Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 102 ge/μL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%–88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%–100%. The specificity was 96%–100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile lab...

La pirazinamida (PZA) es un profármaco utilizado en el tratamiento de la tuberculosis (TB). La pirazinamidasa (pncA) convierte a PZA en ácido pirazinoico (POA). La resistencia a PZA (PZAr) está presente en la mitad de las cepas resistentes a fármacos; sin embargo, PZAr no se prueba rutinariamente debido a la baja confiabilidad de las pruebas de susceptibilidad. El test de Wayne detecta POA como marcador de susceptibilidad PZA. Nos propusimos identificar PZAr cuantificando POA en sobrenadantes de cultivo de esputo mediante el uso de una prueba de Wayne cuantitativa directa.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Despite being considered curable and preventable, the increase of antibiotic resistance is becoming a serious public health problem. Mtb is a pathogen capable of surviving in macrophages, causing long-Term latent infection where the mycobacterial serine/threonine protein kinase G (PknG) plays a protective role. Therefore, PknG is an important inhibitory target to prevent Mtb from entering the latency stage. In this study, we use a pharmacophore-based virtual screening and biochemical assays to identify the compound RO9021 (CHEMBL3237561) as a PknG inhibitor. In detail, 1.5 million molecules were screened using a scalable cloud-based setup, identifying 689 candidates, which were further subjected to additional screening employing molecular docking. Molecular docking spotted 62 compounds with estimated b...

Tuberculosis (TB) remains a significantglobal health challenge, exacerbated by the emergence of drug-resistant strains, such as those resistant to pyrazinamide (PZA). The current scarcity of affordableand precise quantitative diagnostic tests for PZA resistance underscores the urgent need for more accessible diagnostic tools. We evaluated PZA susceptibility in 264 TB-positive samples by quantifying pyrazinoic acid (POA) production, using both the MODS-Wayne qualitative assay and our newly developed quantitative approach (MODS-WQ). The MODS-WQ was assessed in 7H9 medium (MODS-WQ7H9) or citrate buffer(MODS-WQCB), with POA levels measured via spectrophotometry against a calibration curve. PZA susceptibility determinations were based on a composite reference standard. Associations between POA levels and pyrazinamidase mutations were explored. The composite standard detected PZA resistance in...

Most culture-based methods for tuberculosis diagnosis remain low-cost options for low- and mid-income countries. The MODS culture is a rapid and low-cost assay to diagnose tuberculosis and determine drug susceptibility. However, its implementation is limited due to the low accessibility to supplies required for the enriched medium. In this study, we evaluate two alternative culture media: A powder-based mixed (PM) and a lyophilized media (LM). Catalase, PANTA, and gamma irradiation were evaluated as additions to PM and LM. The culture performance of the alternative media was compared with the standard MODS medium (MM) using Mycobacterium tuberculosis isolates and positive acid-fast smear sputum samples. Overall, no significant difference was observed in the bacterial growth between PM and LM with MM. However, PANTA and gamma irradiation combined reduced bacterial growth significantly in ...

La mayoría de los métodos basados en cultivos para el diagnóstico de la tuberculosis siguen siendo opciones de bajo costo para los países de ingresos bajos y medianos. El cultivo MODS es un ensayo rápido y de bajo costo para diagnosticar la tuberculosis y determinar la susceptibilidad a los medicamentos. Sin embargo, su implementación está limitada debido a la baja accesibilidad a los suministros requeridos para el medio enriquecido. En este estudio, evaluamos dos medios de cultivo alternativos: un medio mixto en polvo (PM) y un medio liofilizado (LM). Se evaluaron la catalasa, el PANTA y la irradiación gamma como adiciones a los medios PM y LM. El rendimiento del cultivo de los medios alternativos se comparó con el medio MODS estándar (MM) utilizando aislados de Mycobacterium tuberculosis y muestras de esputo con baciloscopía positiva. En general, no se observó una diferenci...

Tuberculosis phenotypic detection assays are commonly used in low-resource countries. Therefore, reliable detection methods are crucial for early diagnosis and treatment. The microscopic observation drug susceptibility (MODS) assay is a culture-based test to detect Mycobacterium tuberculosis and characterize drug resistance in 7–10 days directly from sputum. The use of MODS is limited by the availability of supplies necessary for preparing the enriched culture. In this study, we evaluated three dry culture media that are easier to produce and cheaper than the standard one used in MODS [1]: an unsterilized powder-based mixed (Boldú et al., 2007) [2], a sterile-lyophilized medium, and (Sengstake et al., 2017) [3] an irradiated powder-based mixed. Mycobacterial growth and drug susceptibility were evaluated for rifampin, isoniazid, and pyrazinamide (PZA). The alternative cultures were eva...

Paenibacillus polymyxa, a Gram-positive bacterium commonly found in soil and plant roots, plays an important role in the environment due to its nitrogen-fixing ability and is renowned for producing antibiotics like polymyxin. In this study, we present a robust framework for investigating the evolutionary and taxonomic connections of strains belonging to P. polymyxa available at the National Center for Biotechnology Information, as well as five new additional strains isolated at the University of Camerino (Italy), through pangenome analysis. These strains can produce secondary metabolites active against Staphylococcus aureus and Klebsiella pneumoniae. Employing techniques such as digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) estimation, OrthoFinder, and ribosomal multilocus sequence typing, we consistently divided these P. polymyxa strains into four clusters, whi...

Los ensayos fenotípicos de detección de tuberculosis son comúnmente utilizados en países con bajos recursos. Por lo tanto, los métodos de detección confiables son cruciales para el diagnóstico y tratamiento temprano. El ensayo de susceptibilidad a los fármacos por observación microscópica (MODS) es una prueba basada en cultivo para detectar Mycobacterium tuberculosis y caracterizar la resistencia a los medicamentos en 7-10 días directamente a partir del esputo. El uso de MODS está limitado por la disponibilidad de suministros necesarios para preparar el cultivo enriquecido. En este estudio, evaluamos tres medios de cultivo secos que son más fáciles de producir y más baratos que el estándar utilizado en MODS [1]: una mezcla en polvo no esterilizada (Boldú et al., 2007) [2], un medio estéril liofilizado y (Sengstake et al., 2017) [3] una mezcla en polvo irradiada. Se eval...