Marsha L. Feske

Polymorphisms in Regulator of Protease B (RopB) Alter Disease Phenotype and Strain Virulence of Serotype M3 Group A Streptococcus

Whole-genome sequencing of serotype M3 group A streptococci (GAS) from oropharyngeal and invasive infections in Ontario recently showed that the gene encoding regulator of protease B (RopB) is highly polymorphic in this population. To test the hypothesis that ropB is under diversifying selective pressure among all serotype M3 GAS strains, we sequenced this gene in 1178 strains collected from different infection types, geographic regions, and time periods. The results confirmed our hypothesis and discovered a significant association between mutant ropB alleles, decreased activity of its major regulatory target SpeB, and pharyngitis. Additionally, isoallelic strains with ropB polymorphisms were significantly less virulent in a mouse model of necrotizing fasciitis. These studies provide a model strategy for applying whole-genome sequencing followed by deep single-gene sequencing to generate new insight to the rapid evolution and virulence regulation of human pathogens.

Enhancement of human antigen-specific memory T-cell responses by interleukin-7 may improve accuracy in diagnosing tuberculosis

ABSTRACT Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-γ) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-γ. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-γ message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-γ responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-γ responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R2 = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-γ by 39% compared to the level with antigen alone. Increased production of IFN-γ induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-γ-based assays might improve TB diagnosis in those populations at highest risk for TB.

Giving TB wheels: Public transportation as a risk factor for tuberculosis transmission

Previous geospatial analysis of the well-defined Houston Tuberculosis Initiative (HTI) database identified an association between the use of city-bus transportation (inclusive of time onboard) and Tuberculosis (TB) incidence in Houston/Harris County census tracts (paper submitted). This paper is an extension of those findings. Contact investigations on school buses have reported a high rate of positive tuberculin skin tests in the persons traveling with the index case and have shown an association with bus ride duration. In Houston, city bus routes are veins connecting even the most diverse of populations within the metropolitan area. Among HTI participants, TB patients who reported weekly bus use were more likely to have demographic and social risk factors associated with poverty, immune suppression and health disparities. An equal proportion of bus riders and non-bus riders were cultured for Mycobacterium tuberculosis (MTB), yet 75% of bus riders were clustered with a mean cluster size of 50.14, indicating recent transmission, compared to 56% of non-bus riders (OR = 2.4, p < 0.001) with a mean cluster size of 28.9 (p < 0.01). Individual bus routes, including one route servicing the local hospitals, were found to be risk factors for endemic MTB clustered strains and the routes themselves geographically connect the census tracts previously identified as having endemic TB.

Including the Third Dimension: A Spatial Analysis of TB Cases in Houston Harris County

To reach the tuberculosis (TB) elimination goals established by the Institute of Medicine (IOM) and the Centers for Disease Control and Prevention (CDC), measures must be taken to speed the currently stagnant TB elimination rate and curtail a future peak in TB incidence. Increases in TB incidence have historically coincided with immigration, poverty, and joblessness; all situations that are currently occurring worldwide. Effective TB elimination strategies will require the geographical elucidation of areas within the U.S. that have endemic TB, and systematic surveillance of the locations and location-based risk factors associated with TB transmission. Surveillance data was used to assess the spatial distribution of cases, the yearly TB incidence by census tract, and the statistical significance of case clustering. The analysis revealed that there are neighborhoods within Houston/Harris County that had a heavy TB burden. The maximum yearly incidence varied from 245/100,000-754/100,000 and was not exclusively dependent of the number of cases reported. Geographically weighted regression identified risk factors associated with the spatial distribution of cases such as: poverty, age, Black race, and foreign birth. Public transportation was also associated with the spatial distribution of cases and census tracts identified as high incidence were found to be irregularly clustered within communities of varied SES.

Counting the Homeless: A Previously Incalculable Tuberculosis Risk and Its Social Determinants

Tuberculosis (TB) surveillance among the homeless is not supported by the political will necessary for TB elimination. We merged the first stakeholder-accepted enumeration of homeless persons with existing surveillance data to assess TB risk among the homeless in Houston, Texas. The average incidence per 100,000 was 411 among homeless and 9.5 among housed persons. The homeless were more likely than the housed to be US-born, clustered, and in a larger-sized cluster. Multivariate analysis revealed that TB rates among the homeless were driven not by comorbidities but by social determinants. Homeless patients were hospitalized more days than the housed and required more follow-up time. Reporting of TB rates for populations with known health disparities could help reframe TB prevention and better target limited funds.

Granzyme B‐ and Fas ligand‐mediated cytotoxic function induced by mitogenic CD28 stimulation of human memory CD4+ T cells

Some human memory CD4+ T cells have cytotoxic functions best understood in the context of viral infections; however, their possible role in pathologic processes is understudied. The novel discovery that mitogenic CD28 antibodies induced proliferation and expansion of Tregs offered therapeutic promise for autoimmune disorders. However, the failed TGN1412 trial forced reassessment of this concept. As memory CD4+ T cells are known to produce toxic molecules, including granzyme B (GrzB) and FasL, we wondered whether mitogenic CD28 was able to induce these cytotoxic molecules. A commercially available mitogenic human CD28 mAb (clone ANC28.1) was used to determine whether mitogenic CD28 induces cytotoxic function from human memory CD4+ T cells. We found that stimulation of memory CD4+ T cells by ANC28.1, as well as by conventional costimulation (CD3/CD28 mAb), robustly induced enzymatically active GrzB, along with increased surface expression of FasL. These functional phenotypes were induced in association with increased expression of T cell activation markers CD69 and CD25, and elimination of target cells by ANC28.1‐activated memory CD4+ T cells involved both GrzB and FasL. Additionally, ANC28.1‐activated memory CD4+ T cells caused disruption of epithelial cell monolayer integrity, which was partially mediated by GrzB. These findings reveal functions of memory CD4+ T cells previously unknown to be induced by mitogenic CD28, and suggest that these pathogenic mechanisms may have been responsible for some of the widespread tissue destruction that occurred in the TGN1412 trial recipients.

Pediatric tuberculosis: the litmus test for tuberculosis control.

Background: The epidemiology of pediatric tuberculosis (TB) from 1995 to 2000 in Harris County, TX, has been previously reported. This study was conducted to evaluate the continued trends of Mycobacterium tuberculosis clustering and the role of genotyping in pediatric TB. Methods: Data came from the Houston Tuberculosis Initiative, a prospective population-based active surveillance and molecular epidemiology project. The study population consisted of TB patients ⩽18 years of age diagnosed in Harris County, TX, from 2000 to 2004. Available Mycobacterium tuberculosis isolates were characterized by insertion sequence 6110 restriction fragment length polymorphism and spoligotyping. Results: One hundred three pediatric TB cases were enrolled in the Houston Tuberculosis Initiative study from 2000 to 2004. Sixty-one (59%) patients had potential source cases. Mycobacterium tuberculosis isolates were available and genotyped for 36 pediatric cases; 27 (75%) were clustered into 22 different genotypes. Of the 20 genotyped patients with a potential source case, 16 (80%) were clustered. Genotypes matched the potential source case in 12 cases. Eleven of the 16 (69%) genotyped patients without a potential source case were clustered. Conclusions: Compared with pediatric cases between 1995 and 2000, there was a significant increase in the number of patients with unknown potential source cases that were clustered within the Houston Tuberculosis Initiative database. Because genotypic clustering is associated with recent transmission, there appears to be a failure in the identification of potential source cases through contact tracing. Reduced funding of public health departments forces more limited TB control activities and therefore could pose a threat to TB control.