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Journal of Clinical Microbiology, November 2008, p. 3591-3594, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.00856-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Identification of Mycobacteria from Smear-Positive Sputum Samples by Nested PCR-Restriction Fragment Length Polymorphism Analysis

Tsu-Lan Wu,^1,2 Ju-Hsin Chia,^1,2 An-Jing Kuo,^1,2 Lin-Hui Su,^1,2 Ting-Shu Wu,^3,4 and Hsin-Chih Lai^1*

Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan,¹ Department of Clinical Pathology,² Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan,³ Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan⁴

Received 5 May 2008/ Returned for modification 24 June 2008/ Accepted 2 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The rapid identification of mycobacteria from smear-positive sputum samples is an important clinical issue. Furthermore, the availability of a cheap, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In the present study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from smear-positive sputum samples. A nested PCR-restriction fragment length polymorphism analysis (nested-PRA) assay that focuses on the analysis of the hsp65 gene was developed and evaluated for its efficiency compared to that of traditional culture methods and 16S rRNA gene sequencing identification. A total of 204 smear-positive and culture-positive sputum specimens were prospectively collected for analysis between November 2005 and May 2006. The samples were classified according to an acid-fast bacillus (AFB) staining scale as rare/1+, 2+, or 3+. The results of the nested-PRA showed that the identification rate for AFB 3+, AFB 2+, and AFB rare/1+ samples was 100, 95, and 53%, respectively, and that the overall identification rate was 89%. All positive results by the nested-PRA method agreed with the results by culture and 16S rRNA gene sequence analysis. The nested-PRA appears to have clinical applicability when used for the direct identification of mycobacterial organisms (both MTBC and NTM) that are present in smear-positive sputum samples, especially for countries in which MTBC is endemic.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The rapid identification of Mycobacterium spp. from smear-positive sputum samples is very important from clinical viewpoints. This is because, in addition to increased clinical infections due to Mycobacterium tuberculosis complex (MTBC) organisms, the percentage of infections with nontuberculous mycobacteria (NTM) also has been increasing recently (1, 6-8, 11, 16, 20, 21, 31), especially among immunocompromised patients (11). Strategies used for the clinical management of patients with MTBC or NTM infections are different. Patients who are suspected to have MTBC infections have to receive proper medication or even be placed in an isolation room immediately. Therefore, the correct and rapid identification of MTBC and NTM organisms represents a clinical emergency that should not be underestimated.

The traditional diagnosis of mycobacterial infections from sputum samples in the mycobacterial laboratory is based primarily on demonstrating the presence of the acid-fast bacilli (AFB) in the smear, followed by a positive culture and the testing of the physiological/biochemical identification of the isolate (19). This approach has a number of defects, including that it is time-consuming, has low sensitivity, and has poor discrimination between closely related NTM species (29). High-performance liquid chromatography is an alternative approach for the identification of mycobacteria, and this approach can identify more than 50 different species (9); however, a concentration of more than 10⁶ bacteria per ml is required. Recently, a paranitrobenzoic acid assay has been applied directly to clinical samples as a rapid screening assay for the detection of and differentiation between MTBC and NTM (32). Nevertheless, this approach does not allow further NTM species differentiation and has a long incubation time (3 weeks) before the results can be read.

Recently, the development of PCR-based methods for the rapid identification and differentiation of mycobacterial organisms has significantly improved the diagnosis efficiency in terms of both sensitivity and specificity (3, 5, 12, 14, 17, 22, 24, 26, 27, 30). We previously developed a multiplex nested PCR combined with lateral-flow technology for the rapid diagnosis of M. tuberculosis and MTBC isolates and the differentiation of these organisms from NTM organisms (28). In addition, a multiplex PCR system for the rapid detection and differentiation of MTBC members from NTM organisms also has been developed recently and evaluated (18). Generally speaking, these methods allow the direct identification of M. tuberculosis or MTBC from sputum samples but provide insufficient information when clinically important NTM infections are encountered. Another problem is that the amount of mycobacterial cells contained in each sputum sample may vary, and the effect on the direct PCR detection of mycobacterial organisms from AFB-positive sputum samples remains to be evaluated.

In the present study, traditional culture and biochemical test methods were used together with 16S rRNA gene sequencing as a standard protocol to evaluate the efficacy of a nested PCR-restriction fragment length polymorphism analysis (nested-PRA) method modified from those described previously by Telenti et al. (30) and Bascuñana and Belák (2). The original assay described by Telenti et al. was able to identify at least 54 Mycobacterium spp., including a range of organisms from the frequently isolated MTBC and NTM groups, such as the M. avium-M. intracellulare complex, the M. chelonae group, M. gordonae, the M. fortuitum group, M. kansasii, M. marinum, etc. (3). In this study, the applicability of the Telenti method in the direct detection of mycobacteria from sputum samples was examined, and to improve the detection limit, a nested PCR protocol was developed for final evaluation. A total of 204 clinical smear-positive and culture-positive sputum samples with different AFB staining scales were prospectively collected between November 2005 and May 2006 for examination. The potential clinical applicability of the nested-PRA system for the rapid identification of mycobacterial organisms from AFB-positive sputum samples, especially in regions with a high prevalence rate, is discussed.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Clinical sputum specimens and bacterial strains. Between November 2005 and May 2006, sputum samples were prospectively collected from the Mycobacteriology Laboratory, Department of Clinical Pathology, Chang Gung Memorial Hospital. Replicate specimens from the same patients were excluded from the study to prevent duplication. To specifically evaluate the efficacy of the nested-PRA method on smear-positive samples and compare them to culture-positive results, sputum samples with discordant smear and culture results were excluded from the present study and will be the target of a future study. The collection of these clinical samples was approved by the Review Board Committee of the Chang Gung Memorial Hospital.

Specimens were processed on receipt according to the standard protocol (19). Briefly, all specimens were treated with 5% oxalic acid before inoculation on Löwenstein Jensen slants (Difco) and Middlebrook 7H11 agar plates (Becton-Dickinson) for the recovery of the mycobacteria. Smears also were made for auramine-rhodamine fluorochrome staining, and these were analyzed by fluorescence microscopy. Positive smears were further confirmed by Kinyoun acid-fast staining and then classified into AFB rare/1+, 2+, or 3+ based on standard procedures (13). The remaining part of each specimen then was stored at –70°C for subsequent PCR analysis.

Identification of Mycobacterium spp. by culture, biochemical methods, and 16S rRNA gene sequencing. The identification of the mycobacterial isolates to the species level is based mainly on routine morphological and biochemical assays (23). The results of species identification were further confirmed by 16S rRNA gene sequence analysis. Briefly, a loopful of mycobacterial cells grown on Middlebrook 7H11 was digested with 200 µl proteinase K (1 mg/ml) solution at 56°C for 2 h. The procedure was followed by sonication at 120 W for 40 min and heating at 94°C for 10 min before being stored at 4°C for PCR. A 16S PCR assay using the primers 8FPL and 1492 then was carried out to amplify a 1,491-bp fragment of the 16S ribosomal gene (25). The PCR product was then purified using a Microcon PCR centrifugal filter device (Millipore) and subjected to sequencing in both directions using the primers 8FPL and 531R (25) on a 3100-Avant genetic analyzer (Applied Biosystems). The first 500 bp of the 16S ribosomal DNA sequence obtained was compared to those deposited in the GenBank database using the basic local alignment search tool on the Internet (http://www.ncbi.nlm.nih.gov/BLAST/). A 99% identity was used to define a specific species.

Preparation of mycobacterial DNA from sputum specimens. DNA was extracted using a QIAamp DNA mini kit (Qiagen, Germany) with some modifications. Briefly, decontaminated sputum samples were washed once with distilled water and centrifuged for 10 min at 5,000 x g. The precipitate then was suspended in ATL buffer (Qiagen, Germany) containing 1 mg/ml proteinase K and incubated at 56°C for 1 h. Afterward, two cycles of freeze-thawing were used to lyse the mycobacterial cells. DNA then was purified through a spin column. To evaluate the detection limit of the nested-PRA assay, the amounts of M. tuberculosis H37Rv and M. chelonae subsp. chelonae ATCC 35749 cells were determined by plate counting on Middlebrook 7H11 agar plates. These bacterial cultures then were serially diluted in normal saline before being spiked into autoclaved decontaminated smear-negative and culture-negative sputum samples. DNA then was extracted using the above-described procedure.

Nested-PRA. The method developed for nested-PRA was derived from Telenti et al. (30) and Bascuñana and Belák (2), with some modifications. The hsp65 gene, which is present in all mycobacterial cells, was PCR amplified and then subjected to restriction fragment length polymorphism analysis. To increase the sensitivity, the nested PCR approach was used. The primer pair M1 (5'-CCCCACGATCACCAACGATG-3') and M4 (5'-CGAGATGTAGCCCTTGTCGAACC-3') (2) was used for the first PCR. Tb11 (5'-ACCAACGATGGTGTGTCCAT-3') and Tb12 (5'-CTTGTCGAACCGCATACCCT-3') were used as inner primers for the second round of amplification. The first PCR amplification was performed in a 50-µl reaction mixture containing 10 µl of the DNA template suspension prepared from the sputum samples, 0.2 mM (each) deoxynucleotide, 20 pmol of each primer (primers M1 and M4), 0.5x Q solution (Qiagen), and 1 U HotStarTaq DNA polymerase (Qiagen) in 1x PCR buffer. The reaction conditions were the following: 95°C for 15 min, followed by 30 cycles of 95°C for 1 min, 60°C for 1 min, 72°C for 1 min, and finally 72°C for 10 min. Subsequently, 5 µl of the PCR mixture was transferred to a new tube containing a 100-µl reaction mixture and Tb11 and Tb12. The conditions of the second PCR were identical to those of the first reaction. The PCRs were performed on a GeneAmp 9700 thermal cycler (Applied Biosystems). The amplified products were digested with BstEII and HaeIII (New England Biolabs) and visualized after 3.5% NuSieve 3:1 agarose gel electrophoresis using ethidium bromide staining. The band sizes, in base pairs, were estimated using BioNumerics software (Applied Maths, Belgium) with a 100-bp ladder and a 20-bp ladder as the molecular size standards. Restriction fragments that were smaller than 60 bp were disregarded. According to the PRA algorithm, the mycobacteria detected were classified to the species level as described in Results and Discussion.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A total of 281 sputum samples (204 smear-positive and culture-positive and 77 smear-negative and culture-negative samples) were examined in this study. Among the 204 smear-positive and culture-positive sputum samples, a total of 151 MTBC and 57 NTM isolates across 14 Mycobacterium species (Table 1) were identified by physiological, biochemical, and 16S rRNA gene sequencing. Mixed infection, in which two mycobacterial species were simultaneously identified from the same specimen, was observed in four specimens.

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TABLE 1. Efficiency of the nested-PRA method evaluated by the direct identification of various Mycobacterium spp. from 204 sputum samples with different AFB scale results

At the preliminary stage of this study, the method described by Telenti et al. (30) was used. The chromosomal DNA extracted from M. tuberculosis H37Rv colonies was amplified with the use of the primer pair Tb11 and Tb12, and a 440-bp DNA fragment was obtained from the hsp65 gene. The detection limit of such a single PCR-restriction enzyme digestion assay was determined to be about 50 pg. When this single-PCR approach was applied to the direct detection of mycobacteria from sputum samples, false-negative or inconclusive results were frequently encountered. This problem was most frequently observed among the AFB rare/1+ samples and occasionally in AFB 2+ samples. It is possible that the amount of target DNA in the sputum samples was not sufficient to produce a reliable result.

To circumvent this difficulty, the Telenti method was modified by a nested PCR approach. The detection limit of the nested-PRA was evaluated by using the chromosomal DNA prepared from M. tuberculosis H37Rv colonies as the template. The primer pair M1 and M4 (2) was used to amplify a 463-bp DNA fragment, which then was used as the template for the second PCR using the primer pair Tb11 and Tb12 (30). Such an approach allowed the DNA detection limit to be increased to 5 fg. Furthermore, to evaluate the detection limit in terms of mycobacterial cells in a sputum sample, negative sputum samples were spiked with serially diluted M. tuberculosis H37Rv or M. chelonae subsp. chelonae ATCC 35749 cells. The DNA then was extracted and subjected to nested-PRA. The nested-PRA was able to detect as few as 10 mycobacterial cells in a reaction mixture with either M. tuberculosis or M. chelonae cells (data not shown). These results are compatible with those obtained by Bascuñana and Belák (2). The nested-PRA method then was applied to the direct identification of mycobacteria from sputum samples.

Negative results were obtained from the nested-PRA when the 77 smear-negative and culture-negative sputum samples were analyzed. The 204 smear-positive and culture-positive sputum samples then were analyzed by the nested-PRA method, and the results were compared to culture results according to the AFB scales (Table 1). All positive results by the nested-PRA method agreed with the results by culture and 16S rRNA gene sequence analysis. However, the efficiency of the nested-PRA method seemed to be correlated with the amounts of mycobacterial cells in the samples. For those sputa scaled as AFB 3+ (65 samples), seven mycobacterial species were identified by culture: MTBC (n = 56), M. abscessus (n = 2), M. intracellulare (n = 1), M. kansasii (n = 2), M. avium (n = 2), M. chelonae (n = 1), and M. malmoense (n = 1). The nested-PRA produced exactly the same results as those by culture methods.

A total of 64 specimens were scaled as AFB 2+, and eight mycobacterial species were identified by culture. Single infection by only one mycobacterial organism was found in 60 specimens: MTBC (n = 43), M. abscessus (n = 5), M. intracellulare (n = 8), M. kansasii (n = 1), M. gordonae (n = 1), M. avium (n = 1), and M. marinum (n = 1). The remaining four samples were mixed infections, and MTBC and NTM (including one M. terrae complex) were isolated simultaneously from three of them. With the use of the nested-PRA method, all MTBC isolates were correctly identified, while 5 of the 21 NTM samples failed to be identified (Table 1).

A total of 75 mycobacterial organisms were isolated from specimens scaled as AFB rare/1+, and 9 mycobacterial species were identified by culture: MTBC (n = 49), M. abscessus (n = 6), M. intracellulare (n = 3), M. kansasii (n = 4), M. gordonae (n = 5), M. fortuitum (n = 3), M. nonchromogenicum (n = 2), M. triviale (n = 2), and M. scrofulaceum (n = 1). Among them, only five species (MTBC, M. abscessus, M. intracellulare, M. gordonae, and M. fortuitum) were correctively identified by the nested-PRA method from 40 (53%) of the specimens.

Overall, for the AFB 3+/2+ samples, the nested-PRA assay showed almost complete congruence with the traditional diagnostic methods and thus provided a satisfactory identification efficacy. In a region in which MTBC is highly endemic, this method has strong potential clinical applicability because it facilitates clinical decisions in terms of patient isolation and treatment or even the design of a prevention strategy.

It appears that the nested-PRA method described in this study has multiple advantages. First, the test is not only accurate but also is fast. The results can be obtained within 8 h, or one working day. This factor is especially important when a decision is to be made on whether to place a patient into an isolation room for the proper treatment of MTBC infections. Second, the approach represents a universal system of identifying clinically frequent mycobacteria, and even some infrequent NTM organisms, to the species level. Third, the technical requirements to carry out the assay are minimal, because it requires neither sequencing nor hybridization to a panel of species-specific probes in order to differentiate the mycobacterial species. Finally, the associated expenditure is low, making it easily tolerated by the general medical insurance system.

However, it also is obvious that, despite the inclusion of a nested-PCR procedure, the efficiency of this nested-PRA assay was greatly reduced when AFB 1+/rare sputum samples were encountered. As the detection limit of this system had been determined to be 10 mycobacterial genome copies, sputum samples with a score of AFB 1+/rare may contain smaller amounts of the required DNA to be amplified. In addition, the presence of different kinds and amounts of polymerase inhibitors in the treated sputum samples might further reduce the sensitivity of this system. For specimens with heavy inhibitors, a further DNA purification step before the nested PCR might help to increase the DNA amplification yields. Another approach would be to increase the efficiency of the DNA extraction from the sputum samples. According to the usual procedure performed in our diagnostic laboratory for sputum samples, a freeze-thaw approach was adopted to break the mycobacterial cells before the DNA was extracted by a commercial kit. The method was not compared to the sonication method that is used for DNA extraction from colonies. It is possible that the method is not efficient enough to extract all DNA from the sputum samples. However, the detection limit of this nested-PRA method already has been determined to be as little as 10 mycobacterial cells in sputum samples. Whether or not an alternative DNA extraction method will provide better efficiency warrants further study. We recently found that a pretreatment with a detergent such as 1% Triton X-100 together with boiling at 100°C for 10 min might increase the nested-PRA-amplified DNA yield up to 10-fold (unpublished results). Because the false-negative samples described in the present study were no longer available for reexamination, the feasibility of this modified DNA extraction method in the nested-PRA procedure will be tested in the future. Alternatively, the addition of PCR inhibitor scavengers or the use of a chemiluminescence approach to improve the detection of the restriction-digested DNA fragments might also have to be considered.

Recently, the global trend has been toward the isolation from sputum samples of more and more NTM organisms in addition to M. tuberculosis (4, 10, 15). Based on the statistical results from the Chang Gung Memorial Hospital, which is the largest tertiary medical center in Taiwan, the isolation of NTM increased significantly from 248 (19%) in 2003 to 407 (30%) in 2006 (P < 0.0005). In 2006, the top five frequently isolated NTM organisms were the M. chelonae group (10.7%), the M. avium-M. intracellulare complex (5.7%), M. gordonae (4.0%), the M. fortuitum group (3.2%), and M. kansasii (2.2%) among the all of the mycobacteria isolated. While the clinical significance of many NTM isolates remains to be determined, the increasing isolation of these NTM may represent an emerging problem that deserves to be closely monitored. Therefore, an accurate, cost-effective, and easy-to-perform approach such as the nested-PRA system described herein may fit well for such a purpose.

 
This work was supported in part by grants CMRPG361231 and CMRPD170101 from the Chang Gung Memorial Hospital and DOH97-DC-1501 from the Department of Health, Taiwan.

 
* Corresponding author. Mailing address: Department of Medical Biotechnology and Laboratory Science, Chang Gung University, No. 259, Wen-Hua 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China. Phone: 886-3-2118800, ext. 3585. Fax: 886-3-2118700. E-mail: hclai{at}mail.cgu.edu.tw

Published ahead of print on 3 September 2008.

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Journal of Clinical Microbiology, November 2008, p. 3591-3594, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.00856-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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