Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of Mycobacterial species from clinical isolates

Background Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. Results Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100% (200 isolates) for the MTC and 91.4% (161 isolates) for the NTM. Among the discordant results (8.6%) for the NTM, one isolate of Mycobacterial species and an isolate of M. flavescens were found as new sub-types in hsp65 PRA. Conclusions This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16 S rDNA sequencing.


Background
Detection and identification of mycobacteria in clinical specimens is a key issue in the therapy of pulmonary diseases because misidentification can lead to inappropriate treatment. Traditionally, mycobacterial species are identified based on their growth rate, presence or absence of pigmentation, and using biochemical assays of the isolates recovered from specimens. The biochemical assays are time-consuming and labor-intensive, usually taking 1 to 2 months to complete, and assays for nontuberculous mycobacteria (NTM) species can have poor reproducibility and provide ambiguous results [1,2].
By contrast, molecular identification, notably PCRrestriction enzyme analysis (PRA), is rapid and simple. The hsp65 PRA method, developed by Telenti et al. in 1993, is a popular DNA-based method for mycobacteria identification [3]. Using hsp65 PRA, Wong et al. [4] reported 100% sensitivity and specificity in identifying Mycobacterium tuberculosis complexes but only 74.5% sensitivity in identifying NTM species. This misidentification may occur because of similarities in band sizes that are critical for species discrimination [3]. An additional contributing factor is a lack of knowledge of all existing PRA profiles, especially among species that are very heterogeneous, such as M. gordonae, M. scrofulaceum, and M. terrae complexes. Recently, capillary electrophoresis (CE) with computer analysis [5][6][7][8][9] has provided more precise band discrimination than analysis by the naked eye.
Previously, we developed an algorithm for mycobacterial species identification from acid-fast bacillus (AFB) smear-positive BACTEC tubes by combining the rpoB duplex PRA (DPRA) [10] and the key phenotypic characters of mycobacteria recovered from the tubes [11]. Using rpoB DPRA, we differentiated Mycobacterium tuberculosis complexes (MTC) from NTM with 235 base pair (bp) and 136 bp PCR amplicons in AFB smearpositive BACTEC cultures. The 136 bp rpoB duplex PCR amplicon was further digested with MspI and HaeIII (rpoB DPRA) to divide the NTM species into eight easily distinguishable groups (A-H) as described by Kim et al. [10]. Using two phenotypic characters (growth rate and photoreactivity on pigment production) and two simple biochemical assays (nitrate reduction test and Tween 80 hydrolysis test) [11], the mycobacterial species were identified. However, the sub-culture and biochemical tests for this algorithm took three weeks.
In the present study, we developed a rapid and effective algorithm for identification of mycobacteria by combined rpoB DPRA and hsp65 PRA with CE.

Development of a species identification algorithm
The results in Tables 1 and 2 and the mycobacterial identification flow chart ( Figure 1) were used to develop a species identification algorithm by combining rpoB duplex PCR [10] and hsp65 PRA [3] using the most common 74 patterns of 40 species in Table 3. In this algorithm (

Discussion
Mycobacterial species identification is generally achieved by standard culture and biochemical methods [12]. Biochemical methods are labor-intensive and time-consuming, and not all are reproducible [2,13]. Recently, PRA and DPRA have been developed for molecular identification of mycobacterial species using different regions of hsp65, 16 S rDNA, 16 S-23 S rDNA spacer, dnaJ, and rpoB as an amplification target [3,[14][15][16][17]. The most common method is hsp65 PRA, and 74 patterns for 40 species are available in the PRASITE database (http://app. chuv.ch/prasite/ index.html).
Previous studies [18,19] have reported that hsp65 PRA is faster and more accurate for species identification than conventional (phenotypic or biochemical) testing. This is because more incorrect and ambiguous results are obtained with conventional methods. The results in our study (Tables 1 and 2) also support this finding. Incorrect and ambiguous results are caused by phenotypic homogeneity among different species and phenotypic variability within species [18]. With by hsp65 PRA, some sub-species, such as M. kansasii, can be identified and rapid-growing mycobacterium can be divided into M. abscessus and M. chelonae, M. fortuitum and M. smegmatis [20], whereas these identifications are difficult with conventional methods [21]. As found in our study (Tables 1 and 2 However, hsp65 PRA limitations have been reported in some articles [22,23]. Failure to identify or incorrect identification of the species may occur because of similarities in band sizes critical for discriminating species, including difficult to distinguish M. tuberculosis complex (M. tuberculosis and M. bovis) [22], and closely related sub-species such as M. avium or M. gordonae, because of sequence heterogeneity [22]. In addition, technical problems can also cause misinterpretation or incorrect identification [23]. Patterns in PRA profiles are complex and difficult to interpret with the naked eye, especially when more detailed sub-types are included [21]. This study combined rpoB DPRA and hsp65 PRA to test both reference strains and clinical respiratory isolates. The mycobacterial identification flow chart ( Figure 1) can identify species to the sub-species level, and final species identification can be obtained instantly with concordant results from the two PRA. M. gordonae has a highly variable gene sequence with 10 sub-types in hsp65 PRA, and there are two groups (G and F) in rpoB DPRA. Most M. gordonae is in the G group, but M. gordonae types 3 and 4 by hsp65 PRA are in the F group (Tables 1 and 2).
In addition, there were different rpoB DPRA results ( Table 2) for M. simaie type 5 (G group but not E group), M. scrofulaceum type 1 (D group but not H group), and M. intracellulare type 3 (F group but not G group). The identities of all of these isolates were finally confirmed by 16 S rDNA sequencing. Variable numbers of restriction sites for HaeIII in these species may derive from genetic sequence mutation. However, these species are included in the species identification algorithm even though they are uncommon isolates.
Using the mycobacteria identification flow chart ( Figure 1) and algorithm (Table 3), M. avium-intracellulare complex (MAC) can be easily divided into M. avium spp. avium and M. intracellulare by both rpoB DPRA and hsp65 PRA. By contrast, this was not possible with the conventional method. Using the results in Table 3, some NTM species with identical or similar hsp65 PRA can be clearly grouped by rpoB DPRA (Table 4). Ambiguous results from hsp65 PRA alone are easier to interpret with combined rpoB DPRA and hsp65 PRA. However, M. intermedium type 1 and M. intracellulare type 3 with identical hsp65 PRA and rpoB DPRA (G group) could not be differentiated further by this species identification algorithm and required 16 S rDNA sequencing for confirmation.
Although 16 S rDNA sequencing is the standard method for mycobacterium species identification, it cannot differentiate some closely related rapid-growing mycobacterium species [24] or slow-growing M. kansasii and M. gastri that had identical 16 S rDNA sequences, but these can be differentiated by hsp65 PRA and rpoB DPRA. There are some reports [6,25] of conflicting results from different methods for mycobacterial species identification, probably caused by a failure of one method to identify all test strains correctly. Combining methods for mycobacterial species identification can improve the accuracy rate, avoid ambiguous results, and save time.   Many CE-based studies [5][6][7][8][9] in PCR-RFLP analysis have investigated improving band size discrimination. In one study by Chang et al. [7], high-resolution CE gave more precise estimates of DNA fragment sizes than analysis by the naked eye, and CE could detect low molecular weight fragments (down to 12 bp). In our study, restriction fragments <50 bp were not selected for analysis to avoid confusion with primer and primer dimer bands. We found that more hsp65 fragment differences than rpoB fragment (data not shown) may explain the size differences with highly variable sequence for species identification but difficult interpretation in hsp65 PRA.
Some sub-types of NTM species are relevant to clinical management, such as the M. kansasii and MAC. M. kansasii type 1 is the most common type associated with human disease [26][27][28] because of its high pathogenicity. However, M. kansasii types 3-7 are most often isolated from the environment and rarely from humans, and have no significant role in clinical management [26,27]. MAC can be divided into M. avium subsp. avium and M. intracellulare because drug sensitivity test and clinical outcomes are different between these two subtypes [29,30].
It is important to identify NTM to the sub-type level both for epidemiologic data and for differentiating potentially pathogenic sub-types [26,27]. Combined rpoB DPRA and hsp65 PRA with capillary electrophoresis provides precise species identification and overcomes problems associated with discrimination by hsp65 PRA band sizes. This combined method takes around 2-3 days to complete in the laboratory once clinical isolates have been received. However, the identification algorithm has some limitations. First, it could not discriminate M. intermedium type 1 from M. intracellulare type 3, and second, not every hospital laboratory will be equipped with the appropriate equipment for this method.

Conclusion
In conclusion, the novel flow chart and identification algorithm obtained by combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can easily differentiate MTC from NTM and identify mycobacterial species to the sub-type level, which is helpful for clinical management. The results are complementary to 16 S rDNA sequencing, and the effective algorithm provides rapid and accurate mycobacterial species identification.

Mycobacterial isolates
Fourteen mycobacterial reference strains including one MTC and 13 NTM strains and 376 clinical respiratory specimens, including sputum, broncho-alveolar lavage, and aspirated secretion from endotracheal tubes, were collected from January to July 2007 from Taichung Veterans General Hospital (Taichung, Taiwan). The respiratory specimens were digested by a N-acetyl-L-cysteine-NaOH decontamination procedure, centrifugal concentration, and sputum dissolving agents [31]. The processed specimens or the concentrated specimens were inoculated into MGIT culture tubes and incubated in the BACTEC 960 instrument at 37°C until a positive signal appeared. Positive BACTEC cultures were smeared on glass slides and Kinyoun staining was used to screen for AFB [31]. Mycobacteria in the positive BACTEC cultures were isolated and identified by conventional methods [12,13] .

Identification of mycobacterial isolates by conventional methods
All 376 isolates from cultures in the AFB smear-positive BACTEC tubes were streaked on Löwenstein-Jensen (LJ) slants and incubated at 37°C under 5% CO 2 . Colonies on the LJ slants were used for species identification by conventional culture and biochemical methods [12,13]. These methods included growth rates, photoreactivity for pigment production, morphology in microcolonies on LJ slants, and biochemical tests, including nitrate reduction, arylsulfatase, Tween 80 hydrolysis, urease, semiquantitative catalase, tolerance to 5% NaCl and niacin production.

Genomic DNA extraction
Mycobacterial DNA was extracted from positive BACTEC cultures using a DTB specimen processing kit (Becton Dickinson, Franklin Lakes, NJ) according to the manufacturer's instructions [11].
For rpoB DPRA, the 136-bp DNA PCR amplicon was further digested with MspI and HaeIII after DPRA, and analyzed by electrophoresis on a 3% agarose gel (NuSieve 3:1 agarose, Cambrex) or CE (eGene). The rpoB restriction fragment length polymorphism (RFLP) patterns were compared to eight groups described by Kim et al. [10].
For hsp65 PRA, the 439-bp DNA hsp65 PCR amplicon was further digested with BstEII and HaeIII after completing hsp65 PCR, and analyzed by electrophoresis on a 3% agarose gel (NuSieve 3:1 agarose, Cambrex) or by CE (eGene). The sizes of the restriction fragment by hsp65 PRA were compared to those reported on the PRASITE database (http://app.chuv.ch/prasite/index.html). Thirteen ATCC NTM reference strains and one MTC reference strain were subjected to hsp65 PRA by CE (eGene).

eGene CE
The restriction fragment sizes were estimated by the naked eye or by eGene CE according to the manufacturer's instructions using size markers (100 bp to 3 kb) and alignment markers (15 bp to 3 kb).

Mycobacterial identification flow chart
The mycobacterial identification flow chart is shown in Figure 1.

S rDNA sequencing
The 16 S rDNA sequencing of mycobacterial DNA as the reference standard method for mycobacterial species identification was carried out using primer pair 8FPL (5' AGTTTGATCCTGGCTCAG 3') and 1492 (5'GGTT ACCTTGTTACGACT T 3') as described by Turenne et al. [32]. The species were identified by comparing the 16 S rDNA sequences with similar sequences from GenBank.