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Direct and Simultaneous Identification of Mycobacteriumtuberculosis complex (MTBC) and Mycobacterium tuberculosis

(MTB) by Rapid Multiplex nested PCR-ICT assay

Po-Chi Soo a ,1 , Yu-Tze Horng a ,c ,1 , Po-Ren Hsueh b , Bin-Jon Shen c , Jann-Yuan Wang b ,Hui-Hsin Tu a , Jun-Rong Wei a , Shang-Chen Hsieh a , Chien-Chung Huang a ,

Hsin-Chih Lai a,b,

a Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, ROC b Department of Laboratory Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine,

Taipei, Taiwan, ROC c Tyson Bioresearch Inc., Taiwan, ROC

Received 29 November 2005; received in revised form 18 January 2006; accepted 23 January 2006Available online 3 March 2006

Abstract

The Mycobacterium tuberculosis (MTB) shows different virulence and host infection range from other members of the M.

tuberculosis complex (MTBC). Differential identification of MTB from MTBC is thus important in certain occasions. Thecurrently commercially available molecular assays which use either IS6110 or 16S rDNA fragment as identification targets aremainly designed for identifying MTBC but not for MTB. Comparative genomic DNA analysis has provided valuable informationon regions of difference (RD) present in MTB but not in other members of the MTBC. RD9 region is further suggested to be a potential target for differential identification of MTB from MTBC. In this study, using IS6110 and Rv3618 (belong to RD9) as thespecific identification targets for MTBC and MTB, respectively, we developed and tested a multiplex nested PCR-ICT (immuno-chromatography test) assay for simultaneously and directly detecting not only MTBC but also MTB from 1500 clinical sputumspecimens. The results were compared with traditional culture and biochemical identification results together with patients' clinicalassessments. This assay showed a 95.5% sensitivity, 97.9% specificity, 2.1% false positive rate and 4.5% false negative ratetowards detection of MTBC, and a 93.0% sensitivity, 99.8% specificity, 0.2% false positive rate and 7.0% false negative rate for detection of MTB. This detection system shows great potential in clinical application. 2006 Elsevier B.V. All rights reserved.

Keywords: Mycobacterium tuberculosis ; Molecular detection; Multiplex nested PCR; Immuno-chromatography test (ICT)

1. Introduction

Tuberculosis (TB) is the leading cause of humanadult death by infectious gents, accounting for approximately two million deaths annually, mainly inthe developing countries. Currently, the global number

Journal of Microbiological Methods 66 (2006) 440 448www.elsevier.com/locate/jmicmeth

Corresponding author. Department of Clinical Laboratory Sciencesand Medical Biotechnology, National Taiwan University College of Medicine, No.1., Chan-Der Street, Taipei 100, Taiwan, ROC.Tel.: +886 2 2312 3456x6931; fax: +886 2 2371 1574.

E-mail address: [email protected] (H.-C. Lai).

0167-7012/$ - see front matter 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2006.01.010
mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.mimet.2006.01.010http://dx.doi.org/10.1016/j.mimet.2006.01.010http://dx.doi.org/10.1016/j.mimet.2006.01.010mailto:[email protected]

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of TB cases is rising at a rate of 2% per year (WorldHealth Organization tuberculosis fact sheet; http:// www.stoptb.org/tuberculosis/ ). While conventionalsmear microscopy and culture methods are widelyused for diagnosis of TB, the former is insensitive

(Caws et al., 2000), and the latter takes up to 6 to8 weeks to provide a result, limiting the value of thesemethods in aiding diagnosis and immediate decisionson treatment. Using IS6110 and 16S rDNA as detectiontargets, many commercially available nucleic acidamplification-based detection systems have also beendeveloped as rapid tests for direct identification of Mycobacterium tuberculosis complex (MTBC) fromclinical specimens (Gardiner and Beavis, 2000; Soiniand Musser, 2001; Woods, 1999). These include PCR- based Amplicor (Roche), ligase chain reaction (Lcx;

Abbott Systems), transcription-mediated amplification(TMA; Gen-Probe), strand displacement amplification(BDProbe; Tec-SDA) and the RAPID-BAP-MTB assay(AsiaGen, Taiwan) (Brown et al., 1999; Eing et al.,1998; Hellyer et al., 1996; Piersimoni et al., 1998;Reischl et al., 1998; Wang et al., 2004; Yuen et al.,1997 ). Variations in the sensitivities and high costs of these tests have hindered these systems from beingwidely used in TB detection.

Although the mycobacteria grouped in the MTBC areclosely related based on DNA DNA hybridization,multilocus enzyme electrophoresis, and 16S rDNA nu-leotide acid sequence level (Boddinghaus et al., 1990;Sreevatsan et al., 1997), MTBC members differ widelyin terms of host tropisms, phenotypes, and pathogenicity.It is intriguing that some are exclusively human ( M.tuberculosis , Mycobacterium africanum , Mycobacteriumcanetti ) or rodent pathogens ( Mycobacterium microti),whereas others either have a wide host spectrum( Mycobacterium bovis) (Brosch et al., 2002) or areused as a vaccine strain ( M. bovis BCG). Differentiationof M. tuberculosis (MTB) from the other members of theMTBC is thus necessary for treatment of individual

patients and for epidemiological study purposes, espe-cially in areas of the world where tuberculosis hasreached epidemic proportions or wherever the transmis-sion of M. bovis between animals or animal products andhumans is a problem.

While current detection methods do not differentiateMTB from other members of the MTBC (Abe et al.,1993; Alcaide et al., 2000; Katila et al., 2000), recent comparative genomic analyses have provided valuableinformation on regions of difference (RD) in the chro-mosome of MTB, and indicated that specific identifi-cation of MTB can be achieved by use of these RDs(Parsons et al., 2002). In this study, to rapidly and

specifically identify MTB from sputum samples, themultiplex nested PCR combined with immuno-chro-matography test (ICT) assay was developed. Rv3618DNA fragment which belongs to RD9 (Behr et al.,1999 ) was selected as a potential target for MTB

diagnosis, and IS6110 as the traditional identificationmarker for MTBC. The results were compared withthose from conventional culture and biochemicalidentification methods in combination with clinicalassessments. Our results showed that the multiplexnested PCR-ICT assay is a convenient, low-cost andeasy-to-use detection system for identification of MTBwith high sensitivity and specificity.

2. Materials and methods

2.1. Specimen collection and processing

A total of 1500 sequential clinical sputum specimenswere collected from the mycobacteriology Laboratory,Department of Laboratory Medicine, National TaiwanUniversity Hospital from the September 2004 to March2005. Collection of these clinical samples was approved by the Review Board Committee in the National TaiwanUniversity Hospital. Specimens were processed onreceipt according to the standard routine diagnosis procedures (Piersimoni et al., 2002; Wang et al., 2004).Briefly, an equal volume of NaOH-citrate- N -acetyl- L-cysteine solution was added into sputum sample at roomtemperature for 15min. After centrifugation, the precip-itate was resuspended in 1ml of phosphate-bufferedsaline (pH 7.4).

2.2. Culture and biochemical methods for diagnosis of MTBC and MTB

The Lowenstein Jensen (LJ) slants (Difco, USA)and Middlebrook 7H11 medium plates (Becton-Dick-inson, USA) were inoculated with 250 l of deconta-

minated sample suspension, incubated at 37C with 5%CO 2 . An inverted light microscope was used for observation of mycobacterial growth during weeks 28 after inoculation. The guidelines of US Center for Disease Control and Prevention (Montenegro et al.,2003 ) were followed for determination of positive my-cobacterial growth. For maximum isolation Mycobac-teria growth indicator tubes, the fluorometric BACTECtechnique (BACTEC MGIT 960 system; Becton-Dickinson Diagnosis Instrument System, USA) wasused for mycobacterial growth before further growth on7H11 medium (O'Sullivan et al., 2002). Identificationof the bacterial strains to be MTBC and MTB is mainly

441 P.-C. Soo et al. / Journal of Microbiological Methods 66 (2006) 440 448
http://www.stoptb.org/tuberculosis/http://www.stoptb.org/tuberculosis/http://www.stoptb.org/tuberculosis/http://www.stoptb.org/tuberculosis/

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controls were within the stipulated limits. For DNAamplification of samples prepared from clinical speci-mens, an internal DNA control in which a partial spnI DNA fragment amplified by SI-1 and SI-2 primers wasincluded (Horng et al., 2002), which rules out the

possibility of false-negative results due to inhibitorsfrom specimen.

2.7. Clinical assessment of TB patients

All medical records, including history, symptoms,signs, radiology, pathology, microbiology results andfollow-up observations were carefully reviewed asdescribed from our previous study (Wang et al., 2004).Basically, the culture results and clinical evaluationswere served as the gold standard for diagnosis. The

multiplex nested PCR-ICT results were evaluated basedon these parameters.

2.8. Detection of RD9 by PCR

A 50 l reaction mixture containing two flanking primers RD9 FF and RD9 FR (10mM each), an internal primer RD9-Int (50 mM), KCl (50 mM), Tris HCl(10mM, pH 8.3), MgCl2 (1.5mM), each deoxynucleo-side triphosphate (200 M each), Taq DNA polymerase(2.5U), and 5 l of crude cell extract (Section 2.4) wereused for PCR. After denaturation at 95C for 5min, thereaction mixtures were subject for reactions of 40 cyclesat 94C for 30s, 65C for 1min, and 72 C for 1min.

3. Results

3.1. Principle of multiplex nested PCR-ICT assay

Design of this assay is described in Fig. 1. For multiplex nested PCR reactions, two set of primer pairs,each containing an external primer pair amplifying alonger DNA fragment and an internal primer pair am-

plifying a shorter internal DNA fragment, were designedfrom the chromosomal DNA sequence of the insertionsequence IS6110 and the Rv3618 open reading frame,respectively. All four external primers were not labeled.Two of the four internal primers were labeled with biotin,and each of the other two was either labeled withfluorescein (for IS6110 ) or digoxigenin (Dig) (for Rv3618 ) at the 5 -terminus, respectively (Fig. 1).

For specifically amplifying Rv3618 , the external primer pair Rv3618F Rv3618R (Table 2) was first usedto amplify a 326bp DNA fragment, which was sub-sequently used as a template for amplifying an internal224bp fragment (Fig. 2A) using the internal primers B-

Rv3618 (biotinylated) and D-Rv3618 (Dig-labeled)(Table 2). For specifically amplifying the IS6110 se-quence, the external primer pair INS1 INS2 (Table 2)and internal primer pair B-INS1 (biotinylated) and F-INS2 (fluorescein-labeled)( Table 2) were used to

amplify a 245 and a 110bp (Fig. 2A) DNA fragment,respectively.

Identification of the labeled amplified DNA productswas achieved by the ICT strip. Under the conditionof formation of a control line where intensified dark brown color deposits were formed, only amplifiedDNA fragments doubly labeled with Dig-biotin and/or fluorescein-biotin could lead to formation of test line(s)

bpM 2 3

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(A)

(B)

224 bp

110 bp

300

100

200

400500

1N

C

T 1

T 2

2 3

1

Fig. 2. Typical results of detection of amplified partial IS6110 andRv3618 DNA fragments by agarose gel electrophoresis and ICT after multiplex nested PCR. (A) Separation of the amplified 110bp(IS6110 ) or 224bp (Rv3618 ) DNA fragment by 2% agarose gelelectrophoresis followed by ethidium bromide staining. M, DNA sizemarker. (B) ICT strip assay. After PCR, 5 l of the solution wasapplied onto the strip. Results were read after 10min incubation at room temperature. N, negative control; C, control line; T1, IS6110detection line; T2, Rv3618 detection line. For both (A) and (B),chromosomal DNAs of Mycobacterium tuberculosis H37Rv (lane 1),

M. bovis (lane 2) and M. avium (lane 3), each at the amount of 1ngwas used as PCR templates.


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(Figs. 1 and 2B). The amplified 110bp DNA product interacted with anti-FITC antibodies, forming IS6110test line (T1), and the amplified 224bp fragment interacted with anti-Dig antibodies, forming Rv3618test line (T2) on the strip (Fig. 2B). Formation of bothtest lines (IS6110 and Rv3618 ) indicated existence of MTB DNA in the specimen, and formation of IS6110test line only indicated MTBC. Formation of no test lines indicated either no Mycobacterium bacteria, or

alternatively existence of non-MTBC (non-tuberculosis Mycobacterium, NTM) organisms, such as Mycobacte-rium avium in the specimen (Fig. 2B). The detectionlimit of this assay system is up to 10CFU per reaction.(Fig. 3).

3.2. Detection of Mycobacterium reference strains

A total of 23 Mycobacterium spp. reference strainsgrown on 7H11 culture plates were first identified by themultiplex nested PCR-ICT assay. While control lines allshowed positive reaction results, both IS6110 andRv3618 test lines were formed in detection of M.

tuberculosis H37Rv. In comparison, only IS6110 test line was present in detection of M. bovis ATCC19210

and M. microtiATCC 19422 which are MTBC members.For the other non-tuberculous Mycobacterium (NTM)strains, negative reactions were observed from both test lines (Table 1 and Fig. 1). Thus besides MTBC, MTBcan further be identified by this assay.

3.3. Screens of clinical isolates

A total of 1500 consecutive clinical sputum speci-mens collected during period September 2004 to March2005 were subject to routine identification of mycobac-teria in National Taiwan University Hospital. In parallel,the spent sediments of specimens were subject for chromosomal DNA extraction and detected by themultiplex nested PCR-ICTassay. The resultsof detectionwere summarized in Tables 3 and 4. Among thespecimens identified by cultures and biochemical meth-ods, a total of 89 specimens were reported to containMTBC strains. Compared with the routine identificationmethods, results from the multiplex nested PCR-ICTassay identified 114 specimens containing MTBCstrains, showing a 95.5% sensitivity and 97.8% speci-ficity (Table 3). Comparatively, a total of 29 specimens

Table 3Comparison of MTBC diagnosis results from consecutive 1500clinical sputum specimens by culture and mutiplex nested PCR-ICT(IS6110 ) assays

Mutiplex nested PCR-ICT (no. of samples) Culturea

Positive NegativePositive(114) 85 29 Negative(1386) 4 1382Overall (1500) 89 1411

a Culture and biochemical diagnosis results were from mycobacter-iology laboratory, National Taiwan University Hospital (NTUH).Sensitivity: 95.5%, Specificity: 97.9%; positive predictive value:74.6%, negative predictive value: 99.7%.

Table 4Comparison of MTB diagnosis results from consecutive 1500 clinicalsputum specimens by culture and mutiplex nested PCR-ICT (Rv3618 )assays

Mutiplex nested PCR-ICT (no. of samples) Culturea

Positive Negative

Positive(83) 80 3 Negative(1417) 6 1411Overall (1500) 86 1414

a Culture and biochemical diagnosis results were from mycobacter-

iology laboratory, NTUH. Sensitivity: 93%, specificity: 99.8%; positive predictive value: 96.4%, negative predictive value: 99.6%.

500

300 200

100

bp M 1

1 0

3

1

1 0

2

1

1 0

0

1

1 0

1

1

1 0 -

1

N

Rv3618

IS6110

C

T1

T2

L

CFU/ml(A)

(B)

Fig. 3. Detection limit of multiplex nested PCR-ICT assay. Thedetection limit of the assay was evaluated by using a serially diluted M.tuberculosis bacteria ranging from 1103 CFU/ml to 110

1 CFU/ml per reaction as the templates. Amplified DNA products were detected by (A) 2% agarose gel electrophoresis followed by ethidium bromidestaining, and (B) the ICT strip assay. M: DNA size marker. N, PCR negative control. L, lateral flow negative control. C, positive controlline, T1, Rv3618 detection line, T2, IS6110 detection line.


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that were MTBC culture-negative were detected asIS6110 -positive. Further clinical assessment showed that 3 out of the 29 patients showed significant clinicalsyndromes of MTB infection. Among the 89 culture positive MTBC specimens, 4 were IS6110 -negative

based on the multiplex nested PCR-ICT assay.Among the 1500 clinical specimens, 86 samples were

MTB positive by culture assay. In comparison, 83specimens were detected Rv3618 positive by the mul-tiplex nested PCR-ICT assay (Table 4). There were 3MTB culture-negative culture samples that weredetected Rv3618 positive by this assay, and all 3 patientsshowed significant clinical syndromes of MTB infec-tion. A total of 6 MTB culture-positive specimens wereRv3618 negative by this assay. Briefly, a sensitivity of 93% and specificity of 99.8% were obtained.

Among the 85 specimens which were IS 6110 positiveand culture confirmed to contain MTBC, 80 containedMTB and 5 did not show Rv3618 positive, suggestingthat these five specimens contain either MTBC but not MTB or MTB deficient in RD9 (Rv3618 ) region.

3.4. Absence of RD9 from two MTB strains

To further confirm these 5 Rv3618 -negative strainswere indeed RD9-deficient, three primers designed fromRD9 region (Table 2) were used in PCR for confirma-tion. Among these, RD9 FF and RD9 FR were designedfrom sequences flanking the RD9 region and RD9-Int from the internal RD9 region (Parsons et al., 2002).Absence of RD9 lead to amplification of a 206bp DNA

product by primer pair RD9 FF/RD9 FR. Comparatively,a 306bp DNA was amplified by primer pair RD9 FF/ RD9-Int, when RD9 is present (Parsons et al., 2002). NoRD9 homologous DNA fragment was detected in these 5Rv3618 -deficient MTBC strains (Fig. 4), which was in

agreement with the multiplex nested PCR-ICT assayresults. Through culture method, morphology observa-tion and biochemical tests, two bacterial isolates werefinally classified as MTB and three as M. bovis. Thisfinding was supported by the results from referencestrains identification (Table 1 and Fig. 1) and the study byParsons et al. (2002) . Briefly, 2.3% (2/86) of clinicallyisolated MTB strains in Taiwan were RD9 absent in thisstudy.

4. Discussion

Prompt diagnosis of pulmonary tuberculosis iscritical for initiating appropriate therapy and facilitatingmeasures to prevent dissemination of this contagiousdisease. While MTB is the main devastating pathogenleading to tuberculosis, other members of the MTBCcontribute to diseases of different host ranges, geograph-ical prevalences and pathogenesis ( Niemann et al.,2004 ). As more and more MTBC strains are isolatedfrom different region, besides diagnosis of MTBC, it isimportant to further distinguish MTB from other members in MTBC. However, the currently commonly usedmolecular diagnosis methods do not achieve suchdistinction, due to use of the target DNA markers suchas IS6110 and 16S rDNA sequences. Comparative geno-mics of the members of MTBC by use of subtractivehybridization (Mahairas et al., 1996), bacterial artificialchromosome arrays (Brosch et al., 1998; Gordon et al.,1999 ) or whole genome DNA microarrays (Behr et al.,1999 ) had identified 16 regions ranging in size from 2 to12.7kb that were present in M. tuberculosis H37Rv but absent in most BCG derivatives and other members of the MTBC. Among the RD regions analyzed, PCR-

based genomic deletion analysis further showed that RD9 seems to be a good DNA marker for specificidentification of M. tuberculosis from other members of MTBC ( Behr et al., 1999; Parsons et al., 2002 ). Based onthese observations, we chose Rv3618 from the RD9region as the potential DNA marker for MTB diagnosis.The results obtained basically agreed with what fromculture and biochemical assays, suggesting Rv3618 is agood marker for MTB. Among the 85 T1-positiveMTBC specimens, 80 were also T2-positive. Three of these 5 specimens were subsequently identified tocontain M. bovis and the remaining 2 to be MTB byculture and biochemical assays. Further confirmation of

M 1 2 3 4 5 6 7

400

100

500

1000

300 200 206 bp

306 bp

bp

Fig. 4. Detection of RD9 by PCR. After PCR using chromosomalDNA as the template and primers listed in the Table 2, DNAs wereseparated in 2% agarose gel electrophoresis followed by ethidium bromide staining. Observation of a 306 bp PCR product indicates presence of RD9 and a 206 bp product indicates deletion of this region.M, DNA size marker; lane 1 2, clinical MTB isolates (IS6110 - positive/Rv3618 -negative); lane 3 5, clinical M. bovis isolates

(IS6110 -positive/Rv 3618 -negative); lane 6, M. tuberculosis H37Rvand lane 7, M. bovis (ATCC 19210).


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absence of RD9 within these 2 MTB strains wasachieved by a PCR protocol described by Parsons et al.(2002) . These data indicated that while RD9 may not exist in other MTBC species, a part of MTB strainsisolated in Taiwan did not contain RD9. This should be

taken into consideration when using Rv3618 as a marker to detect MTB.

Although a range of rapid tests based on nucleic acidamplification techniques have been developed for direct detection of MTBC from clinical samples, it remains to be proven whether they can also fulfill the requirementsof high sensitivity and specificity, simplicity andreasonable cost at the same time. The relative high cost of most molecular methods developed maybe one of themajor reasons that hinder these systems from beingwidely used, especially in developing countries where

TB is much more prevalent than the developed ones. Themultiplex nested PCT-ICT assay developed in thislaboratory is efficient due to characteristics of easy tooperate, no need of sophisticated detection equipment,low cost, time saving (less than 4 h from receipt of sputum specimens to completion of the test), and highsensitivity and specificity. Compared with conventionalculture and biochemical diagnosis methods, the sensi-tivity and specificity of this assay for MTBC diagnosisare 95.5% and 97.9%, respectively, and for MTB are93% and 99.8%, respectively. The results are comparableto, or even better than those obtained by previoussystems we used for MTBC diagnosis (Wang et al.,2004 ). Among the 89 culture identified MTBC speci-mens, 85 were IS6110-positive. There were 4 specimensthat are neither MTBC nor MTB positive by this assay.These might be due to too few bacterial cells within thesputum specimens. There were also 5 specimens whichwere IS6110-positive while Rv3618 -negative. Amongthese, 3 were finally confirmed to contain M. bovis and2 MTB without RD9. On the contrary, there were29 culture-negative but IS6110-positive samples, and 3culture-negative but IS6110 and RV3618 -positive sam-

ples. For the 3 culture-negative but IS6110 and Rv3618 positive samples, retrospective clinical assessment indi-cated that the 3 patients were infected by MTB beforeand had been under drug treatment when samples weretaken. Thus the results might be due to killing of MTBcells by drug or full recovery. The remaining 26 culture-negative but IS6110 -positive samples might be due tonon-specifically amplified DNA products during thePCR-ICT assay.

Although the multiplex nested PCR and ICTtechniques have already been widely used in the areasof molecular and immuno-diagnosis, few reports are oncombination of both techniques into an assay for rapid

diagnosis of specific DNA target sequences. A recent report from Takada et al. (2005) shows a great poten-tiali ty of such technique on direct diagnosis of Porphyromonas gingivalis from periodontitis patients(Takada et al., 2005). Some other techniques which also

detect target nucleic acid sequence by lateral-flow device(strip) are also developed. These include cycling probetechnology (CPT) assay with a lateral-flow device for detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures (Fong et al.,2000 ). Another example is the up-converting phosphor reporters and lateral-flow assay to identify human papillomavirus type16 (Corstjens et al., 2001). Compar-atively, our system has the advantage of direct andsimultaneous detection of two DNA targets, which isconvenient even for use in identification of different

pathogen in one sample. Low cost, simple accurate, the potential of high through detection of target DNAsequences are the main characteristics of these systems.Furthermore, direct detection from clinical samples withcomparable sensitivity and specificity with conventionalor commercial systems are also highlighted. These areespecially important for practical clinical applications.

Acknowledgments

This work was supported by the grant from Tyson bioresearch Inc. (SPA Examination Committee R and DProject, Grand No. 692 ) and the Technology Develop-ment Program for Academia, Ministry of EconomicalAffairs (grant number 91-EC-17-A-10-S1-0013), whichwere really appreciated.

References

Abe, C.,Hirano, K.,Wada, M.,Kazumi, Y., Takahashi, M.,Fukasawa,Y.,Yoshimura, T., Miyagi, C., Goto, S., 1993. Detection of Mycobac-terium tuberculosis in clinical specimens by polymerase chainreaction and Gen-Probe Amplified Mycobacterium TuberculosisDirect Test. J. Clin. Microbiol. 31, 3270 3274.

Alcaide, F., Benitez, M.A., Escriba, J.M., Martin,R.,2000. Evaluation of the BACTEC MGIT 960 and the MB/BacT systems for recovery of mycobacteria from clinical specimens and for species identification by DNA AccuProbe. J. Clin. Microbiol. 38, 398 401.

Behr, M.A., Wilson, M.A., Gill, W.P., Salamon,H., Schoolnik,G.K., Rane,S., Small, P.M., 1999. Comparative genomics of BCG vaccines bywhole-genome DNA microarray. Science 284, 1520 1523.

Boddinghaus, B., Rogall, T., Flohr, T., Blocker, H., Bottger, E.C.,1990. Detection and identification of mycobacteria by amplifica-tion of rRNA. J. Clin. Microbiol. 28, 1751 1759.

Brosch, R., Gordon, S.V., Billault, A., Garnier, T., Eiglmeier, K.,Soravito, C., Barrell, B.G., Cole, S.T., 1998. Use of a Mycobac-terium tuberculosis H37Rv bacterial artificial chromosome library

for genome mapping, sequencing, and comparative genomics.Infect. Immun. 66, 2221 2229.


8/13/2019 1-s2.0-S0167701206000224-main

9/9

Brosch, R., Gordon, S.V., Marmiesse, M., Brodin, P., Buchrieser, C.,Eiglmeier, K., Garnier, T., Gutierrez, C., Hewinson, G., Kremer, K.,Parsons, L.M., Pym, A.S., Samper, S., van Soolingen, D., Cole, S.T.,2002. A newevolutionaryscenario for the Mycobacteriumtuberculosiscomplex. Proc. Natl. Acad. Sci. U. S. A. 99, 3684 3689.

Brown, T.J., Power, E.G., French, G.L., 1999. Evaluation of three

commercial detection systems for Mycobacterium tuberculosiswhere clinical diagnosis is difficult. J. Clin. Pathol. 52, 193 197.

Caws, M., Wilson, S.M., Clough, C., Drobniewski, F., 2000. Role of IS6110 -targeted PCR, culture, biochemical, clinical, and immu-nological criteria for diagnosis of tuberculous meningitis. J. Clin.Microbiol. 38, 3150 3155.

Corstjens, P., Zuiderwijk, M., Brink, A., Li, S., Feindt, H., Niedbala,R.S., Tanke, H., 2001. Use of up-converting phosphor reporters inlateral-flow assays to detect specific nucleic acid sequences: arapid, sensitive DNA test to identify human papillomavirus type16 infection. Clin. Chem. 47, 1885 1893.

Eing, B.R., Becker, A., Sohns, A., Ringelmann, R., 1998. Comparisonof Roche Cobas Amplicor Mycobacterium tuberculosis assay within-house PCR and culture for detection of M. tuberculosis. J. Clin.Microbiol. 36, 2023 2029.

Fong, W.K., Modrusan, Z., McNevin, J.P., Marostenmaki, J., Zin, B.,Bekkaoui, F., 2000. Rapid solid-phase immunoassay for detectionof methicillin-resistant Staphylococcus aureus using cycling probetechnology. J. Clin. Microbiol. 38, 2525 2529.

Gardiner, D.F., Beavis, K.G., 2000. Laboratory diagnosis of mycobac-terial infections. Semin. Respir. Infect. 15, 132 143.

Gordon, S.V., Brosch, R., Billault, A., Garnier, T., Eiglmeier, K., Cole,S.T., 1999. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol.Microbiol. 32, 643 655.

Hellyer,T.J.,Fletcher, T.W., Bates, J.H., Stead, W.W., Templeton, G.L.,Cave, M.D., Eisenach, K.D., 1996. Strand displacement amplifi-cationand the polymerase chain reaction for monitoring response totreatment in patients with pulmonary tuberculosis. J. Infect. Dis.173, 934 941.

Horng, Y.T., Deng, S.C., Daykin, M., Soo, P.C., Wei, J.R., Luh, K.T.,Ho, S.W., Swift, S., Lai, H.C., Williams, P., 2002. The LuxR family protein SpnR functions as a negative regulator of N -acylhomoserine lactone-dependent quorum sensing in Serratiamarcescens . Mol. Microbiol. 45, 1655 1671.

Katila, M.L., Katila, P., Erkinjuntti-Pekkanen, R., 2000. Accelerateddetection and identification of mycobacteria with MGIT 960 andCOBAS AMPLICOR systems. J. Clin. Microbiol. 38, 960 964.

Mahairas, G.G., Sabo, P.J., Hickey, M.J., Singh, D.C., Stover, C.K., 1996.Molecular analysis of genetic differences between Mycobacteriumbovis BCG and virulent M. bovis. J. Bacteriol. 178, 1274 1282.

Montenegro, S.H., Gilman, R.H., Sheen, P., Cama, R., Caviedes, L.,Hopper, T., Chambers, R., Oberhelman, R.A., 2003. Improveddetection of Mycobacterium tuberculosis in Peruvian children byuse of a heminested IS6110 polymerase chain reaction assay. Clin.Infect. Dis. 36, 16 23.

Niemann, S., Kubica, T., Bange, F.C., Adjei, O., Browne, E.N.,Chinbuah, M.A., Diel, R., Gyapong, J., Horstmann, R.D., Joloba,M.L., Meyer, C.G., Mugerwa, R.D., Okwera, A., Osei, I., Owusu-Darbo, E., Schwander, S.K., Rusch-Gerdes, S., 2004. The species Mycobacterium africanum in the light of new molecular markers.J. Clin. Microbiol. 42, 3958 3962.

Noordhoek, G.T., van Embden, J.D., Kolk, A.H., 1996. Reliability of nucleic acid amplification for detection of Mycobacterium

tuberculosis : an international collaborative quality control studyamong 30 laboratories. J. Clin. Microbiol. 34, 2522 2525.

O'Sullivan, C.E., Miller, D.R., Schneider, P.S., Roberts, G.D., 2002.Evaluation of Gen-Probe amplified mycobacterium tuberculosisdirect test by using respiratory and nonrespiratory specimens in atertiary care center laboratory. J. Clin. Microbiol. 40, 1723 1727.

Parsons, L.M., Brosch, R., Cole, S.T., Somoskovi, A., Loder, A.,Bretzel, G., Van Soolingen, D., Hale, Y.M., Salfinger, M., 2002.Rapid and simple approach for identification of Mycobacteriumtuberculosis complex isolates by PCR-based genomic deletionanalysis. J. Clin. Microbiol. 40, 2339 2345.

Piersimoni, C., Callegaro, A., Scarparo, C., Penati, V., Nista, D.,Bornigia, S., Lacchini, C., Scagnelli, M., Santini, G., De Sio, G.,1998. Comparative evaluation of the new gen-probe Mycobacte-rium tuberculosis amplified direct test and the semiautomatedabbott LCx Mycobacterium tuberculosis assay for direct detectionof Mycobacterium tuberculosis complex in respiratory andextrapulmonary specimens. J. Clin. Microbiol. 36, 3601 3604.

Piersimoni, C., Scarparo, C., Piccoli, P., Rigon, A., Ruggiero, G., Nista,D., Bornigia, S., 2002. Performance assessment of two commercialamplification assays for direct detection of Mycobacterium tuber-culosis complex from respiratory and extrapulmonary specimens.J. Clin. Microbiol. 40, 4138 4142.

Reischl, U., Lehn, N., Wolf, H., Naumann, L., 1998. Clinical evaluationof the automated COBAS AMPLICOR MTB assay for testingrespiratory and nonrespiratory specimens. J. Clin. Microbiol. 36,2853 2860.

Salo, W.L., Aufderheide, A.C., Buikstra, J., Holcomb, T.A., 1994. Iden-tification of Mycobacterium tuberculosis DNA in a pre-ColumbianPeruvian mummy. Proc. Natl. Acad. Sci. U. S. A. 91, 2091 2094.

Soini, H., Musser, J.M., 2001. Molecular diagnosis of mycobacteria.Clin. Chem. 47, 809 814.

Sreevatsan, S., Pan, X., Stockbauer, K.E., Connell, N.D., Kreiswirth,B.N., Whittam, T.S., Musser, J.M.,1997. Restricted structural gene polymorphism in the Mycobacterium tuberculosis complexindicates evolutionarily recent global dissemination. Proc. Natl.Acad. Sci. U. S. A. 94, 9869 9874.

Takada, K., Sakaguchi, Y., Oka, C., Hirasawa, M., 2005. New rapid polymerase chain reaction-immunochromatographic assay for Porphyromonas gingivalis . J. Periodontol. 76, 508 512.

Tschopp, J., 1984. Ultrastructure of the membrane attack complex of complement. Heterogeneity of the complex caused by different degree of C9 polymerization. J. Biol. Chem. 259, 7857 7863.

Tschopp, J., Podack, E.R., Muller-Eberhard, H.J., 1982. Ultrastructureof the membrane attack complex of complement: detection of thetetramolecular C9-polymerizing complex C5b-8. Proc. Natl. Acad.Sci. U. S. A. 79, 7474 7478.

Wang, J.Y., Lee, L.N., Chou, C.S., Huang, C.Y., Wang, S.K., Lai, H.C.,Hsueh, P.R., Luh, K.T., 2004. Performance assessment of a nested-PCR assay (the RAPID BAP-MTB) and the BD ProbeTec ETsystem for detection of Mycobacterium tuberculosis in clinicalspecimens. J. Clin. Microbiol. 42, 4599 4603.

Woods, G.L., 1999. Molecular methods in the detection and identifica-tion of mycobacterial infections. Arch. Pathol. Lab Med. 123,1002 1006.

Yuen, K.Y., Yam, W.C., Wong, L.P., Seto, W.H., 1997. Comparison of two automated DNA amplification systems with a manual one-tube nested PCR assay for diagnosis of pulmonary tuberculosis.J. Clin. Microbiol. 35, 1385 1389.


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