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Journal of Clinical Microbiology, October 2007, p. 3387-3389, Vol. 45, No. 10
0095-1137/07/$08.00+0     doi:10.1128/JCM.00580-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Clinical Evaluation of the Microscopic Observation Drug Susceptibility Assay for Detection of Mycobacterium tuberculosis Resistance to Isoniazid or Rifampin

Fernanda C. Q. Mello,^1, Mayra S. Arias,^2, Senia Rosales,³ Anna Grazia Marsico,¹ Ada Pavón,⁴ Carlos Alvarado-Gálvez,⁴ Carlos Leonardo Carvalho Pessôa,¹ Melly Pérez,³ Monica K. Andrade,¹ Afranio L. Kritski,¹ Leila S. Fonseca,¹ Richard E. Chaisson,⁵ Michael E. Kimerling,² and Susan E. Dorman^5*

Federal University of Rio de Janeiro, Rio de Janeiro, Brazil,¹ University of Alabama at Birmingham, Birmingham, Alabama,² Gorgas Tuberculosis Initiative—Honduras,³ National Thorax Institute, Tegucigalpa, Honduras,⁴ Johns Hopkins University School of Medicine and Bloomberg School of Public Health, Baltimore, Maryland⁵

Received 15 March 2007/ Returned for modification 24 June 2007/ Accepted 29 July 2007

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This prospective study evaluated the performance of the microscopic observation drug susceptibility (MODS) assay for the direct detection of Mycobacterium tuberculosis drug resistance. MODS assay sensitivity, specificity, and positive and negative predictive values were 96.7% (95% confidence interval [95% CI], 92.1 to 98.8%), 78.4% (95% CI, 73.5 to 80.6%), 82.4% (95% CI, 78.4 to 84.2%), and 95.8% (95% CI, 89.9 to 98.5%), respectively, for isoniazid resistance and 96.0% (95% CI, 90.3 to 98.6%), 82.9% (95% CI, 78.8 to 84.7%), 80.0% (95% CI, 75.2 to 82.1%), and 96.7% (95% CI, 91.9 to 98.8%), respectively, for rifampin resistance. For both rifampin and isoniazid testing, the likelihood ratio for a negative test was 0.05, indicating that the MODS assay may be useful for ruling out drug resistance.

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It is estimated that approximately 424,000 cases of multidrug-resistant tuberculosis (MDR-TB) occurred in 2004 (12). Accordingly, the new Stop TB Strategy and The Global Plan to Stop TB, 2006-2015 include MDR-TB management as a basic component of TB control (10, 11). The recognition of a cluster of patients with human immunodeficiency virus-associated, extensively drug-resistant TB (XDR-TB) in KwaZulu-Natal province, South Africa, further underscores the need for the prompt diagnosis of drug-resistant TB, since mortality among these patients was nearly 100% (4). The World Health Organization Global Task Force on XDR-TB has recommended timely access to drug susceptibility testing for all patients at risk for or suspected to have MDR- or XDR-TB (9).

The microscopic observation drug susceptibility (MODS) assay is a relatively low-cost, simple liquid culture method that relies on the microscopic detection of cording growth that is characteristic of Mycobacterium tuberculosis (1, 3, 6, 7, 8). Potential advantages of the MODS method are relatively rapid mycobacterial growth in liquid media and reliance on microscopy skills similar to those used for smear microscopy. The MODS assay has been reported to reliably identify M. tuberculosis isolates with resistance to isoniazid (INH) and/or rifampin (RIF) (3, 6, 7, 8).

We undertook a prospective study in two settings to further evaluate the performance of MODS testing for the direct detection of M. tuberculosis drug resistance in specimens from patients with suspected pulmonary TB at risk for drug resistance.

This study was performed as part of a larger study to evaluate the performance of the MODS method for the diagnosis of TB among patients suspected of having pulmonary TB at the National Thorax Institute in Tegucigalpa, Honduras, and the Federal University of Rio de Janeiro in Brazil (1). Susceptibility testing was performed for all suspected TB patients prospectively identified as having one or more of the following risk factors: (i) suspected TB treatment failure, (ii) suspected TB relapse, (iii) treatment default, and (iv) close contact with a patient with MDR-TB (1). Informed consent was obtained from all subjects, and this study was approved by the ethics committees of all involved institutions.

Respiratory specimens submitted for routine care purposes were digested and decontaminated using the N-acetyl-L-cysteine-sodium hydroxide method (5). Pellets were resuspended in a final volume of 2 ml and used immediately for the inoculation of culture media.

INH stock solution (10,000 µg/ml) was prepared by dissolving 100 mg of INH (Sigma) in a final volume of 10 ml of sterile distilled water. RIF stock solution (10,000 µg/ml) was prepared by dissolving 100 mg of RIF (Sigma) in dimethyl sulfoxide and then adding sterile distilled water to a volume of 10 ml. Aliquots were frozen.

MODS liquid medium was prepared as described previously (8). Antibiotic stock solutions were diluted and added to MODS liquid medium to give the following critical concentrations: INH, 0.1 µg/ml (MODS INH medium), and RIF, 2.0 µg/ml (MODS RIF medium). For each respiratory specimen, 1 ml of drug-free MODS medium was dispensed into one well of a 24-well tissue culture plate (Costar, Corning, NY). One milliliter of MODS INH medium was placed into each of two adjacent wells, and 1 ml of MODS RIF medium was placed into each of two additional wells, for a total of five wells per respiratory specimen. Aliquots (0.2 ml) of each decontaminated respiratory specimen were inoculated into one drug-free well, one well containing MODS INH medium, and one well containing MODS RIF medium. In addition, 0.2-ml aliquots of each decontaminated respiratory specimen diluted 1:10 in MODS medium were inoculated into each of the remaining INH- and RIF-containing wells. Specimens obtained from different subjects but processed on the same day were plated into different rows of the same plate, and the plate was placed within a gas-permeable plastic bag. Plates were incubated at 37°C in 10% CO₂. The drug-free well was examined twice weekly for growth and pellicle morphology for 8 weeks by using X40 inverted microscopy. A culture was considered positive for M. tuberculosis if microscopically observed bacterial pellicles had a corded appearance. Drug-containing wells were examined microscopically on the 14th day after cording bacterial growth became visible in the corresponding drug-free well. Drug-containing wells were recorded as positive (indicating drug resistance) if corded growth was visible and negative (indicating susceptibility) if no corded growth was visible. The time interval of 14 days was chosen because our previous work showed that the susceptibility or resistance status indicated by the MODS assay is stable for at least 2 weeks after the detection of growth (8) and because this strategy optimized efficiency by eliminating the need for daily microscopic examination of all drug-containing wells. The MODS technologist was not aware of the Lowenstein-Jensen (LJ) drug susceptibility reference test results.

LJ slants were prepared from a commercially available powder medium base according to the instructions of the manufacturer (Becton Dickinson). Primary cultures were inoculated, examined, and interpreted as described previously (1). For susceptibility testing, several spadesful of growth were transferred from the primary LJ slant to a tube containing glass beads and sterile saline and homogenized for 1 min. After larger particles had been allowed to settle for 30 min, the supernatant was withdrawn, diluted to the turbidity of a 1 McFarland standard, and further diluted to 10^–3 and 10^–5 in saline. For each dilution, 0.2-ml aliquots were inoculated onto three LJ slants, one containing INH at 0.2 µg/ml, one containing RIF at 40 µg/ml, and one drug-free slant (2). Slants were incubated at 37°C, and colonies were enumerated on day 28. Drug resistance was defined as the growth of colonies on the drug-containing slant equal to 1% of that of colonies on the drug-free slant. The LJ technologist was not aware of MODS assay results.

Among 351 individuals at risk for drug-resistant TB, 180 (51.3%) were culture positive for M. tuberculosis by both MODS and LJ methods and were therefore included in this analysis. By the comparator LJ testing method, 69 isolates (38.3%) were resistant to both INH and RIF, 23 (12.8%) were resistant to INH alone, 5 (2.8%) were RIF monoresistant, and 83 (46.1%) were susceptible to both INH and RIF. The performance parameters of the MODS assay are shown in Tables 1 and 2. MODS test results are shown for undiluted MODS inocula and for inocula diluted 1:10. The MODS assay indicated some specimens to be resistant that the comparator method indicated to be susceptible. This trend was slightly more marked when undiluted inocula were used for MODS. For INH testing, specimen dilution resulted in the correct reclassification of four specimens as susceptible. For RIF testing, specimen dilution resulted in the correct reclassification of two specimens as susceptible. Of 69 isolates classified as MDR by LJ testing, the MODS assay (with diluted or undiluted inocula) correctly identified 66 (95.7%) as MDR. Median times to the availability of susceptibility results were 21 days (interquartile range, 17 to 24 days) for the MODS method and 49 days (interquartile range, 46 to 55 days) for the LJ method (P, <0.001 by paired t test).

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TABLE 1. Detection of resistance to INH and RIF by the MODS assay

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TABLE 2. Performance characteristics of MODS for detection of resistance to INH and RIFa

A possible explanation for the disagreement between MODS test results and those of the comparator LJ proportion method lies in the qualitative nature of the MODS assay. In MODS testing, unlike solid-agar methods, there are no discrete colonies to count and, therefore, a resistance proportion cannot be calculated. In the MODS method, any growth in drug-containing medium indicates drug resistance, whereas in solid-agar testing, growth of less than 1% of that on drug-free medium is interpreted as susceptibility. In addition, the bacillary burdens in the inocula appeared to have a small effect on drug susceptibility testing, since the level of agreement between MODS and the reference standard was slightly higher for 1:10-diluted inocula than for undiluted inocula. The impact on MODS assay susceptibility results of the timing of readings and inoculum bacillary burdens warrants further study.

Our results are generally similar to those reported by Caviedes et al. and initially by Moore et al. (3, 6). Caviedes et al. showed that the results of MODS testing for RIF susceptibility had approximately 90% concordance with the results of a microwell Alamar blue comparator assay. The MODS test was sensitive for the detection of resistance to RIF (critical concentration, 0.5 µg/ml), but as in our study, the MODS method misclassified as resistant a number of strains that were classified as susceptible by the comparator method (3). In a retrospective study, Moore et al. analyzed their database of results for 207 pretreatment respiratory specimens collected and cultured by the MODS method as a component of epidemiological studies in Peru (6). The comparator method was either an indirect microwell Alamar blue comparator assay or a tetrazolium microplate assay; critical concentrations for MODS testing were 0.4 µg/ml for INH and 1.0 µg/ml for RIF. MODS assay sensitivities, specificities, positive predictive values (PPVs), and negative predictive values (NPVs) were 81.1%, 96.9%, 85.7%, and 95.7% for INH and 100%, 98.3%, 57.1%, and 100% for RIF. For both drugs, the low PPVs were a consequence of the MODS test's indicating resistance in some isolates that were susceptible by the reference standard method. However, Moore et al. recently evaluated MODS assay performance for direct susceptibility testing using indirect susceptibility testing by the LJ method or the automated MBBacT system as the reference standard and a microwell Alamar blue assay for the resolution of discordant reference method results. For the MODS assay, critical concentrations were 0.4 µg/ml for INH and 1.0 µg/ml for RIF. They reported 100% agreement for RIF results (338 isolates were tested, among which 10.7% were RIF resistant by the reference method) and 96.7% agreement for INH results (334 isolates were tested, among which 19.5% were resistant) (7). Differences among the results of various studies may be attributable in part to differences in MODS critical drug concentrations and comparator methods across studies. In addition, in our study, MODS drug-containing wells were examined microscopically on the 14th day after growth became visible in the corresponding drug free-well, whereas Caviedes et al. and Moore et al. recorded MODS assay results on the same day that growth was observed in the drug-free well (3, 6). In our study, the delayed reading of the MODS wells may have contributed to the misclassification of some strains as resistant.

Taken together, the results of the available studies indicate that the use of the MODS assay as the sole method for drug susceptibility testing may lead, in a small proportion of cases, to the inappropriate classification of isolates as INH and/or RIF resistant. However, the very low likelihood ratio for a negative test indicates that a negative MODS test (i.e., a MODS test that is negative for resistance) is useful for ruling out resistance. This feature, and the relatively short time between specimen acquisition and susceptibility results, indicates that the MODS assay may be useful as a direct drug susceptibility screening tool in order to prioritize M. tuberculosis isolates for subsequent indirect drug susceptibility testing using conventional methods. There is a need for the standardization of MODS methodology for drug susceptibility testing.

 
We thank Hilda Membreño and Nery Almendarez (Central Tuberculosis Laboratory, Tegucigalpa, Honduras) and Gisele Vieira (Hospital Universitario Clementino Fraga Filho) for assistance with drug susceptibility testing, Lygia Kitada for assistance with the recruitment of study subjects at Hospital Universitario Clementino Fraga Filho, Judith Hackman for assistance with data analysis, and Pina Maria Boquin for facilitating the study at the National Thorax Institute.

This study was supported by the Gorgas Tuberculosis Initiative of the U.S. Agency for International Development (GHS A 00 03 00020 00) and the National Institutes of Health (grants K23 AI 51528 to S.E.D. and K24 AI16137 and U19 AI45432 to R.E.C.).

Authors’ specific contributions were as follows: study design, M. S. Arias, F. C. Q. Mello, C. Alvarado-Gálvez, A. L. Kritski, L. S. Fonseca, R. E. Chaisson, M. E. Kimerling, and S. E. Dorman; study implementation and data collection, M. S. Arias, F. C. Q. Mello, A. Pavón, A. G. Marsico, C. Alvarado-Gálvez, S. Rosales, M. Pérez, M. K. Andrade, C. L. C. Pessôa, and L. S. Fonseca; data analysis, M. S. Arias, F. C. Q. Mello, M. E. Kimerling, and S. E. Dorman; manuscript writing, M. S. Arias, F. C. Q. Mello, R. E. Chaisson, M. E. Kimerling, and S. E. Dorman; and manuscript review, all authors. The corresponding author has had full access to all of the data in the study and had final responsibility for the decision to submit for publication. None of the authors have any financial or personal relationships with other people or organizations that may inappropriately influence their work.

 
* Corresponding author. Mailing address: Johns Hopkins University School of Medicine, 1503 East Jefferson St., Room 105, Baltimore, MD 21231. Phone: (410) 502-2717. Fax: (410) 955-0740. E-mail: dsusan1{at}jhmi.edu

Published ahead of print on 15 August 2007.

These authors contributed equally.

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1
1. Arias, M., F. C. Q. Mello, A. Pavon, A. G Marsico, C. Alvarado-Galvez, S. Rosales, C. Pessoa, M. Perez, M. Andrade, A. L. Kritski, L. S. Fonseca, R. E Chaisson, M. E. Kimerling, and S. E. Dorman. 2007. Clinical evaluation of the microscopic-observation drug-susceptibility (MODS) assay for detection of tuberculosis. Clin. Infect. Dis. 44:674-680.[CrossRef][Medline]
2
2. Canetti, G., W. Fox, A. Kohmenko, H. Mahler, N. K. Menon, and D. A. Mitchison. 1969. Advances in techniques of testing mycobacterial drug sensitivity, and the use of sensitivity tests in tuberculosis control programmes. Bull. W. H. O. 41:21-43.[Medline]
3
3. Caviedes, L., T. S. Lee, R. Gilman, P. Sheen, E. Spellman, E. H. Lee, D. E. Berg, S. Montegegro-James, and the Tuberculosis Working Group in Peru. 2000. Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. J. Clin. Microbiol. 38:1203-1208.[Abstract/Free Full Text]
4
4. Ghandi, N. R, A. Moll, R. Pawinski, A. W. Sturm, U. Lalloo, K. Zeller, et al. 2006. High prevalence and mortality from extensively-drug resistant (XDR) TB in TB/HIV coinfected patients in rural South Africa, abstr. THLB0210. Abstr. XVI Int. AIDS Conf., Toronto, Canada, 13 to 18 August 2006.
5
5. Kent, P. T, and G. P. Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. Centers for Disease Control, U.S. Department of Health and Human Services, Atlanta, GA.
6
6. Moore, D. A. J., D. Mendoza, R. H. Gilman, C. A. W. Evans, M.-G. H. Delgado, J. Guerra, L. Caviedes, D. Vargas, E. Ticona, J. Ortiz, G. Soto, J. Serpa, and the Tuberculosis Working Group in Peru. 2004. Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings. J. Clin. Microbiol. 42:4432-4437.[Abstract/Free Full Text]
7
7. Moore, D. A. J., C. A. W. Evans, R. H. Gilman, L. Caviedes, J. Coronel, et al. 2006. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N. Engl. J. Med. 355:1539-1550.[Abstract/Free Full Text]
8
8. Park, W. G., W. R. Bishai, R. E. Chaisson, and S. E. Dorman. 2002. Performance of the microscopic observation drug susceptibility assay in drug susceptibility testing for Mycobacterium tuberculosis. J. Clin. Microbiol. 40:4750-4752.[Abstract/Free Full Text]
9
9. World Health Organization. 2007. Report of the meeting of the WHO Global Task Force on XDR-TB, Geneva, Switzerland, 9-10 October 2006. WHO/HTM/TB/2006.375. World Health Organization, Geneva, Switzerland.
10
10. World Health Organization. 2006. The global plan to stop TB, 2006-2015. WHO/HTM/STB/2006.35. World Health Organization, Geneva, Switzerland.
11
11. World Health Organization. 2006. The stop TB strategy. WHO/HTM/TB/2006.368. World Health Organization, Geneva, Switzerland.
12
12. Zignol, M., M. S. Hosseini, A. Wright, C. L. Weezenbeek, P. Nunn, C. J. Watt, B. G. Williams, and C. Dye. 2006. Global incidence of multidrug-resistant tuberculosis. J. Infect. Dis. 194:479-485.[CrossRef][Medline]

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Journal of Clinical Microbiology, October 2007, p. 3387-3389, Vol. 45, No. 10
0095-1137/07/$08.00+0     doi:10.1128/JCM.00580-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mello, F. C. Q. Articles by Dorman, S. E. Search for Related Content PubMed PubMed Citation Articles by Mello, F. C. Q. Articles by Dorman, S. E.