1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2000, p Vol. 38, No /00/$ Copyright 2000, American Society for Microbiology. All Rights Reserved. Molecular Characterization of Mycobacterium tuberculosis H37Rv/Ra Variants: Distinguishing the Mycobacterial Laboratory Strain P. BIFANI, 1,2 S. MOGHAZEH, 1 B. SHOPSIN, 1,2 J. DRISCOLL, 3 A. RAVIKOVITCH, 1 AND B. N. KREISWIRTH 1 * Public Health Research Institute Tuberculosis Center 1 and Department of Microbiology, New York University School of Medicine, 2 New York, New York 10016, and Wadsworth Center, New York State Department of Health, Albany, New York Received 22 December 1999/Returned for modification 23 February 2000/Accepted 16 May 2000 The Mycobacterium tuberculosis strains H37Rv and H37Ra are the most commonly used controls for M. tuberculosis identification in the clinical and research laboratory setting. To reduce the likelihood of misidentification and possible cross-contamination with this laboratory neotype, it is important to be able to distinguish H37 from clinical isolates. To provide a reference for identifying H37, we used multiple molecular techniques to characterize H37 strains, including 18 of the most frequently used variants available through the American Type Culture Collection. Isolates were genotyped using gene probes to IS6110 and IS1085. In addition, we performed polymorphic GC-rich sequence typing (PGRS), spoligotyping, determination of variable number of tandem repeats (VNTR), and PCR amplification of the mtp40, msx4, and mpp8 polymorphic regions. Southern hybridization with IS6110 provided the most discrimination, differentiating the 18 H37 isolates into 10 discrete patterns made up of 9 H37Rv variants and 1 H37Ra variant. PGRS, IS1085, mpp8, and spoligotyping were not able to distinguish any H37 variants, while VNTR and msx4 discriminated two. Only IS6110 and spoligotyping could distinguish the H37 strain from clinical isolates. In summary, spoligotyping and IS6110 provide a rapid and accurate way to identify H37 contamination, though IS6110 can, in addition, classify many of the H37 variants that would otherwise require phenotypic segregation. The genotyping of Mycobacterium tuberculosis, primarily for outbreak identification, has become a model for the application of strain typing in the field of molecular epidemiology. In the clinical mycobacteriology laboratory, strain typing has been essential in the identification of laboratory cross-contamination (1, 2, 5, 16, 25), an almost impossible task prior to the inception of molecular techniques. The source of laboratory cross-contamination can be a clinical sample (3, 15, 16) or often the M. tuberculosis control strain maintained by the clinical mycobacteriology laboratory (12, 13). In this regard, the virulent and attenuated H37 variants are the most commonly used control isolates and thus are a major source of falsepositive results in M. tuberculosis identification as well as crosscontamination (13). The purpose of this study is to provide the mycobacteriologist with a molecular guide for discriminating H37 from clinical isolates in the genotyping laboratory. The strain H37 was originally isolated in 1905 and gained attention for its noted virulence in the guinea pig model, a distinctive characteristic used in the classification of human tuberculosis in the early 1900s. In 1934, H37 was dissociated into virulent (Rv) and avirulent (Ra) strains (18, 24). The original 1905 H37 isolate was then discontinued, and the H37Rv and H37Ra isolates have been maintained at the Trudeau Institute ever since. Several drug-resistant derivatives have been generated during the years, accounting in part for the 18 H37 variants available through the Trudeau Mycobacterial Collection (TMC) and the American Type Culture Collection (ATCC). * Corresponding author. Mailing address: Public Health Research Institute Tuberculosis Center, 455 First Ave., New York, NY Phone: (212) Fax: (212) Publication 72 from the Public Health Research Institute Tuberculosis Center. Hence, there are 15 H37Rv and 3 H37Ra progenies maintained at the Trudeau Institute and the ATCC. Although H37 variants are widely used as reference strains in mycobacteriology and molecular biology laboratories, their IS6110 patterns are often mistaken for clinical isolates displaying similar fingerprint patterns (unpublished data). In this respect, it is essential for both patient care and tuberculosis control to be able to properly recognize and genotype all possible H37 variants. To do so, we have employed several of the most common M. tuberculosis typing techniques in order to characterize the 18 H37 variants available through the ATCC. The results indicate that both spoligotyping and IS6110 provide a rapid means of distinguishing H37 strains from clinical isolates. In addition, IS6110 DNA fingerprinting analysis further discriminates the collection into 10 distinct H37 variants. MATERIALS AND METHODS M. tuberculosis reference strains. The 18 different catalogued H37 variants were purchased from the ATCC. The strains were deposited at the ATCC as follows: (C. L. Larson, University of Montana, lot ), (A. G. Karlson, Mayo Clinic, lot ), and (G. P. Kubica, Trudeau Laboratories, lot ). All other strains (35820, 35821, 35822, 35823, 35824, 35825, 35826, 35827, 35828, 35829, 35830, 35835, 35836, and 35838) were deposited by the Trudeau Institute in lot (data were kindly provided by the ATCC). In addition, the primary collection of H37 variants was also received, as a kind gift of R. North, from the TMC, Trudeau Institute, Saranac Lake, N.Y. TMC and ATCC reference numbers and susceptibility data are shown in Table 1. M. tuberculosis clinical isolates. A search of the IS6110 fingerprint database maintained at the Public Health Research Institute Tuberculosis Center (TB Center) (n 11,000) identified 131 H37Rv and H37Ra isolates which matched at least one of the nine H37Rv and one H37Ra patterns reported in this study. The fingerprint search was conducted using each of the 10 possible H37Rv and H37Ra patterns as a prototype. The TB Center database includes approximately 8,600 isolates from New York City and New Jersey, while the remaining samples are from seven additional states in the United States and from the former USSR, Singapore, South Africa, Romania, Egypt, Israel, Venezuela, Honduras, Mexico, India, Chile, the Czech Republic, and Kenya. Genotyping by IS6110. Chromosomal DNA extraction and strain typing by IS6110 was performed according to the standard method using the right-side 3200
2 VOL. 38, 2000 H37Rv/Ra IS6110 GENOTYPING 3201 TABLE 1. H37Rv and H37Ra collection a ATCC no. TMC no. Genotype Phenotype Strain origin Source or reference(s) (yr) Rv H37Rv Rv 9 Pan s A. G. Karlson, Mayo Clinic ATCC (1992) (Rv) Rv 7 Pan s Dissociated in , (Rv) Rv 1 Str r Mutant of TMC (Rv) Rv 2 Pas r Mutant of TMC102 W. Steenken (1948) (Rv) Rv 8 Inh r Mutant of TMC (Rv) Rv 3 Str r Inh r Mutant of TMC102 TMC (1954) (Rv) Rv 5 Pas r Str r Mutant of TMC (Rv) Rv 6 Pas r Str r Inh r Mutant of TMC102 TMC (1961) (Rv) Rv 8 Cyc r Mutant of TMC (Rv) Rv 8 Kan r Mutant of TMC (Rv) Rv 8 Pza r Mutant of TMC (Rv) Rv 4 Tac r Mutant of TMC (Rv) Rv 8 Eth r Mutant of TMC (Rv) Rv 7 Emb r Mutant of TMC102 TMC (1970) (Rv) Rv 7 Rif r Mutant of TMC102 TMC (1970) Ra (Ra) Ra 1 Inh r Mutant of TMC201 TMC (1961) (Ra) Ra 1 Str r Mutant of TMC201 TMC (1949) (Ra) Ra 1 Pan s Dissociated in , 24 a H37 variant list. Abbreviations: Cyc, cycloserine; Emb, ethambutol; Eth, ethionamide; Inh, isoniazid; Kan, kanamycin; Pas, para-aminosalicylic acid; PZA, pyrazinamide; Rif, rifampin; Str, streptomycin; Tac, thioacetazone (17 24, 29). Pan s, pan-susceptible. hybridization probe (IS probe) (27). The same membrane was rehybridized with the left-side IS6110 probe (IS probe), the direct repeat (DR) probe, and the insertion sequence IS1085. Genotyping by using PGRS probe. Chromosomal DNA was restricted with AluI and hybridized with the polymorphic GC-rich repetitive sequence (PGRS) probe (GenBank accession no. M95490) (14). All other electrophoretic and hybridization conditions were the same as for IS6110 genotyping (26). Spoligotyping. The DR of the extracted M. tuberculosis DNA was amplified by PCR and analyzed according to the spoligotyping protocol as described by Kamerbeek and colleagues (8). The AluI-digested DNA membranes generated for PGRS typing were also probed with the DR probe to confirm spoligotyping results (27). Determination of VNTR. The variable number of tandem repeat (VNTR) loci ETR-A to ETR-E were determined as described by Frothingham and Meeker- O Connell (7). Briefly, the five selected loci were amplified by PCR and analyzed on a 2% agarose gel. PCR amplification of the polymorphic fragments msx4, mpp8, and mtp40. Amplicons to segments msx4, mpp8, and mtp40 were generated and compared from all 18 H37 variants. The polymorphic segments msx4 and mpp8 containing two DR sequences were PCR amplified with primer pairs SX1-SX2 and PP3- PP4, respectively, as described by Namwat et al. (11). PCR amplification of the mtp40 fragment was accomplished using primers PT1 and PT2 (6). Computer analysis of fingerprint patterns. The IS6110 hybridization patterns were electronically digitized and compared with a pattern-matching computer program on a Sun Sparc5 workstation using a Bioimage Whole Band Analyzer (software version 3.4; Genomic Solutions, Ann Arbor, Mich.). The Jaccard matching method and unweighted-pair-group method using arithmetic averages (UPGMA)-average linkage clustering was used to identify related patterns, in accord with the protocol of the Centers for Disease Control and Prevention, The National Tuberculosis Genotyping and Surveillance Network. hybridizing bands ranged from 14 (Rv 4 )to16(rv 7 ) bands for H37Rv and was 16 bands for H37Ra (Ra 1 ; Fig. 1). The spoligopattern of H37 variants was unique (spoligotype S00001; Fig. 2). The S00001 spoligopattern has only been observed in isolates of H37 variants and clinical samples determined to have been cross-contaminated by H37 strains. VNTR patterns were determined by analysis of the products of the PCR amplification of the five chromosomal loci ETR-A to ETR-E. All 18 ATCC isolates displayed the same VNTR pattern (33433), except for Rv 5. PGRS-typing, spoligotyping, and RESULTS Eighteen H37 variants available through the ATCC and their respective parent strains from the Trudeau Institute were typed by the now standard IS6110 Southern blot hybridization analysis (26). A total of 10 distinct fingerprint patterns were identified (Fig. 1). The nine patterns associated with the H37Rv strains were assigned the genotypes Rv 1 through Rv 9 and all three H37Ra strains (35835, 35836, and 25177) shared the same Ra 1 fingerprint pattern (Table 1). The Rv variants were Rv 1 (strain 35820), Rv 2 (strain 35821), Rv 3 (strain 35823), Rv 4 (strain 35829), Rv 5 (strain 35824), Rv 6 (strain 35825), Rv 7 (strains 35837, 35838, and 27294), Rv 8 (strains 35822, 35826, 35827, 35828, and 35830), and Rv 9 (strain 35618). The number of IS6110 FIG. 1. IS6110 hybridization patterns of H37. Ten different IS6110 patterns were identified from the 18 H37 variants available through the ATCC and the TMC.
3 3202 BIFANI ET AL. J. CLIN. MICROBIOL. with one of the H37Rv or H37Ra variants. The five samples differed from Rv1 to Rv9 and Ra1 by one or two hybridizing bands according to IS6110 analysis (Fig. 3). These samples were designated Rv10 and Rv11 and Ra2 through Ra4 (Fig. 3). Further analysis of all five isolates by the IS1085, VNTR, PGRS, and spoligotype methods failed to distinguish these samples from the reference TMC-ATCC prototypes (Fig. 4). The PGRS profile of H37 Rv1, Rv7, Rv8, Rv10 and Rv11, and Ra1 and Ra4 can be seen in Fig. 4. Two of the strains, designated Rv10 and Ra2 (Fig. 3), were received as part of a large cluster of H37Rv and H37Ra crosscontamination investigations (13). Furthermore, strains Rv10 and Ra2 were very closely related to the standard laboratory reference strains Rv1 and Ra1, respectively. Rv10 and Ra2 differ from Rv1 and Ra1 by the deletion of one IS6110 insertion (Fig. 3). Another variant (Rv11) was routinely used as a reference strain by a clinical laboratory and was supplied for fingerprint analysis as part of a control experiment. Cross-contamination by the remaining two H37 isolates (Ra3 and Ra4) was confirmed by reviewing laboratory and clinical data pertaining to these two cases. Thus, it can be inferred that Rv10, Rv11, Ra2, Ra3, and Ra4 are variants of an H37 reference strain which have further evolved in the lab. DISCUSSION In the speciation and susceptibility testing of M. tuberculosis in the clinical laboratory, suspicion of contamination is heightened by the finding of an unusually high number of positive FIG. 2. Spoligotype pattern of H37 variants. All 18 H37 ATCC and TMC samples, as well as 5 H37 laboratory variants, were found to have the same pattern. This pattern was not seen in a spoligotype database of 2,400 samples other than for H37 control samples. VNTR analysis did not differentiate the 18 H37 variants within the TMC and ATCC isolates (see Fig. 4). PCR amplification of three other polymorphic regions, the two repetitive sequences msx4, mpp8, and mtp40, and hybridization with IS1085, could not be used to distinguish H37 variants from other genetically unrelated M. tuberculosis strains. The msx4 PCR products of all H37 variants were shown to be 437 bp in length, with the exception of strain ATCC 35825, which generated a 720-bp amplicon (data not shown). Genotyping clinical isolates. Searching the IS6110 DNA fingerprint pattern database at the TB Center (n 11,000) using Rv1 to Rv9 and Ra1 as prototypes, identified 131 IS6110 patterns that matched exactly at least 1 of the 18 H37 variants. A total of 102 samples matched H37Ra type Ra1, 27 matched H37Rv type Rv7, and 1 each matched Rv1 and Rv8. Of these, 45 samples identified as H37Ra were previously reported as known cases of cross-contamination following an investigation by the New York City Department of Health (13). Three H37Ra samples were from outside the United States. The New York State Department of Health confirmed an additional 14 H37Ra strains to be cases of laboratory cross-contamination (12). Of the remaining 69 cases, 37 were confirmed to be contamination by the source laboratories, and the remaining are being investigated by the responsible entities. Five additional samples had a similar but not exact match FIG. 3. IS6110 hybridization patterns of Rv7, Ra1, and five H37 polymorphic variants. As indicated, Rv10 and Rv11 (lanes 4 and 5) differ from Rv7 (lane 2) by the loss and addition of an IS6110 insertion, respectively (see arrows). Ra2 (lane 6) has lost an IS6110 insertion, while Ra3 and Ra4 (lanes 7 and 8) have gained an insertion compared to the reference strain Ra1 (lane 3 and 9).
4 VOL. 38, 2000 H37Rv/Ra IS6110 GENOTYPING 3203 FIG. 4. Southern hybridization of AluII-digested chromosomal DNA with the PGRS probe for strains Rv 7,Ra 1, and five H37 polymorphic variants. PGRS grouped all H37 variants but could not differentiate between them. cultures per time period and by inconsistencies between the patient s clinical presentation and his or her laboratory results (10). Suggested cases of contamination are forwarded to a limited number of genotyping laboratories to perform molecular analysis to confirm or reject the possibility of contamination, primarily on a case-by-case basis (1, 2, 5, 16, 25). Often, genotyping and mycobacteriology laboratories are unaware of which H37 variant they are employing as controls, confounding the identification of contaminants. In this study, we have provided a collection of patterns for H37 and its variants for use as a reference by genotyping laboratories. Genotyping analysis of H37 variants. All 18 ATCC H37 variants had the same spoligotype pattern, designated S Although spoligotype pattern S00001 did not discriminate one ATCC H37 variant from another, it proved definitive in distinguishing the ATCC H37 collection from all other clinical isolates analyzed by this technique (n 2,400). Based on the similarity of the IS6110 DNA fingerprint patterns, all H37 variants were grouped as related. IS6110 discriminated the H37 variants, identifying 9 distinct yet similar patterns (Rv 1 to Rv 9 ) of 15 possible phenotypically (drug resistance profile) diverse H37Rv isolates and a single pattern for all 3 H37Ra (Ra 1 ) isolates. The differences in IS6110 fingerprint patterns between H37Rv and H37Ra have been previously examined by restriction analysis using four different endonucleases (9), and the molecular bases for the alternate IS6110 patterning have been investigated by comparative sequence analysis using the bacterial-artificial-chromosome method (4). Unfortunately, since the vast majority of studies involve H37 isolates, the above-mentioned investigations failed to properly identify the H37Rv and H37Ra used (4, 9). The H37 IS6110 patterns shown in this study may be used as a reference for genotyping analysis; however, it is conceivable that cultures that have been maintained over the years in different laboratories or else different culture lots available from the ATCC might have evolved additional related IS6110 patterns. The accuracy of spoligotyping and IS6110 fingerprint analysis in the identification of H37 variants was evaluated by comparison with other well-established molecular techniques. As with spoligotyping, PGRS and VNTR analyses were found to be nondiscriminating within the 18 ATCC H37 variants. Thus, the spoligotyping, PGRS, and VNTR methods can group the ATCC H37 collection as shown but cannot distinguish any variants. In addition, when H37 PGRS and VNTR patterns were compared to our database (PGRS, n 600; VNTR, n 400), a close similarity was noted with a large cluster of clinical isolates (unpublished data), rendering detection of the H37 variants by one of these two techniques unreliable. In agreement with VNTR analysis of an unspecified H37Rv and H37Ra in another work (7), we found that all 18 TMC-ATCC H37 variants (except for Rv 5 ) share the same VNTR pattern for loci ETR-A to ETR-E, i.e., strain Polymorphisms encountered in amplicons, msx4, mpp8, and mtp40 could not be used to discriminate the H37 variants from other clinical isolates, in agreement with other studies (11, 28). Identifying H37 cross-contamination among clinical isolates. The ability to discriminate between the H37 variants and real clinical samples has important public health implications. In this study, the IS6110 fingerprint patterns of 131 clinical isolates, most of which were confirmed contaminants, matched that of one of the reference H37 strains. Random spoligotyping, PGRS, and VNTR analyses confirmed the relatedness of these 131 isolates to the reference ATCC H37 variants. In addition, two H37Rv and three H37Ra IS6110 patterns (Rv 10 and Rv 11 and Ra 2 to Ra 4 ), which are distinct from but related to the ATCC collection, were identified among clinical specimens from our database of 11,000 fingerprints. These five isolates, also confirmed cases of laboratory contamination, were found to have the same spoligotype, PGRS, and VNTR as the TMC-ATCC isolates, and we infer that the isolates have evolved in the clinical laboratory from one of the reference strains. However, investigators should be aware that, given the origins of H37, it is possible that true clinical isolates exist with an identical spoligotype and similar IS6110 pattern as the reference strain H37. Thus, while genotyping may be used to initiate or direct investigation, clinical decisions regarding contamination should be based on a combination of molecular and medical information. Taken together, analysis of our clinical M. tuberculosis collection as well as ATCC isolates indicates that a combination of spoligotyping and IS6110 fingerprinting has proven to be a reliable tool in the proper identification of H37 crosscontamination. Unlike PGRS analysis, the H37 spoligopatterns (S00001) were unambiguous, making interpretation consistent. IS6110 fingerprinting should be used to confirm the proper identification of an H37 isolate. ACKNOWLEDGMENTS This research was supported in part by the Centers for Disease Control and Prevention, National Tuberculosis Genotyping and Surveillance Network cooperative agreement. We are grateful to R. North for providing us with the H37 variants from the TMC. We thank W. Eisner and H. Marasco for assistance in preparing the manuscript. REFERENCES 1. Bauer, J., V. O. Thomsen, S. Poulsen, and A. B. Andersen Falsepositive results from cultures of Mycobacterium tuberculosis due to laboratory
5 3204 BIFANI ET AL. J. CLIN. MICROBIOL. cross-contamination confirmed by restriction fragment length polymorphism. J. Clin. Microbiol. 35: Bhattacharya, M., S. Dietrich, L. Mosher, F. Siddiqui, B. E. Reisberg, W. S. Paul, and J. R. Warren Cross-contamination of specimens with Mycobacterium tuberculosis: clinical significance, causes, and prevention. Am. J. Clin. Pathol. 109: Braden, C. R., G. L. Templeton, W. W. Stead, J. H. Bates, M. D. Cave, and S. E. Valway Retrospective detection of laboratory cross-contamination of Mycobacterium tuberculosis cultures with use of DNA fingerprint analysis. Clin. Infect. Dis. 24: Brosch, R., W. J. Philipp, E. Stavropoulos, M. J. Colston, S. T. Cole, and S. V. Gordon Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain. Infect. Immun. 67: De, C. R. M., H. Soini, G. C. Roscanni, M. Jaques, M. C. Villares, and J. M. Musser Extensive cross-contamination of specimens with Mycobacterium tuberculosis in a reference laboratory. J. Clin. Microbiol. 37: Del Portillo, P., L. A. Murillo, and M. E. Patarroyo Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis. J. Clin. Microbiol. 29: Frothingham, R., and W. A. Meeker-O Connell Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology 144(Pt. 5): Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van Embden Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35: Lari, N., L. Rindi, C. Lami, and C. Garzelli IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and H37Ra. Microb. Pathog. 26: MacGregor, R. R., L. W. Clark, and F. Bass The significance of isolating low numbers of Mycobacterium tuberculosis in culture of sputum specimens. Chest 68: Namwat, W., P. Luangsuk, and P. Palittapongarnpim The genetic diversity of Mycobacterium tuberculosis strains in Thailand studied by amplification of DNA segments containing direct repetitive sequences. Int. J. Tuberc. Lung Dis. 2: Nivin, B., J. Driscoll, T. Glaser, P. Bifani, and S. Munsiff. Use of spoligotype analysis to detect laboratory cross-contamination. Infect. Control Hosp. Epidemiol., in press. 13. Nivin, B., P. I. Fujiwara, J. Hannifin, and B. N. Kreiswirth Crosscontamination with Mycobacterium tuberculosis: an epidemiological and laboratory investigation. Infect. Control Hosp. Epidemiol. 19: Palittapongarnpim, P., P. Luangsook, S. Tansuphaswadikul, C. Chuchottaworn, R. Prachaktam, and B. Sathapatayavongs Restriction fragment length polymorphism study of Mycobacterium tuberculosis in Thailand using IS6110 as probe. Int. J. Tuberc. Lung Dis. 1: Segal-Maurer, S., B. N. Kreiswirth, J. M. Burns, S. Lavie, M. Lim, C. Urban, and J. J. Rahal, Jr Mycobacterium tuberculosis specimen contamination revisited: the role of laboratory environmental control in a pseudooutbreak. Infect. Control Hosp. Epidemiol. 19: Small, P. M., N. B. McClenny, S. P. Singh, G. K. Schoolnik, L. S. Tompkins, and P. A. Mickelsen Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. J. Clin. Microbiol. 31: Steenken, W., and V. Montalbine The antituberculous activity of thiomide in vitro and in the experimental animal (mouse and guinea pig). Am. Rev. Respir. Dis. 81: Steenken, W., W. H. Oatway, and S. A. Petroff Biological studies of the tubercle bacillus. J. Exp. Med. 60: Steenken, W., and E. Wolinsky Cycloserine: antituberculous activity in vitro and in the experimental animal. Am. Rev. Tuberc. 73: Steenken, W., and E. Wolinsky Effect of antimicrobial agents on the tubercle bacillus and on experimental tuberculosis. Am. J. Med. 9: Steenken, W., and E. Wolinsky Streptomycin in experimental tuberculosis. Am. Rev. Tuberc. 58: Steenken, W., E. Wolinsky, M. M. Smith, and V. Montalbine Further observations on pyrazinamide alone and in combination with other drugs in experimental tuberculosis. Am. Rev. Tuberc. 76: Steenken, W., E. Wolinsky, and J. R. Thurston The antituberculous activity of kanamycin in vitro and in the experimental animal (guinea pig). Am. Rev. Tuberc. 79: Steenken, W. J., and L. U. Gardner History of H37 strain of tubercle bacillus. Am. Rev. Tuberc. 54: Van Duin, J. M., J. E. Pijnenburg, C. M. van Rijswoud, P. E. de Haas, W. D. Hendriks, and D. van Soolingen Investigation of cross contamination in a Mycobacterium tuberculosis laboratory using IS6110 DNA fingerprinting. Int. J. Tuberc. Lung Dis. 2: van Embden, J. D. A., M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J. Clin. Microbiol. 31: van Soolingen, D., P. E. de Haas, P. W. Hermans, P. M. Groenen, and J. D. van Embden Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis. J. Clin. Microbiol. 31: Weil, A., B. B. Plikaytis, W. R. Butler, C. L. Woodley, and T. M. Shinnick The mtp40 gene is not present in all strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 34: Wolinsky, E., and W. Steenken Antituberculous activity of hinconstarch, a synthetic polymer of isoniazid, amithiozone, and starch. Am. Rev. Tuberc. 73:7 8.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2005, p. 5034 5043 Vol. 43, No. 10 0095-1137/05/$08.00 0 doi:10.1128/jcm.43.10.5034 5043.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved.
International Journal of Infectious Diseases (2009) 13, 236 242 http://intl.elsevierhealth.com/journals/ijid Strain differentiation of Mycobacterium tuberculosis complex isolated from sputum of pulmonary
Molecular Epidemiology of Tuberculosis Kathy DeRiemer, PhD, MPH School of Medicine University of California, Davis Overview TB transmission and pathogenesis Genotyping methods Genotyping for clinical management
CHEST Emergence of New Forms of Totally Drug-Resistant Tuberculosis Bacilli Original Research Super Extensively Drug-Resistant Tuberculosis or Totally Drug-Resistant Strains in Iran Ali Akbar Velayati,
Original Research LUNG INFECTION Discrepant Results Between Pyrazinamide Susceptibility Testing by the Reference BACTEC 460TB Method and pnca DNA Sequencing in Patients Infected With Multidrug-Resistant
The Molecular Epidemiology of Tuberculosis Barry N. Kreiswirth,, PhD Director, PHRI TB Center Airborne pathogen, Mycobacterium tuberculosis Slow grower; doubles 24hrs; 3-4 weeks to culture 3-4 weeks for
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1999, p. 2607 2618 Vol. 37, No. 8 0095-1137/99/$04.00 0 Copyright 1999, American Society for Microbiology. All Rights Reserved. Comparison of Methods Based on Different
REFERENCES CONTENT ALERTS Genetic Diversity of Mycobacterium africanum Clinical Isolates Based on IS 6110-Restriction Fragment Length Polymorphism Analysis, Spoligotyping, Variable Number of Tandem DNA
MULTIDRUG- RESISTANT TUBERCULOSIS Dean Tsukayama Hennepin County Medical Center Hennepin County Public Health Clinic I have no relevant financial relationships. Discussion includes off label use of: amikacin
Polish Journal of Microbiology 2011, Vol. 60, No 3, 233 241 ORIGINAL PAPER A Two-Step Strategy for Molecular Typing of Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates from Poland TOMASZ
ORIGINAL ARTICLE 10.1111/j.1469-0691.2007.01711.x Association of specific mutations in katg, rpob, rpsl and rrs genes with spoligotypes of multidrug-resistant Mycobacterium tuberculosis isolates in Russia
MIC = Many Inherent Challenges Sensititre MIC for Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis complex Marie Claire Rowlinson, PhD D(ABMM) Florida Bureau of Public Health Laboratories
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2003, p. 5640 5644 Vol. 41, No. 12 0095-1137/03/$08.00 0 DOI: 10.1128/JCM.41.12.5640 5644.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved.
85 Spread of Strain W, a Highly Drug-Resistant Strain of Mycobacterium tuberculosis, Across the United States Tracy B. Agerton, Sarah E. Valway, Richard J. Blinkhorn, Kenneth L. Shilkret, Randall Reves,
1107 7. Bloch AB, Cauthen GM, Onorato IM, et al. Nationwide survey of drug- 11. Dwyer B, Jackson K, Raios K, Sievers A, Wilshire E, Ross B. DNA resistant tuberculosis in the United States. JAMA 1994;271:665
ORIGINAL ARTICLE 10.1111/j.1469-0691.2006.01495.x Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains with a mutation at amino-acid position 315 of katg: a decade of experience
Overview of Mycobacterial Culture, Identification, and Drug Susceptibility Testing 1. Essentials for the Mycobacteriology Laboratory: Promoting Quality Practices 1.1 Overview: Mycobacterial Culture, Identification,
UvA-DARE (Digital Academic Repository) Epidemiological studies on tuberculosis control and respiratory viruses Sloot, R. Link to publication Citation for published version (APA): Sloot, R. (2015). Epidemiological
The treatment of patients with initial isoniazid resistance 2011 INTERTB Meeting, St George s, London Patrick Phillips, MRC Clinical Trials Unit DA Mitchison, AJ Nunn. 21 st October 2011 Outline Background
Tuberculosis in Greenland current situation and future challenges Thomsen VØ 1, Lillebaek T 1, Stenz F 2,*. 1 International Reference Laboratory of Mycobacteriology, Statens Serum Institut (SSI), the National
Journal of Microbiology Research 2014, 4(6A): 25-31 DOI: 10.5923/s.microbiology.201401.04 Mycobacterium tuberculosis and Molecular Epidemiology: An Overview Asho Ali Department of Biology, King Abdul Aziz
TB Laboratory for Nurses Shea Rabley, RN, MN Consultant Mayo Clinic Center for Tuberculosis 2014 MFMER slide-1 Disclosures None 2014 MFMER slide-2 Objectives Participants will be able to: 1. Name 2 safety
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2009, p. 4006 4020 Vol. 47, No. 12 0095-1137/09/$12.00 doi:10.1128/jcm.01270-09 Copyright 2009, American Society for Microbiology. All Rights Reserved. Polymorphic
braz j infect dis. 2013;17(6):667 671 The Brazilian Journal of INFECTIOUS DISEASES www.elsevier.com/locate/bjid Original article A simple, rapid and economic method for detecting multidrug-resistant tuberculosis
CLINICAL MICROBIOLOGY REVIEWS, Oct. 2006, p. 658 685 Vol. 19, No. 4 0893-8512/06/$08.00 0 doi:10.1128/cmr.00061-05 Copyright 2006, American Society for Microbiology. All Rights Reserved. Molecular Epidemiology
TB Nurse Case Management San Antonio, Texas April 1 3, 2014 Diagnosis of TB: Laboratory Ken Jost Tuesday April 1, 2014 Ken Jost, BA has the following disclosures to make: No conflict of interests No relevant
American Journal of EPIDEMIOLOGY Volume 153 Number 12 June 15, 2001 Copyright 2001 by the Johns Hopkins University School of Hygiene and Public Health Sponsored by the Society for Epidemiologic Research
The ABC s of AFB s Laboratory Testing for Tuberculosis Gary Budnick Connecticut Department of Public Health Mycobacteriology Laboratory Laboratory TAT Goals Case Study Specimen Collection Testing Contact
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2010, p. 4102 4106 Vol. 48, No. 11 0095-1137/10/$12.00 doi:10.1128/jcm.00549-10 Copyright 2010, American Society for Microbiology. All Rights Reserved. Spoligotypes
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2002, p. 1417 1424 Vol. 46, No. 5 0066-4804/02/$04.00 0 DOI: 10.1128/AAC.46.5.1417 1424.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved.
Molecular Epidemiology and Clinical Characteristics of Drug-Resistant Mycobacterium tuberculosis in a Tuberculosis Referral Hospital in China Qi Wang 1., Susanna K. P. Lau 2., Fei Liu 1., Yanlin Zhao 3,
TB Nurse Case Management San Antonio, Texas April 9-11, 2013 Diagnosis of TB: Laboratory Ken Jost Tuesday April 9, 2013 Ken Jost has the following disclosures to make: No conflict of interests No relevant
Journal of Infection (2006) 53, 331e336 www.elsevierhealth.com/journals/jinf Prevalence of Haarlem I and Beijing types of Mycobacterium tuberculosis strains in Iranian and Afghan MDR-TB patients Parissa
The image part with relationship ID rid2 was not found in the file. MDR TB Management Review of the Evolution (or Revolution?) Elizabeth A. Talbot MD Assoc Professor, ID and Int l Health Deputy State Epidemiologist,
Original Article SMEAR MICROSCOPY AS SURROGATE FOR CULTURE DURING FOLLOW UP OF PULMONARY MDR-TB PATIENTS ON DOTS PLUS TREATMENT R. Sarin 1, R. Singla 2, P. Visalakshi 3, A. Jaiswal 4, M.M. Puri 4, Khalid
Maha R Farhat, MD MSc Massachusetts General Hospital Harvard Medical School I have no financial or other potential conflicts of interest to disclose Update on the epidemiology of TB drug resistance Success
Original Paper Med Principles Pract 2001;10:129 134 Received: January 31, 2001 Revised: May 14, 2001 Genetic Polymorphism at Codon 463 in the katg Gene in Isoniazid-Sensitive and -Resistant Isolates of
Indian J Med Res 135, May 2012, pp 756-762 Variations in the occurrence of specific rpob mutations in rifampicinresistant Mycobacterium tuberculosis isolates from patients of different ethnic groups in
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2004, p. 5774 5782 Vol. 42, No. 12 0095-1137/04/$08.00 0 DOI: 10.1128/JCM.42.12.5774 5782.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved.
Treatment of Active Tuberculosis Jeremy Clain, MD Pulmonary & Critical Care Medicine Mayo Clinic October 16, 2017 2014 MFMER slide-1 Disclosures No relevant financial relationships No conflicts of interest
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2007, p. 3891 3902 Vol. 45, No. 12 0095-1137/07/$08.00 0 doi:10.1128/jcm.01394-07 Copyright 2007, American Society for Microbiology. All Rights Reserved. Discovery
TB CASE MANAGEMENT AND CONTACT INVESTIGATION INTENSIVE NOVEMBER 1-4, 2016 TB BASICS: PRIORITIES AND CLASSIFICATIONS LEARNING OBJECTIVES Upon completion of this session, participants will be able to: 1.
American Journal of Epidemiology Copyright 2004 by the Johns Hopkins Bloomberg School of Public Health All rights reserved Vol. 159, No. 5 Printed in U.S.A. DOI: 10.1093/aje/kwh065 Diversity of Mycobacterium
JOURNAL OF BACTERIOLOGY, Apr. 2003, p. 2555 2562 Vol. 185, No. 8 0021-9193/03/$08.00 0 DOI: 10.1128/JB.185.8.2555 2562.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Evolutionary
Tuberculosis Disparity Between US-born African-Americans and Caucasians in Houston: A population-based study Jose A. Serpa 1, M.D.; Larry D. Teeter 2, Ph.D., James M. Musser 2, M.D., Ph.D. and Edward A.
A ten-year evolution of a multidrugresistant tuberculosis (MDR-TB) outbreak in an HIV-negative context, Tunisia (2001-2011) Naira Dekhil 1, Besma Mhenni 1, Raja Haltiti 2, and Helmi Mardassi 1 (speaker)
JOURNAL OF CLINICAL MICROBIOLOGY, July 2002, p. 2339 2345 Vol. 40, No. 7 0095-1137/02/$04.00 0 DOI: 10.1128/JCM.40.7.2339 2345.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2003, p. 1520 1524 Vol. 41, No. 4 0095-1137/03/$08.00 0 DOI: 10.1128/JCM.41.4.1520 1524.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved.
Recognizing MDR-TB in Children Ma. Cecilia G. Ama, MD 23 rd PIDSP Annual Convention 17-18 February 2016 Objectives Review the definitions and categorization of drugresistant tuberculosis Understand the
Laboratory Diagnosis for MDR TB Neha Shah MD MPH Centers for Disease Control and Prevention Division of Tuberculosis Elimination California Department of Public Health Guam March 07 Objectives Describe
CDPH - CTCA Joint Guidelines Guideline for Micobacteriology Services In California These guidelines are intended to be used as an educational aid to help clinicians make informed decisions about patient
JOURNAL OF CLINICAL MICROBIOLOGY, May 2004, p. 2234 2238 Vol. 42, No. 5 0095-1137/04/$08.00 0 DOI: 10.1128/JCM.42.5.2234 2238.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved.
WSLH Testing and Surveillance Updates Wisconsin Mycobacteriology Laboratory Network annual conference November 4, 2015, Madison, WI Updates Outline Collection and Transport Smear and Culture Nucleic Acid
AJRCCM Articles in Press. Published on May 18, 2006 as doi:10.1164/rccm.200511-1834oc Lack of Weight Gain and Relapse Risk in a Large Tuberculosis Treatment Trial 1 Awal Khan Ph.D., 1 Timothy R. Sterling
Use of Therapeutic Drug Monitoring for Multidrug-Resistant Tuberculosis Patients* Jiehui Li, MBBS, MS; Joseph N. Burzynski, MD, MPH; Yi-An Lee, MPH; Debra Berg, MD; Cynthia R. Driver, RN, MPH; Renee Ridzon,
Original Article Nepal Medical College Journal 27; 9(1): Rapid differentiation of mycobacterium tuberculosis and mycobacterium leprae from sputum by polymerase chain reaction Bishwa Raj Sapkota, Chaman
JOURNAL OF CLNCAL MCROBOLOGY, OCt. 1994, p. 24252433 951137/94/$4.+ Vol. 32, No. 1 Use of Various Genetic Markers in Differentiation of Mycobacterium bovis Strains from Animals and Humans and for Studying
Molecular typing for surveillance of multidrug-resistant tuberculosis in the EU/EEA January 2016 Summary This report describes the geographical and temporal distribution of multidrug-resistant (MDR) tuberculosis
ORIGINAL ARTICLE BACTERIOLOGY Rapid and accurate detection of rifampin and isoniazid-resistant Mycobacterium tuberculosis using an oligonucleotide array W.-L. Huang 1, Z.-J. Hsu 1, T. C. Chang 2 and R.
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2008, p. 3924 3930 Vol. 46, No. 12 0095-1137/08/$08.00 0 doi:10.1128/jcm.00793-08 Copyright 2008, American Society for Microbiology. All Rights Reserved. Transmission
The Epidemiology of Tuberculosis in Minnesota, 2011 2015 Minnesota Department of Health Tuberculosis Prevention and Control Program (651) 201-5414 Tuberculosis surveillance data for Minnesota are available
Transmission of Mycobacterium tuberculosis Charles Daley, MD National Jewish Health Disclosures Advisory Board Horizon, Johnson and Johnson, Otsuka and Spero Investigator Insmed Objectives After attending
TB 101 Disease, Clinical Assessment and Lab Testing Pacific Islands Tuberculosis Controllers Association Conference (PITCA) Clinical Laboratory Breakout None Disclosure Objectives Be able to list and explain
EPI Case Study 3: Cross-Sectional, Case-Control, and Cohort Studies Identification of TB Risk Time to Complete Exercise: 60 minutes LEARNING OBJECTIVES At the completion of this module, participants should
Use of the Cepheid GeneXpert to Release Patients from Airborne Isolation Dave Warshauer, PhD, D(ABMM) Deputy Director, Communicable Diseases Wisconsin State Laboratory of Hygiene WISCONSIN STATE LABORATORY
Original Article A COMPARATIVE STUDY OF THE DIAGNOSIS OF PULMONARY TUBERCULOSIS USING CONVENTIONAL TOOLS AND POLYMERASE CHAIN REACTION* Kavita Modi Parekh 1, Vikas Inamdar 2, Anagha Jog 3 and Anita Kar
Public Health Agency of Canada Agence de la santé publique du Canada Tuberculosis Drug resistance in Canada Reported susceptibility results of the Canadian Tuberculosis Laboratory Surveillance System Our
JCM Accepts, published online ahead of print on 1 October 2008 J. Clin. Microbiol. doi:10.1128/jcm.01315-08 Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All
Journal of Antimicrobial Chemotherapy (27) 59, 794 798 doi:.93/jac/dkm25 Sputum conversion among patients with pulmonary tuberculosis: are there implications for removal of respiratory isolation? Jesús
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2007, p. 237 240 Vol. 45, No. 1 0095-1137/07/$08.00 0 doi:10.1128/jcm.01429-06 Copyright 2007, American Society for Microbiology. All Rights Reserved. Spoligotype
Tuberculosis (TB) Fundamentals for School Nurses June 9, 2015 Kristin Gall, RN, MSN/Pat Infield, RN-TB Program Manager Marsha Carlson, RN, BSN Two Rivers Public Health Department Nebraska Department of
Kritische Fragen zum Beitrag der kombinierten PCR für den Nachweis von M. tuberculosis und der Identifizierung von Mutationen auf dem rpob-gen Hans L Rieder Union Internationale Contre la Tuberculose et
RESEARCH ARTICLE Open Access Research article Diversity of Mycobacterium tuberculosis genotypes circulating in Ndola, Zambia Chanda Mulenga* 1,2, Isdore C Shamputa 1,2,5, David Mwakazanga 1, Nathan Kapata
HA Convention 2016 : Special Topic Session 3 May 2016 Diagnosis and Management of TB in Adults Dr. Thomas Mok COS(RMD), KH Tuberculosis An airborne infectious disease caused by Mycobacterium tuberculosis
Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Cain KP, McCarthy KD, Heilig CM, et al. An algorithm for tuberculosis
Introduction to TB Nurse Case Management Online February 4, 11, 18 and 25, 2015 Initiation Phase Part 1 Ginny Dowell, RN, BSN February 4, 2015 Ginny Dowell RN, BSN has the following disclosures to make:
Indian J Med Res 143, March 2016, pp 341-347 DOI:10.4103/0971-5916.182625 A study on pre-xdr & XDR tuberculosis & their prevalent genotypes in clinical isolates of Mycobacterium tuberculosis in north India
HLA TYPING AND EXPRESSION: POTENTIAL MARKER FOR IDENTIFYING EARLY DYSPLASIA AND STRATIFYING THE RISK FOR IBD-CANCER Megan Garrity, S. Breanndan Moore, M.D., William Sandborn, M.D., Vernon Pankratz, Ph.D.,
SALSA MLPA KIT P045-B2 BRCA2/CHEK2 Lot 0410, 0609. As compared to version B1, four reference probes have been replaced and extra control fragments at 100 and 105 nt (X/Y specific) have been included. New: