Epidemiology of coccidioidomycosis in Argentina, an update.

The National Reference Laboratory in Clinical Mycology of Argentina conducted a retrospective review of human coccidioidomycosis cases diagnosed by the National Mycology Laboratory Network of Argentina between 2010 and 2022 to determine the burden of the disease in the country. A total of 100 human coccidioidomycosis cases were documented, with a higher prevalence in male patients (male-to-female ratio of 1.9:1), with a median age of 41 years. Comparing the number of cases between two 10-year periods (2000-2009 and 2010-2019), the increase was 36.51% (from 63 to 86 cases). Among the 100 recorded cases, 79 tested positive using the double immunodiffusion test. Spherules were observed in 19 cases through histopathology or direct microscopic examination and the fungus was isolated in 39 cases. Thirty-six isolates were identified as Coccidioides posadasii through partial sequencing of the Ag2/PRA gene. Catamarca province had the highest number of cases, comprising 64% of the total, with an incidence rate above 1.0-2.5/100,000 inhabitants until 2018. However, there has been a recent downward trend in the region from 2018 to 2022. It is concerning that more than half of diagnosed cases were chronic pulmonary or disseminated forms, indicating a lack of early disease detection. To rectify this issue, it is imperative to conduct targeted training programs for healthcare personnel and enhance public awareness within the endemic area. This will contribute to a better understanding of the true burden of coccidioidomycosis and enable the implementation of appropriate sanitary control measures.

Effect of the deletion of lprG and p55 genes in the K10 strain of Mycobacterium avium subspecies paratuberculosis.

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.

Description of a novel adhesin of Mycobacterium avium subsp. paratuberculosis.

The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the µM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding.

Evaluation of cocktails with recombinant proteins of Mycobacterium bovis for a specific diagnosis of bovine tuberculosis.

The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.

Characterization of a Mycobacterium avium subsp. avium operon associated with virulence and drug detoxification.

The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo.