Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Introduction: The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods: The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results: Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identifi cation of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the fi nal diagnosis from the attending physician. Conclusions: Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifi able using conventional biochemical and phenotypic techniques.

The ability of nontuberculous mycobacteria (NTM) to cause disease is clearly described in the literature, and its importance is increasing progressively, with an increasing amount of various species being cultured and isolated in laboratories [1][2][3] .
In Brazil, the NTM species most frequently associated with lung disease are Mycobacterium kasasii and Mycobacterium avium.Other species, such as Mycobacterium xenopi, Mycobacterium malmoense, Mycobacterium lentiflavum, Mycobacterium abscessus and Mycobacterium szulgai, have occasionally been isolated in cultures 4.5 .In 2011 the National Agency for Sanitary Surveillance (ANVISA) published the number of cases of infections by rapidly growing mycobacteria (RGMs) obtained in the last eleven years in Brazil.
Data also suggest that this disease occurs in subjects undergoing invasive procedures, mostly surgical cases guided by video, where the instruments are subjected to high levels of sterilization in glutaraldehyde solution.From 1998 to 2009, there were 2,520 reported cases of postsurgical infections related to rapidly growing mycobacteria, including Mycobacterium fortuitum, Mycobacterium chelonae, M. abscessus and Mycobacterium massiliense, which were distributed predominantly in private hospitals throughout the country.Cases have been reported in 23 states of Brazil, with 97.8% of the cases concentrated within 10 states 6 .The diseases caused by NTMs are not compulsorily notifi able, except where the infection occurred during surgical procedures; however, several studies in Brazil have noted isolated strains of nontuberculous mycobacteria in pulmonary and extrapulmonary clinical samples from patients with suspected tuberculosis 4,7,8.The differential diagnosis between tuberculosis (TB) and diseases caused by NTMs is of great importance because the epidemiology, treatment and prognosis are different.Conventional diagnostics present numerous diffi culties as clinical symptoms are generally similar and non-specifi c smear testing has limited sensitivity and cannot differentiate the species of mycobacteria.
For many years, the identifi cation of mycobacteria was undertaken by gathering phenotypic results and biochemical test results of isolated species in culture medium 9,10 .In the last 20 or 30 years, with the increasing need to identify a greater number of species and also for early diagnosis, new methods

METHODS
have been developed to enable rapid and accurate identifi cation of mycobacterial species [10][11][12] .In Brazil, the PRA-hsp65 technique proposed by Telenti et al. 13 , and Devallois et al. 14 has enabled the identifi cation of several species of NTM, the results of which correlated well with those from biochemical identification [13][14][15][16][17] .Beyond the molecular techniques, the sequencing of the specifi c genes rpoB and hsp65 has become the gold standard for identifying mycobacteria 18,19 .Because of its high discriminatory power, this tool has been used by several groups in epidemiological studies to identify the species involved in human diseases and in outbreaks, and also for taxonomic reclassifi cation 20,21 .
Multiplex polymerase chain reaction (PCR) is a fast tool that allows the simultaneous amplifi cation of more than one sequence of target deoxyribonucleic acid (DNA) in a single reaction, saving time and reagents [22][23][24] .This system, which amplifies two or three different targets, can differentiate M. tuberculosis from the NTMs 25,26 .This molecular approach is used in identifying and differentiating microbes because it is able to supply a simple fi ngerprint of certain bacterial groups when compared to the standard profi les of referent strains.The correct choice of target sequences in the genome is one of the key criteria for detecting and identifying mycobacteria by PCR 27 .
Faced with the need to differentiate tuberculosis from other mycobacteria, the aim of this study was to evaluate a system based on multiplex PCR, already optimized by Poroca et al. 26 , for identifying mycobacterial species isolated from different clinical samples from patients diagnosed with pulmonary and extrapulmonary tuberculosis.This test, in addition to contributing to a rapid and accurate differential diagnosis between tuberculosis and other mycobacteria, will certainly assist in the early and appropriate therapeutic management of the patient.

Study population
Thirty-seven male and female patients over the age of 12 years with pulmonary and extrapulmonary tuberculosis diagnoses, who were referred to public clinics in the metropolitan region of Recife of northeastern Brazil between March 2008 and December 2009, were chosen for this study.The types of biological samples collected were chosen based on the different clinical forms of the disease.The diagnosis of tuberculosis was made by the attending physician in accordance with the standards of the American Thoracic Society (ATS), in 2007, based on the isolation of Mycobacterium in culture from different samples and visualization of acid fast bacilli (AFB) in the smear microscopy 28 .

Smear microscopy
The presence of acid fast bacilli in different clinical specimens was determined by Ziehl-Neelsen staining (Stewart,  1953) in the Public Health Laboratory of Recife (Unifi ed Health System -SUS), in accordance with the guidelines of the Brazilian Ministry of Health 29 .

Specimen processing
In total, of 1-5 ml of various clinical specimens (except for the sterile samples collected) was processed using the modifi ed Petroff method in 4% NaOH 29,30 .

Culture
Cultures were performed in a Lowenstein-Jensen medium 29,31 and mycobacterium species were identifi ed using the following biochemical tests: selective inhibition by para-nitrobenzoic (PNB) acid and thiophene-2-carboxylic hydrazide (TCH) acid, niacin accumulation and heat-stable catalase at 68°C 29 .The cultures analyzed using the biochemical tests in this study were considered as references for comparison with the results obtained by multiplex PCR and PRA-hsp65.

DNA extraction
DNA was extracted and purifi ed from reference strains of Mycobacterium tuberculosis (H37Rv).In all, 40 species of mycobacteria were isolated on a Lowenstein-Jensen medium from clinical samples obtained from 37 patients diagnosed with pulmonary and extrapulmonary tuberculosis using the conventional method described by Sambrook et al. using a mixture of solvents and phenol chloroform 32 .

Polymerase chain reaction and restriction enzyme analysis
The polymerase chain reaction and restriction enzyme analysis (PRA-hsp65) were performed using the techniques described in Telenti et al. 13 The identifi cation was determined by comparing the sizes of the fragments with the algorithm described in the PRASITE site (http://app.chuv.ch/prasite/index.html) 17.

Multiplex polymerase chain reaction
In multiplex PCR, there are 3 pairs of primers, 1 pair for each target.The gene encoding the antigen of Mycobacterium tuberculosis 65KDa, the dnaJ gene and the insertion element IS6110 were used in a single reaction with a mixture containing 10mM Tris-HCl (pH 8.3); 50mM KCl; 2.5mM MgCl 2; 2mM dNTP; 2.5U of Taq DNA Polymerase (Invitrogen); and 20pmol, 50pmol and 10pmol of each oligonucleotide pair, respectively.A total of 2µL of DNA at a concentration of 20ng/µL for each strain of mycobacteria was added to the reaction mixture for

Species identifi cation using restriction enzyme analysis
When the mycobacterial species were identifi ed by analyzing the banding pattern obtained by the PRA-hsp65 technique, 29 (72.5%) of the species were identifi ed as the Mycobacterium tuberculosis complex; 4 (10%) of the species as Mycobacterium fortuitum 2; one (2.5%) of the species as Mycobacterium abscessus 2, Mycobacterium bolletii/Mycobacterium massiliense; one (2.5%) of the species as Mycobacterium gastritis 2/M.kansasii 6 and one (2.5%) of the species as Mycobacterium parmense type 1, whereas 4 (10%) of the results were inconclusive.None of the results indicated the presence of the M. avium complex or a fi nal volume of 50µL.These reaction parameters are in accordance with the conditions described by Poroca et al. 26 .

Statistical analysis
The value of Kappa was determined using SPSS statistical software for Windows (version 18; SPSS, Chicago, IL).

Ethical considerations
The research was approved by the Ethics Committee of the Aggeu Magalhães Research Center (CPqAM) and the respective ethics committees of the participating hospitals under protocol No. 39/2009.

Clinical, epidemiological and laboratory fi ndings
Of the 37 patients diagnosed with tuberculosis, 48.7% (n=18) of the cases originated from the ward, and 59.5% (n=22) were males aged 12 to 64 years (median 39 years).The clinical results showed that 70.3% (n=26) of the patients had pulmonary tuberculosis.The main clinical symptoms found were weight loss (90%), cough (80%) and fever (62.5%).Among the patients' pre-existing diseases, 24.3% (n=9) were infected with the human immunodefi ciency virus (HIV) virus and 13.5% (n=5) previously had tuberculosis.In laboratory tests, 82% had chest X-rays and 21.7% had the Mantoux tuberculin skin test.A smear of lung samples was taken from 73% (n =19) of the patients; 17 were sputum samples and 2 were bronchoalveolar lavages, and 94.4% (n=18) of the specimens lung specimens were positive for The presence of acid fast bacilli in different clinical specimens was determined by Ziehl-Neelsen staining (Stewart, 1953) in the Public Health Laboratory of Recife (Unified Health System -SUS), in accordance with the guidelines of the Brazilian Ministry of Health.Mycobacterial strains were isolated in Lowenstein-Jensen culture medium for the 40 biological specimens analyzed (Table 1).

Identifi cation of the mycobacterial species by culture and biochemical testing (reference test)
With respect to the reference test, of the 40 mycobacterial strains isolated by culture from the specimens obtained from the 37 patients diagnosed with tuberculosis, 57.5% (n=23) of the strains were identifi ed as belonging to the M. tuberculosis complex, 20% (n=8) of the strains were identifi ed as nontuberculous mycobacteria (NTM) and 22.5% (n=9) of the results were inconclusive.

Evaluation of multiplex PCR using mycobacterial species
Of the 40 mycobacterial isolates, 27 (67.5%)showed the molecular pattern for M. tuberculosis with the simultaneous amplifi cation of the three targets IS6110 (541bp), dnaJ (365bp) and hsp65 (165bp).In 13 of the samples (32.5%), amplifi cation of a fragment sized 20 to 40 base pairs less than 165bp was observed, which characterized these samples as NTM, according to Poroca et al. 26 (Figure 1).M. intracellulare among the strains isolated via culture from the pulmonary and extrapulmonary tuberculosis samples.

Results of the multiplex PCR and PRA-hsp65 with strains whose results were inconclusive by the reference test (culture and biochemical testing)
In this study, 9 (22.5%) of the mycobacteria samples isolated in culture from the clinical specimens of patients diagnosed with tuberculosis could not be identifi ed by culture and biochemical tests; however, 5 (55.5%) of these strains were identifi ed as M. tuberculosis by multiplex PCR, and 4 (44.4%) were identifi ed as NTM.When using the PRA-hsp65, 6 (66.6%) strains were identifi ed as an M. tuberculosis complex, and 3 (33.3%)could not be identifi ed by PRA-hsp65 .The diagnosis of tuberculosis in these patients was performed by the medical health service based on smear and/or clinical symptoms.

Comparison of the reference test with multiplex PCR and PRA-hsp65
The results of the multiplex PCR and PRA-hsp65 were compared to the results of the reference test (culture and biochemical assays) and are summarized in Table 2.The correlation calculation between the two tests was conducted using the results from 30 strains identifi ed by culture and biochemical tests, as these results were not considered inconclusive in the reference tests and PRA-hsp65.

Data from subjects who had mycobacteria identifi ed as NTM by multiplex PCR and PRA-hsp65
We found that 7 (17.5%)strains of mycobacteria were identified as NTM using multiplex PCR and PRA-hsp65 in 6 individuals who were diagnosed with pulmonary or extrapulmonary tuberculosis.Among those individuals, only one subject had two different isolates derived from clinical urine (non-sterile) and pleural fl uid (sterile) specimens.The other 5 patients had only one species of NTM isolated in culture from the clinical urine, sputum or BAL specimens.None of these cases were diagnosed as an NTM infection.

DISCUSSION
Advances in molecular biology techniques and the information provided by sequencing the complete genome of M. tuberculosis prompted the development of new tools for the rapid diagnosis of tuberculosis, including the differentiation of M. tuberculosis from other mycobacteria 33,34 .Multiplex PCR has the ability to amplify different targets simultaneously and has been used to detect and identify mycobacteria from the Mycobacterium tuberculosis complex and nontuberculous mycobacteria 26,27,35,36 .
In this study, when the multiplex PCR was compared with the reference tests (cultures and phenotypic and biochemical tests) to identify 40 mycobacterial isolates, a signifi cant agreement was observed: 22 (100%) of the 37 cases with a fi nal diagnoses of pulmonary or extrapulmonary tuberculosis presented species of the Mycobacterium tuberculosis complex (MTBC) using the reference tests.Multiplex PCR identifi ed the presence of the M. tuberculosis complex in 27 (67.5%) of the cases.In 13 (32.5%) of the cases, multiplex PCR showed a molecular pattern consistent with an NTM, thus disagreeing with the fi nal diagnosis made by the attending physician.It is important to be cautious when making a differential diagnosis between tuberculosis and other mycobacteria because the clinical symptoms and X-ray images may be similar.In Pernambuco in 2010, approximately 26.7% of the patients were treated without bacteriological confi rmation of pulmonary tuberculosis; the diagnosis was based only on clinical and X-ray fi ndings, which are often inconclusive and provide inferior data relative to those obtained in this study 37 .
The results of the PRA-hsp65 technique, when compared with the reference tests, showed the same degree of agreement obtained with the multiplex PCR for the Mycobacterium tuberculosis complex and NTM.It is notable that the multiplex PCR, because of its ability to amplify three different, specifi c targets in the same reaction, has the ability to identify the Mycobacterium tuberculosis complex and differentiate it from the species M. tuberculosis, M. bovis and M. avium 26,27 .In contrast, although PRA-hsp65 does not differentiate between the species of the Mycobacterium tuberculosis complex, it is able to identify most of the NTM species 5,38 .This tool is recommended when the culture and phenotypic and biochemical tests suggest the presence of NTM species, but it is costly and time-intensive 5 .
During this study, 3 patients diagnosed with pulmonary tuberculosis were beginning their second dose of anti-TB treatment when the species of mycobacteria was identifi ed as NTM by multiplex PCR.In 2 cases, only 1 isolation from a lung sample was obtained, and in one of the cases, the species was identifi ed as M. gastritis 2/M.kansassi 6 using PRA-hsp65.The presence of NTM in a single sample from a non-sterile source requires careful investigation, including requests for new samples to eliminate the possibility of contamination or transitory colonization 11,39 .In this study, no patients were diagnosed by the health service professionals as infected with NTM; therefore, there is a need for patient follow-up using a new collection of biological specimens, as the diagnosis may be suspect.The present study showed that, despite the high prevalence of tuberculosis in Brazil, the presence of NTM should strongly be considered prior to growing mycobacterial cultures in sterile clinical samples as part of the investigation when patients have symptoms such as an undefi ned fever.
The fi ndings in this study suggest the need to investigate the presence of NTM in diagnosed lung diseases, following from the recommendations of other studies, which showed that NTM species can be detected in cases where TB is being re-treated 7,8 .The most frequently isolated species belong to the M. avium (MAC) complex and M. kansassi 40,41 .Ueki et al. 11 demonstrated that the most prevalent species in the lung compartment was M. kansassi and MAC in disseminated diseases 11 .For the treatment of pulmonary NTM infections, laboratory support is needed both to identify species and to determine the in vitro profi le of resistance to antimicrobial agents.M. kansasii remains the most easily treatable form of pulmonary infections; there is a strong correlation between the in vitro susceptibility and the in vivo response to rifampicin, macrolides and fl uoroquinolones.The greatest risk is the emergence of drug-resistant strains, similar to what is observed with M. tuberculosis 42 , when patients fail to follow the correct treatment regimen.
Among the cases diagnosed as extrapulmonary tuberculosis, we isolated the same species of mycobacteria (M.fortuitum 2) in Lowenstein Jensen (LJ) culture medium from samples of urine and pleural fl uid, where the bacteriological diagnosis for NTM can be defi ned according to ATS criteria because the pleural fl uid is considered a sterile sample 28 .The M. fortuitum 2 species were identifi ed by phenotypic and biochemical tests, multiplex PCR and PRA-hsp65.M. fortuitum is a rapidly growing mycobacterium.Although its pathogenic potential is very low, it is often acquired in hospitals and can infect immunocompromised patients.One patient was HIV-positive and died as soon as he began treatment for tuberculosis, which indicates the increasing clinical importance of identifying mycobacterial species 43,44 .Several studies have demonstrate the importance of M. fortuitum infections in immunocompromised patients and found that the most prevalent species in these cases include the Mycobacterium avium complex, Mycobacterium kansasii and Mycobacterium fortuitum 10,11,45 .
It can therefore be concluded that multiplex PCR has the ability to identify and differentiate species of M. tuberculosis and NTMs and thus can be used as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by NTMs.However, multiplex PCR should not be used as a sole laboratory method; it is important that the differential diagnosis be based on the joint analysis of various parameters using PRA-hsp65 tests and the sequencing of specifi c genes that may identify the NTM species.
The speed and accuracy of multiplex PCR to differentiate between the M. tuberculosis complex and NTMs enables it to be used as an alternative to phenotypic and biochemical tests when identifying species of the M. tuberculosis complex and when screening to determine the NTM species present by sequencing specifi c genes.
Thus, molecular methods (multiplex PCR, PRA-hsp65 and sequencing of specifi c genes) can support diagnoses based on clinical determinations regarding mycobacterial infections, and the implementation of these methods, particularly in reference to services for those patients with tuberculosis (TB), Multidrug-resistant tuberculosis (MDR-TB) and Human Immunodeficiency Virus/ Acquired Immunodeficiency Syndrome (HIV/AIDS), reduces the possibility of an inadequate diagnosis and treatment.

FIGURE 1 -
FIGURE 1 -Multiplex PCR of mycobacterial isolates from patients diagnosed with tuberculosis.Bands 1, 2, 5 and 7 represent the mycobacterial species displaying the molecular pattern of M. tuberculosis; Bands 3, 4, 6 and 8 represent the mycobacterial species displaying the molecular pattern of NTM; Bands 9 and 10 represent genomic DNA for M. tuberculosis (H37Rv); Band 11 represents a negative control for the reaction; Band M represents a 50bp DNA ladder (New England BioLabs, Hitchin, Hertfordshire, UK).

TABLE 2 -
Concordance between the results of multiplex PCR and PRA-hsp65 with the biochemical and phenotypic tests considered for 30 different isolates of mycobacteria differentiated by phenotypic and biochemical tests.