Oladunni, Fatai S, Jun-Gyu Park, Paula A Pino, Olga Gonzalez, Anwari Akhter, Anna Allué-Guardia, Angélica Olmo-Fontánez, et al. (2020) 2020. “Lethality of SARS-CoV-2 Infection in K18 Human Angiotensin-Converting Enzyme 2 Transgenic Mice”. Nature Communications 11 (1): 6122.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

García, Juan Ignacio, Johanna Meléndez, Rosa Álvarez, Carlos Mejía-Chew, Holden Kelley V, Sabeen Sidiki, Alejandra Castillo, et al. (2020) 2020. “Accuracy of the Tuberculosis Point-of-Care Alere Determine Lipoarabinomannan Antigen Diagnostic Test Using α-Mannosidase Treated and Untreated Urine in a Cohort of People Living With HIV in Guatemala”. AIDS Research and Therapy 17 (1): 62.

BACKGROUND: Improved point-of-care diagnostic tests for tuberculosis (TB) in severe immune suppressed people living with HIV (PLWH) are needed to decrease morbidity and mortality outcomes. The aim of the study is to evaluate the performance of the lipoarabinomannan antigen test (LAM-test) with and without α-mannosidase pre-treated urine in a cohort of PLWH in primary care clinics in Guatemala. We further determined TB incidence, and mortality rates and its risk factors in PLWH with TB symptoms.

METHODS: Prospective longitudinal study of PLWH with TB symptoms. Urine samples were collected at 2 HIV sites to test the sensitivity of the LAM-test in urine with and without α-mannosidase pre-treatment. A composite reference standard of either a positive Mycobacterium tuberculosis complex culture and/or GeneXpert® MTB/RIF (Xpert, Cepheid, Sunnyvale, CA, USA) results was used in the LAM-test diagnostic accuracy studies. Cox proportional hazards regression was used to study mortality predictors.

RESULTS: The overall sensitivity of the LAM-test was of 56.1% with 95% CI of (43.3-68.3). There were no differences in the LAM-test sensitivity neither by hospital nor by CD4 T cell values. LAM-test sensitivity in PLWH with < 200 CD4 T cells/µl was of 62.2% (95% CI 46.5-76.2). There were no significant differences in sensitivity when comparing LAM-test results obtained from untreated vs. α-mannosidase treated urine [55.2% (95% CI 42.6-67.4) vs. 56.9% (95% CI 44-69.2), respectively]. TB incidence in our cohort was of 21.4/100 person years (PYs) (95% CI 16.6-27.6), and mortality rate was of 11.1/100 PYs (95% CI 8.2-15.0). Importantly, PLWH with a positive LAM-test result had an adjusted hazard ratio (aHR) of death of 1.98 (1.0-3.8) with a significant p value of 0.044 when compared to PLWH with a negative LAM-test result.

CONCLUSIONS: In this study, α-mannosidase treatment of urine did not significantly increase the LAM-test performance, however; this needs to be further evaluated in a large-scale study due to our study limitations. Importantly, high rates of TB incidence and mortality were found, and a positive LAM-test result predicted mortality in PLWH with TB clinical symptoms.


Moliva, Juan I, Austin P Hossfeld, Sabeen Sidiki, Cynthia H Canan, Varun Dwivedi, Gillian Beamer, Joanne Turner, and Jordi B Torrelles. (2019) 2019. “Selective Delipidation of Mycobacterium Bovis BCG Enables Direct Pulmonary Vaccination and Enhances Protection Against Mycobacterium Tuberculosis”. Mucosal Immunology 12 (3): 805-15.

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.

Scordo, J M, A M Olmo-Fontánez, H Kelley V, S Sidiki, J Arcos, A Akhter, M D Wewers, and J B Torrelles. (2019) 2019. “The Human Lung Mucosa Drives Differential Mycobacterium Tuberculosis Infection Outcome in the Alveolar Epithelium”. Mucosal Immunology 12 (3): 795-804.

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.

Moliva, Juan I, Michael A Duncan, Angélica Olmo-Fontánez, Anwari Akhter, Eusondia Arnett, Julia M Scordo, Russell Ault, et al. (2019) 2019. “The Lung Mucosa Environment in the Elderly Increases Host Susceptibility to Mycobacterium Tuberculosis Infection”. The Journal of Infectious Diseases 220 (3): 514-23.

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.

Garcia-Vilanova, Andreu, John Chan, and Jordi B Torrelles. (2019) 2019. “Underestimated Manipulative Roles of Mycobacterium Tuberculosis Cell Envelope Glycolipids During Infection”. Frontiers in Immunology 10: 2909.

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.

García, Juan Ignacio, Holden Kelley V, Johanna Meléndez, Rosa Alejandra Alvarez de León, Alejandra Castillo, Sabeen Sidiki, Kizil A Yusoof, et al. (2019) 2019. “Improved Alere Determine Lipoarabinomannan Antigen Detection Test for the Diagnosis of Human and Bovine Tuberculosis by Manipulating Urine and Milk”. Scientific Reports 9 (1): 18012.

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.

Shibabaw, Agumas, Baye Gelaw, Holden Kelley, Joan Miquel Balada-Llasat, Carlton Evans, Shu-Hua Wang, Jordi B Torrelles, and Belay Tessema. (2019) 2019. “Accuracy of the Color Plate Micro-Colony Detection for the Diagnosis of Mycobacterium Tuberculosis Complex in Northwest Ethiopia”. Tuberculosis (Edinburgh, Scotland) 114: 54-60.

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.

Zewude, Aboma, Temesgen Mohammed, Lemma Terfassa, Garrett Hunt, Xueliang Pan, Joan Miquel Balada-Llasat, Wondwossen Gebreyes, Jordi B Torrelles, Shu-Hua Wang, and Gobena Ameni. (2019) 2019. “Evaluation of Mycobacterium Tuberculosis Lipoarabinomannan Antigen Assay and Rapid Serology Blood Test for the Diagnosis of Bovine Tuberculosis in Ethiopia”. BMC Veterinary Research 15 (1): 359.

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.

Lafuse, William P, Murugesan S Rajaram V, Qian Wu, Juan I Moliva, Jordi B Torrelles, Joanne Turner, and Larry S Schlesinger. (2019) 2019. “Identification of an Increased Alveolar Macrophage Subpopulation in Old Mice That Displays Unique Inflammatory Characteristics and Is Permissive to Mycobacterium Tuberculosis Infection”. Journal of Immunology (Baltimore, Md. : 1950) 203 (8): 2252-64.

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.