The WHO states that TB control is lacking precisely in countries where the disease is most prevalent (41
). The presence of TB, and especially drug-resistant TB, in these developing nations is a concern for all, since we are in an era of global commerce and migrations. In the United States, for example, a major contributor to the rise in TB cases in the 1990s has been the influx of foreign-born cases (4
). Thus attention to TB control throughout the world should be a priority for the developed world as much as for the developing world.
Part of the problem in TB control in developing nations is inadequate resources to perform drug susceptibility test and ensure appropriate therapy for all patients. We have previously shown that the luciferase reporter phage assay is useful for determining antibiotic susceptibilities of clinical strains of M. tuberculosis
, with sensitivity (5
) and specificity (28
), in a timely and a relatively “low-tech” manner. To further expedite the use of this assay in developing countries where most tuberculosis occurs, we have developed a photographic detection apparatus, designated the Bronx Box, which greatly simplifies the detection protocol and removes the requirement for the most costly component in the protocol, a luminometer. The semiquantitative output which results is simple to interpret and accurate compared to that from the luminometer or conventional AST assays. Furthermore, we show here that our method can be applied directly to primary isolates of M. tuberculosis
grown on solid agar, available in 3 to 4 weeks from the time of sample acquisition (11
) and still the most common means of cultivating M. tuberculosis
Significantly, our method uses concentrations of antibiotics which have been validated in the other major liquid AST methodology, the BACTEC method (32
). We are thus able to correctly identify even low-level resistant strains (some of the MICs for the reference strains are close to the breakpoint) and in the case of the one rifampin-resistant “misdiagnosis,” may actually be more sensitive for early emergence of resistance. In fact, if the assay is applied earlier to primary cultures, slower-growing resistant subpopulations could be detected more readily than by other growth-based methods. Furthermore, subtle differences from wild-type patterns of susceptibility can be evaluated by applying a range of antibiotic concentrations in calculating an MIC (42
). Though not common in our data set, the issue of intermediate susceptibility needs to be considered, whether for a heterogeneous population of strains or for a homogeneous population which has a slightly elevated MIC relative to that for wild-type isolates. We have observed that phage-induced light production (measured by a luminometer) is reduced by at least 90% after antibiotic treatment of strains considered to be susceptible by conventional assays (29
). Conversely, resistant strains in the presence of ineffective antibiotics produce at least 10% of the light observed without any antibiotic (29
). In order to be certain that this 10% threshold can be visualized by the Bronx Box assay, we are currently including an additional control, a 1:10 dilution of the no-antibiotic, phage-infected cells. If this well produces any spot at all, we are confident that we will be able to visualize any relevant resistance, should it be present, as spots of similar or greater intensity in the antibiotic-containing wells.
While not reported here, other antimycobacterial agents can be tested or screened for activity by using photographic detection. Our preliminary data with ethambutol and streptomycin in this data set, while still being optimized, reveal a sensitivity and specificity of 50 and 97% for ethambutol resistance and 89 and 67% for streptomycin resistance, respectively. Notably these antibiotics have been difficult to standardize for AST (41
) and have undergone modification of their breakpoints even with conventional methods (32
Our efforts to improve the sensitivity of photographic detection were largely empirical. Though it is not clear why DTT and Mg affect the kinetics of luciferase-generated light output, it can be postulated that the DTT stabilizes the luciferin and Mg stabilizes the ATP (18
). Although it has been shown that high ATP levels can interfere with light production (1
), it is still surprising that at low pH, modest levels of extrinsic ATP can dampen light production in our in vivo luciferase detection system. Understanding these phenomena may help guide us in developing superior reaction conditions. In the meantime, there are ongoing efforts in our laboratory to improve the sensitivity of the film detection system by using more sophisticated optics and newer phages. In addition, we hope to further simplify the protocol (i) to be able to add phage and luciferin at the same time and (ii) to be able to add phage and antibiotics at the same time, with a lysogenic phage (10
). We plan to extend the methods directly to processed sputum samples. Finally, we aim to verify that a phage-infected mycobacterial culture is far less virulent than an uninfected culture, given that excess phage in the assay ultimately will lyse all the mycobacterial cells, thus providing an important extra measure of laboratory safety.
In summary, the Bronx Box adaptation of the luciferase reporter phage assay is a rapid, reliable means for performing antibiotic susceptibility testing of cultures M. tuberculosis in a simple, low-tech manner well-suited to the challenge of drug-resistant TB in the developing world. While further validation of the method is necessary, clinical trials in TB-ravaged countries are currently being initiated.