Mycobacterium tuberculosis ( Mtb ) is a major threat to global health, and there is an urgent need for affordable, simple tuberculosis (TB) diagnosis in underresourced areas. Here, we combine recombinase polymerase amplification with Cas13a and Cas12a detection to create two parallelized one-pot assays that detect two conserved elements of Mtb ( IS6110 and IS1081 ) and a human DNA internal control. These assays are compatible with lateral flow and can be readily lyophilized. Our final assay showed a limit of detection of 69.0 CFU per milliliter for Mtb H37Rv and 80.5 CFU per milliliter for Mycobacterium bovis BCG in spiked sputum, with no cross-reactivity to diverse bacterial or fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (six of six) sensitivity and 100% (seven of seven) specificity compared to culture. SHINE-TB streamlines TB diagnosis from sample to answer by combining amplification and detection while being compatible with lateral flow and lyophilization.

The quality and magnitude of the immune and inflammatory responses determine the clinical outcome of Leishmania infection, and contribute to the efficacy of antileishmanial treatments. However, the precise immune mechanisms involved in healing or in the chronic immunopathology of human cutaneous leishmaniasis (CL) are not well understood. Through sequential transcriptomic profiling of blood monocytes, neutrophils, and eosinophils over the course of systemic treatment with meglumine antimoniate, we revealed that a heightened and sustained Type-I interferon response signature is a hallmark of treatment failure (TF) in CL patients infected with Leishmania (Viannia) panamensis and L.V. braziliensis. The transcriptomes of pre-treatment, mid-treatment and end-of-treatment samples were interrogated to identify predictive and prognostic biomarkers of TF. A composite score derived from the expression of 11 differentially expressed genes (common between monocytes, neutrophils and eosinophils) is predictive of TF. Similarly, machine learning models constructed using data from pre-treatment as well as post-treatment samples, accurately classify treatment outcome into cure and TF. Results from this study instigate the evaluation of Type-I interferon responses as immunological targets for host-directed therapies for the treatment of CL, and highlight the feasibility of using transcriptional signatures as predictive biomarkers of outcome for therapeutic decision making.

Background: Rapid, accurate, nonsputum tests are needed to close gaps in tuberculosis (TB) detection. We evaluated the diagnostic performance of molecular testing on saliva and oral swabs. Methods: We conducted a nested case–control study with 1:1 incidence-density sampling within a prospective cohort of adults and children undergoing evaluation for pulmonary TB at primary care centers in Colombia (July 2023–August 2024). Participants provided a sputum sample for liquid mycobacterial culture and paired saliva and nylon-flocked oral swabs for storage at −80°C. A microbiologist blinded to clinical and culture data performed Xpert MTB/RIF Ultra on thawed saliva and on swab eluate, each mixed 1:1 with sample reagent. We calculated the sensitivity and specificity of saliva and swab against sputum culture and compared them using McNemar's test. Results: Among 648 enrolled participants, we tested saliva and swabs from all 95 individuals with culture-confirmed TB and 95 matched culture-negative controls (n = 190). Saliva sensitivity was 90.5% (95% confidence interval [CI], 82.8–95.6), and specificity was 95.8% (95% CI, 89.6–98.8). Swab sensitivity was 71.6% (95% CI, 61.4–80.4), and specificity was 99% (95% CI, 94.3–100). Saliva sensitivity exceeded that of swab by an absolute difference of 18.9% (95% CI, +10.0 to +27.9, P < .001), but there was no significant difference in specificity (−3.2%, 95% CI, −7.7 to +1.4, P = .25). Over 95% of participants found saliva and swab collection procedures acceptable. Conclusions: Both saliva and swabs were highly sensitive and specific for culture-confirmed pulmonary TB. Saliva sensitivity exceeded the World Health Organization's ≥80% target for a low-complexity, nonsputum TB diagnostic test.

The global spread of high-risk clo1nes (HRCs) of multidrug-resistant (MDR) Pseudomonas aeruginosa has hindered infection control and treatment strategies worldwide. In Colombia, globally relevant HRCs such as ST235 and ST111 have been widely reported. In this study, we evaluated phenotypic and metabolic variations associated with intracellular survival and dissemination in P. aeruginosa. A total of 100 clinical isolates were collected from 22 hospitals in Colombia. The isolates had been previously characterized and classified as MDR or susceptible strains (SSs), and their sequence types (STs) had been earlier determined. Based on this prior characterization, isolates were grouped in this study as multidrug-resistant high-risk clones (HRC, n = 50; corresponding to sequence types ST235 and ST111), multidrug-resistant non-high-risk clones (NHRCs, n = 27; non-ST235/ST111), and susceptible strains (SS, n = 23; also, non-ST235/ST111). Phenotypic traits, including motility, spontaneous mutation frequency, biofilm formation, and pigment production, were evaluated. In addition, a subset of 30 isolates was assessed for intracellular survival in vitro and metabolomic profiling using liquid chromatography-mass spectrometry. HRC isolates exhibited significantly reduced motility compared with NHRC and SS isolates (swarming: HRC vs. NHRC, p = 0.0032; HRC vs. SS, p = 0.010; swimming: HRC vs. NHRC and SS, p < 0.0001; twitching: HRC vs. SS, p = 0.0004), as well as lower pigment production (pyocyanin: HRC vs. NHRC and SS, p < 0.0001; pyoverdine: HRC vs. NHRC, p < 0.0001). Metabolomic analysis revealed increased concentrations of metabolites associated with iron acquisition and siderophore-related pathways in HRC isolates. Overall, these findings suggest that P. aeruginosa HRCs display distinct phenotypic and metabolic patterns that may contribute to persistence and dissemination in clinical settings, contributing to their epidemiological success.

Within-host SARS-CoV-2 diversity during acute infection is largely characterized by low-frequency intra-host single-nucleotide variants (iSNVs), but whether early iSNV patterns differ by immune status remains unclear. We analyzed biobanked diagnostic specimens from adults with RT-qPCR-confirmed SARS-CoV-2 infection collected in Cali, Colombia, between 2020 and 2022. Immunocompromised cases sampled during the acute phase were classified into four clinical categories, and immunocompetent cases from the same catchment area were used for comparison. Whole-genome sequencing (Illumina) yielded 102 high-quality genomes (≥85% coverage; immunocompetent, n = 60; immunocompromised, n = 42) from 114 samples. iSNVs were identified using iVar with stringent thresholds (depth ≥400× , Phred ≥20, allele frequency 3%–97%). Primary outcomes included iSNV counts, allele-frequency distributions, within-host complexity (Shannon entropy), normalized polymorphic sites, and Hamming distance. Analyses were conducted across all circulating lineages and within the Mµ (B.1.621) variant. Across all lineages, normalized polymorphic sites differed by immune status, with higher values in immunocompetent individuals ( P = 0.0064), whereas iSNV counts, allele-frequency distributions, and Shannon entropy were comparable between groups. When restricted to Mµ, immunocompetent individuals exhibited higher iSNV allele frequencies in the Spike region ( P = 0.0016). Within Mµ, mutational profiles differed by immune status: S-region iSNVs were predominantly synonymous in immunocompromised individuals and non-synonymous in immunocompetent individuals. Recurrent iSNVs revealed a shared core mutational signature across immune groups, with no Mµ-specific recurrent iSNVs exclusive to immunocompromised patients. Overall, intra-host diversity during acute infection was broadly similar by immune status, while lineage- and region-specific signals in the S region highlight nuanced early sub-consensus dynamics warranting functional investigation in immunophenotype-informed studies. IMPORTANCE: Understanding how immune status shapes within-host SARS-CoV-2 diversity is key for interpreting transmission risk and evolutionary potential. Immunocompromised patients are often presumed to harbor more diverse viral populations, yet evidence early in infection remains limited. Analyzing samples, we compared minor variant (intra-host single-nucleotide variant [iSNV]) patterns between immunocompromised and immunocompetent individuals and evaluated lineage-specific effects. We found comparable within-host diversity, but normalized polymorphic sites were higher in immunocompetent than in immunocompromised individuals when considering all lineages. We observed an Mµ-restricted S-region signal: immunocompetent hosts showed higher S-region iSNV allele frequencies, whereas immunocompromised hosts exhibited a shift toward synonymous S-region iSNVs. These results refine assumptions about early infection in immunocompromised hosts, emphasize the value of reporting synonymous and non-synonymous changes, and highlight that conclusions can depend on viral lineage and the diversity metric considered. Our study underscores the need for immunophenotype-informed studies to test whether synonymous iSNVs modulate viral expression, replication, and transmission.