Tuberculosis (TB) is the leading cause of death worldwide from a single infectious agent. An ability to detect the Mycobacterium tuberculosis complex (MTC) in clinical material while simultaneously differentiating its members is considered important. This allows for the gathering of epidemiological information pertaining to the prevalence, transmission and geographical distribution of the MTC, including those MTC members associated with zoonotic TB infection in humans. Also differentiating between members of the MTC provides the clinician with inherent MTC specific drug susceptibility profiles to guide appropriate chemotherapy.
The aim of this study was to develop a multiplex real-time PCR assay using novel molecular targets to identify and differentiate between the phylogenetically closely related M. bovis, M. bovis BCG and M. caprae. The lpqT gene was explored for the collective identification of M. bovis, M. bovis BCG and M. caprae, the lepA gene was targeted for the specific identification of M. caprae and a Region of Difference 1 (RD1) assay was incorporated in the test to differentiate M. bovis BCG. The multiplex real-time PCR assay was evaluated on 133 bacterial strains and was determined to be 100% specific for the members of the MTC targeted.
The multiplex real-time PCR assay developed in this study is the first assay described for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. Future validation of this multiplex assay should demonstrate its potential in the rapid and accurate diagnosis of TB caused by these three mycobacteria. Furthermore, the developed assay may be used in conjunction with a recently described multiplex real-time PCR assay for identification of the MTC and simultaneous differentiation of M. tuberculosis, M. canettii resulting in an ability to differentiate five of the eight members of the MTC.
Citation: Reddington K, O'Grady J, Dorai-Raj S, Niemann S, van Soolingen D, Barry T (2011) A Novel Multiplex Real-Time PCR for the Identification of Mycobacteria Associated with Zoonotic Tuberculosis. PLoS ONE 6(8): e23481. https://doi.org/10.1371/journal.pone.0023481
Editor: Tanya Parish, Queen Mary University of London, United Kingdom
Received: May 30, 2011; Accepted: July 18, 2011; Published: August 9, 2011
Copyright: © 2011 Reddington et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: College of Sciences, National University of Ireland Galway funded KR Postgraduate Scholarship (http://www.nuigalway.ie/science/scholarshipsandprizes.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Tuberculosis (TB) in humans is mostly caused by members of the Mycobacterium tuberculosis complex (MTC), although infections caused by nontuberculous mycobacteria (NTM) can mimic this disease . M. tuberculosis is considered to be the primary pathogen of TB in humans . Other members of the MTC associated with human TB infection include M. canettii and M. africanum, with the highest prevalence in various parts of Africa . The remaining members of the MTC including M. bovis, M. caprae, M. microti and M. pinnipedii are considered predominantly animal pathogens, apart from the, for vaccination purposes, attenuated M. bovis BCG strain. However, all members of the MTC have been shown to cause human TB infection . For example, the major etiological agents of zoonotic TB in humans are the phylogenetically related M. bovis and M. caprae . These members of the MTC occur worldwide and there are indications which suggest the true prevalence of zoonotic human TB infection may be underrepresented, especially because of the extrapulmonary manifestation of the disease generally caused by these bacteria , , . In developed countries it has been suggested that the burden of bovine TB in humans ranges from 0.5 to 7.2% of TB cases, while in developing countries, where very little data is available, this figure may be as high as 15% , .
The diagnosis of TB in humans in high prevalence setting usually still relies on smear microscopy, although this is insensitive, especially in patients co-infected with HIV , .The gold standard culture method is both time consuming and laborious . Therefore, in vitro nucleic acid amplification tests (NAATs) offer a rapid and sensitive alternative to traditional methods for the detection of TB infection, with an increasing number of commercially available TB diagnostics kits becoming available , , , in addition to NAAT methods described in the literature , . However, most of these rapid and traditional diagnostics methods detect the presence of the MTC but do not identify the specific etiological agent of infection.
Since the introduction of pasteurisation and bovine TB eradication programmes in developed countries there has been a significant reduction in the number of zoonotic TB cases in humans , with the majority of such cases occurring as a result of endogenous reactivation of a previous infection , . However, recent reports have identified TB in humans caused by M. bovis in countries officially free from bovine TB  and suggest that the true prevalence of zoonotic TB may be underestimated clinically . In addition, recent reports show that M. bovis infection in younger patient groups in the Western world are still common, most likely owing to consumption of raw milk (products) in the country of origin . Moreover, zoonotic TB remains a significant threat to human health in developing countries where its prevalence is currently unknown, as differentiation between the members of the MTC is not routinely performed , .
The accurate differentiation of the MTC is also clinically important, due to varying natural resistances of members of the MTC to anti-TB drugs. . As such, the ability to differentiate between members of the MTC could potentially allow for the monitoring of zoonotic exposure leading to human TB infection, in addition to providing accurate epidemiological information for the clinician, guiding contact tracing and source case finding.
In this study, we report the design, development and testing of a multiplex real-time PCR assay, using novel nucleic acid molecular targets, to identify and simultaneously differentiate between M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. This assay can be used independently, or in combination with, a recently described multiplex PCR assay for the identification of the MTC, M. tuberculosis and M. canettii .
Materials and Methods
Diagnostics target identification
The diagnostics target genes used in this study were identified using a number of experimental and bioinformatics approaches. Potential target regions were identified using the Mycobacterial Genome Divergence Database (MGDD) (http://mirna.jnu.ac.in/mgdd/), which allowed for identification of insertions, deletions and single nucleotide polymorphisms between M. tuberculosis, M. bovis and M. bovis BCG. Other potential target regions were identified using the web based version of the Artemis comparison tool, WebACT (http://www.webact.org/WebACT/home). Nucleotide sequence information for M. africanum, M. microti and M. canettii was retrieved from the Welcome Trust Sanger Institute website (www.sanger.ac.uk/resources/downloads/bacteria/mycobacterium- currently being sequenced and annotated) to determine, in silico, if the candidate diagnostics target sequences for M. bovis identification were specific. As there was no nucleotide sequence information available for M. caprae or M. pinnipedii at the time of this study, specificity of the developed assays against these members of the MTC was determined empirically and further validated by nucleotide sequencing.
Since the diagnostics target used in this study for the differentiation of M. bovis from the remaining members of the MTC was experimentally determined to also identify M. caprae and M. bovis BCG, alternative nucleic acid diagnostics targets were required to differentiate this sub-group of the MTC.
For each putative diagnostics target identified, alignments were performed using clustalW multiple sequence alignment programme (http://www.ebi.ac.uk/Tools/clustalw2/index.html), from which real-time PCR primers and probes were designed (Table 1).
Bacterial strains, culture media and growth conditions
Seventy two MTC isolates (26 M. tuberculosis, 14 M. bovis, 7 M. bovis BCG, 5 M. canettii, 5 M. caprae, 5 M. africanum, 5 M. microti and 5 M. pinnipedii), 44 non tuberculosis mycobacteria (NTM) and 17 other bacterial species were used in this study (supplementary Table S1 and Table S2). Of the 72 MTC isolates, 36 strains previously characterised by a variety of methods , , , ,  were provided by The National Institute for Public Health and the Environment (RIVM), the Netherlands. Three M. caprae, 3 M. bovis and 2 M. pinnipedii strains were supplied by the National Reference Centre for Mycobacteria, Borstel, Germany and were previously characterised using a variety of methods , . All other MTC strains, provided by Professor Mario Vaneechoutte (University of Ghent, Ghent, Belgium), were clinical isolates collected over a ten year period from reference laboratories in Belgium and the Netherlands. These isolates were characterised on the basis of custom techniques available at the time. Twenty eight NTM were purchased from the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ) and grown on Middlebook agar/broth at the appropriate temperature (either 30°C or 37°C). Fast growing mycobacteria were cultured for 3-6 days and slow growing mycobacteria were incubated for six weeks, or until sufficient growth was visible. All media were purchased from BD Biosciences (Oxford, United Kingdom). DNA was supplied from the 16 remaining NTM and 4 of the 5 Norcardia strains by Professor Vaneechoutte and these species were characterised using techniques previously described , , .
DNA isolation and quantification
Genomic DNA from 28 NTM and 2 M. bovis BCG cultures was isolated from 1 ml of culture (Middlebrook 7H9 broth, Becton Dickinson), using a technique previously described in the literature . Total genomic DNA samples provided by the RIVM were extracted according to Van Soolingen et al. , whereas total genomic DNA samples from the National Reference Centre for Mycobacteria, Borstel were extracted as described by van Embden et al. , while genomic DNA provided by Professor Vaneechoutte was extracted as described by De Baere et al. . For all other bacterial species tested, DNA was provided from stocks held within this laboratory (Microbiology, NUIG). DNA concentrations for all species and strains used in this study were determined using the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Eugene, Oregon, USA) and the TBS-380 mini-fluorometer (Invitrogen Corporation, California, USA). DNA samples were stored at −20°C before use.
Conventional and real-time PCR primers and hydrolysis probe design
Oligonucleotide primers and hydrolysis probes were designed in accordance with general recommendations and guidelines , , following alignments of each of the nucleic acid diagnostics target genes identified in this study. Primers and probes (Table 1) were supplied by MWG-BIOTECH AG (Essenberg, Germany) or TIB MOLBIOL (Berlin, Germany). Primers were designed to have a melting temperature (Tm) of 58–61°C and the probes were designed to have a Tm 4–7°C higher. The probe designed to identify M. bovis, M. bovis BCG and M. caprae had a higher Tm (69.8°C) due to a particularly G/C rich target region. Hydrolysis probes were designed to be specific for each target following published design guidelines . Assay oligonucleotides were designed with similar properties such that monoplex assays could be easily multiplexed after specificity and sensitivity testing was complete .
The M. caprae specific assay PCR primers, MTC_Fw (position 618–634 bp of the lepA gene of M. tuberculosis H37Rv) and MTC_Rv (position 754–772 bp), were designed to amplify a 155 bp fragment of the lepA gene. lpqT_FW and lpqT_RV were designed to amplify a 141 bp fragment of the lpqT gene for the identification of M. bovis, M. bovis BCG and M. caprae (positions 17–34 bp and 146–162 bp of the M. tuberculosis H37RV lpqT gene). The RD1 assay was designed to amplify a 117 bp region of the Rv3876 gene, a conserved hypothetical protein, part of RD1, absent in all M. bovis BCG strains. The RD1_Fw primer was located at position 1416–1433 bp and the reverse primer RD1_Rv between 1516–1532 bp of the M. tuberculosis H37 Rv 3876 gene. The internal amplification control (IAC) PCR primers, IAC_Fw and IAC_Rv, were designed to amplify a 157 bp region of the M. smegmatis MSMEG_0660 gene. The IAC_Fw primer was located at positions 497–513 bp and the reverse primer between positions 636–653 bp.
All real-time PCR assays were initially tested for in a monoplex format, evaluating their specificity and sensitivity using probes labelled with FAM and Black Hole Quencher 1 (BHQ1). After optimisation of the monoplex real-time PCR assays, three of the four assay probes were labelled with different fluorescent dyes to allow for incorporation into a multiplex real-time PCR format. The M. caprae specific probe was labelled with cyan500 and BBQ, the M. bovis/M. bovis BCG/M. caprae specific probe with FAM and BHQ1, the M. bovis/M. caprae probe with HEX and BHQ2 and the IAC probe with Cy5 and BHQ2.
Conventional PCR was performed using the sequencing primers outlined in Table 1 on the iCycler iQ thermal cycler (Bio-Rad Laboratories Inc., California, USA). All reactions were carried out in a final volume of 50 µl, containing 5 µl 10× buffer (15 mM MgCl2), 1 µl forward and reverse primers (0.2 µM final conc.), 2 µl Taq DNA polymerase (1 U/µl, Roche Diagnostics, Basel, Switzerland), 1 µl dNTP mix [(10 mM deoxynucleoside triphosphate set (Roche Diagnostics)], 2 µl of template DNA and 38 µl nuclease free water (Applied Biosystems/Ambion, Texas, USA). The cycling parameters consisted of initial denaturation at 95°C for 5 mins, followed by 35 cycles of denaturation at 95°C (1 min), annealing at 50°C (1 min), and extension at 72°C (1 min), with a final elongation step at 72°C for 10 min.
Nucleotide sequence data for real-time PCR assay design was generated in this study or was obtained from the National Centre for Biotechnology Information (NCBI- http://www.ncbi.nlm.nih.gov/), or the Sanger website (where partial nucleotide sequences for M. canettii, M. africanum and M. microti were available). Real-time PCR assay primers were used to generate nucleotide sequence information for each of the assays developed. Sequencing primers were also designed to span the full gene sequence of lpqT, lepA and a number of other genes to identify potential candidate nucleic acid diagnostics targets for the specific identification of M. caprae.
PCR products were generated according to section 2.5, purified using the High Pure PCR Product Purification Kit (Roche Diagnostics) and sequenced commercially (Sequiserve, Vaterstetten, Germany).
Development of an IAC for real-time PCR
A non-competitive IAC  was designed and incorporated into the multiplex real-time PCR. The M. smegmatis MSMEG_0660 gene was chosen as the target as it is specific to M. smegmatis, avoiding any possible cross reaction. Titration of IAC DNA was performed to determine the optimum concentration per reaction that could be reliably detected in the presence of MTC DNA, yet not inhibit detection of low concentrations of the primary assay targets. An IAC concentration of 100 genome equivalents per reaction was determined as optimum.
Monoplex real-time PCR was performed on the LightCycler 2.0 Instrument (Roche Diagnostics) using the LightCycler® TaqMan® Master kit (Roche Diagnostics). Each reaction contained 5× master mix, forward and reverse primers (0.5 µM final conc.), FAM labelled probe (0.2 µM final conc.), template DNA (2 µl) and nuclease free dH2O to a final volume of 20 µl. The cycling parameters consisted of 10 min incubation at 95°C to activate the Taq, 50 cycles of 95°C for 10 s and 60°C for 30 s, followed by a cooling step at 40°C for 10 s. The temperature transition rate for all cycling steps was 20°C/s.
Multiplex real-time PCR reactions were carried out on the LightCycler 480 using LightCycler® 480 Probes Master kit (Roche Diagnostics). The optimised PCR mix contained 2× LightCycler 480 Probes Master (6.4 mM MgCl2), forward and reverse primer (0.5 µM final conc.), Cyan500, FAM, HEX and CY5 labelled probes (0.2 µM final conc.), dimethyl sulfoxide (4%, Sigma-Aldrich Missouri, USA), template DNA (MTC: 2 µl; IAC: 2 µl; NTM: 10 µl) adjusted to a final volume of 40 µl with the addition of nuclease free dH2O. The M. smegmatis internal control DNA was diluted to contain 100 genome equivalents per 2 µl and NTM DNA was diluted to contain ∼104 genome equivalents per 10 µl.
The cycling parameters used were the same as those used on the LightCycler 2.0. The temperature transition rate, referred to as the ramp rate on the LightCycler 480 was 4.4°C/s while heating and 2.2°C/s while cooling. Prior to experimental analysis on the LightCycler 480, a colour compensation file was generated, to avoid fluorescence leaking from channel to channel, using the technical note outlined in the Advanced Software Functionalities of the operator manual .
Diagnostics target identification
The diagnostics target used for the specific identification of M. caprae was the lepA (Rv 2404c) gene. In-silico analysis of nucleotide sequences generated in this study revealed a single nucleotide polymorphorism (SNP) at position 690 bp of the M. tuberculosis H37Rv lepA gene which was specific for M. caprae. LepA is an essential bacterial elongation factor. The lepA gene codes for a highly conserved protein present in all bacteria sequenced to date, with a homologue (Guf1) found in higher organisms . The M. caprae specific SNP, identified from nucleotide sequence data generated in this study, was a C to T substitution which was conserved in all 5 strains of M. caprae tested.
The diagnostics target identified in this study for collective identification of M. bovis, M. bovis BCG and M. caprae was the lpqT (Rv1016c) gene. LpqT belongs to a group of lipoproteins which are present in all bacteria  and is thought to be required for optimal growth of mycobacteria in vivo . In-silico analysis of publicly available lpqT nucleotide sequences, performed in this study, revealed a 5 bp region of lpqT, deleted in M. bovis and M. bovis BCG but present in M. tuberculosis, M. canettii, M. africanum and M. microti. Sequence information was generated for the lpqT gene in M. caprae and M. pinnipedii which confirmed the deletion was also present in M. caprae but not M. pinnipedii.
The diagnostics target used to differentiate M. bovis BCG from M. bovis and M. caprae was the Rv3876 gene, which is part of Region of Difference 1 (RD1). Regions of Difference (RD) in the Mycobacterium tuberculosis complex refer to regions of the genome present in M. tuberculosis but deleted from M. bovis BCG . The particular RD targeted in this study (RD1), has been shown to be deleted in all M. bovis BCG strains and has been linked to the attenuation of this strain . Any region of RD1 could potentially be incorporated into this multiplex real-time PCR and used with the novel diagnostics targets described above.
Assay design and development
While the guidelines for primer and probe design were adhered to as closely as possible, the high G/C content (60–65%) of the Mycobacterium species had an impact on assay design. The lpqT specific probe was designed spanning the deletion junction of a region deleted in M. bovis, M. bovis BCG and M. caprae and present in the other members of the MTC, that was very G/C rich, making probe design difficult. The lpqT probe, therefore, had a relatively high Tm, however this did not impact on assay performance. The M. caprae specific probe targeted an SNP in the lepA gene. Avoiding cross reaction of the M. caprae probe with other members of the MTC proved challenging. A number of probes were designed and tested and the optimum probe was chosen empirically based on specificity and sensitivity results. The optimum probe was designed complementary to the + strand of the lepA gene as the resulting G/A mismatch, that occurred in the presence of non-target MTC DNA, was more destabilising to the probe than the C/T mismatch, hence improving specificity. The probe was designed to have a Tm of 60.1°C, only slightly above the annealing temperature of the assay (60°C), allowing the probe to hybridise to exactly matched sequence only, therefore maximising the specificity effect of the SNP. This did, however, slightly reduce probe binding efficiency, leading to a small reduction in sensitivity.
Internal amplification control
In order for a result to be considered valid using this assay, a positive signal must be obtained in the Cy5 detection channel on the LightCycler 480. If the IAC is not detected, the result is considered invalid and must be repeated , . For the purposes of this study, M. smegmatis DNA was spiked into the PCR master mix to act as an internal control target. Alternatively, M. smegmatis cultured cells could be used as a process control, spiked into patient samples before genomic DNA isolation. In this design, the M. smegmatis cells would serve as both an extraction control and an amplification control and could be used when testing patient samples.
Specificity of the diagnostic assays
The specificity of each real-time PCR assay was confirmed both in monoplex and multiplex formats using the specificity panel listed in supplementary Table S1 and Table S2. The lepA assay was specific for the detection of the 5 M. caprae strains in our collection (Figure 1 A). The remaining members of the MTC, NTM and other bacteria tested were not detected. The M. bovis, M. bovis BCG and M. caprae assay was also specific, detecting only target species (Figure 1 B). The diagnostics assay targeting RD1 did not detect any of the 7 M. bovis BCG strains tested for (Figure 1 C) but detected all M. bovis and M. caprae strains tested for, allowing for the specific identification of M. bovis BCG. All remaining members of the MTC (with the exception of the 5 M. microti and one M. canettii) were detected. None of the NTM or other bacteria on the specificity panel were detected using the RD1 assay.
Figure 1 A. Real-time amplification curves for M. caprae (circle) using lepA gene in Cyan500 channel (450–500). Figure 1 B Amplification curves for M. caprae (circle), M. bovis (triangles) and M. bovis BCG (diamonds) using the lpqT gene in FAM channel (483–533). Figure 1 C Amplification curves for all members of the MTC with the exception of M. bovis BCG (diamonds) and M. microti in the HEX channel (523–568). Figure 1 D Amplification curves for IAC in Cy5 channel (615–670) with the no template control highlighted with stars through amplification curve.
The IAC assay targeted the M. smegmatis MSMEG_0660 gene. This assay was tested against all members of the MTC and NTM and was specific for the detection of M. smegmatis. One hundred genome equivalents of M. smegmatis DNA was added to all samples tested, to control for PCR inhibition, and was detected in all samples (Figure 1 D). Table 2 describes how to interpret the results of the multiplex real-time PCR assay.
Sensitivity of the assays
The limit of detection (LOD) of each assay was evaluated in a monoplex real-time PCR format. Genomic DNA was quantified and serial dilutions were prepared from 200,000 to 2 genome equivalents (with an estimated genome size of 4.5 million base pairs, one M. caprae cell contains approximately 5.0 fg DNA ). Assay sensitivity testing was performed using M. caprae DNA as it is detected in all of the multiplex assays.
In a monoplex format, the dilution series was run in duplicate and a sensitivity of 2- 20 M. caprae genome equivalents was determined for each assay. In multiplex format the lower limit of detection was established using probit regression analysis. Twelve replicates of each of 20, 15, 12, 10, 7.5, 4, 2 and 0.2 M. caprae genome equivalents were tested. LOD's of 3.95 and 3.55 genome equivalents were determined (95% probability) for the M. bovis/M. bovis BCG/M. caprae and the M. bovis/M. caprae assays respectively. However, these dilutions were not appropriate to determine the M. caprae specific assay LOD. The analysis was repeated using concentrations of 200, 100, 75, 50, 25, 15, 10 and 5 genome equivalents of M. caprae DNA. An LOD of 14.77 genome equivalents (95% probability) was determined for the M. caprae specific assay when tested as part of the multiplex. The IAC, at a concentration of 100 genome equivalents per reaction, was included in all samples during sensitivity testing and was detected as expected.
The WHO  defines zoonoses as infections and diseases which can be transmitted naturally from animals to humans. A recent WHO report highlights the difficulties in the diagnosis of such diseases and suggests that the true incidence of many neglected zoonoses, including zoonotic TB, may be greatly underestimated . Zoonotic TB in humans is most commonly caused by M. bovis and to a lesser extent M. caprae (prevalent in certain regions in Europe ). Human TB infection caused by the zoonotic transmission of M. microti and M. pinnipedii has also been reported , , . While bovine TB in humans is considered rare in developed countries, it has been described as an ‘endemic neglected zoonoses’ in developing countries . In these settings, the true incidence of zoonotic TB in humans remains largely unknown due to inabilities in hospital laboratories to distinguish between the causative agent of infection, a scarcity of trained personnel, financial constraints in addition to limited control measures in place , .
M. bovis is the predominant member of the MTC associated with bovine TB infection. However, it also has been isolated from and is the causative agent of TB in a wide range of mammalian hosts including humans, goats, dogs, deer and ferrets . Transmission to and infection of human hosts with M. bovis can result from zoonotic exposure to TB infected animals or to consumption of unpasteurised dairy products . Recent studies have also shown that human to human transmission of M. bovis can also occur, but is considered rare .
Since its transfer from M. tuberculosis subsp caprae to M. bovis subsp caprae in 2002 , this member of the MTC has been recognised as the cause of a number of outbreaks of TB in animals such as goats, cattle and red deer . In addition, M. caprae has been associated with outbreaks of TB in humans in a number of European countries , ,  and was shown to be the true causative agent of one third of human M. bovis associated TB cases in Germany between 1999–2001 .
M. bovis BCG, the commonly used vaccine strain, has also been implicated in human disease particularly in immunocompromised patients, such as HIV positive and cancer patients, where it has been shown to cause disseminated disease , and in children who have been vaccinated in endemic TB areas .
While the members of the MTC discussed above share many phenotypic characteristics, in addition to natural host preference, M. bovis, M. bovis BCG and M. caprae exhibit a differential intrinsic susceptibility profile to pyrazinamide (PZA), an important first line anti-TB drug responsible for reducing patient treatment time . M. bovis and M. bovis BCG are intrinsically resistant to PZA whereas M. caprae is sensitive to PZA . This highlights the need for a rapid test that can detect the MTC and differentiate between its members so that the clinician can choose the appropriate chemotherapy for the patient. More accurate diagnosis and treatment will protect against the development of drug resistant TB and reduce morbidity and mortality . Furthermore, accurate identification of these organisms allows monitoring of these neglected members of the MTC with respect to prevalence, transmission and global distribution . Also, differentiation of the complex could contribute to the better understanding of the clinical relevance of these members of the MTC.
Currently, there is only one commercially available diagnostics kit for the differentiation of the MTC, the GenoType MTBC line probe assay (Hain Lifesciences). This assay differentiates members of the MTC utilising highly conserved SNP's in the gyrB gene , however it is limited as it does not differentiate M. canettii and M. pinnipedii. Literature describing NAAT's for the complete differentiation of the MTC , , ,  are hindered by the complexity of the multiple reactions required, the inability to differentiate between some members of the MTC, in addition to the complex interpretation of the results generated. While SNP's have commonly been used for accurate differentiation of members of the MTC ,  further validation of the SNP identified in this study for the specific detection of M. caprae is required.
The multiplex real-time PCR assay described in this study is the first description of a real-time PCR diagnostics assay for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae using novel diagnostics targets in one internally controlled reaction. This diagnostics assay takes approximately one hour to perform after DNA extraction. The assay has been validated against a large panel of well characterised clinical isolates. In its current format, this multiplex real-time PCR assay would be applied to MTC clinical isolates for the identification of the most common causes of zoonotic TB. Monitoring bovine TB in humans is valuable for guiding public health policy and is important for the study of zoonotic TB epidemiology.
The developed assay also has the potential to be used directly in sputum samples following identification of an efficient sputum sample preparation method. If performing direct sputum testing, the assay developed in this study would be used as a follow on test in combination with a real-time PCR assay for identification of the MTC and differentiation of M. tuberculosis and M. canettii, which was previously developed by this group. More specifically, if a patient sample is identified as positive for the presence of the MTC but M. tuberculosis or M. canettii negative, the assay developed in this study can then be used to determine if sample contains M. bovis, M. bovis BCG or M. caprae, the most common causes of zoonotic TB in humans.
The study presented here focuses on accurately differentiating members of the MTC most commonly associated with zoonotic human TB infection. However, there is a need to differentiate between all members of the MTC to supply unambiguous epidemiological data as to the true prevalence of each member of the MTC causing TB infection . Work has begun in this group on the development of a final multiplex real-time PCR diagnostics assay for the accurate identification of M. africanum, M. microti and M. pinnipedii. This will complete a cassette of three multiplex real-time PCR diagnostics assays for the identification and differentiation of all members of the MTC.
Description of Mycobacterium tuberculosis complex isolates used in this study.
We thank Kristen Kremer (RIVM) and Michaela Projahn (Research center Borstel) for supplying genomic DNA samples for some MTC strains used in this study.
Conceived and designed the experiments: KR JO SDR TB. Performed the experiments: KR SDR. Analyzed the data: KR JO TB. Contributed reagents/materials/analysis tools: SN DvS TB. Wrote the paper: KR JO SDR SN DvS TB.
- 1. Gopinath K, Singh S (2010) Non-Tuberculous Mycobacteria in TB-Endemic Countries: Are We Neglecting the Danger? PLoS Negl Trop Dis 4: e615.K. GopinathS. Singh2010Non-Tuberculous Mycobacteria in TB-Endemic Countries: Are We Neglecting the Danger?PLoS Negl Trop Dis4e615
- 2. Das S, Das SC, Verma R (2007) Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis. Microbiol Immunol 51: 231–234.S. DasSC DasR. Verma2007Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis.Microbiol Immunol51231234
- 3. Djelouadji Z, Raoult D, Daffé M, Drancourt M (2008) A Single-Step Sequencing Method for the Identification of Mycobacterium tuberculosis Complex Species. PLoS Negl Trop Dis 2: e253.Z. DjelouadjiD. RaoultM. DafféM. Drancourt2008A Single-Step Sequencing Method for the Identification of Mycobacterium tuberculosis Complex Species.PLoS Negl Trop Dis2e253
- 4. Kjeldsen MK, Bek D, Rasmussen EM, Priemé A, Thomsen VØ (2009) Line probe assay for differentiation within Mycobacterium tuberculosis complex. Evaluation on clinical specimens and isolates including Mycobacterium pinnipedii. Scandinavian Journal of Infectious Diseases 41: 635–641.MK KjeldsenD. BekEM RasmussenA. PrieméVØ Thomsen2009Line probe assay for differentiation within Mycobacterium tuberculosis complex. Evaluation on clinical specimens and isolates including Mycobacterium pinnipedii.Scandinavian Journal of Infectious Diseases41635641
- 5. Niemann S, Richter E, Rusch-Gerdes S (2002) Biochemical and genetic evidence for the transfer of Mycobacterium tuberculosis subsp. caprae Aranaz et al. 1999 to the species Mycobacterium bovis Karlson and Lessel 1970 (Approved Lists 1980) as Mycobacterium bovis subsp. caprae comb. nov. Int J Syst Evol Microbiol 52: 433–436.S. NiemannE. RichterS. Rusch-Gerdes2002Biochemical and genetic evidence for the transfer of Mycobacterium tuberculosis subsp. caprae Aranaz et al. 1999 to the species Mycobacterium bovis Karlson and Lessel 1970 (Approved Lists 1980) as Mycobacterium bovis subsp. caprae comb. nov.Int J Syst Evol Microbiol52433436
- 6. Ojo O, Sheehan S, Corcoran GD, Okker M, Gover K, et al. (2008) Mycobacterium bovis Strains Causing Smear-Positive Human Tuberculosis, Southwest Ireland. Emerging Infectious Diseases: Centers for Disease Control & Prevention (CDC). pp. 1931–1934.O. OjoS. SheehanGD CorcoranM. OkkerK. Gover2008Mycobacterium bovis Strains Causing Smear-Positive Human Tuberculosis, Southwest Ireland.Emerging Infectious Diseases: Centers for Disease Control & Prevention (CDC)19311934
- 7. Cicero R, Olivera H, Hernández-Solis A, Ramírez-Casanova E, Escobar-Gutiérrez A (2009) Frequency of Mycobacterium bovis as an etiologic agent in extrapulmonary tuberculosis in HIV-positive and -negative Mexican patients. European Journal of Clinical Microbiology & Infectious Diseases 28: 455–460.R. CiceroH. OliveraA. Hernández-SolisE. Ramírez-CasanovaA. Escobar-Gutiérrez2009Frequency of Mycobacterium bovis as an etiologic agent in extrapulmonary tuberculosis in HIV-positive and -negative Mexican patients.European Journal of Clinical Microbiology & Infectious Diseases28455460
- 8. Allix-Béguec C, Fauville-Dufaux M, Stoffels K, Ommeslag D, Walravens K, et al. (2010) Importance of identifying Mycobacterium bovis as a causative agent of human tuberculosis. European Respiratory Journal 35: 692–694.C. Allix-BéguecM. Fauville-DufauxK. StoffelsD. OmmeslagK. Walravens2010Importance of identifying Mycobacterium bovis as a causative agent of human tuberculosis.European Respiratory Journal35692694
- 9. de la Rua-Domenech R (2006) Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis. Tuberculosis 86: 77–109.R. de la Rua-Domenech2006Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis.Tuberculosis8677109
- 10. Kubica T, Rusch-Gerdes S, Niemann S (2003) Mycobacterium bovis subsp. caprae Caused One-Third of Human M. bovis-Associated Tuberculosis Cases Reported in Germany between 1999 and 2001. J Clin Microbiol 41: 3070–3077.T. KubicaS. Rusch-GerdesS. Niemann2003Mycobacterium bovis subsp. caprae Caused One-Third of Human M. bovis-Associated Tuberculosis Cases Reported in Germany between 1999 and 2001.J Clin Microbiol4130703077
- 11. Steingart KR, Ng V, Henry M, Hopewell PC, Ramsay A, et al. (2006) Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. The Lancet Infectious Diseases 6: 664–674.KR SteingartV. NgM. HenryPC HopewellA. Ramsay2006Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review.The Lancet Infectious Diseases6664674
- 12. Reid MJA, Shah NS (2009) Approaches to tuberculosis screening and diagnosis in people with HIV in resource-limited settings. The Lancet Infectious Diseases 9: 173–184.MJA ReidNS Shah2009Approaches to tuberculosis screening and diagnosis in people with HIV in resource-limited settings.The Lancet Infectious Diseases9173184
- 13. Huard R, de Oliveira Lazzarini L, Butler W, van Soolingen D, Ho J (2003) PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis Complex on the basis of genomic deletions. J Clin Microbiol 41: 1637–1650.R. HuardL. de Oliveira LazzariniW. ButlerD. van SoolingenJ. Ho2003PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis Complex on the basis of genomic deletions.J Clin Microbiol4116371650
- 14. Blakemore R, Story E, Helb D, Kop J, Banada P, et al. (2010) Evaluation of the Analytical Performance of the Xpert(R) MTB/RIF Assay. J Clin Microbiol JCM.00128-00110.R. BlakemoreE. StoryD. HelbJ. KopP. Banada2010Evaluation of the Analytical Performance of the Xpert(R) MTB/RIF Assay.J Clin MicrobiolJCM.00128-00110
- 15. Coll P, Garrigó M, Moreno C, Martí N (2003) Routine use of Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) Test for detection of Mycobacterium tuberculosis with smear-positive and smear-negative specimens. The International Journal of Tuberculosis and Lung Disease 7: 886–891.P. CollM. GarrigóC. MorenoN. Martí2003Routine use of Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) Test for detection of Mycobacterium tuberculosis with smear-positive and smear-negative specimens.The International Journal of Tuberculosis and Lung Disease7886891
- 16. Hofmann-Thiel S, Turaev L, Hoffmann H (2010) Evaluation of the hyplex(R) TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples. BMC Microbiology 10: 95.S. Hofmann-ThielL. TuraevH. Hoffmann2010Evaluation of the hyplex(R) TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples.BMC Microbiology1095
- 17. Cleary TJ, Roudel G, Casillas O, Miller N (2003) Rapid and Specific Detection of Mycobacterium tuberculosis by Using the Smart Cycler Instrument and a Specific Fluorogenic Probe. J Clin Microbiol 41: 4783–4786.TJ ClearyG. RoudelO. CasillasN. Miller2003Rapid and Specific Detection of Mycobacterium tuberculosis by Using the Smart Cycler Instrument and a Specific Fluorogenic Probe.J Clin Microbiol4147834786
- 18. Richardson ET, Samson D, Banaei N (2009) Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex, Real-Time PCR. J Clin Microbiol 47: 1497–1502.ET RichardsonD. SamsonN. Banaei2009Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex, Real-Time PCR.J Clin Microbiol4714971502
- 19. Thoen C, LoBue P, de Kantor I (2006) The importance of Mycobacterium bovis as a zoonosis. Veterinary Microbiology 112: 339–345.C. ThoenP. LoBueI. de Kantor2006The importance of Mycobacterium bovis as a zoonosis.Veterinary Microbiology112339345
- 20. Majoor CJ, Magis-Escurra C, van Ingen J, Boeree MJ, van Soolingen D (2011) Epidemiology of Mycobacterium bovis disease in humans, the Netherlands, 1993–1997. Emerg Infect Dis 17: CJ MajoorC. Magis-EscurraJ. van IngenMJ BoereeD. van Soolingen2011Epidemiology of Mycobacterium bovis disease in humans, the Netherlands, 1993–1997.Emerg Infect Dis17
- 21. Cadmus S, Hill V, van Soolingen D, Rastogi N (2011) Spoligotype Profile of Mycobacterium tuberculosis Complex Strains from HIV-Positive and -Negative Patients in Nigeria: a Comparative Analysis. J Clin Microbiol 49: 220–226.S. CadmusV. HillD. van SoolingenN. Rastogi2011Spoligotype Profile of Mycobacterium tuberculosis Complex Strains from HIV-Positive and -Negative Patients in Nigeria: a Comparative Analysis.J Clin Microbiol49220226
- 22. Somoskovi A, Dormandy J, Mayrer AR, Carter M, Hooper N, et al. (2009) “Mycobacterium canettii” Isolated from a Human Immunodeficiency Virus-Positive Patient: First Case Recognized in the United States. J Clin Microbiol 47: 255–257.A. SomoskoviJ. DormandyAR MayrerM. CarterN. Hooper2009“Mycobacterium canettii” Isolated from a Human Immunodeficiency Virus-Positive Patient: First Case Recognized in the United States.J Clin Microbiol47255257
- 23. Reddington K, O'Grady J, Dorai-Raj S, Maher M, van Soolingen D, et al. (2011) Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains. J Clin Microbiol 49: 651–657.K. ReddingtonJ. O'GradyS. Dorai-RajM. MaherD. van Soolingen2011Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains.J Clin Microbiol49651657
- 24. Kremer K, van Soolingen D, Frothingham R, Haas WH, Hermans PWM, et al. (1999) Comparison of Methods Based on Different Molecular Epidemiological Markers for Typing of Mycobacterium tuberculosis Complex Strains: Interlaboratory Study of Discriminatory Power and Reproducibility. J Clin Microbiol 37: 2607–2618.K. KremerD. van SoolingenR. FrothinghamWH HaasPWM Hermans1999Comparison of Methods Based on Different Molecular Epidemiological Markers for Typing of Mycobacterium tuberculosis Complex Strains: Interlaboratory Study of Discriminatory Power and Reproducibility.J Clin Microbiol3726072618
- 25. Kremer K, Arnold C, Cataldi A, Gutierrez MC, Haas WH, et al. (2005) Discriminatory Power and Reproducibility of Novel DNA Typing Methods for Mycobacterium tuberculosis Complex Strains. J Clin Microbiol 43: 5628–5638.K. KremerC. ArnoldA. CataldiMC GutierrezWH Haas2005Discriminatory Power and Reproducibility of Novel DNA Typing Methods for Mycobacterium tuberculosis Complex Strains.J Clin Microbiol4356285638
- 26. van Soolingen D, Hermans PW, de Haas PE, van Embden JD (1992) Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG. J Clin Microbiol 30: 1772–1777.D. van SoolingenPW HermansPE de HaasJD van Embden1992Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.J Clin Microbiol3017721777
- 27. van Soolingen D, Hoogenboezem T, De Haas PEW, Hermans PWM, Koedam MA, et al. (1997) A Novel Pathogenic Taxon of the Mycobacterium tuberculosis Complex, Canetti: Characterization of an Exceptional Isolate from Africa. Int J Syst Bacteriol 47: 1236–1245.D. van SoolingenT. HoogenboezemPEW De HaasPWM HermansMA Koedam1997A Novel Pathogenic Taxon of the Mycobacterium tuberculosis Complex, Canetti: Characterization of an Exceptional Isolate from Africa.Int J Syst Bacteriol4712361245
- 28. van Soolingen D, van der Zanden AGM, de Haas PEW, Noordhoek GT, Kiers A, et al. (1998) Diagnosis of Mycobacterium microti Infections among Humans by Using Novel Genetic Markers. J Clin Microbiol 36: 1840–1845.D. van SoolingenAGM van der ZandenPEW de HaasGT NoordhoekA. Kiers1998Diagnosis of Mycobacterium microti Infections among Humans by Using Novel Genetic Markers.J Clin Microbiol3618401845
- 29. Allix-Beguec C, Harmsen D, Weniger T, Supply P, Niemann S (2008) Evaluation and Strategy for Use of MIRU-VNTRplus, a Multifunctional Database for Online Analysis of Genotyping Data and Phylogenetic Identification of Mycobacterium tuberculosis Complex Isolates. J Clin Microbiol 46: 2692–2699.C. Allix-BeguecD. HarmsenT. WenigerP. SupplyS. Niemann2008Evaluation and Strategy for Use of MIRU-VNTRplus, a Multifunctional Database for Online Analysis of Genotyping Data and Phylogenetic Identification of Mycobacterium tuberculosis Complex Isolates.J Clin Microbiol4626922699
- 30. Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rusch-Gerdes S, et al. (2006) Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis. J Clin Microbiol 44: 4498–4510.P. SupplyC. AllixS. LesjeanM. Cardoso-OelemannS. Rusch-Gerdes2006Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis.J Clin Microbiol4444984510
- 31. De Baere T, de Mendonca R, Claeys G, Verschraegen G, Mijs W, et al. (2002) Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory. BMC Microbiology 2: 4.T. De BaereR. de MendoncaG. ClaeysG. VerschraegenW. Mijs2002Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory.BMC Microbiology24
- 32. Vaneechoutte M, De Beenhouwer H, Claeys G, Verschraegen G, De Rouck A, et al. (1993) Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J Clin Microbiol 31: 2061–2065.M. VaneechoutteH. De BeenhouwerG. ClaeysG. VerschraegenA. De Rouck1993Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis.J Clin Microbiol3120612065
- 33. Wauters G, Avesani V, Charlier J, Janssens M, Vaneechoutte M, et al. (2005) Distribution of Nocardia Species in Clinical Samples and Their Routine Rapid Identification in the Laboratory. J Clin Microbiol 43: 2624–2628.G. WautersV. AvesaniJ. CharlierM. JanssensM. Vaneechoutte2005Distribution of Nocardia Species in Clinical Samples and Their Routine Rapid Identification in the Laboratory.J Clin Microbiol4326242628
- 34. Van Soolingen D, de Hass PEW, Kremer K (2001) Restriction fragment length polymorphism typing of mycobacteria, p. 165-203. In T. Parish and N. G. Stoker (ed.), Mycobacterium tuberculosis protocols. Humana Press Inc., Totowa NJ. D. Van SoolingenPEW de HassK. Kremer2001Restriction fragment length polymorphism typing of mycobacteria, p. 165-203.In T. Parish and N. G. Stoker (ed.), Mycobacterium tuberculosis protocols. Humana Press Inc., Totowa NJ
- 35. van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, et al. (1993) Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 31: 406–409.JD van EmbdenMD CaveJT CrawfordJW DaleKD Eisenach1993Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology.J Clin Microbiol31406409
- 36. Dorak MT (2006) In M. T. Dorak (ED.), Real-time PCR,. MT Dorak2006In M. T. Dorak (ED.), Real-time PCR,<http://www.dorak.info/genetics/realtime.html>. <http://www.dorak.info/genetics/realtime.html>.
- 37. Robertson JM, Walsh-Weller J (1998) An Introduction to PCR Primer Design and Optimization of Amplification Reactions. Forensic DNA Profiling Protocols. pp. 121–154.JM RobertsonJ. Walsh-Weller1998An Introduction to PCR Primer Design and Optimization of Amplification Reactions.Forensic DNA Profiling Protocols121154
- 38. Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, et al. (2004) Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays. J Clin Microbiol 42: 1863–1868.J. HoorfarB. MalornyA. AbdulmawjoodN. CookM. Wagner2004Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays.J Clin Microbiol4218631868
- 39. Anonymous (2008a) LightCycler® 480 Instrument Operator's Manual. pp. 248–256.Anonymous2008aLightCycler® 480 Instrument Operator's Manual248256
- 40. Qin Y, Polacek N, Vesper O, Staub E, Einfeldt E, et al. (2006) The Highly Conserved LepA Is a Ribosomal Elongation Factor that Back-Translocates the Ribosome. Cell 127: 721–733.Y. QinN. PolacekO. VesperE. StaubE. Einfeldt2006The Highly Conserved LepA Is a Ribosomal Elongation Factor that Back-Translocates the Ribosome.Cell127721733
- 41. Rezwan M, Grau T, Tschumi A, Sander P (2007) Lipoprotein synthesis in mycobacteria. Microbiology 153: 652–658.M. RezwanT. GrauA. TschumiP. Sander2007Lipoprotein synthesis in mycobacteria.Microbiology153652658
- 42. Sassetti CM, Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proceedings of the National Academy of Sciences of the United States of America 100: 12989–12994.CM SassettiEJ Rubin2003Genetic requirements for mycobacterial survival during infection.Proceedings of the National Academy of Sciences of the United States of America1001298912994
- 43. Behr MA, Wilson MA, Gill WP, Salamon H, Schoolnik GK, et al. (1999) Comparative Genomics of BCG Vaccines by Whole-Genome DNA Microarray. Science 284: 1520–1523.MA BehrMA WilsonWP GillH. SalamonGK Schoolnik1999Comparative Genomics of BCG Vaccines by Whole-Genome DNA Microarray.Science28415201523
- 44. O' Grady J, Sedano-Balbás S, Maher M, Smith T, Barry T (2008) Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target. Food Microbiology 25: 75–84.J. O' GradyS. Sedano-BalbásM. MaherT. SmithT. Barry2008Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target.Food Microbiology257584
- 45. Yun JJ, Heisler LE, Hwang IIL, Wilkins O, Lau SK, et al. (2006) Genomic DNA functions as a universal external standard in quantitative real-time PCR. Nucleic Acids Research 34: e85.JJ YunLE HeislerIIL HwangO. WilkinsSK Lau2006Genomic DNA functions as a universal external standard in quantitative real-time PCR.Nucleic Acids Research34e85
- 46. WHO (2006) The control of neglected zoonotic diseases: a route to poverty alleviation. Report of a joint WHO/DFIAD-AHP Meeting, 20-21 September 2005, WHO Headquarters Geneva, with the participation of FAO and OEI, p. 62. Geneva, Switzerland. WHO. WHO2006The control of neglected zoonotic diseases: a route to poverty alleviation.Report of a joint WHO/DFIAD-AHP Meeting, 20-21 September 2005, WHO Headquarters Geneva, with the participation of FAO and OEI, p. 62. Geneva, Switzerland. WHO<http://www.who.int/zoonoses/Report_Sept06.pdf>. <http://www.who.int/zoonoses/Report_Sept06.pdf>.
- 47. Prodinger WM, Brandstatter A, Naumann L, Pacciarini M, Kubica T, et al. (2005) Characterization of Mycobacterium caprae Isolates from Europe by Mycobacterial Interspersed Repetitive Unit Genotyping. J Clin Microbiol 43: 4984–4992.WM ProdingerA. BrandstatterL. NaumannM. PacciariniT. Kubica2005Characterization of Mycobacterium caprae Isolates from Europe by Mycobacterial Interspersed Repetitive Unit Genotyping.J Clin Microbiol4349844992
- 48. Kiers A, Klarenbeek A, Mendelts B, Van Soolingen D, Koëter G (2008) Transmission of Mycobacterium pinnipedii to humans in a zoo with marine mammals. The International Journal of Tuberculosis and Lung Disease 12: 1469–1473.A. KiersA. KlarenbeekB. MendeltsD. Van SoolingenG. Koëter2008Transmission of Mycobacterium pinnipedii to humans in a zoo with marine mammals.The International Journal of Tuberculosis and Lung Disease1214691473
- 49. Panteix G, Gutierrez MC, Boschiroli ML, Rouviere M, Plaidy A, et al. (2010) Pulmonary tuberculosis due to Mycobacterium microti: a study of six recent cases in France. J Med Microbiol 59: 984–989.G. PanteixMC GutierrezML BoschiroliM. RouviereA. Plaidy2010Pulmonary tuberculosis due to Mycobacterium microti: a study of six recent cases in France.J Med Microbiol59984989
- 50. Huard RC, Fabre M, de Haas P, Claudio Oliveira Lazzarini L, van Soolingen D, et al. (2006) Novel Genetic Polymorphisms That Further Delineate the Phylogeny of the Mycobacterium tuberculosis Complex. J Bacteriol 188: 4271–4287.RC HuardM. FabreP. de HaasL. Claudio Oliveira LazzariniD. van Soolingen2006Novel Genetic Polymorphisms That Further Delineate the Phylogeny of the Mycobacterium tuberculosis Complex.J Bacteriol18842714287
- 51. Maudlin I, Eisler MC, Welburn SC (2009) Neglected and endemic zoonoses. Philosophical Transactions of the Royal Society B: Biological Sciences 364: 2777–2787.I. MaudlinMC EislerSC Welburn2009Neglected and endemic zoonoses.Philosophical Transactions of the Royal Society B: Biological Sciences36427772787
- 52. Cosivi O, Grange JM, Daborn CJ, Raviglione MC, Fujikura T, et al. (1998) Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 4: 59–70.O. CosiviJM GrangeCJ DabornMC RaviglioneT. Fujikura1998Zoonotic tuberculosis due to Mycobacterium bovis in developing countries.Emerg Infect Dis45970
- 53. Evans JT, Smith EG, Banerjee A, Smith RMM, Dale J, et al. (2007) Cluster of human tuberculosis caused by Mycobacterium bovis: evidence for person-to-person transmission in the UK. The Lancet 369: 1270–1276.JT EvansEG SmithA. BanerjeeRMM SmithJ. Dale2007Cluster of human tuberculosis caused by Mycobacterium bovis: evidence for person-to-person transmission in the UK.The Lancet36912701276
- 54. Cvetnic Z, Katalinic-Jankovic V, Sostaric B, Spicic S, Obrovac M, et al. (2007) Mycobacterium caprae in cattle and humans in Croatia. The International Journal of Tuberculosis and Lung Disease 11: 652–658.Z. CvetnicV. Katalinic-JankovicB. SostaricS. SpicicM. Obrovac2007Mycobacterium caprae in cattle and humans in Croatia.The International Journal of Tuberculosis and Lung Disease11652658
- 55. Prodinger WM, Eigentler A, Allerberger F, Schonbauer M, Glawischnig W (2002) Infection of Red Deer, Cattle, and Humans with Mycobacterium bovis subsp. caprae in Western Austria. J Clin Microbiol 40: 2270–2272.WM ProdingerA. EigentlerF. AllerbergerM. SchonbauerW. Glawischnig2002Infection of Red Deer, Cattle, and Humans with Mycobacterium bovis subsp. caprae in Western Austria.J Clin Microbiol4022702272
- 56. Rodríguez E, Sánchez LP, Pérez S, Herrera L, Jimínez MS, et al. (2009) Human tuberculosis due to Mycobacterium bovis and M. caprae in Spain, 2004-2007. The International Journal of Tuberculosis and Lung Disease 13: 1536–1541.E. RodríguezLP SánchezS. PérezL. HerreraMS Jimínez2009Human tuberculosis due to Mycobacterium bovis and M. caprae in Spain, 2004-2007.The International Journal of Tuberculosis and Lung Disease1315361541
- 57. Pinsky B, Banaei N (2008) Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level. J Clin Microbiol 46: 2241–2246.B. PinskyN. Banaei2008Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level.J Clin Microbiol4622412246
- 58. Ereqat S, Bar-Gal GK, Nasereddin A, Azmi K, Qaddomi SE, et al. (2010) Rapid Differentiation of Mycobacterium tuberculosis and M. bovis by High-Resolution Melt Curve Analysis. J Clin Microbiol 48: 4269–4272.S. EreqatGK Bar-GalA. NasereddinK. AzmiSE Qaddomi2010Rapid Differentiation of Mycobacterium tuberculosis and M. bovis by High-Resolution Melt Curve Analysis.J Clin Microbiol4842694272
- 59. Somoskovi A, Dormandy J, Parsons LM, Kaswa M, Goh KS, et al. (2006) Sequencing of the pncA gene in members of the Mycobacterium tuberculosis complex has important diagnostic applications: Identification of a species-specific pncA Mutation in Mycobacterium canettii, and the Reliable and Rapid Predictor of Pyrazinamide Resistance. J Clin Microbiol JCM.01454-01406.A. SomoskoviJ. DormandyLM ParsonsM. KaswaKS Goh2006Sequencing of the pncA gene in members of the Mycobacterium tuberculosis complex has important diagnostic applications: Identification of a species-specific pncA Mutation in Mycobacterium canettii, and the Reliable and Rapid Predictor of Pyrazinamide Resistance.J Clin MicrobiolJCM.01454-01406
- 60. Richter E, Weizenegger M, Fahr A, Rusch-Gerdes S (2004) Usefulness of the GenoType MTBC assay for differentiating species of the Mycobacterium tuberculosis complex in cultures obtained from clinical specimens. J Clin Microbiol 42: 4303–4306.E. RichterM. WeizeneggerA. FahrS. Rusch-Gerdes2004Usefulness of the GenoType MTBC assay for differentiating species of the Mycobacterium tuberculosis complex in cultures obtained from clinical specimens.J Clin Microbiol4243034306
- 61. Brosch R, Gordon SV, Pym A, Eiglmeier K, Garnier T, et al. (2000) Comparative genomics of the mycobacteria. Int J Med Microbiol 290: 143–152.R. BroschSV GordonA. PymK. EiglmeierT. Garnier2000Comparative genomics of the mycobacteria.Int J Med Microbiol290143152
- 62. Cole ST (2002) Comparative and functional genomics of the Mycobacterium tuberculosis complex. Microbiology 148: 2919–2928.ST Cole2002Comparative and functional genomics of the Mycobacterium tuberculosis complex.Microbiology14829192928
- 63. Parsons L, Brosch R, Cole S, Somoskovi A, Loder A, et al. (2002) Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis. J Clin Microbiol 40: 2339–2345.L. ParsonsR. BroschS. ColeA. SomoskoviA. Loder2002Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis.J Clin Microbiol4023392345