Microscopic-observation drug-susceptibility assay for the diagnosis of TB

Abstract

Background: New diagnostic tools are urgently needed to interrupt the transmission of tuberculosis and multidrug-resistant tuberculosis. Rapid, sensitive detection of tuberculosis and multidrug-resistant tuberculosis in sputum has been demonstrated in proof-of-principle studies of the microscopic-observation drug-susceptibility (MODS) assay, in which broth cultures are examined microscopically to detect characteristic growth.

Methods: In an operational setting in Peru, we investigated the performance of the MODS assay for culture and drug-susceptibility testing in three target groups: unselected patients with suspected tuberculosis, prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, and unselected hospitalized patients infected with the human immunodeficiency virus. We compared the MODS assay head-to-head with two reference methods: automated mycobacterial culture and culture on Löwenstein-Jensen medium with the proportion method.

Results: Of 3760 sputum samples, 401 (10.7%) yielded cultures positive for Mycobacterium tuberculosis. Sensitivity of detection was 97.8% for MODS culture, 89.0% for automated mycobacterial culture, and 84.0% for Löwenstein-Jensen culture (P<0.001); the median time to culture positivity was 7 days, 13 days, and 26 days, respectively (P<0.001), and the median time to the results of susceptibility tests was 7 days, 22 days, and 68 days, respectively. The incremental benefit of a second MODS culture was minimal, particularly in patients at high risk for tuberculosis or multidrug-resistant tuberculosis. Agreement between MODS and the reference standard for susceptibility was 100% for rifampin, 97% for isoniazid, 99% for rifampin and isoniazid (combined results for multidrug resistance), 95% for ethambutol, and 92% for streptomycin (kappa values, 1.0, 0.89, 0.93, 0.71, and 0.72, respectively).

Conclusions: A single MODS culture of a sputum sample offers more rapid and sensitive detection of tuberculosis and multidrug-resistant tuberculosis than the existing gold-standard methods used.

Figure 1. Recruitment of Patients and Culture Results

For Löwenstein–Jensen (L-J) and automated mycobacterial cultures, positive cultures were those that were positive according to either method or both; negative cultures were those that were negative according to both methods or negative according to one method and indeterminate according to the other; and indeterminate cultures were those that were indeterminate according to both methods. For patients with suspected tuberculosis, the 242 reference-standard culture-positive samples included 4 false positive samples arising from cross-contamination (1 in Löwenstein–Jensen culture alone, 1 in automated mycobacterial culture alone, and 2 in both automated mycobacterial culture and MODS culture), and the 32 culture-positive samples in MODS culture alone included 7 that were false positive owing to cross-contamination. For the prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, the 87 reference-standard culture-positive samples included 1 false positive sample in both automated mycobacterial culture and MODS culture, and the 7 samples that were culture-positive in MODS culture alone included 2 false positive cultures. The indeterminate cultures for the prescreened patients and the hospitalized patients with HIV infection were designated as such because cultures from all three methods were repeatedly contaminated by bacterial overgrowth (two with rapid-growing nontuberculous mycobacteria).

Figure 2. Incremental Benefit of One and Two Sputum Cultures as Compared with Sputum-Smear Microscopy

The percentage of cases detected during examination of the first smear or culture is shown, as well as the additional (not total) percentage detected during examination of a second smear or culture. For patients with suspected tuberculosis (Panel A) and prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis (Panel B), examination of smears stained with the Ziehl–Neelsen (Z-N) stain was performed at the local laboratory of the National TB Programme before the sample was retrieved for study purposes. For samples from the hospitalized patients with HIV infection (Panel C), microscopical examination of sputum smears stained with auramine but not Z-N stain was performed at Universidad Peruana Cayetano Heredia, because these patients were not recruited through the National TB Programme. L-J denotes Löwenstein–Jensen.

Figure 3. Cumulative Percentages of the Time to Culture Positivity for 325 Culture-Positive Samples According to Culture Method (Panel A) and the Effect of the Quantitative Status of Sputum Smears for Acid-Fast Bacilli (Panels B, C, and D)

The percentages of cultures that were positive at days 7, 14, and 21 were 74%, 99%, and 100%, respectively, in MODS culture; 7%, 62%, and 89% in automated mycobacterial culture; and 0%, 5%, and 28% in Löwenstein–Jensen (L-J) culture (Panel A). In Panels B, C, and D, “auramine smear–” was defined by the presence of fewer than 10 acid-fast bacilli per 100 fields, “1+” 10 to 99 acid-fast bacilli per 100 fields, “2+” 1 to 10 acid-fast bacilli per field, and “3+” more than 10 acid-fast bacilli per field. One field was equivalent to the examination of one carbol-fuchsin–stained smear at a magnification of 1000×.