| Home | E-Submission | Sitemap | Contact Us |  
Korean J Intern Med > Volume 24(2); 2009 > Article
Cho, Han, Chin, Choi, Lee, Kim, Kim, Choi, Song, and Kim: Diagnosis and Species Identification of Mycobacterial Infections by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of Sterile Body Fluids

Abstract

Background/Aims

The development of effective, accurate, and rapid diagnostic methods for Mycobacterium infection and mycobacterial species identification is required. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is an easy, rapid and inexpensive technique for identifying Mycobacterium spp.

Methods

We performed PCR-RFLP to detect and identify Mycobacterium spp. from 10 sterile body fluids, including ascites, cerebrospinal fluid, pleural fluid, synovial fluid, and peritoneal dialysis fluid. Clinical samples were collected from patients with diagnoses of definite, probable or suspected mycobacterial infection. The conserved RNA polymerase genes of Mycobacterium spp. were amplified by PCR.

Results

The amplified 360-bp region of rpoB was digested with the restriction enzyme MspI or HaeIII. The PCR-RFLP results for the clinical samples were identical to those for M. tuberculosis, M. fortuitum, M. intracellulare, and M. avium. In addition, the results of the PCR-RFLP were identical to those obtained by DNA sequencing.

Conclusions

PCR-RFLP analysis of sterile body fluids may be a useful method for the diagnosis of mycobacterial infections and for the differentiation of mycobacterial species.

INTRODUCTION

Identification of Mycobacterium spp. in clinical specimens is based primarily on culturing and biochemical tests. These conventional tests take several weeks and sometimes fail to provide precise identification [1]. Mycobacterial identification at the species level is a critical step in patient management, as the results obtained influence decisions on both the optimal therapy and the need for patient isolation. Therefore, the development of effective diagnostic methods that allow accurate and rapid diagnosis of mycobacterial infections and differentiation among species are urgently required. Although molecular diagnostic methods, high-performance liquid chromatography, and DNA sequence analysis have been investigated, these methods require substantial effort and are difficult to implement in the clinical setting [2]. Molecular biological tests using DNA amplification and probe hybridization have also been developed. Using these methods, the diagnosis and differentiation of bacterial species can be rapidly accomplished. However, the high costs associated with these methods make them less attractive in practice [2-7].
In contrast to these techniques, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) offers an easy, rapid, and inexpensive way to identify Mycobacterium spp [7,8]. Therefore, we attempted to diagnose mycobacterial infections at the early clinical stages and to differentiate between Mycobacterium species using PCR-RFLP.

METHODS

In the present study, we performed PCR-RFLP to detect and identify Mycobacterium spp. in sterile body fluids, including ascites, cerebrospinal fluid, pleural fluid, synovial fluid, and continuous ambulatory peritoneal dialysis (CAPD) fluid. Clinical samples were collected from patients who were diagnosed with mycobacterial infections. A definite tuberculosis (TB) case was defined as a positive result for M. tuberculosis on culture testing. A probable TB case was defined as a positive result in an acid-fast bacilli (AFB) smear, chronic granulomatous inflammation seen on histopathology, and/or other positive findings consistent with TB (i.e., a high level of adenosine deaminase or a positive TB PCR result for appropriate samples). A suspected TB case was defined as having typical TB findings by radiology or clinical features of TB, and showing improvement after treatment. A non-tuberculous mycobacterial infection (NTMI) was defined as repeatedly positive results for non-tuberculous mycobacteria and clinical features associated with mycobacterial infection.
Between May, 2001 and July, 2003, sterile specimens from synovial fluid (n=2), CAPD fluid (n=1), ascites (n=1), pleural fluid (n=4), and cerebrospinal fluid (n=2) were obtained. The clinical diagnoses of those patients were: definite TB (n=1); probable TB (n=3); suspected TB (n=1); and NTMI (n=5).
An aliquot (5-20 mL) of each specimen was centrifuged at 14,000 rpm for 5 min and the supernatant was used for bacterial DNA extraction. Bacterial genomic DNA was extracted from clinical specimens using the DNeasy Tissue Kit (Qiagen, Hilden, Germany). The 360-bp rpoB gene was amplified by PCR using the following primers: forward, 5'-TCAAGGAGAAGCGCTACGA-3'; and reverse, 5'-GGATGTTGATCAGGGTCTGC-3'. The PCR consisted of 35 cycles of preheating at 94℃ for 5 minutes, denaturation at 94℃ for 1 minute, annealing at 60℃ for 1 minute, and extension at 72℃ for 1 minute, followed by a final extension step of 72℃ for 7 minutes. The 360-bp amplified product of rpoB was detected by 2.5% agarose gel electrophoresis; the gel samples included a 100-bp DNA ladder, a negative control (distilled water), and a positive control (360-bp amplified product of rpoB of M. tuberculosis). The amplified products were digested separately with MspI (Boehringer Mannheim Biochemicals, Mannheim, Germany) and HaeIII (Boehringer Mannheim Biochemicals) at 37℃ for 2 hours. The digested products were separated by 4% agarose gel electrophoresis at 50 V for 2 hours; the gel samples included a 50-bp DNA ladder, a negative control (distilled water), and the digested products of M. avium, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, and M. tuberculosis (Fig. 1). Reference strains that had been identified by conventional biochemical testing and rpoB sequencing were provided by the Department of Microbiology, Yonsei University College of Medicine [9]. For each specimen, the results of the culture tests, histologic tests, and clinical diagnoses were compared with the results of the PCR-RFLP analysis.
The DNA sequence of the PCR-amplified rpoB gene was analyzed using an rpoB primer and a DNA sequence analyzer. Species identification by DNA sequence analysis was compared with that achieved by PCR-RFLP.

RESULTS

PCR-RFLP results using 10 clinical samples such as ascites, cerebrospinal fluid, pleural fluid, synovial fluid, and CAPD fluid from subjects who were suspected of having mycobacterial infections were identical to the PCR-RFLP results of M. tuberculosis, M. fortuitum, M. intracellulare or M. avium (Table 1). PCR-RFLP results using the three culture-proven samples coincided with the results of culture test including M. tuberculosis, M. fortuitum and M. avium. In the other seven samples that were not identified by culture, the PCR-RFLP results provided the precise identification of each Mycobacterium spp. All of 2 probable and 2 suspected TB cases showed PCR-RFLP results consistent with M. tuberculosis. PCR-RFLP results of 3 NTMI cases known partially as atypical mycobacterium by culture test were consistent with M. avium, M. intracellulare or M. fortuitum respectively. In all cases the results of PCR-RFLP were also identical with those from sequencing.

DISCUSSION

Currently, laboratory diagnosis of Mycobacterium spp. infection is made primarily using smear tests and culturing. The advantage of the smear test is that the results can be obtained rapidly. However, since mycobacterial concentrations of 5×103 to 1×104/mL are required for the isolation procedure, the sensitivity of this test is low and the results may vary depending on the examiner [8-11]. It has been reported that 30-50% of culture-positive sputum samples show positive results [8-11]. Although the sensitivity of the Mycobacterium spp. culture test is relatively high, it takes 4-8 weeks to obtain the results, which means that treatment may be delayed and the bacteria may be transmitted to other people. Other shortcomings of these tests are that: 1) during the pretreatment process, Mycobacterium spp. may be killed; and 2) the pathogen isolation rate is relatively low in treated patients. In recent years, molecular methods for identifying bacteria (e.g., PCR) have been introduced. These methods overcome the shortcomings of the smear and culture tests, while maintaining high levels of sensitivity and specificity [8-11].
In the present study, we examined whether PCR-RFLP using the rpoB gene could detect and differentiate between mycobacterial strains present in sterile body fluids. PCR-RFLP was performed on 10 clinical specimens, which included synovial fluid, CAPD fluid, ascites, pleural fluid, and cerebrospinal fluid. All of the patients were diagnosed as having or suspected of having Mycobacterium infection, as confirmed by PCR of the rpoB gene. With regard to the differentiation of mycobacterial strains, for some patients who were diagnosed by culture testing, the results of the culture and PCR-RFLP were in agreement. Therefore, the detection and differentiation of strains by PCR-RFLP is believed to be clinically useful. However, as the PCR-RFLP method may produce a high number of false-positives, the overall clinical profile should be considered when assessing the results for patients.
There have been some reports on the use of PCR-RFLP for the diagnosis of mycobacterial infections. Kim et al. [5] reported that by analyzing the rpoB gene, 49 mycobacterial strains could be differentiated. In addition, PCR-RFLP analysis of bronchoalveolar lavage specimens enabled early diagnosis of mycobacterial infection, and the mycobacterial strains could be differentiated from each other. Similarly, Kontos et al. [12] reported that Mycobacterium spp. infection could be diagnosed effectively by culturing clinical specimens in the Bactec MGIT 960 system, followed by PCR-RFLP analysis. However, to our knowledge, there have been no studies using sterile body fluids to evaluate the usefulness of PCR-RFLP for the diagnosis of patients with mycobacterial infections and mycobacterial species identification.
The present study has some limitations. The number of subjects used was low. Therefore, we could not test the sensitivity, specificity, and predictive values of the assay. Moreover, culture-positive results were obtained for only six patients, and the infecting mycobacterial species was identified in only three patients. We cannot exclude the possibility of contamination by environmental mycobacteria. Furthermore, the false-positive rate of PCR-RFLP may be high. Finally, the positive control used for PCR-RFLP did not contain all the mycobacterial species that could be present in clinical specimens. In spite of these limitations, to our knowledge, this is the first investigation to focus on the usefulness of PCR-RFLP using various sterile body fluids.
In conclusion, PCR-RFLP analysis using sterile body fluids may be a useful method for diagnosing mycobacterial infections and for differentiating among mycobacterial strains. In the present study, the number of specimens and strains was low, which meant that the usefulness of PCR-RFLP analysis could not be compared in a statistically meaningful way with that of culture testing. Nonetheless, further studies on the usefulness of PCR-RFLP using more specimens and species are warranted.

NOTES

This work was supported in part by a grant from the Korea Health 21 R&D Project (01-PJ10-PG6-01GM03-0002) and the Brain Korea 21 Project for Medical Science, Yonsei University.

References

1. Arnadottir T. Tuberculosis: trends and the twenty-first century. Scand J Infect Dis 2001;33:563–567PMID : 11525347.
crossref pmid
2. Glickman SE, Kilburn JO, Butler WR, Ramos LS. Rapid identification of mycolic acid patterns of mycobacteria by high-performance liquid chromatography using pattern recognition software and a Mycobacterium library. J Clin Microbiol 1994;32:740–745PMID : 8195387.
pmid pmc
3. Hines ME 2nd, Frazier KS. Differentiation of mycobacteria on the basis of chemotype profiles by using matrix solid-phase dispersion and thin-layer chromatography. J Clin Microbiol 1993;31:610–614PMID : 8458954.
pmid pmc
4. Musial CE, Tice LS, Stockman L, Roberts GD. Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex. J Clin Microbiol 1988;26:2120–2123PMID : 3141459.
pmid pmc
5. Kim BJ, Lee KH, Park BN, et al. Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB). J Clin Microbiol 2001;39:2102–2109PMID : 11376042.
crossref pmid pmc
6. Kim BJ, Lee SH, Lyu MA, et al. Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol 1999;37:1714–1720PMID : 10325313.
pmid pmc
7. Lee H, Park HJ, Cho SN, Bai GH, Kim SJ. Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene. J Clin Microbiol 2000;38:2966–2971PMID : 10921960.
pmid pmc
8. Hidaka E, Honda T, Ueno I, Yamasaki Y, Kubo K, Katsuyama T. Sensitive identification of mycobacterial species using PCR-RFLP on bronchial washings. Am J Respir Crit Care Med 2000;161:930–934PMID : 10712345.
crossref pmid
9. Lee H, Bang HE, Bai GH, Cho SN. Novel polymorphic region of the rpoB gene containing Mycobacterium species-specific sequences and its use in identification of mycobacteria. J Clin Microbiol 2003;41:2213–2218PMID : 12734283.
crossref pmid pmc
10. Chen KH, Sheu MM, Lin SR. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis: a case report of corneal ulcer. Kaohsiung J Med Sci 1997;13:583–588PMID : 9348738.
pmid
11. Sion C, Degraux J, Delmee M. Early identification of Mycobacterium tuberculosis and Mycobacterium avium using the polymerase chain reaction on samples positive by a rapid commercial culture system. Eur J Clin Microbiol Infect Dis 1999;18:346–351PMID : 10421042.
crossref pmid
12. Kontos F, Petinaki E, Gitti Z, et al. Combined use of the fully automated Bactec MGIT 960 system and a PCR-restriction fragment length polymorphism analysis for routine detection and identification of mycobacteria from clinical samples. J Microbiol Methods 2003;52:137–140PMID : 12401236.
crossref pmid
Figure 1
PCR-RFLP analyses of mycobacterial species. (A) PCR-RFLP patterns with MspI. (B) PCR-RFLP patterns with HaeIII. Lane 1, DNA size marker (50-bp ladder); lane 2, M. avium; lane 3, M. chelonae; lane 4, M. fortuitum; lane 5, M. gordonae; lane 6, M. intracellulare; lane 7, M. kansasii; lane 8, M. tuberculosis.
kjim-24-135-g001.jpg
Table 1
Results of the culture, biopsy, PCR-RFLP, and sequencing analyses of sterile body fluids from patients
kjim-24-135-i001.jpg

TB, tuberculosis; NTMI, non-tuberculous mycobacterial infection.

Editorial Office
101-2501, Lotte Castle President, 109 Mapo-daero, Mapo-gu, Seoul 04146, Korea
Tel: +82-2-2271-6792   Fax: +82-2-790-0993    E-mail: kaim@kams.or.kr
Copyright © 2019 The Korean Association of Internal Medicine. All rights reserved.
About |  Browse Articles |  Current Issue |  For Authors and Reviewers
powerd by m2community