Culture of T. whipplei from the Stool of a Patient with Whipple's Disease
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《新英格兰医药杂志》
To the Editor: Tropheryma whipplei (formerly T. whippelii) causes Whipple's disease, an infectious disorder involving the digestive tract.1 The organism can be seen within macrophages in the duodenum, but T. whipplei was first isolated from specimens of human mitral valve.1 Molecular analysis of stool samples with the use of the polymerase chain reaction (PCR) has shown DNA of T. whipplei in patients with Whipple's disease. The DNA of the organism has also been found in unaffected people2 and in wastewater samples.2,3
T. whipplei has not been cultured from stool samples because of the large number of contaminating bacteria. While investigating the possibility of the transmission of Whipple's disease during endoscopy, we found that T. whipplei was resistant to decontamination with glutaraldehyde.4
We report the culture of T. whipplei from experimentally and naturally infected stool samples in a specific axenic medium5 after a decontamination procedure (Figure 1).4 Infected stool was obtained from a 36-year-old woman who first received a diagnosis of Whipple's disease in March 1995; she had PCR-positive stools during relapses in 2000 and 2004. Glutaraldehyde slowed the growth of T. whipplei (Figure 1) in our control cultures, and growth of T. whipplei was observed in experimentally infected stools after 7 months. The number of copies of DNA in the culture of stool from the patient did not increase after 7 months, so we centrifuged the medium and reinoculated the pellet into fresh specific axenic medium 8 and 9 months after inoculation. At months 10 and 11, the growth of T. whipplei, evaluated by PCR and immunofluorescence, became exponential.5 The strain we isolated from the patient is now established and has been subcultured three times. We used genotyping to confirm that the isolated strain was that found in the duodenal-biopsy specimen from the patient and that no other bacteria were associated with the sample (see supplementary data 1 in the Supplementary Appendix, available with the full text of this letter at www.nejm.org).
Figure 1. Culture of Tropheryma whipplei from a Stool Sample from the Patient and from Experimentally Infected Stool Samples (Samples A, B, and C) in a Specific Axenic Medium (SAM) and the Effect of Gluteraldehyde on T. whipplei.
Because the number of copies of DNA in the culture of stool from the patient plateaued at 7 months, we centrifuged the medium and reinoculated the pellet into fresh specific axenic medium 8 and 9 months after inoculation. The growth became exponential at month 10. I bars denote standard deviations. See the Supplementary Appendix (available with the full text of this letter at www.nejm.org) for details of the culture methods and PCR assays.
Although T. whipplei DNA has previously been found in feces, we found viable bacteria in feces, and we speculate that Whipple's disease may be linked to fecal–oral contamination. Our study also confirms that T. whipplei is resistant to glutaraldehyde, a unique feature for a bacterium, since this product is widely used for disinfection.5 Our study has revealed a method of isolating strains of T. whipplei from patients, asymptomatic carriers, and the environment. Comparison of the genomic content of these strains could lead to the identification of pathogenic factors.
Didier Raoult, M.D., Ph.D.
Florence Fenollar, M.D., Ph.D.
Marie-Laure Birg
Université de la Méditerranée
13385 Marseille CEDEX 5, France
didier.raoult@gmail.com
References
Raoult D, Birg ML, La Scola B, et al. Cultivation of the bacillus of Whipple's disease. N Engl J Med 2000;342:620-625.
Maibach RC, Dutly F, Altwegg M. Detection of Tropheryma whipplei DNA in feces by PCR using a target capture method. J Clin Microbiol 2002;40:2466-2471.
Maiwald M, Schuhmacher F, Ditton HJ, von Herbay A. Environmental occurrence of the Whipple's disease bacterium (Tropheryma whippelii). Appl Environ Microbiol 1998;64:760-762.
La Scola B, Rolain JM, Maurin M, Raoult D. Can Whipple's disease be transmitted by gastroscopes? Infect Control Hosp Epidemiol 2003;24:191-194.
Renesto P, Crapoulet N, Ogata H, et al. Genome-based design of a cell-free culture medium for Tropheryma whipplei. Lancet 2003;362:447-449.
T. whipplei has not been cultured from stool samples because of the large number of contaminating bacteria. While investigating the possibility of the transmission of Whipple's disease during endoscopy, we found that T. whipplei was resistant to decontamination with glutaraldehyde.4
We report the culture of T. whipplei from experimentally and naturally infected stool samples in a specific axenic medium5 after a decontamination procedure (Figure 1).4 Infected stool was obtained from a 36-year-old woman who first received a diagnosis of Whipple's disease in March 1995; she had PCR-positive stools during relapses in 2000 and 2004. Glutaraldehyde slowed the growth of T. whipplei (Figure 1) in our control cultures, and growth of T. whipplei was observed in experimentally infected stools after 7 months. The number of copies of DNA in the culture of stool from the patient did not increase after 7 months, so we centrifuged the medium and reinoculated the pellet into fresh specific axenic medium 8 and 9 months after inoculation. At months 10 and 11, the growth of T. whipplei, evaluated by PCR and immunofluorescence, became exponential.5 The strain we isolated from the patient is now established and has been subcultured three times. We used genotyping to confirm that the isolated strain was that found in the duodenal-biopsy specimen from the patient and that no other bacteria were associated with the sample (see supplementary data 1 in the Supplementary Appendix, available with the full text of this letter at www.nejm.org).
Figure 1. Culture of Tropheryma whipplei from a Stool Sample from the Patient and from Experimentally Infected Stool Samples (Samples A, B, and C) in a Specific Axenic Medium (SAM) and the Effect of Gluteraldehyde on T. whipplei.
Because the number of copies of DNA in the culture of stool from the patient plateaued at 7 months, we centrifuged the medium and reinoculated the pellet into fresh specific axenic medium 8 and 9 months after inoculation. The growth became exponential at month 10. I bars denote standard deviations. See the Supplementary Appendix (available with the full text of this letter at www.nejm.org) for details of the culture methods and PCR assays.
Although T. whipplei DNA has previously been found in feces, we found viable bacteria in feces, and we speculate that Whipple's disease may be linked to fecal–oral contamination. Our study also confirms that T. whipplei is resistant to glutaraldehyde, a unique feature for a bacterium, since this product is widely used for disinfection.5 Our study has revealed a method of isolating strains of T. whipplei from patients, asymptomatic carriers, and the environment. Comparison of the genomic content of these strains could lead to the identification of pathogenic factors.
Didier Raoult, M.D., Ph.D.
Florence Fenollar, M.D., Ph.D.
Marie-Laure Birg
Université de la Méditerranée
13385 Marseille CEDEX 5, France
didier.raoult@gmail.com
References
Raoult D, Birg ML, La Scola B, et al. Cultivation of the bacillus of Whipple's disease. N Engl J Med 2000;342:620-625.
Maibach RC, Dutly F, Altwegg M. Detection of Tropheryma whipplei DNA in feces by PCR using a target capture method. J Clin Microbiol 2002;40:2466-2471.
Maiwald M, Schuhmacher F, Ditton HJ, von Herbay A. Environmental occurrence of the Whipple's disease bacterium (Tropheryma whippelii). Appl Environ Microbiol 1998;64:760-762.
La Scola B, Rolain JM, Maurin M, Raoult D. Can Whipple's disease be transmitted by gastroscopes? Infect Control Hosp Epidemiol 2003;24:191-194.
Renesto P, Crapoulet N, Ogata H, et al. Genome-based design of a cell-free culture medium for Tropheryma whipplei. Lancet 2003;362:447-449.