Anton Stoltz

The Mycobacterium tuberculosis cell wall component mycolic acid elicits pathogen-associated host innate immune responses.

Recognition of conserved pathogen‐associated molecular patterns constitutes a crucial step in the initiation of innate immune responses. We studied the contribution to the host‐pathogen interaction of mycolic acid (MA), a major lipid component of the cell envelope of the macrophage intracellular pathogen Mycobacterium tuberculosis and other mycobacteria. MA administered to the peritoneal cavity or to the airways induced a unique macrophage morphotype, similar to the foamy macrophage derivatives observed in tuberculous granulomas and characterized by intracellular accumulation of neutral lipids and entry into mitosis. When assayed for production of inflammatory mediators, a conditioning rather than a direct activation of the MA‐elicited foamy macrophages was observed. MA enabled production of IFN‐γ and myeloperoxidase, enhanced TNF‐α production and suppressed IL‐10 upon renewed exposure to innate triggers. Intratracheal instillation of MA mimicked additional features of the airway response to M. tuberculosis infection, namely a rapid but transient neutrophil influx and IL‐6 production and a chronic IL‐12 production. These MA‐elicited cellular innate defenses and the accompanying formation of foamy macrophages identify for the first time the foamy macrophage morphotype as part of the host response to a pathogen‐associated structure. Furthermore, these results characterize MA as a direct trigger of innate immunity, distinct from Toll‐like receptor ligands.

Dual time-point FDG PET-CT for differentiating benign from malignant solitary pulmonary nodules in a TB endemic area

OBJECTIVE Fluorodeoxyglucose (FDG)-positron emission tomography (PET) is an accurate non-invasive imaging test for differentiating benign from malignant solitary pulmonary nodules (SPNs). We aimed to assess its diagnostic accuracy for differentiating benign from malignant SPNs in a tuberculosis (TB)-endemic area. METHODS Thirty patients, 22 men and 8 women, mean age 60 years, underwent dual time point FDG-PET/computed tomography (CT) imaging, followed by histological examination of the SPN. Maximum standard uptake values (SUVmax) with the greatest uptake in the lesion were calculated for two time points (SUV1 and SUV2), and the percentage change over time per lesion was calculated (%DSUV). Routine histological findings served as the gold standard. RESULTS Histological examination showed that 14 lesions were malignant and 16 benign, 12 of which were TB. SUVmax for benign and malignant lesions were 11.02 (standard deviation (SD) 6.6) v. 10.86 (SD 8.9); however, when tuberculomas were excluded from the analysis, a significant difference in mean SUV1max values between benign and malignant lesions was observed (p=0.0059). Using an SUVmax cut-off value of 2.5, a sensitivity of 85.7% and a specificity of 25% was obtained. Omitting the TB patients from analysis resulted in a sensitivity of 85.7% and a specificity of 100%. Mean %DSUV of benign lesions did not differ significantly from mean %DSUV of malignant lesions (17.1% (SD 16.3%) v. 19.4% (SD 23.7%)). Using a cut-off of %DSUV>10% as indicative of malignancy, a sensitivity of 85.7% and a specificity of 50% was obtained. Omitting the TB patients from the analysis yielded a sensitivity of 85.7% and a specificity of 75%. CONCLUSION Our findings suggest that FDG-PET cannot distinguish malignancy from tuberculoma and therefore cannot reliably be used to reduce futile biopsy/thoracotomy.

Surgical face masks worn by patients with multidrug-resistant tuberculosis: impact on infectivity of air on a hospital ward.

RATIONALE Drug-resistant tuberculosis transmission in hospitals threatens staff and patient health. Surgical face masks used by patients with tuberculosis (TB) are believed to reduce transmission but have not been rigorously tested. OBJECTIVES We sought to quantify the efficacy of surgical face masks when worn by patients with multidrug-resistant TB (MDR-TB). METHODS Over 3 months, 17 patients with pulmonary MDR-TB occupied an MDR-TB ward in South Africa and wore face masks on alternate days. Ward air was exhausted to two identical chambers, each housing 90 pathogen-free guinea pigs that breathed ward air either when patients wore surgical face masks (intervention group) or when patients did not wear masks (control group). Efficacy was based on differences in guinea pig infections in each chamber. MEASUREMENTS AND MAIN RESULTS Sixty-nine of 90 control guinea pigs (76.6%; 95% confidence interval [CI], 68-85%) became infected, compared with 36 of 90 intervention guinea pigs (40%; 95% CI, 31-51%), representing a 56% (95% CI, 33-70.5%) decreased risk of TB transmission when patients used masks. CONCLUSIONS Surgical face masks on patients with MDR-TB significantly reduced transmission and offer an adjunct measure for reducing TB transmission from infectious patients.

Use of 18F-FDG PET to Predict Response to First-Line Tuberculostatics in HIV-Associated Tuberculosis

This prospective pilot study examined the relationship between the severity and extent of tuberculosis as assessed by 18F-FDG PET at the time of diagnosis and response to treatment or treatment failure at 4 mo. Methods: Twenty-four consecutive HIV patients with newly diagnosed tuberculosis were prospectively included in the study after providing written informed consent. Seventeen patients had pulmonary tuberculosis, and 7 patients had extrapulmonary tuberculosis. All patients underwent whole-body 18F-FDG PET; none were receiving tuberculostatics at the time of the PET investigation. After undergoing 18F-FDG PET, the patients were given tuberculosis treatment (the classic triad: isoniazid, rifampicin, and ethambutol) and reevaluated for treatment response: monthly assessment of sputum, smears, and cultures in patients who proved positive at the time of diagnosis, and clinical and radiologic (when relevant) assessment 4 mo after treatment instigation in all patients. Quantitative 18F-FDG PET results (averaged 18F-FDG maximum standardized uptake value [SUVmax] derived from early and delayed imaging), percentage change in SUVmax, and number of involved lymph node bastions were related to treatment response or failure. Results: Age, sex, viral load, CD4 status, duration of HIV treatment, SUVmax of lung and splenic lesions (early and delayed), and percentage change in SUVmax of lymph nodes were not significantly different between responders and nonresponders (P ≥ 0.3). In contrast, SUVmax of involved lymph node bastions (both early and delayed) and number of involved lymph node bastions were significantly higher in nonresponders than in responders (respective P values were 0.03, 0.04, and 0.002). Using a cutoff of 5 or more involved lymph node bastions, responders could be separated from nonresponders with a sensitivity, specificity, and positive and negative predictive value of, respectively, 88%, 81%, 70%, and 93%. Using a cutoff of 8.15 for early SUVmax of lymph node bastions and of 10 for late SUVmax of lymph node bastions, a comparable sensitivity of 88% came at the cost of a lower specificity: 73% and 67%, respectively. Conclusion: In this pilot study, a cutoff of 5 or more involved lymph node bastions allowed for separation of tuberculostatic responsive and nonresponsive tuberculosis-infected HIV patients with a sensitivity of 88%, a specificity of 81%, and a negative predictive value of 93%. These findings warrant confirmation by additional studies on larger cohorts of patients.

Rapid impact of effective treatment on transmission of multidrug-resistant tuberculosis

BACKGROUND Effective treatment for drug-susceptible tuberculosis (TB) rapidly renders patients non-infectious, long before conversion of sputum acid-fast smear or culture to negative. Multidrug-resistant TB (MDR-TB) patients on treatment are currently assumed to remain infectious for months. While the resources required for prolonged hospitalization are a barrier to the scale-up of MDR-TB treatment, the safety of community treatment is clear. OBJECTIVES To estimate the impact of treatment on infectiousness among MDR-TB patients. METHODS A series of five human-to-guinea pig TB transmission studies was conducted to test various interventions for infection control. Guinea pigs in adjacent chambers were exposed to exhaust air from a hospital ward occupied by mostly sputum smear- and culture-positive MDR-TB patients. The guinea pigs then underwent tuberculin skin testing for infection. Only the control groups of guinea pigs from each study (no interventions used) provide the data for this analysis. The number of guinea pigs infected in each study is reported and correlated with Mycobacterium tuberculosis drug susceptibility relative to treatment. RESULTS Despite exposure to presumably infectious MDR-TB patients, infection percentages among guinea pigs ranged from 1% to 77% in the five experiments conducted. In one experiment in which guinea pigs were exposed to 27 MDR-TB patients newly started on effective treatment for 3 months, there was minimal transmission. In four other experiments with greater transmission, guinea pigs had been exposed to patients with unsuspected extensively drug-resistant tuberculosis who were not on effective treatment. CONCLUSIONS In this model, effective treatment appears to render MDR-TB patients rapidly non-infectious. Further prospective studies on this subject are needed.

Structure-function relationships of the antigenicity of mycolic acids in tuberculosis patients.

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

Impact of FDG PET on the management of TBC treatment: a pilot study

UNLABELLED The aim of this study is to assess the potential impact of double-phase FDG PET versus routine staging in HIV-negative patients suffering from tuberculosis. PATIENTS, METHODS 16 consecutive patients suffering from tuberculosis underwent contrast-enhanced CT and double-phase FDG PET imaging (45 min, 120 min). Early (E) and delayed (D) SUVmax values were determined for all identified lesions and % change in SUV calculated (DeltaSUV). RESULTS Seven patients presented with lung lesions on PET as well as CT (mean SUVmaxE 8.2, mean SUVmaxD 11.1, (p = 0.002), DeltaSUV 35%. In two patients, lesions were judged as non-active on CT. In nine patients, 18 sites of LN involvement were identified on both early and delayed FDG PET images (mean SUVmaxE 6.3, mean SUVmaxD 7.9, (p = 0.0001), DeltaSUV: 25%). 9 out of 18 sites of LN involvement, occurring in five patients, were missed on CT. In four of these five patients, sites of LN involvement were the only sites of extra-pulmonary involvement identified. In 6 out of 16 patients, pleural involvement was identified, respectively in 5 on FDG PET and in 6 on CT imaging (mean SUVmaxE 1.3, mean SUVmaxD 1.7, (p = 0.06), DeltaSUV 21%). In 4 patients, osseous involvement was identified by both FDG PET and CT (mean SUVmaxE 7.2, mean SUVmaxD 10.7, (p = 0,06), DeltaSUV 45%). Finally, in 3 patients, joint involvement was identified on both FDG PET as well as on CT imaging (mean SUVmaxE 4.7, mean SUVmaxD 5.2, DeltaSUV 23%). FDG PET did not identify CT-additional sites of involvement that would have resulted in a prolonged treatment. CONCLUSION In HIV-negative patients suffering from tuberculosis, FDG PET images suggested a more extensive involvement by Mycobacterium tuberculosis when compared to contrast enhanced CT.

Einfluss der FDG-PET auf das therapeutische Vorgehen bei TBC – eine Pilotstudie

UNLABELLED The aim of this study is to assess the potential impact of double-phase FDG PET versus routine staging in HIV-negative patients suffering from tuberculosis. PATIENTS, METHODS 16 consecutive patients suffering from tuberculosis underwent contrast-enhanced CT and double-phase FDG PET imaging (45 min, 120 min). Early (E) and delayed (D) SUVmax values were determined for all identified lesions and % change in SUV calculated (DeltaSUV). RESULTS Seven patients presented with lung lesions on PET as well as CT (mean SUVmaxE 8.2, mean SUVmaxD 11.1, (p = 0.002), DeltaSUV 35%. In two patients, lesions were judged as non-active on CT. In nine patients, 18 sites of LN involvement were identified on both early and delayed FDG PET images (mean SUVmaxE 6.3, mean SUVmaxD 7.9, (p = 0.0001), DeltaSUV: 25%). 9 out of 18 sites of LN involvement, occurring in five patients, were missed on CT. In four of these five patients, sites of LN involvement were the only sites of extra-pulmonary involvement identified. In 6 out of 16 patients, pleural involvement was identified, respectively in 5 on FDG PET and in 6 on CT imaging (mean SUVmaxE 1.3, mean SUVmaxD 1.7, (p = 0.06), DeltaSUV 21%). In 4 patients, osseous involvement was identified by both FDG PET and CT (mean SUVmaxE 7.2, mean SUVmaxD 10.7, (p = 0,06), DeltaSUV 45%). Finally, in 3 patients, joint involvement was identified on both FDG PET as well as on CT imaging (mean SUVmaxE 4.7, mean SUVmaxD 5.2, DeltaSUV 23%). FDG PET did not identify CT-additional sites of involvement that would have resulted in a prolonged treatment. CONCLUSION In HIV-negative patients suffering from tuberculosis, FDG PET images suggested a more extensive involvement by Mycobacterium tuberculosis when compared to contrast enhanced CT.

Institutional Tuberculosis Transmission. Controlled Trial of Upper Room Ultraviolet Air Disinfection: A Basis for New Dosing Guidelines

RATIONALE Transmission is driving the global tuberculosis epidemic, especially in congregate settings. Worldwide, natural ventilation is the most common means of air disinfection, but it is inherently unreliable and of limited use in cold climates. Upper room germicidal ultraviolet (UV) air disinfection with air mixing has been shown to be highly effective, but improved evidence-based dosing guidelines are needed. OBJECTIVES To test the efficacy of upper room germicidal air disinfection with air mixing to reduce tuberculosis transmission under real hospital conditions, and to define the application parameters responsible as a basis for proposed new dosing guidelines. METHODS Over an exposure period of 7 months, 90 guinea pigs breathed only untreated exhaust ward air, and another 90 guinea pigs breathed only air from the same six-bed tuberculosis ward on alternate days when upper room germicidal air disinfection was turned on throughout the ward. MEASUREMENTS AND MAIN RESULTS The tuberculin skin test conversion rates (>6 mm) of the two chambers were compared. The hazard ratio for guinea pigs in the control chamber converting their skin test to positive was 4.9 (95% confidence interval, 2.8-8.6), with an efficacy of approximately 80%. CONCLUSIONS Upper room germicidal UV air disinfection with air mixing was highly effective in reducing tuberculosis transmission under hospital conditions. These data support using either a total fixture output (rather than electrical or UV lamp wattage) of 15-20 mW/m(3) total room volume, or an average whole-room UV irradiance (fluence rate) of 5-7 μW/cm(2), calculated by a lighting computer-assisted design program modified for UV use.

Mycolic acids, a promising mycobacterial ligand for targeting of nanoencapsulated drugs in tuberculosis

The appearance of drug-resistant strains of Mycobacterium tuberculosis (Mtb) poses a great challenge to the development of novel treatment programmes to combat tuberculosis. Since innovative nanotechnologies might alleviate the limitations of current therapies, we have designed a new nanoformulation for use as an anti-TB drug delivery system. It consists of incorporating mycobacterial cell wall mycolic acids (MA) as targeting ligands into a drug-encapsulating Poly dl-lactic-co-glycolic acid polymer (PLGA), via a double emulsion solvent evaporation technique. Bone marrow-derived mouse macrophages, either uninfected or infected with different mycobacterial strains (Mycobacterium avium, Mycobacterium bovis BCG or Mtb), were exposed to encapsulated isoniazid-PLGA nanoparticles (NPs) using MA as a targeting ligand. The fate of the NPs was monitored by electron microscopy. Our study showed that i) the inclusion of MA in the nanoformulations resulted in their expression on the outer surface and a significant increase in phagocytic uptake of the NPs; ii) nanoparticle-containing phagosomes were rapidly processed into phagolysosomes, whether MA had been included or not; and iii) nanoparticle-containing phagolysosomes did not fuse with non-matured mycobacterium-containing phagosomes, but fusion events with mycobacterium-containing phagolysosomes were clearly observed.