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Journal of Clinical Microbiology, June 2007, p. 1898-1903, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02253-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Comparison of Three Methods for Rapid Identification of Mycobacterial Clinical Isolates to the Species Level

Xueqiong Wu,^1* Junxian Zhang,¹ Jianqin Liang,¹ Yang Lu,¹ Hongmin Li,¹ Chuihuan Li,² Jun Yue,³ Lishui Zhang,⁴ and Zhihui Liu⁵

Tuberculosis Research Laboratory, Tuberculosis Center, The 309th Hospital of PLA, 100091 Beijing,¹ Thorax Disease Hospital of Hebei Province, 050041 Shijiazhuang,² Pulmonary Disease Hospital of Shanghai, 200433 Shanghai,³ Pulmonary Disease Hospital of Fujian Province, 350008 Fuzhou,⁴ Thorax Disease Hospital of Guangzhou, 510095 Guangzhou, China⁵

Received 3 November 2006/ Returned for modification 10 January 2007/ Accepted 6 March 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A new PCR-reverse dot blot hybridization (RDBH) assay was developed for the rapid identification of Mycobacterium species in clinical isolates. The assay, which targets the 16S rRNA, was evaluated for 27 mycobacterial reference strains and 340 clinical isolates that were simultaneously identified by DNA sequencing and conventional methods, including growth characteristics, pigment production, colony morphology, and biochemical tests. All reference strains and clinical isolates hybridized to the Mycobacterium genus probe (probe M) on the membrane (100% sensitivity). Each probe had only one hybridization signal with the corresponding Mycobacterium species or complex (100% specificity). Compared with DNA sequencing, the RDBH assay correctly identified 337 (99.1% accuracy) of the 340 isolates tested. One M. asia isolate and one M. neoaurum isolate were not identified by the RDBH assay due to the absence of specific probes for the two species on the membrane. Three isolates with different nucleotide sequences from M. intracellulare reference strains had a negative hybridization signal with probe c, which is specific for M. intracellulare. The whole procedure can be completed within 2.5 h post-PCR processing. A total of 32 of 340 isolates were erroneously identified by conventional methods (90.6% accuracy). Molecular identification based on the 16S rRNA sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the RDBH assay can be considered a rapid, simple, and reliable method for routine identification of frequently occurring and clinically relevant mycobacteria.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Mycobacterium tuberculosis complex (MTC) and Mycobacterium leprae are important pathogens that cause diseases in both humans and animals. Other mycobacterial species, such as M. intracellulare, M. kansasii, M. gordonae, and M. chelonae, have been identified as opportunistic pathogens. The incidence of severe disease caused by nontuberculous mycobacteria (NTM) has gradually increased over the past decade. According to a report of a nationwide random survey in China for the epidemiology of tuberculosis in 2000, 11.1% of 441 strains isolated from patient sputa were identified as NTM (8). Clinical diseases caused by NTM are often hard to distinguish from tuberculosis caused by MTC. Because most NTM are naturally resistant to first-line antituberculosis drugs, a rapid and accurate diagnostic tool for the identification of mycobacterial species is essential for designing correct regimens at clinics.

Conventional methods for the identification of Mycobacterium species rely on growth characteristics, pigment production, colony morphology, and biochemical tests. Although the tests are easy to perform, they are costly (4). They are also time-consuming, taking 4 to 8 weeks to complete due to the slow growth of mycobacteria in cultures. Experience in the interpretation of the results of biochemical tests is required, and it is sometimes difficult to identify clinical isolates to the species level. As a result, conventional methods are not widely used in the majority of clinical laboratories in China. There is an urgent need for the development of a rapid, simple, and accurate method for Mycobacterium species identification.

The 16S rRNA gene sequences of most Mycobacterium species are well known and can be found in online databases (6, 15). They have been used to identify the species of all mycobacteria, including known and novel mycobacteria (2, 5, 7). In this study, we report the results of a PCR-reverse dot blot hybridization (RDBH) assay developed in our laboratory for the rapid identification of Mycobacterium species by comparison to the 16S rRNA sequences. The species assignments of 27 mycobacterial reference strains and 340 mycobacterial clinical isolates were determined in parallel by the PCR-RDBH assay, DNA sequencing, and conventional methods. The sensitivity and specificity of the PCR-RDBH assay were evaluated compared to the results from the conventional methods, with DNA sequencing data used as the standard.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains and clinical isolates. A total of 367 mycobacterial strains belonging to 27 different species (27 reference strains and 340 clinical strains) and 9 nonmycobacterial strains were used in this study. Reference strains of M. tuberculosis (H37Rv), M. bovis, M. avium (ATCC 25291), M. intracellulare (ATCC 13950), M. kansasii (ATCC 12478), M. scrofulaceum (ATCC 19981), M. simiae (ATCC 25275), M. nonchromogenicum (TMC1481), M. terrae (ATCC 15755), M. xenopi (ATCC 19250), M. gordonae (ATCC 14470), M. szulgai (ATCC 35799), M. marinum (TMC1218), M. flavescens (ATCC 14474), M. chelonae subsp. chelonae (TMC1544), M. chelonae subsp. abscessus (ATCC 19977), M. smegmatis (ATCC 19420), M. thermoresistibile (ATCC 19527), M. fortuitum (ATCC 6841), M. phlei (ATCC 11758), M. gilvum (ATCC 43909), M. aurum (ATCC 23366), M. triviale (TMC1453), M. vaccae (TMC1526), M. diernhoferi (ATCC19340), M. chubuense (ATCC 27278), M. aichiense (ATCC 27280), Pseudomonas aeruginosa, Klebsiella pneumoniae, Rhodococcus rubrum, Streptococcus pneumoniae, Staphylococcus epidermidis, Diplococcus lanceolatus, Bacterium diphtheriae, Escherichia coli, and Micrococcus catarrhalis were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Three hundred forty mycobacterial clinical isolates were obtained from patients with tuberculosis or other mycobacterial diseases by the tuberculosis departments of the 309th Hospital of PLA, Thorax Disease Hospital of Hebei Province, Pulmonary Disease Hospital of Shanghai, Pulmonary Disease Hospital of Fujian Province, and Thorax Disease Hospital of Guangzhou, China, between 1999 and 2005.

Conventional identification. Stock cultures of mycobacteria were grown on Lowenstein-Jensen slants medium at 37°C for 4 weeks. The cultures were subjected to phenotypic identification based on growth and colony characteristics, pigment production, and biochemical tests. Growth in the presence of PNB (p-nitrobenzoic acid) and TCH (thiophene-2-carboxylic acid hydrazide); growth rates at temperatures of 28, 37, and 45°C; pigmentation; and colony characteristics were assessed. Biochemical tests included the following: heat-stable (68°C) catalase, nitrate reduction, Tween 80 hydrolysis (3, 5, and 10 days), urease, arylsulfatase (3 and 10 days), iron uptake, tellurite reduction, and niacin production. The exact methods used in our laboratory were in accord with those described in Chinese Laboratory Science Procedure of Diagnostic Bacteriology in Tuberculosis (1).

DNA extraction. Cultured bacteria (about 5 mg) were transferred to microcentrifuge tubes containing 500 µl of TE buffer (10 mM Tris-1 mM EDTA, pH 8.0), heat killed at 80°C for 30 min, and harvested by centrifugation at 2,000 x g for 30 min. After the supernatant was removed, cells were resuspended in 1 ml TE buffer containing 2 mg lysozyme and incubated at 37°C for 2 h. Sodium dodecyl sulfate (SDS) and protease K were added to final concentrations of 1% and 50 µg/ml, respectively, and the mixture was incubated at 55°C for 2 h. Cell lysates were extracted with phenol and chloroform. DNA was precipitated with 2 volumes of ethanol and a 1/10 volume of 3 M sodium acetate (pH 5.2), dissolved in TE buffer, and stored at –20°C.

PCR amplification. DNA was amplified in a 25-µl PCR mixture containing 50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl₂, 200 µM (each) deoxynucleoside triphosphates (dTTP, dATP, dCTP, and dGTP) (Saibaisheng, Beijing, China), 0.4 µM (each) primer, 10 to 100 µg of genomic DNA, and 1 unit of Taq polymerase (Saibaisheng, Beijing, China). Special primers (forward primer, biotinylated at the 5' end, 5'-bio-CGA GTG GCG AAC GGG TGA G-3'; reverse primer, 5'-TTG TGC AAT ATT CCC CAC TGC TG-3') were used to amplify the 16S rRNA fragments, 268 to 282 bp in length, with genomic DNA extracted from mycobacterial isolates used as templates. The reaction mixtures were subjected to amplification, including 35 cycles of denaturation at 94°C for 1 min, primer annealing at 58°C for 1 min, and extension at 72°C for 1 min, with a final extension at 72°C for 7 min. For all strains tested, the presence of amplified products was verified on ethidium bromide-stained 2% agarose gels. The amplified target was visualized as a single band corresponding to a length of 268 to 282 bp by UV transillumination.

RDBH assay. The RDBH assay was developed based on the reverse hybridization principle (12). A series of oligonucleotide probes based on the 16S rRNA sequences of 22 Mycobacterium species were synthesized and then selected with PCR products from 22 Mycobacterium species by RDBH. The oligonucleotide probes specific for the detection of different Mycobacterium species are listed in Table 1. One microliter (3 pmol) of DNA probes was immobilized onto Protran nitrocellulose transfer membranes (pore size, 0.2 µm; Schleicher & Schuell BioScience GmbH, Germany) by UV radiation. The membranes were prehybridized in a 2-ml solution containing 6x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhardt's solution, 0.1 mg/ml calf thymus, and 0.5% SDS at 37°C for 30 min. The biotinylated PCR products were denatured at 95°C for 10 min and cooled on ice for 10 min. Ten microliters of the heat-denatured single-stranded PCR products were used to hybridize the membranes at 42°C for 30 min. The membranes were then washed with gentle shaking in 100 ml of 2x SSC-0.1% SDS for 5 to 10 min at 42°C, followed by a second wash in 100 ml of 0.2x SSC-0.1% SDS solution at the same temperature for 5 min. Hybridization was determined first by incubating the membrane at 37°C in 100 ml of a 1:1,000 dilution of streptavidin-alkaline phosphatase conjugate prepared in 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM MgCl₂, and 0.05% Triton X-100 for 30 min; washing once with the same buffer at room temperature for 5 min; and following that with a second wash with buffer containing 100 mM Tris-HCl (pH 9.0), 150 mM NaCl, and 1 mM MgCl₂ at room temperature for 5 min. The colorimetric hybridization signals were visualized by adding 2 ml of nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate substrate solution. Lastly, the membranes were washed with 100 ml of distilled water. The presence of clearly visible purple-blue spots on the membrane was considered a positive hybridization reaction. Each hybridization membrane includes a positive control spot representing a Mycobacterium genus hybridization probe. This spot is used to detect the presence of amplified product after hybridization, and it must always be positive when a mycobacterial species is present. Moreover, the membrane includes a spot representing the conjugate control spot, which must always be visible. Only those spots whose intensities were as strong as or stronger than that of the Mycobacterium positive control spot were considered positive.

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TABLE 1. Mycobacterium species identified by different probes

DNA sequencing. Each 16S rRNA gene fragment was sequenced by Canada Sangon Ltd., Beijing, China, with the primers used for PCR amplification to confirm the results obtained with the PCR-RDBH analysis. Sequences were compared with those registered in the GenBank database by BLAST analyses. Only 100% identities with mycobacteria were determined to be Mycobacterium species.

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DNA sequencing. Three hundred forty mycobacterial clinical isolates were identified by DNA sequencing (Table 2). Based on the 16S rRNA sequences, 151 strains were MTC, 1 was M. asia, 2 were M. avium, 37 were M. intracellulare, 29 were M. kansasii complex, 1 was M. terrae, 15 were M. gordonae, 3 were M. marinum complex, 73 were M. chelonae complex, 26 were M. fortuitum, 1 was M. neoaurum, and 1 was M. diernhoferi.

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TABLE 2. Identification of 340 mycobacterial clinical isolates by RDBH, DNA sequencing, and conventional methods

PCR-RDBH assay. Twenty nanograms of purified chromosomal DNA from each reference strain was amplified. Of the nine nonmycobacterial DNAs, only Streptococcus pneumoniae DNA was not amplified. Other nonmycobacterial DNAs were amplified and produced DNA fragments. None of the non-Mycobacterium reference strains hybridized to the Mycobacterium probes on the membrane (100% specificity). All of the Mycobacterium reference strains and clinical isolates were amplified. The resulting specific DNA fragments hybridized to the Mycobacterium genus probe (M) on the membrane (100% sensitivity). While all of the reference strains hybridized to the Mycobacterium genus probe, each reacted only to the corresponding species-specific probes on the membrane. No cross-species reaction was observed. Figure 1 shows the hybridization patterns of the Mycobacterium reference strains; only one signal per membrane was detected.

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FIG. 1. Identification of Mycobacterium reference strains by the RDBH assay. This figure shows representative examples obtained after hybridization of the membranes with different amplified gene fragments from 23 Mycobacterium reference strains. M. bovis, M. simiae, M. chelonae subsp. chelonae, and M. thermoresistibile, which were absent, had the same hybridization patterns as M. tuberculosis, M. kansasii, M. chelonae subsp. abscessus, and M. smegmatis, respectively. Line 1: the first spot is the conjugate control, the second spot is probe M, the third spot is probe a, the fourth spot is probe b, and the fifth spot is probe c. Line 2: the first spot is probe d, the second spot is probe h, the third spot is probe i, the fourth spot is probe j, and the fifth spot is probe k. Line 3: the first spot is probe l, the second spot is probe m, the third spot is probe n, the fourth spot is probe p, and the fifth spot is probe q. Line 4: the first spot is probe r, the second spot is probe s, the third spot is probe t, the fourth spot is probe u, and the fifth spot is probe w. Line 5: the first spot is probe y, the second spot is probe z, the third spot is probe ab, and the fourth spot is probe ac.

Compared to the results from DNA sequencing, the RDBH assay correctly identified 337 isolates (99.1% accuracy) of the 340 samples tested (Table 2). Among the mismatches, one M. asia isolate and one M. neoaurum isolate were not identified by the RDBH assay simply because no M. asia- and M. neoaurum-specific probes were included on the membrane. The remaining three isolates, identified as M. intracellulare by sequencing analysis, had negative hybridization signals to the specific probe, probe c, for M. intracellulare. This discrepancy is likely due to the one-nucleotide mismatch between probe c and the 16S rRNA fragments amplified for these isolates (Fig. 2).

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FIG. 2. Alignment of two parts of the 16S rRNA genes of M. intracellulare and M. intracellulare isolate FZ34 by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/BLAST.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Semiauto&ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&CLIENT=web&DESCRIPTIONS=100&ENTREZ_QUERY=%28none%29&EXPECT=10&FILTER=L&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&NCBI_GI=off&PAGE=Nucleotides&PROGRAM=blastn&SERVICE=plain&SET_DEFAULTS.x=34&SET_DEFAULTS.y=8&SHOW_OVERVIEW=on&END_OF_HTTPGET=Yes&SHOW_LINKOUT=yes&GET_SEQUENCE=yes&NEW_VIEW=yes&SEARCH_NAME=bn). The single mismatch is indicated in gray. Two other isolates bearing the same mismatched base pairs did not hybridize to probe c on the membrane.

Conventional identification of mycobacterial clinical isolates. In comparison with the sequence analysis results, 326 of the 340 clinical isolates were correctly assigned to MTC or NTM by TCH and PNB cultures (95.9% accuracy). A series of biochemical tests were able to further correctly match only 124 (87.3%) of 142 NTM isolates to the species level. Table 2 lists the species identification results obtained from the RDBH assay, DNA sequencing, and the conventional methods. For MTC, the RDBH assay correctly identified all 151 isolates, based on DNA sequencing, whereas only 143 were identified as MTC by the conventional methods. Among the misdiagnosed isolates, eight were mismatched to the NTM group. As shown by the data in Table 2, species identification based on the RDBH assay is more consistent with the results obtained by DNA sequencing than is that of the conventional methods for the entire list of mycobacterial species analyzed in this study.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
At present, the diagnosis of NTM diseases relies on multiple assays with poor accuracy and long periods of laboratory testing. In addition, not all clinical laboratories are equipped to conduct the assays. Considering the impact of rapid diagnosis on the development of timely and appropriate treatment regimens, as well as on epidemiological research, any rapid, simple, and accurate laboratory method that can be used to differentiate various species of mycobacteria is essential and urgently needed in developing countries, where mycobacterial infections are still a serious public health problem. Recently, a high-performance liquid chromatography (HPLC) technique and several newer commercial nucleic acid probe kits, such as Accuprobe (Gen-Probe, Inc., San Diego, CA), INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium), and GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), have been used for the identification of mycobacterial clinical isolates (3, 9, 13, 14). The HPLC technique, which analyzes the high-molecular-weight mycolic acids of the cell wall, can provide a species-specific, rapid, and reproducible method of identification (14). Other nucleic acid probe methods, which depend on unique DNA sequences for identification, have the advantage of providing rapid and accurate results. But they are costly for the developing countries. However, HPLC requires special equipment, software, and technicians for identification. Accuprobe targeting of the rRNA requires the use of a special instrument and can identify only five species or groups: MTC, M. avium complex, M. avium, M. intracellulare, M. gordonae, and M. kansasii (3). INNO-LiPA Mycobacteria and GenoType Mycobacterium utilize a reverse hybridization approach similar to that of our RDHB assay, with different DNA amplification targets. INNO-LiPA uses the mycobacterial 16S-23S rRNA spacer regions for 16 species (9), while GenoType targets the 23S rRNAs of 13 species (11). In comparison, our RDHB assay needs no special instrument and targets the 16S rRNA regions of 22 different mycobacterial species. The 16S rRNA genes of most Mycobacterium species have been sequenced. These sequences differ among mycobacterial species; thus, they are good candidate targets for species identification.

Multiple studies have confirmed that sequence analysis can obtain useful and accurate information from isolates with common, aberrant, or new species (2, 10, 16). It has been accepted as the "gold standard" for bacterial strain identification. However, sequence analysis requires a costly automated sequencer and specially trained technicians to conduct the analysis. At present, this method has not been widely implemented as a routine test in the clinical laboratories of developing countries. In contrast, our RDBH assay does not require a sequencer and is easy to perform in laboratories where a PCR cycler is available. It takes only 2.5 h post-PCR amplification to complete the assay. Hybridization technology allows us to incorporate as many unique mycobacterial target sequences as possible on membranes in order to cover more species than the currently commercially available detection kits.

Compared with DNA sequencing, 337 (99.1%) strains were correctly identified by the RDBH assay, demonstrating excellent agreement. Among the species that were misidentified, one was M. asia and another was M. neoaurum, which had been isolated from a tuberculosis patient in Hong Kong, China (16). The specific probes for these species were not included in our collection. A nucleotide mismatch present in the amplified 16S rRNA fragments found in certain M. intracellulare clinical isolates resulted in negative reactions to all of the species probes (three isolates [Table 2]). Negative detection due to the lack of specific probes can be easily corrected by adding the appropriate probes on the membrane.

In comparison, 14 of 340 strains were incorrectly differentiated as MTC or NTM by the TCH and PNB culture method, and 18 of 142 NTM isolates were further incorrectly identified to the species level by the traditional phenotypic method. Together, 32 isolates were misdiagnosed by the conventional methods. This result is similar to the study of Cloud et al., who obtained 87% concordance between sequence analysis and the traditional phenotypic method (2). The mistakes of conventional identification may be caused by bacterial aberrance or by misreading of the results from a series of biochemistry tests. If tuberculosis should be misdiagnosed as NTM disease, a patient would be treated with more than four first- and second-line antituberculous drugs for more than 1 year, treatment which is not necessary and might induce side effects. In addition, different species of NTM are sensitive to different antituberculous drugs. Therefore, it is necessary to further discriminate NTM species. If NTM disease should be misdiagnosed as tuberculosis in clinics, the patient would be treated with the standard antituberculous regimen for several months, which is not effective against NTM infection. Correct identification is important for effective therapy and minimizing of side effects. Molecular identification methods based on the 16S rRNA sequences were superior to conventional identification in sensitivity and specificity. However, current methods using molecular identification cannot distinguish subspecies of MTC and other mycobacterial species, such as M. chelonae, M. kansasii, M. scrofulaceum, M. gastri, M. simiae, M. marinum, M. ulcerans, M. smegmatis, and M. thermoresistibile, as the subspecies in each group have identical rRNA sequences. To further identify the subspecies, conventional approaches will continue to play a role in confirming and supplementing the results of molecular methods. By selecting a second unique mycobacterial target, we anticipate that in the near future we will be able to advance our RDBH assay to the next level, at which the identification of subspecies becomes possible.

The results presented above reveal that RDBH identification had 100% sensitivity and specificity for the reference strains. For the clinical isolates, RDBH identification had 99.1% accuracy, while conventional identification had only 90.6% accuracy. Molecular identification based on the 16S rRNA sequences demonstrated excellent sensitivity and specificity relative to the conventional approach. The RDBH assay is easy to perform and time saving. The data presented in this study have demonstrated the value of this assay in the identification of mycobacterial species in clinical settings.

In conclusion, the RDBH assay is a rapid, simple, and reliable method. It is a promising approach for the routine identification of frequently encountered mycobacteria in the professional mycobacterial laboratory.

 
This work was supported by the Army Research Foundation for the Outstanding Person with Ability of China (no. 01J020) and by the "973" Infectious Diseases Special Foundation of China (no. 2005CB523102).

 
* Corresponding author. Mailing address: Tuberculosis Research Laboratory, Tuberculosis Center, The 309th Hospital of PLA, Beijing 100091, China. Phone: 86-13671334568. Fax: (8610) 62582972. E-mail: wu-xueqiong{at}263.net

Published ahead of print on 14 March 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Clinical Microbiology, June 2007, p. 1898-1903, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02253-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wu, X. Articles by Liu, Z. Search for Related Content PubMed PubMed Citation Articles by Wu, X. Articles by Liu, Z.