Molecular Evidence for the Aerial Route of Infection of Mycobacterium leprae and the Role of Asymptomatic Carriers in the Persistence of Leprosy.

BACKGROUND: Leprosy persists as a public health problem. The chain of transmission and mechanism of infection are not completely understood. In the current study, we investigated the route of infection and of disease onset, from airway exposure, colonization, and bloodstream dissemination. METHODS: Mycobacterium leprae DNA was detected through quantitative polymerase chain reaction in nasal vestibule, nasal turbinate mucosa, and peripheral blood samples, along with anti-phenolic glycolipid I serology and skin tests from the same individual, from 113 leprosy patients and 104 household contacts of patients (HHCs). Bivariate statistics and multiple correspondence analysis were employed. RESULTS: The rates of DNA positivity among patients were 66.4% (75 of 113) for nasal swab samples, 71.7% (81 of 113) for nasal turbinate biopsy samples, 19.5% (22 of 113) for blood samples, with seropositivity of 62.8% (71 of 113 samples) and with increasing incidences toward the multibacillary pole of the clinical spectrum. Positivity among HHCs were as follows: 49% (51 of 104) for nasal swab samples, 53.8% (56 of 104) for nasal biopsy samples, 6.7% (7 of 104) for blood samples, and 18.3% (19 of 104 samples) for anti-phenolic glycolipid I serology. During the follow-up of 5-7 years, out of 104 HHCs, 7 developed leprosy (6.7%). Risk for the disease outcome was estimated by comparing results in HHCs who develop leprosy with those not affected. Neither nasal passage nor mucosa positivity was determinant of later disease onset; however, blood presence increased the risk for disease development (relative risk/positive likelihood ratio, 5.54; 95% confidence interval, 1.30-23.62), as did seropositivity (positive likelihood ratio, 3.69 [1.67-8.16]; relative risk, 5.97 [1.45-24.5]). CONCLUSIONS: Our findings strongly suggest that the aerosol route of infection and transmission is predominant and that HHCs contribute to the infection risk to themselves and probably to others.

Enhancement in Secondary Organic Aerosol Formation in the Presence of Preexisting Organic Particle.

Atmospheric models of secondary organic aerosol (SOA) typically assume organic species form a well-mixed phase. As a result, partitioning of semivolatile oxidation products into the particle phase to form SOA is thought to be enhanced by preexisting organic particles. In this work, the physicochemical properties that govern such enhancement in SOA yield were examined. SOA yields from α-pinene ozonolysis were measured in the presence of a variety of organic seeds which were chosen based on polarity and phase state at room temperature. Yield enhancement was only observed with seeds of medium polarities (tetraethylene glycol and citric acid). Solid hexadecanol seed was observed to enhance SOA yields only in chamber experiments with longer mixing time scales, suggesting that the mixing process for SOA and hexadecanol may be kinetically limited at shorter time scales. Our observations indicate that, in addition to kinetic limitations, intermolecular interactions also play a significant role in determining SOA yields. Here we propose for the first time to use the Hansen solubility framework to determine aerosol miscibility and predict SOA yield enhancement. These results highlight that current models may overestimate SOA formation, and parametrization of intermolecular forces is needed for accurate predictions of SOA formation.

Vaccination with a Sindbis virus-based DNA vaccine expressing antigen 85B induces protective immunity against Mycobacterium tuberculosis.

To improve DNA vaccination against Mycobacterium tuberculosis, we evaluated the effectiveness of a Sindbis virus-based DNA construct expressing the tuberculosis antigen 85B (Sin85B). The protective efficacy of Sin85B was initially assessed by aerogenically challenging immunized C57BL/6 mice with virulent Mycobacterium tuberculosis. At 1 and 7 months postinfection, the lung bacterial burdens were considerably reduced and the lung pathology was improved in vaccinated mice compared to naive controls. Furthermore, the mean survival period for Sin85B-immunized mice (305 +/- 9 days) after the tuberculous challenge was extended 102 days relative to the naive mice (203 +/- 13 days) and was essentially equivalent to the survival time of Mycobacterium bovis BCG-vaccinated mice (294 +/- 15 days). The essential role of gamma interferon (IFN-gamma) in Sin85B-mediated protection was established by showing that significantly increased levels of IFN-gamma mRNA were present postinfection in lung cells from vaccinated mice relative to control mice and by demonstrating that IFN-gamma depletion prior to challenge abolished the vaccine-induced protection. The substantial antituberculosis protective responses induced by Sin85B immunization of CD4-/- mice strongly suggested that CD8 cells partially mediate Sin85B-induced protective immunity. Interestingly, Sin85B vaccination did not protect RNase L-/- (a key enzyme in the innate antiviral response) mice while significant protection was detected in RNase L-/- mice immunized with either BCG or a conventional DNA plasmid expressing antigen 85B. These data show that immunization with Sin85B offers protection similar to BCG in a murine model of pulmonary tuberculosis and suggest that Sin85B-induced protection is dependent upon both innate and acquired immune mechanisms.

Effect of amino acids on the dispersion of disodium cromoglycate powders.

Modified disodium cromoglycate powders were prepared by co-spray drying with different concentrations of leucine, phenylalanine, tryptophan, methionine, asparagine, and arginine. Amorphous spherical particles of the same size and density where obtained which, however, exhibited different surface properties as measured by the inverse gas chromatography (IGC) and X-ray photoelectron spectroscopy (XPS) techniques. The surface energy parameters, such as the dispersive component of surface free energy of the sample, gammaSD, and the total solubility parameter, delta, were significantly lower in the presence of nonpolar chain amino acids, particularly with leucine and phenylalanine, than pure DSCG. However no quantitative relationship between these parameters, the additive concentrations, and the fine particle fractions, FPF, determined for different inhalers and air flow rates, was observed. The FPF significantly increased with addition of leucine and this effect was attributed to reduced intermolecular interactions between leucine and disodium cromoglycate molecules, as indicated by the difference in corresponding Hansen solubility parameters. Decrease of interparticle interactions for leucine-containing powders also led to a lesser dependence of FPF on the flow rate and inhaler type.

Contribution of the Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance.

The genome sequence of Mycobacterium tuberculosis revealed the presence of 12 membrane proteins proposed to have a function in the transport of lipids. Insertional inactivation of 11 of these has revealed that only 1 (MmpL3) is apparently essential for viability. Five of these proteins are conserved within the genome of Mycobacterium leprae. The drug susceptibilities of these 11 mutants to a broad spectrum of agents are unaltered, suggesting that unlike their function in other organisms, these proteins do not play a significant role in intrinsic drug resistance. Each of these mutants was assessed for growth kinetics and lethality in a murine low-dose aerosol model of tuberculosis, and four were found to be impaired in one or both measures of virulence. Two of these, with mutations of MmpL4 and the previously characterized MmpL7, which transports phthiocerol dimycocerosate, were found to have both impaired growth kinetics and impaired lethality. Mutants with inactivation of MmpL8, which transports a precursor of the sulfatides, or MmpL11, which transports an unknown substrate, were found to establish infection normally but to be significantly attenuated for lethality in time-to-death studies. These studies support the concept that MmpL-mediated lipid secretion both contributes to the innate ability of the pathogen to survive intracellularly and also contributes directly to the host-pathogen dialogue that determines the ultimate outcome of infection.

Airborne infection with Mycobacterium leprae in mice.

Although the portal of entry and mode of spread of M. leprae in human leprosy are still uncertain, it is widely held that direct person-to-person skin contact is important. This assumption has ignored the fact that patients with highly bacilliferous leprosy have nasal as well as dermal infection and that, since M. leprae is shed predominantly from the nose, leprosy might be an airborne infection. The present study was designed to investigate this possibility with mice exposed to airborne infection with M. leprae. The conditions are described in which thymectomised-irradiated CBA strain mice exposed to M. leprae aerosols sustained an immediate lung retention of 1 X 10(5) bacteria. Fourteen to 24 months later, 33% (10 of 30) of the mice had countable numbers of acid-fast bacilli (greater than 2 X 10(4)) with the characteristics of M. leprae in one or more homogenates prepared from ears, foot pads, nose or lungs. Evidence is presented from the distribution of M. leprae that the infection had arisen from systemic spresd of bacilli initially entering the lungs rather than from multiplication of organisms locally retained there, or in the nose, at the time of airborne infection. The relevance of these results to the possible route of infection of leprosy in man is discussed.