Publications by Year: 2019

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is the leading killer due to an infectious organism. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved against TB, however, its efficacy against pulmonary TB is poor. While BCG is currently inoculated intradermally, the natural route of M.tb infection is through the lung. Excessive lung pathology caused by pulmonary inoculation of BCG has prevented the use of this immunization route. Here, we show that selective chemical treatment of BCG with petroleum ether removes inflammatory lipids from the bacterial surface while keeping BCG viable. Pulmonary vaccination using this modified BCG attenuated inflammatory responses, prevented immunopathology of the lung, and significantly increased protection against M.tb infection in mice. We further directly linked IL-17A as the responsible contributor of improved immunity against M.tb infection. These results provide evidence that selective removal of cytotoxic lipids from the BCG surface attenuates inflammation and offers a safer and superior vaccine against TB causing less damage post-infectious challenge with M.tb.

Mycobacterium tuberculosis (M.tb) is deposited into the alveolus where it first encounters the alveolar lining fluid (ALF) prior contacts host cells. We demonstrated that M.tb-exposure to human ALF alters its cell surface, driving better M.tb infection control by professional phagocytes. Contrary to these findings, our results with non-professional phagocytes alveolar epithelial cells (ATs) define two distinct subsets of human ALFs; where M.tb exposure to Low (L)-ALF or High(H)-ALF results in low or high intracellular bacterial growth rates in ATs, respectively. H-ALF exposed-M.tb growth within ATs was independent of M.tb-uptake, M.tb-trafficking, and M.tb-infection induced cytotoxicity; however, it was associated with enhanced bacterial replication within LAMP-1+/ABCA1+ compartments. H-ALF exposed-M.tb infection of ATs decreased AT immune mediator production, decreased AT surface adhesion expression, and downregulated macrophage inflammatory responses. Composition analysis of H-ALF vs. L-ALF showed H-ALF with higher protein tyrosine nitration and less functional ALF-innate proteins important in M.tb pathogenesis. Replenishment of H-ALF with functional ALF-innate proteins reversed the H-ALF-M.tb growth rate to the levels observed for L-ALF-M.tb. These results indicate that dysfunctionality of innate proteins in the H-ALF phenotype promotes M.tb replication within ATs, while limiting inflammation and phagocyte activation, thus potentiating ATs as a reservoir for M.tb replication and survival.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.

The Mycobacterium tuberculosis cell envelope has been evolving over time to make the bacterium transmissible and adaptable to the human host. In this context, the M. tuberculosis cell envelope contains a peripheral barrier full of lipids, some of them unique, which confer M. tuberculosis with a unique shield against the different host environments that the bacterium will encounter at the different stages of infection. This lipid barrier is mainly composed of glycolipids that can be characterized by three different subsets: trehalose-containing, mannose-containing, and 6-deoxy-pyranose-containing glycolipids. In this review, we explore the roles of these cell envelope glycolipids in M. tuberculosis virulence and pathogenesis, drug resistance, and further, how these glycolipids may dictate the M. tuberculosis cell envelope evolution from ancient to modern strains. Finally, we address how these M. tuberculosis cell envelope glycolipids are impacted by the host lung alveolar environment, their role in vaccination and masking host immunity, and subsequently the impact of these glycolipids in shaping how M. tuberculosis interacts with host cells, manipulating their immune response to favor the establishment of an infection.

Tuberculosis (TB) disease still kills 1-person every 21-seconds. Few TB diagnostic tests are considered truly appropriate for point of care settings. The WHO-endorsed immunodiagnostic Alere Determine Lipoarabinomannan Ag-test (LAM-test) detects Mycobacterium tuberculosis complex LAM in urine, and its use is recommended for TB diagnosis among HIV co-infected individuals with low CD4 T-cell counts. Here we found that a simple 15-minute enzymatic treatment at room temperature of LAM-spiked urine with α-mannosidase (for human TB), and LAM-spiked milk with combined lactase and caseinase (for bovine TB), enhanced 10-fold the detection levels of the LAM-test and thus, improved the detection of LAM by the LAM-test in urine and milk that otherwise could be missed in the field. Future separate clinical research studies specifically designed to address the potential of these findings are required.

BACKGROUND: Accurate and timely tuberculosis diagnosis is the primary step for initiating effective treatment. The color plate agar-based culture test (TB-CX test) is low cost, simple to use and detects Mycobacterium tuberculosis faster. Therefore, the main objective of this study was to compare the diagnostic accuracy and time to detection of positive cultures using color test and Lӧwenstein Jensen culture.

METHODS: A comparative cross-sectional study was conducted at University of Gondar Hospital. A total of 200 sputum samples were collected from TB patients and processed for direct smear microscopy and cultures.

RESULTS: Sixty-five percent were found positive on both methods and 4 (2%) were positive on LJ culture and negative on the color plate. The median time for detection of MTB growth was significantly shorter using color plate test (Median 12 days) than LJ culture (Median 21 days) (P < 0.0001). The overall sensitivity and specificity of the color test compared to LJ culture were 97% (95% CI: 93-99) and 100% (95% CI: 94-100), respectively.

CONCLUSIONS: The color plate test for micro-colonies allows early and accurate MTB diagnosis in a median time of 12 days. This rapid method could be an option for diagnosis of pulmonary TB in resource limited settings.

BACKGROUND: Bovine tuberculosis (bTB) is prevalent in dairy cattle in Ethiopia. Currently used diagnostic tools such as the single intradermal comparative tuberculin test (SICTT) are time consuming and labor intensive. A rapid, easy-to-use and cost-effective diagnostic test would greatly contribute to the control of bTB in developing countries like Ethiopia. In the present study, two point-of-care diagnostic tests were evaluated for the detection of bTB: LIONEX® Animal TB Rapid test, a membrane-based test for the detection of antibodies to Mycobacterium bovis in blood and ALERE® Determine TB Lipoarabinomannan (LAM) Ag, an immunoassay for the detection of lipoarabinomannan (LAM) antigen (Ag) of mycobacteria in urine. A combination of the SICTT and gamma interferon (IFN-γ) test was used as the gold standard for the validation of these point-of-care tests, as it was not feasible to slaughter the study animals to carry out the historical gold standard of mycobacterial culture. A total of 175 heads of cattle having three different bTB infection categories (positive SICTT, negative SICTT, and unknown SICTT status) were used for this study.

RESULT: The sensitivity and specificity of TB LAM Ag were 72.2% (95% CI = 62.2, 80.4) and 98.8% (95% CI = 93.6, 99.7), respectively, while the sensitivity and specificity of the LIONEX Animal TB rapid test assay were 54% (95% CI = 44.1 64.3) and 98.8% (95% CI = 93.6, 99.7) respectively. The agreement between TB LAM Ag and SICTT was higher (κ = 0.85; 95% CI = 0.65-0.94) than between TB LAM Ag and IFN-γ (κ = 0.67; 95% CI = 0.52-0.81). The agreement between LIONEX Animals TB Rapid blood test and SICTT was substantial, (κ = 0.63; 95% CI = 0.49-0.77) while the agreement between LIONEX Animal TB rapid blood test and IFN-γ test was moderate (κ = 0.53; 95% CI = 0.40-0.67). Analysis of receiver operating curve (ROC) indicated that the area under the ROC curve (AUC) for TB LAM Ag was 0.85 (95% CI = 0.79-0.91) while it was 0.76 (95% CI; =0.69-0.83) for LIONEX Animal TB rapid test assay.

CONCLUSION: This study showed that TB LAM Ag had a better diagnostic performance and could potentially be used as ancillary either to SICTT or IFN-γ test for diagnosis of bTB.

The elderly population is more susceptible to pulmonary infections, including tuberculosis. In this article, we characterize the impact of aging on the phenotype of mouse alveolar macrophages (AMs) and their response to Mycobacterium tuberculosis. Uninfected AMs were isolated from bronchoalveolar lavage of young (3 mo) and old (18 mo) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNF-α, and MIF than young mice, and old mice contained higher levels of CCL2, IL-1β, IFN-β, and MIF in their alveolar lining fluid. We identified two distinct AM subpopulations, a major CD11c+ CD11b- population and a minor CD11c+ CD11b+ population; the latter was significantly increased in old mice (4-fold). Expression of CD206, TLR2, CD16/CD32, MHC class II, and CD86 was higher in CD11c+ CD11b+ AMs, and these cells expressed monocytic markers Ly6C, CX3CR1, and CD115, suggesting monocytic origin. Sorted CD11c+ CD11b+ AMs from old mice expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c+ CD11b- AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c+ CD11b+ AMs phagocytosed significantly more M. tuberculosis, which expressed higher RNA levels of genes required for M. tuberculosis survival. Our studies identify two distinct AM populations in old mice: a resident population and an increased CD11c+ CD11b+ AM subpopulation expressing monocytic markers, a unique inflammatory signature, and enhanced M. tuberculosis phagocytosis and survival when compared with resident CD11c+ CD11b- AMs, which are more immune regulatory in nature.

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.