PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp.

Kim, Hong; Kim, Sun Hyun; Shim, Tae Sun; Kim, Seung Up; Bai, Gill Han; Park, Young Gil; Lee, Sueng Hyun; Chen, Yong; Kook, Yoon Hoh; Kim, Bum Joon

doi:10.1016/j.mimet.2005.02.010

Cited by 36 publications

(37 citation statements)

References 21 publications

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“…Moreover, this strain may be unusually resistant to disinfectants 2 . The pathogenic potential of M. massiliense has been demonstrated by human infections in both immunocompetent and immunocompromised hosts, and by its intracellular survival and growth 2,[6][7][8] . In the present case, repeated incision and drainage at a local clinic failed to eradicate M. massiliense.…”

Section: Discussionmentioning

confidence: 99%

“…The following primer pairs were used: 285 (5'-GAG AGT TTG ATC CTG GCT CAG-3') and 244 (5'-CCC ACT GCT GCC TCC CGT AG-3') for the 16S rRNA gene (351 bp) 12 , RGMF (5'-GAC GAC ATC GAC CAC TTC GG-3') and RGMR (5'-GGG GTC TCG ATC GGG CAC AT-3') for rpoB (365 bp) 8 , and HSPF3 (5'-ATC GCC AAG GAG ATC GAG CT-3') and HSPR4 (5'-AAG GTG CCG CGG ATC TTG TT-3') for hsp65 (644 bp) 13 . PCR products were purified using QIAEX II gel extraction kits (Qiagen, Hilden, Germany) and sequenced directly using forward and reverse primers with an Applied Biosystems automated sequencer (model 377) and BigDye Terminator cycle sequencing kits (Perkin-Elmer Applied Biosystems, Warrington, UK 7,8,10 . The isolate was also susceptible to amikacin (MIC=4μg/ml) and cefoxitin (MIC=16μg/ml), and it showed intermediate susceptibility to imipenem (MIC=8μg/ml) and tobramycin (MIC=8μg/ ml).…”

Section: Case Reportmentioning

confidence: 99%

“…M. massiliense is very closely related to M. abscessus and M. chelonae 7 . It has been isolated from a patient with pneumonia, a patient with pacemaker pocket infection, an outbreak associated with intramuscular injections of antimicrobial agents, and outbreaks associated with laparoscopic surgeries and cosmetic procedures 2,5,[7][8][9][10] . Here we report the case of a cutaneous M. massiliense infection associated with a surgical procedure in an immunocompetent patient.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Cutaneous Mycobacterium massiliense Infections Associated with Repeated Surgical Procedures

et al. 2010

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Section: Discussionmentioning

confidence: 99%

Section: Case Reportmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of Cutaneous Mycobacterium massiliense Infections Associated with Repeated Surgical Procedures

et al. 2010

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“…With respect to the observations regarding the inaccuracy of 16S rRNA sequencing results of non-tuberculous mycobacteria made by Turenne and others, 24 sequencing of the two additional regions of the M. ulcerans genome served as a quality-assurance measure. [23][24][25][26] Sequence analysis. Sequences were analyzed using DNASIS Max software (MiraiBio, San Francisco, CA ) and aligned with the M. ulcerans wild-type sequence (Agy99) of the respective gene.…”

Section: 15mentioning

confidence: 99%

A Genotypic Approach for Detection, Identification, and Characterization of Drug Resistance in Mycobacterium ulcerans in Clinical Samples and Isolates from Ghana

Beißner

Awua-Boateng

Thompson

et al. 2010

The American Society of Tropical Medicine and Hygiene

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Abstract. Standardized antimycobacterial therapy is considered the treatment of choice for Buruli ulcer disease. To assess the prevalence of drug resistance among clinical Mycobacterium ulcerans isolates in Ghana, we conducted a sequence-based approach to detect mutations associated with drug resistance. We subjected clinical samples to direct DNA sequencing of rpoB and rpsL genes and compared culture and whole-genome extracts regarding the efficiency of sequence analysis; 99.1% ( rpoB ) and 100% ( rpsL ) of the patients harbored M. ulcerans wild type. In one isolate (0.9%), a point mutation of the rpoB gene at codon Ser522 leading to an amino acid change was detected. Culture extracts yielded a significantly higher sequencing efficiency than whole-genome extracts. Our data suggest a low level of drug resistance in Ghana. However, mutations associated with drug resistance do occur and require monitoring. Improved techniques are necessary to enhance the efficiency of sequence analysis of whole-genome extracts.

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“…T were used as a positive control for M. tuberculosis from sputum samples, and as described in a previous report (12), the presence of the two fragments, 471 and 164 bp, in gel was regarded as indicative of M. tuberculosis positivity by hsp65 PRA (Fig. 1).…”

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confidence: 99%

Direct Application of AvaII PCR Restriction Fragment Length Polymorphism Analysis (AvaII PRA) Targeting 644 bp Heat Shock Protein 65 (hsp65) Gene to Sputum Samples

Mun

Kim

et al. 2007

Microbiology and Immunology

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Of the validated species in the genus Mycobacterium, M. tuberculosis is the most common and important pathogen and causes 2 million deaths and over 8 million cases of tuberculosis annually worldwide (2-4, 7). In addition to the multidrug resistant strains of M. tuberculosis, NTM (non-tuberculosis mycobacteria) infection can cause other serious clinical problems. Moreover, because the drugs of choice for the treatment of NTM infection are heavily dependent on taxonomic statuses (15,19,20), it is of importance that we are able to differentiate these at the species level during early diagnostic procedures.The classical identification of mycobacteria based on cultural and biochemical tests may take several weeks, and sometimes, even then, may fail to provide a precise identification. To resolve such disadvantages of conventional methods, various PCR-mediated methods have been applied for the rapid detection and identification of mycobacteria species. Among these, PCRrestriction analysis (PRA) is preferred, since it offers an easy, rapid, and inexpensive means of identifying mycobacteria species. Thus, a number of PRA methods targeting several chronometers, such as, 16S rDNA (6, 8), hsp65 (5, 17), dnaJ (16), and rpoB (11), have been developed. However, although these PRA techniques have been successfully applied to mycobacteria identification at the culture level, problems are encountered when they are applied directly to clinical specimens, especially sputum samples, which also contain numbers of commensal bacteria from the respiratory tract, because these bacteria produce complex PRA patterns that are difficult to interpret.Previously, we reported that a novel PRA method that targets 644 bp hsp65 DNA is useful for the differentiation of mycobacterial species at the culture level (12). In particular, by only the use of a single restriction enzyme, AvaII, it can differentiate the three most clinically important mycobacteria species, i.e., M. tuberculosis and two MACs, M. avium and M. intracellulare. Moreover, the simplicity of this PRA method might enable the direct detection of pathogenic mycobacteria from clinical specimens such as sputum.In the present study, to evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 sequence Abstract: To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS6110 PCR. Although this method showed a sensitivity slightly lower than IS6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.

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PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp.

Cited by 36 publications

References 21 publications

Identification of Cutaneous Mycobacterium massiliense Infections Associated with Repeated Surgical Procedures

Identification of Cutaneous Mycobacterium massiliense Infections Associated with Repeated Surgical Procedures

A Genotypic Approach for Detection, Identification, and Characterization of Drug Resistance in Mycobacterium ulcerans in Clinical Samples and Isolates from Ghana

Direct Application of AvaII PCR Restriction Fragment Length Polymorphism Analysis (AvaII PRA) Targeting 644 bp Heat Shock Protein 65 (hsp65) Gene to Sputum Samples

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