spectrophotometric interpretations. There was no apparent difference between stool samples received in transport media and those that were received fresh. Excellent agreement was also found between the manual and spectrophotometric interpretations. Because one stool specimen was negative on initial testing but positive on repeat testing, we recommend that stool specimens be thoroughly mixed prior to testing. We also suggest that specimens that generate an indeterminate result be retested. Repetitively indeterminate results were never encountered in this assessment. The ProSpecT Campylobacter microplate assay appears to be a reliable method for the detection of C. jejuni and C. coli.
Acknowledgments
This work would not have been possible without the technical assistance of Amanda Fares, Margaret LaSalvia, Suzanne Schroeder, Parul Shah, Kathiann Smith, and Rita Tolcin. Their dedication and hard work on this project are greatly appreciated.
REFERENCES
- 1.Altekruse S F, Stern N J, Fields P I, Swerdlow D L. Campylobacter jejuni—an emerging foodborne pathogen. Emerg Infect Dis. 1999;5:28–35. doi: 10.3201/eid0501.990104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Altekruse S F, Cohen M L, Swerdlow D L. Emerging foodborne diseases. Emerg Infect Dis. 1997;3:285–293. doi: 10.3201/eid0303.970304. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Blaser M J. Principles and practice of infectious diseases. 5th ed. Philadelphia, Pa: Churchill Livingstone; 2000. Campylobacter jejuni and related species; pp. 2276–2285. [Google Scholar]
- 4.Blaser M J, Wells J G, Feldman R A, Pollard R A, Allen J R the Collaborative Diarrheal Disease Study Group. Campylobacter enteritis in the United States: a multicenter study. Ann Intern Med. 1983;98:360–365. doi: 10.7326/0003-4819-98-3-360. [DOI] [PubMed] [Google Scholar]
- 5.Bolton F J, Robertson L. A selective medium for isolating Campylobacter jejuni/coli. J Clin Pathol. 1982;35:462–467. doi: 10.1136/jcp.35.4.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Bolton F J, Coates D, Hinchliffe P M, Robertson L. Comparison of selective media for isolation of Campylobacter jejuni/coli. J Clin Pathol. 1983;36:78–83. doi: 10.1136/jcp.36.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bolton F J, Coates D. A comparison of microaerobic systems for the culture of Campylobacter jejuni and Campylobacter coli. Eur J Clin Microbiol. 1983;2:105–110. doi: 10.1007/BF02001574. [DOI] [PubMed] [Google Scholar]
- 8.Centers for Disease Control. Outbreak of Campylobacter enteritis associated with cross-contamination of food—Oklahoma, 1996. Morb Mortal Wkly Rep. 1998;47:129–131. [PubMed] [Google Scholar]
- 9.Centers for Disease Control. Epidemiologic notes and reports: Campylobacter outbreak associated with raw milk provided on a dairy tour—California. Morb Mortal Wkly Rep. 1986;35:311–312. [PubMed] [Google Scholar]
- 10.Centers for Disease Control. Campylobacter isolates in the United States, 1982–1986. Morb Mortal Wkly Rep. 1988;37(SS-2):1–13. [PubMed] [Google Scholar]
- 11.Centers for Disease Control. Campylobacteriosis associated with raw milk consumption—Pennsylvania. Morb Mortal Wkly Rep. 1983;32:337–338. [PubMed] [Google Scholar]
- 12.Centers for Disease Control. Surveillance for waterborne-disease outbreaks—United States, 1993–1994. Morb Mortal Wkly Rep. 1996;45(SS):1–33. [PubMed] [Google Scholar]
- 13.Centers for Disease Control. Premature labor and neonatal sepsis caused by Campylobacter fetus subsp. fetus—Ontario. Morb Mortal Wkly Rep. 1984;33:483–484. [PubMed] [Google Scholar]
- 14.Chan R, Hannan B, Munro R. Use of a selective enrichment broth for isolation of Campylobacter species from human feces. Pathology. 1985;17:640–641. doi: 10.3109/00313028509084768. [DOI] [PubMed] [Google Scholar]
- 15.Council for Agricultural Science and Technology. Foodborne pathogens: risks and consequences. Task Force Report No. 122. Ames, Iowa: Council for Agricultural Science and Technology; 1994. [Google Scholar]
- 16.Fermer C, Engvall E O. Specific PCR identification and differentiation of the thermophilic campylobacters Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis. J Clin Microbiol. 1999;37:3370–3373. doi: 10.1128/jcm.37.10.3370-3373.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Gilchrist M J R, Grewell C M, Washington J A., II Evaluation of media for isolation of Campylobacter fetus subsp. jejuni from fecal specimens. J Clin Microbiol. 1981;14:393–395. doi: 10.1128/jcm.14.4.393-395.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Goossens H, De Boeck M, Butzler J P. A new selective medium for the isolation of Campylobacter jejuni from human faeces. Eur J Clin Microbiol. 1983;2:389–394. doi: 10.1007/BF02019476. [DOI] [PubMed] [Google Scholar]
- 19.Griffiths P L, Moreno G S, Park R W. Differentiation between thermophilic Campylobacter species by species-specific antibodies. J Appl Bacteriol. 1992;72:467–474. doi: 10.1111/j.1365-2672.1992.tb01860.x. [DOI] [PubMed] [Google Scholar]
- 20.Guerrant R L, Lima A A M. Principles and practice of infectious diseases. 5th ed. Philadelphia, Pa: Churchill Livingstone; 2000. Inflammatory enteritides; pp. 1126–1136. [Google Scholar]
- 21.Guerrant R L, Bobak D A. Bacterial and protozoal gastroenteritis. N Engl J Med. 1991;325:327–340. doi: 10.1056/NEJM199108013250506. [DOI] [PubMed] [Google Scholar]
- 22.Guerrant R L, et al. Evaluation and diagnosis of acute infectious diarrhea. Am J Med. 1985;78:91–98. doi: 10.1016/0002-9343(85)90370-5. [DOI] [PubMed] [Google Scholar]
- 23.Helmick C G, Griffin P M, Addiss D G, Tauxe R V, Juranek D D. Infectious diarrheas. In: Everhart J E, editor. Digestive diseases in the United States: epidemiology and impact. Bethesda, Md: National Institutes of Health; 1994. pp. 85–120. [Google Scholar]
- 24.Ho D D, Ault M J, Ault M A, Murata G H. Campylobacter enteritis: early diagnosis with Gram's stain. Arch Intern Med. 1982;142:1858–1860. doi: 10.1001/archinte.142.10.1858. [DOI] [PubMed] [Google Scholar]
- 25.Hopkins R S, Scott A S. Handling raw chicken as a source for sporadic Campylobacter jejuni infections. J Infect Dis. 1983;148:770. doi: 10.1093/infdis/148.4.770. [DOI] [PubMed] [Google Scholar]
- 26.Lawson A J, Shafi M S, Pathak K, Stanley J. Detection of Campylobacter in gastroenteritis: comparison of direct PCR assay of faecal samples with selective culture. Epidemiol Infect. 1998;121:547–553. doi: 10.1017/s0950268898001630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Linton D, Owen R J, Stanley J. Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals. Res Microbiol. 1996;147:707–718. doi: 10.1016/s0923-2508(97)85118-2. [DOI] [PubMed] [Google Scholar]
- 28.Lopez L, Castillo F J, Clavel A, Rubio M C. Use of a selective medium and a membrane filter method for isolation of Campylobacter species from Spanish paediatric patients. Eur J Clin Microbiol. 1998;17:489–492. doi: 10.1007/BF01691131. [DOI] [PubMed] [Google Scholar]
- 29.Lu P, Brooks B W, Robertson R H, Nielsen K H, Garcia M M. Characterization of monoclonal antibodies for the rapid detection of foodborne campylobacters. Int J Food Microbiol. 1997;37:87–91. doi: 10.1016/s0168-1605(97)00053-6. [DOI] [PubMed] [Google Scholar]
- 30.Mathewson J J, Keswick B H, DuPont H L. Evaluation of filters for recovery of Campylobacter jejuni from water. Appl Environ Microbiol. 1983;46:985–987. doi: 10.1128/aem.46.5.985-987.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Metherell L A, Logan J M, Stanley J. PCR-enzyme-linked immunosorbent assay for detection and identification of Campylobacter species: application to isolates and stool samples. J Clin Microbiol. 1999;37:433–435. doi: 10.1128/jcm.37.2.433-435.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Mishu B, Blaser M J. Role of infection due to Campylobacter jejuni in the initiation of Guillain-Barre syndrome. Clin Infect Dis. 1993;17:104–108. doi: 10.1093/clinids/17.1.104. [DOI] [PubMed] [Google Scholar]
- 33.Mitchell J E, Skelton M M. Diarrheal infections. Am Fam Physician. 1988;37:195–207. [PubMed] [Google Scholar]
- 34.Nachamkin I. Campylobacter and Arcobacter. In: Murray P R, Baron E J, Pfaller M A, Tenover F C, Yolken R H, editors. Manual of clinical microbiology. 7th ed. Washington, D.C.: American Society for Microbiology; 1999. pp. 716–726. [Google Scholar]
- 35.Nachamkin I, Allos B M, Ho T. Campylobacter species and Guillain-Barré syndrome. Clin Microbiol Rev. 1998;11:555–567. doi: 10.1128/cmr.11.3.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Norkrans G, Svedhem A. Epidemiologic aspects of Campylobacter jejuni enteritis. J Hyg. 1982;89:163–70. doi: 10.1017/s0022172400070662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.On S L W. Identification methods for campylobacters, helicobacters, and related organisms. Clin Microbiol Rev. 1996;9:405–422. doi: 10.1128/cmr.9.3.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Perlman D J, Ampel N M, Schifman R B, Cohn D L, Patton C M, Aguirre M L, et al. Persistent Campylobacter jejuni infections in patients infected with the human immunodeficiency virus (HIV) Ann Intern Med. 1988;108:540–546. doi: 10.7326/0003-4819-108-4-540. [DOI] [PubMed] [Google Scholar]
- 39.Rollins D M, Coolbaugh J C, Walker R I, Weiss E. Biphasic culture system for rapid Campylobacter cultivation. Appl Environ Microbiol. 1983;45:284–289. doi: 10.1128/aem.45.1.284-289.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Sorvillo F J, Lieb L E, Waterman S H. Incidence of campylobacteriosis among patients with AIDS in Los Angeles County. J Acquir Immune Defic Syndr Hum Retrovirol. 1991;4:598–602. [PubMed] [Google Scholar]
- 41.Steele T W, McDermott S N. The use of membrane filters applied directly to the surface of agar plates for the isolation of Campylobacter jejuni from feces. Pathology. 1984;16:263–265. doi: 10.3109/00313028409068535. [DOI] [PubMed] [Google Scholar]
- 42.Tauxe R V. Epidemiology of Campylobacter jejuni infections in the United States and other industrial nations. In: Nachamkin I, Blaser M J, Tompkins L S, editors. Campylobacter jejuni: current and future trends—1992. Washington, D.C.: American Society for Microbiology; 1992. pp. 9–12. [Google Scholar]
- 43.Totten P A, Fennell C L, Tenover F C, et al. Campylobacter cinaedi and Campylobacter fennelliae: two new Campylobacter species associated with enteric disease in homosexual men. J Infect Dis. 1985;151:131–139. doi: 10.1093/infdis/151.1.131. [DOI] [PubMed] [Google Scholar]
- 44.van Doorn L J, Verschuuren-van Haperen A, Burnens A, Huysmans M, Vandamme M, Giesendorf B A, Blaser M J, Quint W G. Rapid identification of thermotolerant Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis from various geographic locations by a GTPase-based PCR-reverse hybridization assay. J Clin Microbiol. 1999;37:1790–1796. doi: 10.1128/jcm.37.6.1790-1796.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Vanniasinkam T, Lanser J A, Barton M D. PCR for the detection of Campylobacter spp. in clinical specimens. Lett Appl Microbiol. 1999;28:52–56. doi: 10.1046/j.1365-2672.1999.00474.x. [DOI] [PubMed] [Google Scholar]