Absence of Helicobacter pylori within the Oral Cav
http://www.100md.com
微生物临床杂志 2006年第2期
ABSTRACT
Our study aimed to evaluate the oral cavity as a reservoir from where Helicobacter pylori may be transmitted. Histology and PCR amplification were performed. Eighty-four percent of the stomach biopsies tested positive; however, H. pylori was not detected in dental samples, indicating the absence of H. pylori within the oral cavity.
TEXT
It has been suggested that the oral cavity may play a role in the transmission of Helicobacter pylori. Increased H. pylori prevalence rates have been reported in Chinese immigrants who use chopsticks and in African infants whose mothers premasticate their food (4, 16). H. pylori has been detected in dental plaque and saliva by culture and PCR methods, with the results obtained ranging from 0 to 100% for culture and 0 to 90% for PCR (1-3, 5, 7, 9, 11, 12, 14, 15, 17, 18, 20, 21). The role of the oral cavity as a permanent reservoir for H. pylori infection in healthy nonhospitalized individuals is still unclear (6, 10).
Seventy-nine (25 men and 54 women; mean age, 27 years; range, 5 to 65 years) healthy individuals from a rural community in South Africa were included in this study. We previously showed that this community had a high H. pylori seroprevalence rate (23, 24). The ethics committee of the University of Pretoria and the Review Board of Unitas Hospital approved this study.
Cumulative dental plaque samples were obtained from the oral cavity, and biopsies were taken from two sites in the stomach (two from the antrum and two from the corpus). One biopsy specimen from each site was placed in 10% formalin, cut, and stained with hematoxylin-eosin and methylene blue. DNA was extracted from the remainder of the samples by using the QIAamp DNA Mini kit (QIAGEN, Hilden, Germany) per the manufacturer's instructions and stored at –20°C. The quality and concentration of the extracted DNA was assessed by means of a Nanodrop Spectrophotometer (A260/A280, 1.6 to 2.00).
A single-step PCR directed toward the urease AB gene and a heminested PCR directed toward the phosphoglucosamine mutase (glmM) gene of H. pylori were performed on dental plaque and biopsy samples (8). A third nested PCR directed toward the 860-bp DNA region of H. pylori was performed on dental plaque samples (21, 22). Negative and positive controls (H. pylori type strain Hp115.90) were included in each batch of amplifications. Amplification was performed on the GeneAmp 9700 thermocycler (Applied Biosystems, Foster City, Calif.). To exclude the possibility of PCR amplifications being negative due to the presence of inhibitors, the single-step PCR amplifying the urease AB gene was repeated for dental plaque samples spiked with DNA isolated from the positive control.
The sensitivities, specificities, and percentages of infected individuals are represented in Table 1. Sensitivity and specificity were calculated compared to the gold standard of histology. Dental plaque samples were not collected for five individuals, and biopsy samples were not collected for one individual included in the study. Five samples were excluded due to the fact that the A260/A280 ratio of the extracted DNA did not fall between 1.6 and 2.00. Eighty-four percent of the individuals tested positive for H. pylori infection by means of histology, confirming the previously reported high prevalence of infection within this community (23, 24). None of the dental plaque samples showed amplification of the urease AB gene; however, all of the dental plaque samples spiked with the positive control showed amplification.
Controversy still exists regarding the role of the oral cavity as a possible reservoir for H. pylori infection in healthy individuals due to the varying results obtained by different research groups throughout the world (6, 21). It has been suggested that possible reasons for the lack of uniform positive findings of H. pylori within the oral environment are the sample collection procedure, methodology, and type of population studied (21).
Song and coworkers recently reported finding a characteristic distribution pattern of H. pylori within the oral cavity (21). If H. pylori has a preferred oral niche, sample collection procedures could potentially give rise to false-negative results. To exclude this possibility, cumulative dental plaque samples were collected from six different sites in the mouth.
The most commonly used PCR methods for the detection of H. pylori in dental plaque and saliva were evaluated by Song and coworkers. They reported that the nested PCR directed toward an 860-bp fragment of H. pylori genomic DNA was the most sensitive and specific for the detection of H. pylori in dental plaque and saliva (22). We included three different PCR methods: (i) single-step amplification of a 113-bp fragment of the urease AB gene, (ii) heminested PCR directed toward the glmM gene, and (iii) nested PCR directed toward the 860-bp fragment of H. pylori. The heminested PCR showed greater sensitivity and specificity than the single-step PCR for the DNA extracted from biopsy samples. No positive results were obtained when these PCR methods were performed on the dental plaque samples. The amplification of the 860-bp product as described by Song and coworkers also yielded no positive results for the dental plaque samples.
Most previously reported studies which have attempted to assess the presence of H. pylori within the oral environment have been conducted in symptomatic, often hospitalized patients (1-3, 5, 7, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21). H. pylori has been successfully cultured and detected by means of PCR from vomitus by various groups (13, 19). This indicates that transient colonization of the mouth with H. pylori may occur after vomiting. Our study was based on a healthy study population in an attempt to exclude the possibility of detecting H. pylori in the oral environment due to transient colonization.
From our findings we cannot exclude that the oral cavity does not act as a reservoir for the transmission of H. pylori within the setting of symptomatic patients or even healthy individuals from the developed world. However, this study indicates that the oral cavity does not favor prolonged colonization of H. pylori in a healthy black South African population with a high prevalence of infection, indicating that another mode of transmission exists in which the oral cavity is unlikely to contribute to the spread of this organism.
(This work forms the basis of the study "A population genetics pedigree perspective on the familial transmission of Helicobacter pylori," which was presented by S. van der Merwe at a distinguished plenary session at Digestive Disease Week, May 2004.)
ACKNOWLEDGMENTS
We acknowledge the contributions and suggestions of Johannes Kusters and Ernst J. Kuipers in supplying the primers for the urease AB gene and for valuable comments during the initial versions of the manuscript. We also extend our sincere gratitude to hospital manager Johan Geertsema and the nursing staff of the gastrointestinal unit of the Unitas Hospital for the exceptional efforts made in collection of the clinical data. We also acknowledge the contributions of Christel Veldboer, Marleen Lips, and Justin Fransen in manuscript preparation.
S.v.d.M. is a recipient of a Astra-Zeneca/South African Gastroenterology Society Fellowship in Gastroenterology.
REFERENCES
Allaker, R. P., J. K. Young, J. M. Hardie, P. Domizio, and N. J. Meadows. 2002. Prevalence of Helicobacter pylori at oral and gastrointestinal sites in children: evidence of possible oral-oral transmission. J. Med. Microbiol. 51:312-317.
Bernader, S., J. Dalen, B. Gastrin, L. Hedenborg, L. O.
Lamke, and R. Ohrn. 1993. Abscence of Helicobacter pylori in dental plaques in Helicobacter pylori positive dyspeptic patients. Eur. J. Clin. Microbiol. Infect. Dis. 12:282-285.
Cheng, H. H., M. Webberly, M. Evans, N. Hanson, and R. Brown. 1996. Helicobacter pylori in dental plaque and gastric mucosa. Oral Surg. Oral Med. Oral Pathol. Oral Radio. Endod. 81:421-423.
Chow, T. K., J. R. Lambert, and M. L. Wahlqvist. 1995. Helicobacter pylori in Melbourne Chinese immigrants: evidence for oral-oral transmission via chopsticks. J. Gastroenterol. Hepatol. 10:562-569.
Dore-Davin, C., M. Heitz, H. Yang, M. Herranz, A. L. Blum, and I. Corthesy-Theulaz.1999. Helicobacter pylori in the oral cavity reflects handling of contaminants but not gastric infection. Digestion 60:196-202.
Dowsett, S. A., and M. J. Kowolik. 2003. Oral Helicobacter pylori: can we stomach it? Crit. Rev. Oral Biol. Med. 14:226-233.
Gebara, E. C., C. Pannuti, C. M. Faria, L. Chehter, M. P. Mayer, and L. A. Lima. 2004. Prevalence of Helicobacter pylori detected by polymerase chain reaction in the oral cavity of periodontitis patients. Oral Microbiol. Immunol. 19:277-280.
Goosen, C., J. Theron, M. Ntsala, F. F. Maree, A. Olckers, S. J. Botha, A. J. Lastovica, and S. W. van der Merwe. 2002. Evaluation of a novel heminested PCR assay based on the phosphoglucosamine mutase gene for detection of Helicobacter pylori in saliva and dental plaque. J. Clin. Microbiol. 40:205-209.
Ishihara, K., T. Miura, R. Kimizuka, Y. Ebihara, Y. Mizuno, and K. Okuda. 1997. Oral bacteria inhibit Helicobacter pylori growth. FEMS Microbiol. Lett. 152:355-361.
Kabir, S. 2004. Detection of Helicobacter pylori DNA in feces and saliva by polymerase chain reaction: a review. Helicobacter. 9:115-123.
Khandakar, K., K. R. Palmer, M. A. Eastwood, A. C. Scott, M. Desai, and R. J. Owens. 1993. DNA fingerprints of Helicobacter pylori from mouth and antrum of patients with chronic ulcer dyspepsia. Lancet 342:751.
Kim, N., S. H. Lim, K. H. Lee, J. Y. You, J. M. Kim, N. R. Lee, H. C. Jung, I. S. Song, and C. Y. Kim. 2000. Helicobacter pylori in dental plaque and saliva. Korean J. Int. Med. 15:187-194.
Leung, W. K., K. L. Siu, K. L., C. K. L. Kwok, S. Y. Chan, R. Sung, and J. J. Sung. 1999. Isolation of Helicobacter pylori from vomitus in children and its implication in gastro-oral transmission. Am. J. Gastroenterol. 94:2881-2884.
Luman, W., A. M. Alkout, C. C. Blackwell, D. M. Weir, and K. R. Plamer. 1996. Helicobacter pylori in the mouth - negative isolation from dental plaque and saliva. Eur. J. Gastroenterol. Hepatol. 8:11-14.
Majmudar, P., D. M. Shah, K. R. Dhunjbhoy, and H. G. Desai. 1990. Isolation of Helicobacter pylori from dental plaques in healthy volunteers. Indian J. Gastroenterol. 9:271-272.
Megraud, F. 1995. Transmission of Helicobacter pylori: faecal-oral versus oral-oral route. Aliment. Pharmacol. Ther. 9(Suppl.):85-91.
Namavar, F., R. Roosendaal, E. J. Kuipers, P. de Groot, M. W. van der Bijl, A. S. Pena, and J. de Graaff. 2001. Presence of Helicobacter pylori in the oral cavity, oesophagus, stomach and faeces of patients with gastritis. Eur. J. Clin. Microbiol. Infect. Dis. 14:234.
Oshowo, A., M. Tunio, D. Gillam, A. J. Botha, J. Holton, P. Boulos, and M. Hobsley. 1998. Oral colonisation is unlikely to play an important role in Helicobacter pylori infection. Br. J. Surg. 85:850-852.
Parsonnet, J., H. Shmuely, and B. S. Haggerty. 1999. Fecal and oral shedding of Helicobacter pylori from healthy infected adults. J. Am. Med. Assoc. 282:2240-2245.
Sahin, F. I., A. C. Tinaz, I. S. Simsek, S. Menevse, and A. Gorgul. 2001. Detection of Helicobacter pylori in dental plaque and gastric biopsy of Turkish patients by PCR-RFLP. Acta Gastroenetrol. Belg. 64:150-152.
Song, Q., T. Lange, A. Sphar, et al. 2000. Characteristic distribution pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR. J. Med. Microbiol. 49:349-353.
Song, Q., B. Haller, R. M. Schmid, et al. 1999. Helicobacter pylori in dental plaque: a comparison of different PCR primer sets. Dig. Dis. Sci. 44:479-484.
Van der Merwe, S. W., M. Cunningham, O. Preisig, and B. Olivier. 2004. A population genetics pedigree perspective on familial transmission of Helicobacter pylori (abstract). Gastroenterology 126(Suppl. 2):A641.Van der Merwe, S. W., C. Goosen, and R. Bond. 2002.
Evaluation of the mouth as a reservoir in H. pylori infection using a novel double hemi-nested PCR method for the detection of the glmM gene in dental plaque (abstract). Gut 51(Suppl. III):A109.
Department of Chemical Pathology
1 Hepatology and GI-Research Laboratory
2 Department of Internal Medicine and Gastroenterology
3 Department of Medical Virology, University of Pretoria
4 Ampath Pathology,
5 Department of Internal Medicine and Gastroenterology, Unitas Hospital, Netcare, Pretoria
6 Department of Medical Microbiology, University of Cape Town, Cape Town, South Africa(Brenda J. Olivier, Robert)
Our study aimed to evaluate the oral cavity as a reservoir from where Helicobacter pylori may be transmitted. Histology and PCR amplification were performed. Eighty-four percent of the stomach biopsies tested positive; however, H. pylori was not detected in dental samples, indicating the absence of H. pylori within the oral cavity.
TEXT
It has been suggested that the oral cavity may play a role in the transmission of Helicobacter pylori. Increased H. pylori prevalence rates have been reported in Chinese immigrants who use chopsticks and in African infants whose mothers premasticate their food (4, 16). H. pylori has been detected in dental plaque and saliva by culture and PCR methods, with the results obtained ranging from 0 to 100% for culture and 0 to 90% for PCR (1-3, 5, 7, 9, 11, 12, 14, 15, 17, 18, 20, 21). The role of the oral cavity as a permanent reservoir for H. pylori infection in healthy nonhospitalized individuals is still unclear (6, 10).
Seventy-nine (25 men and 54 women; mean age, 27 years; range, 5 to 65 years) healthy individuals from a rural community in South Africa were included in this study. We previously showed that this community had a high H. pylori seroprevalence rate (23, 24). The ethics committee of the University of Pretoria and the Review Board of Unitas Hospital approved this study.
Cumulative dental plaque samples were obtained from the oral cavity, and biopsies were taken from two sites in the stomach (two from the antrum and two from the corpus). One biopsy specimen from each site was placed in 10% formalin, cut, and stained with hematoxylin-eosin and methylene blue. DNA was extracted from the remainder of the samples by using the QIAamp DNA Mini kit (QIAGEN, Hilden, Germany) per the manufacturer's instructions and stored at –20°C. The quality and concentration of the extracted DNA was assessed by means of a Nanodrop Spectrophotometer (A260/A280, 1.6 to 2.00).
A single-step PCR directed toward the urease AB gene and a heminested PCR directed toward the phosphoglucosamine mutase (glmM) gene of H. pylori were performed on dental plaque and biopsy samples (8). A third nested PCR directed toward the 860-bp DNA region of H. pylori was performed on dental plaque samples (21, 22). Negative and positive controls (H. pylori type strain Hp115.90) were included in each batch of amplifications. Amplification was performed on the GeneAmp 9700 thermocycler (Applied Biosystems, Foster City, Calif.). To exclude the possibility of PCR amplifications being negative due to the presence of inhibitors, the single-step PCR amplifying the urease AB gene was repeated for dental plaque samples spiked with DNA isolated from the positive control.
The sensitivities, specificities, and percentages of infected individuals are represented in Table 1. Sensitivity and specificity were calculated compared to the gold standard of histology. Dental plaque samples were not collected for five individuals, and biopsy samples were not collected for one individual included in the study. Five samples were excluded due to the fact that the A260/A280 ratio of the extracted DNA did not fall between 1.6 and 2.00. Eighty-four percent of the individuals tested positive for H. pylori infection by means of histology, confirming the previously reported high prevalence of infection within this community (23, 24). None of the dental plaque samples showed amplification of the urease AB gene; however, all of the dental plaque samples spiked with the positive control showed amplification.
Controversy still exists regarding the role of the oral cavity as a possible reservoir for H. pylori infection in healthy individuals due to the varying results obtained by different research groups throughout the world (6, 21). It has been suggested that possible reasons for the lack of uniform positive findings of H. pylori within the oral environment are the sample collection procedure, methodology, and type of population studied (21).
Song and coworkers recently reported finding a characteristic distribution pattern of H. pylori within the oral cavity (21). If H. pylori has a preferred oral niche, sample collection procedures could potentially give rise to false-negative results. To exclude this possibility, cumulative dental plaque samples were collected from six different sites in the mouth.
The most commonly used PCR methods for the detection of H. pylori in dental plaque and saliva were evaluated by Song and coworkers. They reported that the nested PCR directed toward an 860-bp fragment of H. pylori genomic DNA was the most sensitive and specific for the detection of H. pylori in dental plaque and saliva (22). We included three different PCR methods: (i) single-step amplification of a 113-bp fragment of the urease AB gene, (ii) heminested PCR directed toward the glmM gene, and (iii) nested PCR directed toward the 860-bp fragment of H. pylori. The heminested PCR showed greater sensitivity and specificity than the single-step PCR for the DNA extracted from biopsy samples. No positive results were obtained when these PCR methods were performed on the dental plaque samples. The amplification of the 860-bp product as described by Song and coworkers also yielded no positive results for the dental plaque samples.
Most previously reported studies which have attempted to assess the presence of H. pylori within the oral environment have been conducted in symptomatic, often hospitalized patients (1-3, 5, 7, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21). H. pylori has been successfully cultured and detected by means of PCR from vomitus by various groups (13, 19). This indicates that transient colonization of the mouth with H. pylori may occur after vomiting. Our study was based on a healthy study population in an attempt to exclude the possibility of detecting H. pylori in the oral environment due to transient colonization.
From our findings we cannot exclude that the oral cavity does not act as a reservoir for the transmission of H. pylori within the setting of symptomatic patients or even healthy individuals from the developed world. However, this study indicates that the oral cavity does not favor prolonged colonization of H. pylori in a healthy black South African population with a high prevalence of infection, indicating that another mode of transmission exists in which the oral cavity is unlikely to contribute to the spread of this organism.
(This work forms the basis of the study "A population genetics pedigree perspective on the familial transmission of Helicobacter pylori," which was presented by S. van der Merwe at a distinguished plenary session at Digestive Disease Week, May 2004.)
ACKNOWLEDGMENTS
We acknowledge the contributions and suggestions of Johannes Kusters and Ernst J. Kuipers in supplying the primers for the urease AB gene and for valuable comments during the initial versions of the manuscript. We also extend our sincere gratitude to hospital manager Johan Geertsema and the nursing staff of the gastrointestinal unit of the Unitas Hospital for the exceptional efforts made in collection of the clinical data. We also acknowledge the contributions of Christel Veldboer, Marleen Lips, and Justin Fransen in manuscript preparation.
S.v.d.M. is a recipient of a Astra-Zeneca/South African Gastroenterology Society Fellowship in Gastroenterology.
REFERENCES
Allaker, R. P., J. K. Young, J. M. Hardie, P. Domizio, and N. J. Meadows. 2002. Prevalence of Helicobacter pylori at oral and gastrointestinal sites in children: evidence of possible oral-oral transmission. J. Med. Microbiol. 51:312-317.
Bernader, S., J. Dalen, B. Gastrin, L. Hedenborg, L. O.
Lamke, and R. Ohrn. 1993. Abscence of Helicobacter pylori in dental plaques in Helicobacter pylori positive dyspeptic patients. Eur. J. Clin. Microbiol. Infect. Dis. 12:282-285.
Cheng, H. H., M. Webberly, M. Evans, N. Hanson, and R. Brown. 1996. Helicobacter pylori in dental plaque and gastric mucosa. Oral Surg. Oral Med. Oral Pathol. Oral Radio. Endod. 81:421-423.
Chow, T. K., J. R. Lambert, and M. L. Wahlqvist. 1995. Helicobacter pylori in Melbourne Chinese immigrants: evidence for oral-oral transmission via chopsticks. J. Gastroenterol. Hepatol. 10:562-569.
Dore-Davin, C., M. Heitz, H. Yang, M. Herranz, A. L. Blum, and I. Corthesy-Theulaz.1999. Helicobacter pylori in the oral cavity reflects handling of contaminants but not gastric infection. Digestion 60:196-202.
Dowsett, S. A., and M. J. Kowolik. 2003. Oral Helicobacter pylori: can we stomach it? Crit. Rev. Oral Biol. Med. 14:226-233.
Gebara, E. C., C. Pannuti, C. M. Faria, L. Chehter, M. P. Mayer, and L. A. Lima. 2004. Prevalence of Helicobacter pylori detected by polymerase chain reaction in the oral cavity of periodontitis patients. Oral Microbiol. Immunol. 19:277-280.
Goosen, C., J. Theron, M. Ntsala, F. F. Maree, A. Olckers, S. J. Botha, A. J. Lastovica, and S. W. van der Merwe. 2002. Evaluation of a novel heminested PCR assay based on the phosphoglucosamine mutase gene for detection of Helicobacter pylori in saliva and dental plaque. J. Clin. Microbiol. 40:205-209.
Ishihara, K., T. Miura, R. Kimizuka, Y. Ebihara, Y. Mizuno, and K. Okuda. 1997. Oral bacteria inhibit Helicobacter pylori growth. FEMS Microbiol. Lett. 152:355-361.
Kabir, S. 2004. Detection of Helicobacter pylori DNA in feces and saliva by polymerase chain reaction: a review. Helicobacter. 9:115-123.
Khandakar, K., K. R. Palmer, M. A. Eastwood, A. C. Scott, M. Desai, and R. J. Owens. 1993. DNA fingerprints of Helicobacter pylori from mouth and antrum of patients with chronic ulcer dyspepsia. Lancet 342:751.
Kim, N., S. H. Lim, K. H. Lee, J. Y. You, J. M. Kim, N. R. Lee, H. C. Jung, I. S. Song, and C. Y. Kim. 2000. Helicobacter pylori in dental plaque and saliva. Korean J. Int. Med. 15:187-194.
Leung, W. K., K. L. Siu, K. L., C. K. L. Kwok, S. Y. Chan, R. Sung, and J. J. Sung. 1999. Isolation of Helicobacter pylori from vomitus in children and its implication in gastro-oral transmission. Am. J. Gastroenterol. 94:2881-2884.
Luman, W., A. M. Alkout, C. C. Blackwell, D. M. Weir, and K. R. Plamer. 1996. Helicobacter pylori in the mouth - negative isolation from dental plaque and saliva. Eur. J. Gastroenterol. Hepatol. 8:11-14.
Majmudar, P., D. M. Shah, K. R. Dhunjbhoy, and H. G. Desai. 1990. Isolation of Helicobacter pylori from dental plaques in healthy volunteers. Indian J. Gastroenterol. 9:271-272.
Megraud, F. 1995. Transmission of Helicobacter pylori: faecal-oral versus oral-oral route. Aliment. Pharmacol. Ther. 9(Suppl.):85-91.
Namavar, F., R. Roosendaal, E. J. Kuipers, P. de Groot, M. W. van der Bijl, A. S. Pena, and J. de Graaff. 2001. Presence of Helicobacter pylori in the oral cavity, oesophagus, stomach and faeces of patients with gastritis. Eur. J. Clin. Microbiol. Infect. Dis. 14:234.
Oshowo, A., M. Tunio, D. Gillam, A. J. Botha, J. Holton, P. Boulos, and M. Hobsley. 1998. Oral colonisation is unlikely to play an important role in Helicobacter pylori infection. Br. J. Surg. 85:850-852.
Parsonnet, J., H. Shmuely, and B. S. Haggerty. 1999. Fecal and oral shedding of Helicobacter pylori from healthy infected adults. J. Am. Med. Assoc. 282:2240-2245.
Sahin, F. I., A. C. Tinaz, I. S. Simsek, S. Menevse, and A. Gorgul. 2001. Detection of Helicobacter pylori in dental plaque and gastric biopsy of Turkish patients by PCR-RFLP. Acta Gastroenetrol. Belg. 64:150-152.
Song, Q., T. Lange, A. Sphar, et al. 2000. Characteristic distribution pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR. J. Med. Microbiol. 49:349-353.
Song, Q., B. Haller, R. M. Schmid, et al. 1999. Helicobacter pylori in dental plaque: a comparison of different PCR primer sets. Dig. Dis. Sci. 44:479-484.
Van der Merwe, S. W., M. Cunningham, O. Preisig, and B. Olivier. 2004. A population genetics pedigree perspective on familial transmission of Helicobacter pylori (abstract). Gastroenterology 126(Suppl. 2):A641.Van der Merwe, S. W., C. Goosen, and R. Bond. 2002.
Evaluation of the mouth as a reservoir in H. pylori infection using a novel double hemi-nested PCR method for the detection of the glmM gene in dental plaque (abstract). Gut 51(Suppl. III):A109.
Department of Chemical Pathology
1 Hepatology and GI-Research Laboratory
2 Department of Internal Medicine and Gastroenterology
3 Department of Medical Virology, University of Pretoria
4 Ampath Pathology,
5 Department of Internal Medicine and Gastroenterology, Unitas Hospital, Netcare, Pretoria
6 Department of Medical Microbiology, University of Cape Town, Cape Town, South Africa(Brenda J. Olivier, Robert)