Research Article: Evaluation of the tuberculosis culture color plate test for rapid detection of drug susceptible and drug-resistant Mycobacterium tuberculosis in a resource-limited setting, Addis Ababa, Ethiopia

Date Published: May 28, 2019

Publisher: Public Library of Science

Author(s): Biruk Mekonnen, Adane Mihret, Muluwork Getahun, Tsegaye Hailu, Sabeen Sidiki, Holden V. Kelley, Julia M. Scordo, W. Garrett Hunt, Xueliang Pan, Joan-Miquel Balada-Llasat, Wondwossen Gebreyes, Carlton A. Evans, Abraham Aseffa, Jordi B. Torrelles, Shu-Hua Wang, Tamrat Abebe, Hasnain Seyed Ehtesham.


Timely diagnosis of tuberculosis (TB) is limited in Ethiopia. We evaluated the performance of a low technology, thin layer agar, Mycobacterium tuberculosis (M.tb) culture color plate (TB-CX) test with concurrent drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), and pyrazinamide (PZA) directly from sputum specimens. Patients undergoing examination for TB and multidrug-resistant (MDR)-TB were enrolled in Addis Ababa, Ethiopia from March 2016 to February 2017. All subjects received a GeneXpert MTB/RIF PCR test. TB-CX test results were compared to reference Löwenstein–Jensen (LJ) culture for M.tb detection and DST for susceptibility to INH and RIF. Kappa statistic was applied to test agreement between results for TB-CX test and the reference methods, a cut-off Kappa value of 0.75 was considered as high level of agreements. A total of 137 participants were analyzed: 88 (64%) were new TB cases, 49 (36%) were re-treatment cases. The TB-CX test detected M.tb and DST in an average of 13 days compared to 50 days for the conventional DST result. The sensitivity and specificity of the TB-CX test for detecting M.tb were 94% and 98%, respectively (concordance, 96%; kappa 0.91). The sensitivity of the TB-CX test to detect drug resistance to INH, RIF, and MDR-TB was 91%, 100%, and 90% respectively. The specificity of the TB-CX test for detecting INH, RIF, and MDR-TB was 94%, 40%, and 94% respectively. Overall agreement between TB-CX test and LJ DST for detection of MDR-TB was 93%. The TB-CX test showed strong agreement with the GeneXpert test for detecting M.tb (89%, kappa 0.76) but low agreement for the detection of RIF resistance (57%, kappa 0.28). The TB-CX test was found to be a good alternative method for screening of TB and selective drug resistant-TB in a timely and cost-efficient manner.

Partial Text

Tuberculosis (TB) is the single highest infection-associated cause of mortality in the world, surpassing either HIV/AIDS or malaria [1]. In 2017, the World Health Organization (WHO) estimated that approximately 4,000 people die of TB every day. Although TB is curable, the emergence of drug-resistant-TB is a recognized challenge for global TB prevention [1, 2, 3]. The WHO estimates approximately half a million new MDR-TB cases annually, defined as exhibiting resistance to both isoniazid (INH) and rifampin (RIF) [1].

Rapid determination of M.tb drug susceptibility is important for patient management and public health. Unfortunately, the utility of current TB diagnostic modalities in low and middle-income countries is limited either by turnaround time (traditional solid media) or by affordability, infrastructure, and/or technical expertise (liquid media systems and molecular diagnostics). Our present study revealed that an inexpensive TB-CX test had rapid TAT and comparable sensitivity for the detection of M.tb as well as isoniazid, rifampin, and pyrazinamide susceptibility when compared to LJ culture/indirect proportional method/MIGT and the GeneXpert test.

This study presented several limitations. An internal quality control was done against physical appearance and sterility of all the TB-CX plates. Despite the fact that concentrations (stability) of drugs in TB-CX plates were not concurrently validated (culturing the plate while quality control was underway), few TB-CX plates (3%) were tested with control strains just after transit in the field. Since the plates had been stored up to 4 months prior to inoculation, degradation of the drugs on TB-CX plates with long term suboptimal temperature storage conditions and during transport (overseas and in-country transport to testing sites) could be possible. Sputum specimens were not cultured right away but frozen (-20°C) for 14 days which may also decrease the sensitivity of the TB-CX test. Unlike conventional culture method, a diluted sputum-disinfectant mixture was used as the inoculum. All of these factors may lead to decrease the sensitivity of TB-CX test. The other drawback of the study was the lack of comparative PZA DST by MGIT for all LJ culture positive isolates due to resource limitations.

In this study, we evaluated a rapid DST method that concurrently detects M.tb and susceptibilities to INH, RIF and PZA, the three key first-line anti-TB drugs used in the treatment of TB. Despite the suboptimal performance for detection of RIF resistance (40%), the TB-CX test showed similar sensitivity for detecting M.tb and selective drug resistant-TB in a timely way as compared to solid media and GeneXpert. Limitations of this test need to be further evaluated to increase the power of the analysis by increasing the number of samples tested before further implementation in clinical settings.