Shu-Hua Wang

Characterization of Host and Microbial Determinants in Individuals with Latent Tuberculosis Infection Using a Human Granuloma Model

ABSTRACT Granulomas sit at the center of tuberculosis (TB) immunopathogenesis. Progress in biomarkers and treatment specific to the human granuloma environment is hindered by the lack of a relevant and tractable infection model that better accounts for the complexity of the host immune response as well as pathogen counterresponses that subvert host immunity in granulomas. Here we developed and characterized an in vitro granuloma model derived from human peripheral blood mononuclear cells (PBMCs) and autologous serum. Importantly, we interrogated this model for its ability to discriminate between host and bacterial determinants in individuals with and without latent TB infection (LTBI). By the use of this model, we provide the first evidence that granuloma formation, bacterial survival, lymphocyte proliferation, pro- and anti-inflammatory cytokines, and lipid body accumulation are significantly altered in LTBI individuals. Moreover, we show a specific transcriptional signature of Mycobacterium tuberculosis associated with survival within human granuloma structures depending on the host immune status. Our report provides fundamentally new information on how the human host immune status and bacterial transcriptional signature may dictate early granuloma formation and outcome and provides evidence for the validity of the granuloma model and its potential applications. IMPORTANCE In 2012, approximately 1.3 million people died from tuberculosis (TB), the highest rate for any single bacterial pathogen. The long-term control of TB requires a better understanding of Mycobacterium tuberculosis pathogenesis in appropriate research models. Granulomas represent the characteristic host tissue response to TB, controlling the bacilli while concentrating the immune response to a limited area. However, complete eradication of bacteria does not occur, since M. tuberculosis has its own strategies to adapt and persist. Thus, the M. tuberculosis-containing granuloma represents a unique environment for dictating both the host immune response and the bacterial response. Here we developed and characterized an in vitro granuloma model derived from blood cells of individuals with latent TB infection that more accurately defines the human immune response and metabolic profiles of M. tuberculosis within this uniquely regulated immune environment. This model may also prove beneficial for understanding other granulomatous diseases. In 2012, approximately 1.3 million people died from tuberculosis (TB), the highest rate for any single bacterial pathogen. The long-term control of TB requires a better understanding of Mycobacterium tuberculosis pathogenesis in appropriate research models. Granulomas represent the characteristic host tissue response to TB, controlling the bacilli while concentrating the immune response to a limited area. However, complete eradication of bacteria does not occur, since M. tuberculosis has its own strategies to adapt and persist. Thus, the M. tuberculosis-containing granuloma represents a unique environment for dictating both the host immune response and the bacterial response. Here we developed and characterized an in vitro granuloma model derived from blood cells of individuals with latent TB infection that more accurately defines the human immune response and metabolic profiles of M. tuberculosis within this uniquely regulated immune environment. This model may also prove beneficial for understanding other granulomatous diseases.

Molecular composition of the alveolar lining fluid in the aging lung

As we age, there is an increased risk for the development of pulmonary diseases, including infections, but few studies have considered changes in lung surfactant and components of the innate immune system as contributing factors to the increased susceptibility of the elderly to succumb to infections. We and others have demonstrated that human alveolar lining fluid (ALF) components, such as surfactant protein (SP)-A, SP-D, complement protein C3, and alveolar hydrolases, play a significant innate immune role in controlling microbial infections. However, there is a lack of information regarding the effect of increasing age on the level and function of ALF components in the lung. Here we addressed this gap in knowledge by determining the levels of ALF components in the aging lung that are important in controlling infection. Our findings demonstrate that pro-inflammatory cytokines, surfactant proteins and lipids, and complement components are significantly altered in the aged lung in both mice and humans. Further, we show that the aging lung is a relatively oxidized environment. Our study provides new information on how the pulmonary environment in old age can potentially modify mucosal immune responses, thereby impacting pulmonary infections and other pulmonary diseases in the elderly population.

Completion of therapy for latent tuberculosis in children of different nationalities.

We performed a retrospective review of prospectively stored data on 545 children from 54 different birth countries with latent tuberculosis cared for in a pediatric tuberculosis clinic between August 1, 2005 and July 31, 2006. For analysis, patients were grouped into 6 geographic regions. The overall rate of completion of therapy was 54.4%. There were no significant differences among regions in the rate of completion of therapy by age, duration in the United States, or exposure to active tuberculosis. However, no children from Eastern Europe completed therapy compared with 67.9% from Central and South America. Of those children who did not complete therapy, parental refusal to start medication accounted for 54% and 80% of Eastern European and Asian children compared with <10% of children from Sub-Saharan Africa, Central and South America, and the United States.

Evaluation of a modified interferon-gamma release assay for the diagnosis of latent tuberculosis infection in adult and paediatric populations that enables delayed processing

Abstract The objective of the study was to evaluate the specificity of a modified interferon-gamma release assay (IGRA) procedure that allows storage of blood samples for up to 32 h before processing. A total of 116 subjects were enrolled in the study. Two blood samples were collected from each volunteer; 1 specimen was processed within 8 h and analyzed using the T-SPOT®.TB test and the second specimen was stored overnight and processed 23–32 h later after addition of the T-Cell Xtend™ reagent and then analyzed using the T-SPOT.TB test. A total of 108 paired T-SPOT.TB and T-SPOT.TB plus T-Cell Xtend tests were analyzed on specimens from 97 adults and 11 children. The median age of the subjects was 28 y with 68.5% female and 78.7% white. The overall agreement between the 2 tests was 98.2% (106/108). The specificity of the T-SPOT.TB test was 99.1% (107/108) and for T-SPOT.TB plus T-Cell Xtend was 97.2%. The 2 tests were comparable in results. Increasing storage time of the collected blood specimen prior to processing provides flexibility for clinicians and laboratories. Additional studies in larger and diverse patient populations including immunocompromised and paediatric patients, and patients with active TB disease or latent tuberculosis infection are needed.

Methicillin-resistant Staphylococcus aureus sequence type 239-III, Ohio, USA, 2007-2009.

Identification of virulent strains emphasizes the need for molecular surveillance.

Characterization of "Mycobacterium paraffinicum" associated with a pseudo-outbreak.

ABSTRACT We describe the first “Mycobacterium paraffinicum” (unofficial taxon) pseudo-outbreak in a tertiary-care medical center. Fifteen clinical nontuberculous mycobacterium isolates from 10 patients were initially identified by biochemical tests and high-performance liquid chromatography as Mycobacterium scrofulaceum. However, further testing by molecular analysis revealed “M. paraffinicum.” Epidemiological and environmental investigation determined that the ice machine was the source of the pseudo-outbreak.

Mycobacterial Skin and Soft Tissue Infection

Mycobacterial skin and soft tissue infection (SSTI) includes nontuberculous mycobacterial (NTM) infections, tuberculosis (TB), and leprosy. Diagnosis of mycobacterial SSTI can be challenging due to diverse clinical presentation, low yield from cultured specimens, and nonspecific histopathology on tissue biopsy. In addition, immunosuppressed patients may present with atypical or disseminated disease. Despite aggressive medical treatment and often with surgical intervention, results may be suboptimal with poor outcomes. Regimens typically require multiple antibiotics for extended periods of time and are often complicated by medication side effects and drug-drug interactions. Biopsy with culture is the gold standard for diagnosis, but newer molecular diagnostics and proteomics such as matrix-assisted laser desorption ionization-time of flight mass spectrometry have improved diagnosis with increased identification of clinically significant mycobacteria species in clinically relevant time frames. We will review updates in diagnostic tests along with clinical presentation and treatment of mycobacterial SSTI for NTM, TB, and leprosy.

Tuberculin skin testing and T-SPOT.TB in internationally adopted children.

Background: Diagnosis of latent tuberculosis infection is a problem in children because of lack of a diagnostic standard and potential impact of previous Bacille Calmette–Guérin vaccination and exposure to environmental mycobacteria. Effectiveness and usefulness of interferon-gamma release assays in infants and younger children have yet to be clearly demonstrated. Methods: Prospective cohort study including 109 children (4 months to 16 years) seen in an international adoption clinic at Nationwide Children’s Hospital, Columbus, OH. Children were adopted from 14 countries, mostly (72.5%) from China, Russia and Ethiopia. Correspondence between tuberculin skin test (TST) and the T-SPOT.TB assay was evaluated. Factors associated with positive results on the TST and T-SPOT.TB were determined, and the impact of age on test performance was specifically addressed. Results: TST was positive in 23.4% (25 of 107). T-SPOT.TB was positive in 4.6% (5 of 109). Overall agreement between TST and T-SPOT.TB was 71%, with prevalence-adjusted, bias-adjusted Kappa of 0.68. History of Mycobacterium tuberculosis exposure was associated with positive results on TST (odds ratio: 25.4, 95% confidence interval: 4.8–261.6, exact logistic regression) and T-SPOT.TB (odds ratio: 78.9, 95% confidence interval: 9.7–∞). All 5 children with positive T-SPOT.TB had TST induration ≥15 mm. No patient less than 1 year of age (n = 17) had positive TST or T-SPOT.TB. Positive TST was not associated with Bacille Calmette–Guérin vaccination or scar. Conclusions: TST was positive in a significant percentage of international adoptees. T-SPOT.TB was rarely positive and discordant results reflected negative T-SPOT.TB with positive TST. In this population latent tuberculosis infection may be over-estimated by TST. Regardless, in our context at the time of the study, treatment decisions were based upon TST results, not results of the T-SPOT.TB assay. Age was consistently associated with findings on TST and T-SPOT.TB with no positive result on either test for any child <1 year of age.

High Rate of Treatment Completion in Program Settings With 12-Dose Weekly Isoniazid and Rifapentine for Latent Mycobacterium tuberculosis Infection

Background Randomized controlled trials have demonstrated that the newest latent tuberculosis (LTBI) regimen, 12 weekly doses of directly observed isoniazid and rifapentine (3HP), is as efficacious as 9 months of isoniazid, with a greater completion rate (82% vs 69%); however, 3HP has not been assessed in routine healthcare settings. Methods Observational cohort of LTBI patients receiving 3HP through 16 US programs was used to assess treatment completion, adverse drug reactions, and factors associated with treatment discontinuation. Results Of 3288 patients eligible to complete 3HP, 2867 (87.2%) completed treatment. Children aged 2-17 years had the highest completion rate (94.5% [155/164]). Patients reporting homelessness had a completion rate of 81.2% (147/181). In univariable analyses, discontinuation was lowest among children (relative risk [RR], 0.44 [95% confidence interval {CI}, .23-.85]; P = .014), and highest in persons aged ≥65 years (RR, 1.72 [95% CI, 1.25-2.35]; P < .001). In multivariable analyses, discontinuation was lowest among contacts of patients with tuberculosis (TB) disease (adjusted RR [ARR], 0.68 [95% CI, .52-.89]; P = .005) and students (ARR, 0.45 [95% CI, .21-.98]; P = .044), and highest with incarceration (ARR, 1.43 [95% CI, 1.08-1.89]; P = .013) and homelessness (ARR, 1.72 [95% CI, 1.25-2.39]; P = .001). Adverse drug reactions were reported by 1174 (35.7%) patients, of whom 891 (76.0%) completed treatment. Conclusions Completion of 3HP in routine healthcare settings was greater overall than rates reported from clinical trials, and greater than historically observed using other regimens among reportedly nonadherent populations. Widespread use of 3HP for LTBI treatment could accelerate elimination of TB disease in the United States.

Molecular and Clinical Characteristics of Hospital and Community Onset Methicillin-Resistant Staphylococcus aureus Strains Associated with Bloodstream Infections

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSI) are classified epidemiologically as health care-associated hospital onset (HAHO)-, health care-associated community onset (HACO)-, or community-associated (CA)-MRSA. Clinical and molecular differences between HAHO- and HACO-MRSA BSI are not well known. Thus, we evaluated clinical and molecular characteristics of MRSA BSI to determine if distinct features are associated with HAHO- or HACO-MRSA strains. Molecular genotyping and medical record reviews were conducted on 282 MRSA BSI isolates from January 2007 to December 2009. MRSA classifications were 38% HAHO-, 54% HACO-, and 8% CA-MRSA. Comparing patients with HAHO-MRSA to those with HACO-MRSA, HAHO-MRSA patients had significantly higher rates of malignancy, surgery, recent invasive devices, and mortality and longer hospital stays. Patients with HACO-MRSA were more likely to have a history of renal failure, hemodialysis, residence in a long-term-care facility, long-term invasive devices, and higher rate of MRSA relapse. Distinct MRSA molecular strain differences also were seen between HAHO-MRSA (60% staphylococcal cassette chromosome mec type II [SCCmec II], 30% SCCmec III, and 9% SCCmec IV) and HACO-MRSA (47% SCCmec II, 35% SCCmec III, and 16% SCCmec IV) (P < 0.001). In summary, our study reveals significant clinical and molecular differences between patients with HAHO- and HACO-MRSA BSI. In order to decrease rates of MRSA infection, preventive efforts need to be directed toward patients in the community with health care-associated risk factors in addition to inpatient infection control.