PCR Amplification of the IS6110 Insertion Element of Mycobacterium tuberculosis in Fecal Samples from Patients with Intestinal Tuberculosis
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微生物临床杂志 2006年第5期
Departments of Gastrointestinal Sciences
Community Health, Christian Medical College, Vellore, India
ABSTRACT
PCR amplification of insertion element IS6110 of Mycobacterium tuberculosis in fecal samples was evaluated in the diagnosis of intestinal tuberculosis (ITB). The numbers of samples that tested positive by PCR with SalI digestion were 16/18 untreated-ITB samples, 0/8 treated-ITB samples, 12/14 smear-positive pulmonary tuberculosis samples, and 0/30 control samples. The sensitivity, specificity, positive predictive value, and negative predictive value of fecal PCR were 88.8%, 100%, 100%, and 93.7%, respectively.
TEXT
Tuberculosis (TB) infection affects a third of the world's population (4). Gastrointestinal TB mimics many conditions, including inflammatory bowel disease, malignancy, and infectious diarrhea, and is often difficult to diagnose (7, 17). With colonic disease, endoscopy and biopsy may confirm the diagnosis (13, 16), while surgical biopsy is necessary with disease confined to the small intestine. By use of mucosal biopsies, Mycobacterium tuberculosis is demonstrated by culture to occur in one-third (2) and by PCR in two-thirds (6) of patients with colonic TB. In countries with a high incidence of TB, gastrointestinal tuberculosis is often diagnosed and treated on empirical grounds and confirmed by resolution following therapy. This study evaluated the ability of PCR of feces to amplify the IS6110 insertion element of M. tuberculosis in the diagnosis of gastrointestinal TB.
Consecutive patients with intestinal tuberculosis seen in the Department of Gastrointestinal Sciences between April 2003 and January 2005 were included. The diagnosis was made on the basis of one of the following criteria: (i) intestinal biopsies showing caseating granulomas, (ii) intestinal granulomas accompanied by confirmed extraintestinal tuberculosis, (iii) endoscopic findings of TB with noncaseating granulomas on biopsy and complete resolution after antituberculosis therapy, or (iv) culture of M. tuberculosis from intestinal biopsy specimens. All patients were negative for acid-fast bacilli in induced-sputum specimens. Twenty-four subjects with irritable bowel syndrome (all with normal hematology and colonoscopy) undergoing colonoscopy during this period and six healthy adult volunteers served as controls. Fourteen patients with pulmonary TB and acid-fast bacilli on Ziehl-Neelsen stain of sputum smears were recruited from the DOTS Clinic of Christian Medical College, Vellore, India. Eight patients with intestinal tuberculosis who completed 9 months of antituberculosis therapy and were clinically symptom free with normal hemoglobin, sedimentation rate, serum albumin, and globulin were included. The protocol and consent forms were approved by the Research Committee of the Christian Medical College, Vellore, India.
Fresh feces were collected after consent and stored at –20°C. Feces (250 mg) were homogenized in 2 ml sterile phosphate-buffered saline and heated at 90°C for 15 min. After centrifugation (500 x g, 3 min), the supernatant was mixed with lysozyme (10 mg/ml) (Genei, India) and incubated at 37°C for 1 h; then, proteinase K (Genei, India) (30 mg/ml) and 20% sodium dodecyl sulfate (Sigma, India) were added, and the mixture was incubated at 55°C for 2 h. Following centrifugation (9,000 x g, 10 min) nucleic acid was extracted from the supernatant by using equal volumes of equilibrated phenol (pH 8.0; Ambion) and chloroform-isoamyl alcohol (25:24:1). DNA was precipitated with ethanol and 2.5 M ammonium acetate (2.0:0.1, –20°C, 1 h), pelleted (18,000 x g, 20 min), washed with 70% ethanol, air dried, resuspended in 50 μl sterile water, and stored at –20°C (12). A positive control was prepared from killed culture of Mycobacterium tuberculosis strain H37Rv. The primers used (MTB1, 5' CCT GCG AGC GTA GGC GTC GG 3', and MTB2, 5' CTC GTC CAG CGC CGC TTC GG 3') amplified a 123-bp fragment of IS6110 (5), which is specific for M. tuberculosis and contains an internal endonuclease site that allows confirmation of the product by digestion with SalI. PCR amplification in 10 μl used titanium Taq polymerase (BD Biosciences Clontech) and 10 pmol of each primer (Sigma, Bangalore, India). The final PCR conditions consisted of an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation (94°C, 30 s), annealing (68°C, 30 s), and extension (72°C, 30 s), with a final extension for 10 min. The PCR product was subjected to electrophoresis on 2% agarose, stained with ethidium bromide, and imaged (Bio-Rad). The PCR products were digested with 10 U/μl of SalI (MBI Fermentas) at 37°C overnight, followed by electrophoresis on 3% agarose. Specificity of the primers was tested by sequence searches against prokaryotic DNA databases, in silico PCRs, and PCR using DNA from atypical mycobacteria—M. avium, M. kansasii, and M. fortuitum. Serial 10-fold dilutions of M. tuberculosis H37Rv (3) were added to PCR-negative fecal samples to establish the detection limit of PCR. All samples that tested positive were reconfirmed using two additional DNA extracts.
PCR using M. tuberculosis H37Rv, but not atypical mycobacteria, produced a 123-bp amplicon (Fig. 1, top left). With serial dilutions, PCR was able to detect >10 copies of M. tuberculosis. SalI digestion of the PCR product from positive cases and the positive control produced two bands, one of 66 bp and one of 57 bp, respectively (Fig. 1, bottom left). The specific PCR product was noted for 16 of 18 patients with untreated intestinal tuberculosis (Fig. 1, top right). Samples were negative by PCR for all 30 control subjects (Fig. 1, top left) and all eight treated gastrointestinal TB patients, five of whom were tested prior to therapy and were positive and three of whom were not tested prior to therapy. Twelve of 14 patients with smear-positive pulmonary tuberculosis tested positive (Fig. 1, bottom right), indicating fecal excretion of swallowed organisms. The sensitivity of fecal PCR for the diagnosis of intestinal TB was 88.8%, and the specificity was 100% (Table 1). In comparison to results with fecal PCR, 9/18 and 6/18 patients with intestinal TB tested positive by histopathology and culture, indicating sensitivities of 50% and 33.3%, respectively.
PCR is used increasingly in the diagnosis of tuberculosis (1). For smear-positive pulmonary TB, PCR confirms or excludes the diagnosis in 48 h, compared to culture, which takes 2 to 8 weeks (8). PCR of mucosal biopsy specimens diagnoses colonic TB in 45 to 64% of cases (6, 9). Unlike endoscopic biopsy, fecal PCR is noninvasive, and it is also less subject to sampling error. Stool PCR for atypical mycobacteria has been used for patients with human immunodeficiency virus (10). The IS6110 sequence is present in most clinical isolates of M. tuberculosis; however, 4/378 (1%) isolates and 19/80 (24%) isolates in two studies from southern India showed an absence of even a single copy of IS6110 (11, 14). As with intestinal TB samples, untreated-pulmonary-TB samples were positive by fecal PCR, likely due to swallowed M. tuberculosis. Gastric lavage is used widely to diagnose infection in adults and children with pulmonary TB who do not expectorate sputum (15, 18). This study suggests that fecal PCR will achieve the same end. Fecal PCR for the IS6110 sequence is thus an attractive test for the diagnosis and follow-up of intestinal TB. Its utility in distinguishing patients with Crohn's disease from those with TB and in identifying patients with active pulmonary TB needs further study.
ACKNOWLEDGMENTS
R.B. was supported by a Senior Research Fellowship from the Indian Council of Medical Research. This work was supported by a grant from the Fluid Research Fund of the Christian Medical College, Vellore.
REFERENCES
Abe, C., K. Hirano, M. Wada, Y. Kazumi, M. Takahashi, Y. Fukasawa, T. Yoshimura, C. Miyagi, and S. Goto. 1993. Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J. Clin. Microbiol. 31:3270-3274.
Bhargava, D. K., H. D. Tandon, T. C. Chawla, Shriniwas, B. N. Tandon, and B. M. Kapur. 1985. Diagnosis of ileocecal and colonic tuberculosis by colonoscopy. Gastrointest. Endosc. 31:68-70.
Bollela, V. R., D. N. Sato, and B. A. L. Fonseca. 1999. McFarland nephelometer as a simple method to estimate the sensitivity of the polymerase chain reaction using Mycobacterium tuberculosis as a research tool. Braz. J. Med. Biol. Res. 32:1073-1076.
DeAngelis, C. D., and A. Flanagin. 2005. Tuberculosis—a global problem requiring a global solution. JAMA 293:2793-2794.
Eisenach, K. D., M. D. Cave, J. H. Bates, and J. T. Crawford. 1990. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J. Infect. Dis. 161:977-981.
Gan, H. T., Y. Q. Chen, Q. Ouyang, H. Bu, and X. Y. Yang. 2002. Differentiation between intestinal tuberculosis and Crohn's disease in endoscopic biopsy specimens by polymerase chain reaction. Am. J. Gastroenterol. 97:1446-1451.
Jadvar, H., R. E. Mindelzun, E. W. Olcott, and D. B. Levitt. 1997. Still the great mimicker: abdominal tuberculosis. Am. J. Roentgenol. 168:1455-1460.
Katoch, V. M. 2004. Newer diagnostic techniques for tuberculosis. Indian J. Med. Res. 120:418-428.
Kim, K. M., A. Lee, K. Y. Choi, K. Y. Lee, and J. J. Kwak. 1998. Intestinal tuberculosis: clinicopathologic analysis and diagnosis by endoscopic biopsy. Am. J. Gastroenterol. 93:606-609.
Li, Z., G. H. Bai, C. F. von Reyn, P. Marino, M. J. Brennan, N. Gine, and S. L. Morris. 1996. Rapid detection of Mycobacterium avium in stool samples from AIDS patients by immunomagnetic PCR. J. Clin. Microbiol. 34:1903-1907.
Narayanan, S., S. Das, R. Garg, L. Hari, V. B. Rao, T. R. Frieden, and P. R. Narayanan. 2002. Molecular epidemiology of tuberculosis in a rural area of high prevalence in south India: implications for disease control and prevention. J. Clin. Microbiol. 40:4785-4788.
Noordhoek, G. T., J. D. van Embden, and A. H. Kolk. 1996. Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories. J. Clin. Microbiol. 34:2522-2525.
Pulimood, A., S. Peter, B. S. Ramakrishna, A. Chacko, R. Jeyamani, L. Jeyaseelan, and G. Kurian. 2005. Segmental colonoscopic biopsies in the differentiation of ileocolic tuberculosis from Crohn's disease. J. Gastroenterol. Hepatol. 20:688-696.
Radhakrishnan, I., Y. K. Manju, R. A. Kumar, and S. Mundayoor. 2001. Implications of low frequency of IS6110 in fingerprinting field isolates of Mycobacterium tuberculosis from Kerala, India. J. Clin. Microbiol. 39:1683.
Rizvi, N., N. A. Rao, and M. Hussain. 2000. Yield of gastric lavage and bronchial wash in pulmonary tuberculosis. Int. J. Tuberc. Lung Dis. 4:147-151.
Shah, S., V. Thomas, M. Mathan, A. Chacko, G. Chandy, B. S. Ramakrishna, and D. D. Rolston. 1992. Colonoscopic study of 50 patients with colonic tuberculosis. Gut 33:347-351.
Sheer, T. A., and W. J. Coyle. 2003. Gastrointestinal tuberculosis. Curr. Gastroenterol. Rep. 5:273-278.
Somu, N., S. Swaminathan, C. N. Paramasivan, D. Vijayasekaran, A. Chandrabhooshanam, V. K. Vijayan, and R. Prabhakar. 1995. Value of bronchoalveolar lavage and gastric lavage in the diagnosis of pulmonary tuberculosis in children. Tuber. Lung Dis. 76:295-299.(Ramadass Balamurugan, Sub)
Community Health, Christian Medical College, Vellore, India
ABSTRACT
PCR amplification of insertion element IS6110 of Mycobacterium tuberculosis in fecal samples was evaluated in the diagnosis of intestinal tuberculosis (ITB). The numbers of samples that tested positive by PCR with SalI digestion were 16/18 untreated-ITB samples, 0/8 treated-ITB samples, 12/14 smear-positive pulmonary tuberculosis samples, and 0/30 control samples. The sensitivity, specificity, positive predictive value, and negative predictive value of fecal PCR were 88.8%, 100%, 100%, and 93.7%, respectively.
TEXT
Tuberculosis (TB) infection affects a third of the world's population (4). Gastrointestinal TB mimics many conditions, including inflammatory bowel disease, malignancy, and infectious diarrhea, and is often difficult to diagnose (7, 17). With colonic disease, endoscopy and biopsy may confirm the diagnosis (13, 16), while surgical biopsy is necessary with disease confined to the small intestine. By use of mucosal biopsies, Mycobacterium tuberculosis is demonstrated by culture to occur in one-third (2) and by PCR in two-thirds (6) of patients with colonic TB. In countries with a high incidence of TB, gastrointestinal tuberculosis is often diagnosed and treated on empirical grounds and confirmed by resolution following therapy. This study evaluated the ability of PCR of feces to amplify the IS6110 insertion element of M. tuberculosis in the diagnosis of gastrointestinal TB.
Consecutive patients with intestinal tuberculosis seen in the Department of Gastrointestinal Sciences between April 2003 and January 2005 were included. The diagnosis was made on the basis of one of the following criteria: (i) intestinal biopsies showing caseating granulomas, (ii) intestinal granulomas accompanied by confirmed extraintestinal tuberculosis, (iii) endoscopic findings of TB with noncaseating granulomas on biopsy and complete resolution after antituberculosis therapy, or (iv) culture of M. tuberculosis from intestinal biopsy specimens. All patients were negative for acid-fast bacilli in induced-sputum specimens. Twenty-four subjects with irritable bowel syndrome (all with normal hematology and colonoscopy) undergoing colonoscopy during this period and six healthy adult volunteers served as controls. Fourteen patients with pulmonary TB and acid-fast bacilli on Ziehl-Neelsen stain of sputum smears were recruited from the DOTS Clinic of Christian Medical College, Vellore, India. Eight patients with intestinal tuberculosis who completed 9 months of antituberculosis therapy and were clinically symptom free with normal hemoglobin, sedimentation rate, serum albumin, and globulin were included. The protocol and consent forms were approved by the Research Committee of the Christian Medical College, Vellore, India.
Fresh feces were collected after consent and stored at –20°C. Feces (250 mg) were homogenized in 2 ml sterile phosphate-buffered saline and heated at 90°C for 15 min. After centrifugation (500 x g, 3 min), the supernatant was mixed with lysozyme (10 mg/ml) (Genei, India) and incubated at 37°C for 1 h; then, proteinase K (Genei, India) (30 mg/ml) and 20% sodium dodecyl sulfate (Sigma, India) were added, and the mixture was incubated at 55°C for 2 h. Following centrifugation (9,000 x g, 10 min) nucleic acid was extracted from the supernatant by using equal volumes of equilibrated phenol (pH 8.0; Ambion) and chloroform-isoamyl alcohol (25:24:1). DNA was precipitated with ethanol and 2.5 M ammonium acetate (2.0:0.1, –20°C, 1 h), pelleted (18,000 x g, 20 min), washed with 70% ethanol, air dried, resuspended in 50 μl sterile water, and stored at –20°C (12). A positive control was prepared from killed culture of Mycobacterium tuberculosis strain H37Rv. The primers used (MTB1, 5' CCT GCG AGC GTA GGC GTC GG 3', and MTB2, 5' CTC GTC CAG CGC CGC TTC GG 3') amplified a 123-bp fragment of IS6110 (5), which is specific for M. tuberculosis and contains an internal endonuclease site that allows confirmation of the product by digestion with SalI. PCR amplification in 10 μl used titanium Taq polymerase (BD Biosciences Clontech) and 10 pmol of each primer (Sigma, Bangalore, India). The final PCR conditions consisted of an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation (94°C, 30 s), annealing (68°C, 30 s), and extension (72°C, 30 s), with a final extension for 10 min. The PCR product was subjected to electrophoresis on 2% agarose, stained with ethidium bromide, and imaged (Bio-Rad). The PCR products were digested with 10 U/μl of SalI (MBI Fermentas) at 37°C overnight, followed by electrophoresis on 3% agarose. Specificity of the primers was tested by sequence searches against prokaryotic DNA databases, in silico PCRs, and PCR using DNA from atypical mycobacteria—M. avium, M. kansasii, and M. fortuitum. Serial 10-fold dilutions of M. tuberculosis H37Rv (3) were added to PCR-negative fecal samples to establish the detection limit of PCR. All samples that tested positive were reconfirmed using two additional DNA extracts.
PCR using M. tuberculosis H37Rv, but not atypical mycobacteria, produced a 123-bp amplicon (Fig. 1, top left). With serial dilutions, PCR was able to detect >10 copies of M. tuberculosis. SalI digestion of the PCR product from positive cases and the positive control produced two bands, one of 66 bp and one of 57 bp, respectively (Fig. 1, bottom left). The specific PCR product was noted for 16 of 18 patients with untreated intestinal tuberculosis (Fig. 1, top right). Samples were negative by PCR for all 30 control subjects (Fig. 1, top left) and all eight treated gastrointestinal TB patients, five of whom were tested prior to therapy and were positive and three of whom were not tested prior to therapy. Twelve of 14 patients with smear-positive pulmonary tuberculosis tested positive (Fig. 1, bottom right), indicating fecal excretion of swallowed organisms. The sensitivity of fecal PCR for the diagnosis of intestinal TB was 88.8%, and the specificity was 100% (Table 1). In comparison to results with fecal PCR, 9/18 and 6/18 patients with intestinal TB tested positive by histopathology and culture, indicating sensitivities of 50% and 33.3%, respectively.
PCR is used increasingly in the diagnosis of tuberculosis (1). For smear-positive pulmonary TB, PCR confirms or excludes the diagnosis in 48 h, compared to culture, which takes 2 to 8 weeks (8). PCR of mucosal biopsy specimens diagnoses colonic TB in 45 to 64% of cases (6, 9). Unlike endoscopic biopsy, fecal PCR is noninvasive, and it is also less subject to sampling error. Stool PCR for atypical mycobacteria has been used for patients with human immunodeficiency virus (10). The IS6110 sequence is present in most clinical isolates of M. tuberculosis; however, 4/378 (1%) isolates and 19/80 (24%) isolates in two studies from southern India showed an absence of even a single copy of IS6110 (11, 14). As with intestinal TB samples, untreated-pulmonary-TB samples were positive by fecal PCR, likely due to swallowed M. tuberculosis. Gastric lavage is used widely to diagnose infection in adults and children with pulmonary TB who do not expectorate sputum (15, 18). This study suggests that fecal PCR will achieve the same end. Fecal PCR for the IS6110 sequence is thus an attractive test for the diagnosis and follow-up of intestinal TB. Its utility in distinguishing patients with Crohn's disease from those with TB and in identifying patients with active pulmonary TB needs further study.
ACKNOWLEDGMENTS
R.B. was supported by a Senior Research Fellowship from the Indian Council of Medical Research. This work was supported by a grant from the Fluid Research Fund of the Christian Medical College, Vellore.
REFERENCES
Abe, C., K. Hirano, M. Wada, Y. Kazumi, M. Takahashi, Y. Fukasawa, T. Yoshimura, C. Miyagi, and S. Goto. 1993. Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J. Clin. Microbiol. 31:3270-3274.
Bhargava, D. K., H. D. Tandon, T. C. Chawla, Shriniwas, B. N. Tandon, and B. M. Kapur. 1985. Diagnosis of ileocecal and colonic tuberculosis by colonoscopy. Gastrointest. Endosc. 31:68-70.
Bollela, V. R., D. N. Sato, and B. A. L. Fonseca. 1999. McFarland nephelometer as a simple method to estimate the sensitivity of the polymerase chain reaction using Mycobacterium tuberculosis as a research tool. Braz. J. Med. Biol. Res. 32:1073-1076.
DeAngelis, C. D., and A. Flanagin. 2005. Tuberculosis—a global problem requiring a global solution. JAMA 293:2793-2794.
Eisenach, K. D., M. D. Cave, J. H. Bates, and J. T. Crawford. 1990. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J. Infect. Dis. 161:977-981.
Gan, H. T., Y. Q. Chen, Q. Ouyang, H. Bu, and X. Y. Yang. 2002. Differentiation between intestinal tuberculosis and Crohn's disease in endoscopic biopsy specimens by polymerase chain reaction. Am. J. Gastroenterol. 97:1446-1451.
Jadvar, H., R. E. Mindelzun, E. W. Olcott, and D. B. Levitt. 1997. Still the great mimicker: abdominal tuberculosis. Am. J. Roentgenol. 168:1455-1460.
Katoch, V. M. 2004. Newer diagnostic techniques for tuberculosis. Indian J. Med. Res. 120:418-428.
Kim, K. M., A. Lee, K. Y. Choi, K. Y. Lee, and J. J. Kwak. 1998. Intestinal tuberculosis: clinicopathologic analysis and diagnosis by endoscopic biopsy. Am. J. Gastroenterol. 93:606-609.
Li, Z., G. H. Bai, C. F. von Reyn, P. Marino, M. J. Brennan, N. Gine, and S. L. Morris. 1996. Rapid detection of Mycobacterium avium in stool samples from AIDS patients by immunomagnetic PCR. J. Clin. Microbiol. 34:1903-1907.
Narayanan, S., S. Das, R. Garg, L. Hari, V. B. Rao, T. R. Frieden, and P. R. Narayanan. 2002. Molecular epidemiology of tuberculosis in a rural area of high prevalence in south India: implications for disease control and prevention. J. Clin. Microbiol. 40:4785-4788.
Noordhoek, G. T., J. D. van Embden, and A. H. Kolk. 1996. Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories. J. Clin. Microbiol. 34:2522-2525.
Pulimood, A., S. Peter, B. S. Ramakrishna, A. Chacko, R. Jeyamani, L. Jeyaseelan, and G. Kurian. 2005. Segmental colonoscopic biopsies in the differentiation of ileocolic tuberculosis from Crohn's disease. J. Gastroenterol. Hepatol. 20:688-696.
Radhakrishnan, I., Y. K. Manju, R. A. Kumar, and S. Mundayoor. 2001. Implications of low frequency of IS6110 in fingerprinting field isolates of Mycobacterium tuberculosis from Kerala, India. J. Clin. Microbiol. 39:1683.
Rizvi, N., N. A. Rao, and M. Hussain. 2000. Yield of gastric lavage and bronchial wash in pulmonary tuberculosis. Int. J. Tuberc. Lung Dis. 4:147-151.
Shah, S., V. Thomas, M. Mathan, A. Chacko, G. Chandy, B. S. Ramakrishna, and D. D. Rolston. 1992. Colonoscopic study of 50 patients with colonic tuberculosis. Gut 33:347-351.
Sheer, T. A., and W. J. Coyle. 2003. Gastrointestinal tuberculosis. Curr. Gastroenterol. Rep. 5:273-278.
Somu, N., S. Swaminathan, C. N. Paramasivan, D. Vijayasekaran, A. Chandrabhooshanam, V. K. Vijayan, and R. Prabhakar. 1995. Value of bronchoalveolar lavage and gastric lavage in the diagnosis of pulmonary tuberculosis in children. Tuber. Lung Dis. 76:295-299.(Ramadass Balamurugan, Sub)