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Mycobacteriology and Aerobic Actinomycetes

Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology

Danica Helb, Martin Jones, Elizabeth Story, Catharina Boehme, Ellen Wallace, Ken Ho, JoAnn Kop, Michelle R. Owens, Richard Rodgers, Padmapriya Banada, Hassan Safi, Robert Blakemore, N. T. Ngoc Lan, Edward C. Jones-López, Michael Levi, Michele Burday, Irene Ayakaka, Roy D. Mugerwa, Bill McMillan, Emily Winn-Deen, Lee Christel, Peter Dailey, Mark D. Perkins, David H. Persing, David Alland
Danica Helb
1Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey
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Martin Jones
2Cepheid, Sunnyvale, California
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Elizabeth Story
1Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey
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Catharina Boehme
3Foundation for Innovative New Diagnostics, Geneva, Switzerland
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Ellen Wallace
2Cepheid, Sunnyvale, California
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Ken Ho
2Cepheid, Sunnyvale, California
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JoAnn Kop
2Cepheid, Sunnyvale, California
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Michelle R. Owens
2Cepheid, Sunnyvale, California
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Richard Rodgers
2Cepheid, Sunnyvale, California
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Padmapriya Banada
1Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey
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Hassan Safi
1Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey
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Robert Blakemore
1Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey
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N. T. Ngoc Lan
4Pham Ngoc Thach Hospital, Ho Chi Minh City, Vietnam
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Edward C. Jones-López
1Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey
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Michael Levi
5Montefiore Medical Center, Bronx, New York
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Michele Burday
6Department of Pathology, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey
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Irene Ayakaka
7Makerere University-University of Medicine and Dentistry, New Jersey, Research Collaboration, Kampala, Uganda
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Roy D. Mugerwa
8Department of Medicine, Makerere University School of Medicine, Kampala, Uganda
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Bill McMillan
2Cepheid, Sunnyvale, California
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Emily Winn-Deen
2Cepheid, Sunnyvale, California
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Lee Christel
2Cepheid, Sunnyvale, California
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Peter Dailey
2Cepheid, Sunnyvale, California
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Mark D. Perkins
3Foundation for Innovative New Diagnostics, Geneva, Switzerland
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David H. Persing
2Cepheid, Sunnyvale, California
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David Alland
1Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey
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  • For correspondence: allandda@umdnj.edu
DOI: 10.1128/JCM.01463-09
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  • FIG. 1.
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    FIG. 1.

    Limit of detection for the in-cartridge heminested PCR. (A) DNA detection. M. tuberculosis DNA at final concentrations of 0, 1, 1.5, 2.5, 3, or 7.5 genomes per PCR was loaded into cartridges and processed according to the Xpert MTB/RIF protocol. For each genomic concentration tested (n = 5 to 7), the percentage of M. tuberculosis-positive cartridges was plotted. As determined by logistic regression, there is a 95% probability of detecting M. tuberculosis in samples containing at least 4.5 genomes per PCR (95% CI, 3.3 to 9.7). (B) Detection of M. tuberculosis cells in clinical sputum samples. M. tuberculosis cells were added to 1 ml of M. tuberculosis-negative sputum to final concentrations of 10, 50, 100, 150, or 300 CFU/ml (n = 20) and then processed according to the Xpert MTB/RIF protocol. The percentage of assays where M. tuberculosis was detected was then plotted for each concentration of cells. As determined by logistic regression, there was a 95% probability of detecting M. tuberculosis in samples containing at least 131 CFU/ml (95% CI, 106.2 to 176.4).

  • FIG. 2.
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    FIG. 2.

    Detection of RRDR mutations. Genomic M. tuberculosis DNA or artificial targets containing clinically relevant mutations were added to the wash buffer of cartridges that were then run with M. tuberculosis-negative sputum. Typical results from six rifampin-susceptible (wild-type RRDR) isolates and 23 RRDR mutants are shown. The results produced by each sample are indicated by a single vertical line on which the CT of each of the five rpoB-specific molecular beacons (probes A to E) is plotted. ΔCT values ≥3.5 cycles indicate rifampin resistance. All 23 RRDR mutants were correctly identified as rifampin resistant.

  • FIG. 3.
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    FIG. 3.

    Effect of sample reagent. (A) Biohazard reduction. Sputum samples spiked with high concentrations of M. tuberculosis cells were treated with 2 volumes of SR and incubated for 15 min to 5 days (n = 3). M. tuberculosis cells remaining viable after SR treatment were measured by quantitative culture. The log decrease in M. tuberculosis viability, compared to an untreated control, was plotted for each incubation time tested. (B) Assay performance. Sputum spiked with 150 CFU/ml of M. tuberculosis cells was treated with 2 volumes of SR and incubated for 15 min to 5 days and then processed in the Xpert MTB/RIF assay. Average CT values were plotted for each incubation time (n = 3 to 4).

  • FIG. 4.
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    FIG. 4.

    Assay performance with nontuberculosis mycobacteria. (A) Lack of cross-reaction by NTM. A total of 106 CFU/ml of relevant NTM species was added into M. tuberculosis-negative sputum and processed according to the Xpert MTB/RIF protocol. The results produced by each species are indicated by a single vertical line on which the CT of each of the five rpoB-specific molecular beacons (probes A to E) is plotted. For tuberculosis detection, the GeneXpert software requires a sample to have at least two positive molecular beacon probes with a ΔCT of <2 cycles. In the example shown, the assay correctly identified members of the M. tuberculosis complex (M. tuberculosis, M. bovis BCG, and Mycobacterium africanum) as M. tuberculosis positive. However, none of the NTM samples produced ΔCTs that fulfilled the criteria for M. tuberculosis detection. (B) Lack of interference by NTM. Possible interactions between low concentrations of M. tuberculosis and high concentrations of NTM species were investigated. Sputum containing both 200 CFU/ml of M. tuberculosis and 106 CFU/ml of an NTM species was processed according to the Xpert MTB/RIF protocol, and cycle thresholds for all five rpoB probes were plotted. M. tuberculosis was accurately identified in all of these samples. However, the combination of high levels of M. malmoense with low concentrations of M. tuberculosis resulted in a false rifampin resistance result. Rifampin resistance was not seen when 200 CFU/ml of H37Rv was tested in combination with 105 CFU/ml of M. malmoense or when sputum contained 300 CFU/ml of H37Rv and 106 CFU/ml of M. malmoense (data not shown).

Tables

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  • TABLE 1.

    Clinical characteristics of study patients

    CharacteristicValue in the indicated group
    Vietnamese patients (n = 107)aUgandan patients (n = 64)a
    Patient characteristics
        Median age (yr [range])34 (18-76)34 (18-60)
        No. of males (%)74 (69)38 (59)
        No. of HIV-positive patients (%)1 (0.9)b20 (40)
        No. of unavailable HIV result(s)014
        No. of previous TB episodes (no. of patients [%])
            12 (1.9)54 (84)
            2-307 (11)
            4-503 (5)
        Median duration of cough (days [range]28 (14-336)90 (21-365)
    AFB smear grade (no. [%])b0
        Negative53 (65)c
        Scanty (1-9/100 fields)d8 (10)
        1+ (10-99/100 fields)d8 (10)
        2+ (1-10/field)d10 (12)
        3+ (>10/field)d3 (4)
        1+ (1-9/100 fields)e2 (3)
        2+ (1-9/10 fields)e8 (13)
        3+ (1-9/field)e4 (6)
        4+ (>9/field)e50 (78)
    Days to positive BACTEC culture (no. [%])f
        <75 (6)54 (84)
        7-2173 (89)8 (13)
        >214 (5)2 (3)
    Extent of disease on chest radiograph (no. [%])
        Normal07 (12)
        Minimal21 (26)2 (4)
        Moderate34 (41)9 (16)
        Severe27 (33)38 (68)
    Presence of cavities (no. [%])17 (21)35 (63)
    No radiograph available (no.)08
    • ↵ a Except where indicated otherwise.

    • ↵ b Culture-positive subjects only.

    • ↵ c Eight subjects classified as AFB negative had one scanty sputum and one smear-negative sputum.

    • ↵ d Vietnam study smear definition.

    • ↵ e Uganda study smear definition.

    • ↵ f Days until growth was flagged by the instrument. BACTEC MGIT 960 was used in the Vietnam study; BACTEC 460 TB was used in the Uganda study.

  • TABLE 2.

    Performance of Xpert MTB/RIF assay

    Sample originSmear sample typeXpert MTB resultDetection in solid medium cultureDetection in liquid medium cultureDetection in liquid and solid medium cultureSpecificitya (95% CI)
    No. of samples by culture typeSensitivity (95% CI)No. of samples by culture typeSensitivity (95% CI)No. of samples by culture typeSensitivity (95% CI)
    +−+−+−
    VietnamPositive+290100 (85.4-100)290100 (85.4-100)290100 (85.4-100)
    −000000
    Negative+33084.6 (68.8-93.6)37071.1 (56.7-82.4)38071.7 (57.4-82.8)100 (83.4-100)
    −62515251525
    UgandaPositive+63098.4 (90.5-99.9)63098.4 (90.5-99.9)63098.4 (90.5-99.9)100 (80.0-100)
    −120120120
    • ↵ a Specificity values represent the proportions of culture-negative samples that were correctly identified as giving negative results by the Xpert MTB/RIF assay.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 - Methods including details of the Xpert MTB/RIF assay protocol, killing conditions, and culture conditions and Tables S1 (Primer and molecular beacon sequences used in the assay) and S2 (Mycobacterial species used in this study.)
      MS Word document, 91K.
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Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology
Danica Helb, Martin Jones, Elizabeth Story, Catharina Boehme, Ellen Wallace, Ken Ho, JoAnn Kop, Michelle R. Owens, Richard Rodgers, Padmapriya Banada, Hassan Safi, Robert Blakemore, N. T. Ngoc Lan, Edward C. Jones-López, Michael Levi, Michele Burday, Irene Ayakaka, Roy D. Mugerwa, Bill McMillan, Emily Winn-Deen, Lee Christel, Peter Dailey, Mark D. Perkins, David H. Persing, David Alland
Journal of Clinical Microbiology Jan 2010, 48 (1) 229-237; DOI: 10.1128/JCM.01463-09

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Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology
Danica Helb, Martin Jones, Elizabeth Story, Catharina Boehme, Ellen Wallace, Ken Ho, JoAnn Kop, Michelle R. Owens, Richard Rodgers, Padmapriya Banada, Hassan Safi, Robert Blakemore, N. T. Ngoc Lan, Edward C. Jones-López, Michael Levi, Michele Burday, Irene Ayakaka, Roy D. Mugerwa, Bill McMillan, Emily Winn-Deen, Lee Christel, Peter Dailey, Mark D. Perkins, David H. Persing, David Alland
Journal of Clinical Microbiology Jan 2010, 48 (1) 229-237; DOI: 10.1128/JCM.01463-09
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  • Article
    • ABSTRACT
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KEYWORDS

Antitubercular Agents
bacteriological techniques
Drug Resistance, Bacterial
Mycobacterium tuberculosis
Point-of-Care Systems
rifampin
tuberculosis

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