Tuberculosis (TB) remains an impactful infectious disease, leading to millions of deaths every year. Mycobacterium tuberculosis causes the formation of granulomas, which will determine, through the host–pathogen relationship, if the infection will remain latent or evolve into active disease. Early TB diagnosis is life-saving, especially among immunocompromised individuals, and leads to proper treatment, preventing transmission. This review addresses different approaches to diagnosing TB, from traditional methods such as sputum smear microscopy to more advanced molecular techniques. Integrating these techniques, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), has significantly improved the sensitivity and specificity of M. tuberculosis identification. Additionally, exploring novel biomarkers and applying artificial intelligence in radiological imaging contribute to more accurate and rapid diagnosis. Furthermore, we discuss the challenges of existing diagnostic methods, including limitations in resource-limited settings and the emergence of drug-resistant strains. While the primary focus of this review is on TB diagnosis, we also briefly explore the challenges and strategies for diagnosing non-tuberculous mycobacteria (NTM). In conclusion, this review provides an overview of the current landscape of TB diagnostics, emphasizing the need for ongoing research and innovation. As the field evolves, it is crucial to ensure that these advancements are accessible and applicable in diverse healthcare settings to effectively combat tuberculosis worldwide.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

Since ancient times, plants have been used in folk medicine to treat different diseases. Plants offer exceptional chemical diversity with a wide range of biological activities, and have therefore been the most promising sources for the discovery and development of drugs, including antimicrobial agents. This study reports the antibacterial effect of geraniol (GER), alone and in combination with biogenic silver nanoparticles (bioAgNPs), produced using the aqueous extract of Trichilia catigua bark, against planktonic and sessile cells of methicillin-resistant Staphylococcus aureus (MRSA), one of the main opportunistic and potentially fatal human pathogens. GER had a time-dependent bactericidal effect on planktonic cells, impairing the cell membrane integrity. In addition, GER inhibited the staphyloxanthin production, and molecular docking analyses supported the in silico affinity of GER to dehydrosqualene synthase (CrtM) and 4,4′-diaponeurosporen-aldehyde dehydrogenase (AldH), which are key enzymes within the pigment biosynthesis pathway in S. aureus. GER treatment increased the sensitivity of MRSA to hydrogen peroxide killing. GER displayed synergism with bioAgNPs against planktonic and sessile cells, inhibiting bacterial adhesion and the viability of biofilms formed on abiotic surfaces. MRSA planktonic and sessile cells treated with GER or GER/bioAgNPs displayed severe morphological and ultrastructural alterations. Notably, neither GER nor its combination caused in vitro and in vivo toxicity in mammalian cells and Galleria mellonella larvae, respectively. These findings suggest that the combination of GER/bioAgNPs may be a promising strategy to control MRSA infections.

Candida tropicalis is regarded as an opportunistic pathogen, causing diseases ranging from superficial infections to life-threatening disseminated infections. The ability of this yeast to form biofilms and develop resistance to antifungals represents a significant therapeutic challenge. Herein, the effect of geraniol (GER), alone and combined with fluconazole (FLZ), was evaluated in the planktonic and sessile cells of azole-resistant C. tropicalis. GER showed a time-dependent fungicidal effect on the planktonic cells, impairing the cell membrane integrity. Additionally, GER inhibited the rhodamine 6G efflux, and the molecular docking analyzes supported the binding affinity of GER to the C. tropicalis Cdr1 protein. GER exhibited a synergism with FLZ against the planktonic and sessile cells, inhibiting the adhesion of the yeast cells and the viability of the 48-h biofilms formed on abiotic surfaces. C. tropicalis biofilms treated with GER, alone or combined with FLZ, displayed morphological and ultrastructural alterations, including a decrease in the stacking layers and the presence of wilted cells. Moreover, neither GER alone nor combined with FLZ caused toxicity, and both treatments prolonged the survival of the Galleria mellonella larvae infected with azole-resistant C. tropicalis. These findings indicate that the combination of GER and FLZ may be a promising strategy to control azole-resistant C. tropicalis infections.

Candida species are the primary fungal pathogens of invasive infections associated with high morbidity and mortality. The identification of these microorganisms is critical for therapeutic management and control of hospital infection. Herein, assays targeting the Intergenic Spacer 2 (IGS2) and Internal Transcribed Spacer 1 (ITS1) from the rDNA locus were developed to differentiate Candida species. Based on consensus nucleotide sequences, specific primers and positive controls were designed, and standard PCR and real-time PCR (qPCR) assays were performed. All primers resulted in specific amplification of the molecular targets of each species with no amplifications of the negative template control. Furthermore, the primers were highly specific when tested with a range of fungal DNAs and no cross-reactivity was observed among Candida species. The assays presented a limit of detection (LoD) of 10 copies of positive control per reaction for all specific primers designed. Overall, our results showed that qPCR assays employing primers targeting the regions IGS2 and ITS1 completely differentiated between Candida albicans, Candida auris, Candida parapsilosis, Candida tropicalis, and Nakazeomyces glabratus, with great accuracy and no amplification of DNA from other fungal species.

Introduction: Candida infections represent a major public health concern and the intrinsic and acquired resistance of Candida species to antifungals underscores the need for new therapeutic approaches. In this context, natural products represent a valuable source of bioactive compounds with potential antifungal activity. Methods: The antifungal activity of the Ethyl Acetate Fraction (EAF) of Poincianella pluviosa (DC.) L. P. Queiroz stem bark was evaluated against planktonic Candida spp. by broth microdilution method. The effect of EAF combined with Fluconazole (FLZ) was assessed using the checkerboard assay. The effects of EAF alone and combined with FLZ on Candida albicans morphology and membrane integrity were investigated by scanning electron microscopy and fluorescence microscopy, respectively. The antibiofilm activity of EAF against developing and mature biofilms, as well as its effects on mammalian cell viability, was assessed using tetrazolium-based assays. Results: To our knowledge, this is the first study to investigate the antifungal activity of P. pluviosa stem bark EAF against planktonic and biofilm-associated Candida cells and its interaction with FLZ. EAF exhibited fungistatic activity against all tested strains, including FLZ-resistant isolates, with Minimum Inhibitory Concentrations (MICs) ranging from 31.2 to 500 µg/mL. EAF also reduced the metabolic activity of developing and mature biofilms. The combination of EAF and FLZ showed synergistic activity against most tested strains, reducing the MICs of both compounds. Microscopic analyses revealed reduced metabolic activity in cells treated with EAF alone or combined with FLZ, without detectable changes in cell morphology or cytoplasmic membrane integrity. EAF showed no cytotoxic effects on mammalian cells at the antifungal concentrations tested. Conclusion: These findings highlight the antifungal potential of the stem bark EAF of P. pluviosa, alone and in combination with FLZ, supporting further investigation of EAF as a potential source of bioactive compounds for developing alternative strategies to control Candida infections.