Detection of Mtb and NTM: preclinical validation of a new asymmetric PCR-binary deoxyribozyme sensor assay.

Tuberculosis (TB) and infectious diseases caused by non-tuberculous mycobacteria (NTM) are global concerns. The development of a rapid and accurate diagnostic method, capable of detecting and identifying different mycobacteria species, is crucial. We propose a molecular approach, the BiDz-TB/NTM, based on the use of binary deoxyribozyme (BiDz) sensors for the detection of Mycobacterium tuberculosis (Mtb) and NTM of clinical interest. A panel of DNA samples was used to evaluate Mtb-BiDz, Mycobacterium abscessus/Mycobacterium chelonae-BiDz, Mycobacterium avium-BiDz, Mycobacterium intracellulare/Mycobacterium chimaera-BiDz, and Mycobacterium kansasii-BiDz sensors in terms of specificity, sensitivity, accuracy, and limit of detection. The BiDz sensors were designed to hybridize specifically with the genetic signatures of the target species. To obtain the BiDz sensor targets, amplification of a fragment containing the hypervariable region 2 of the 16S rRNA was performed, under asymmetric PCR conditions using the reverse primer designed based on linear-after-the-exponential principles. The BiDz-TB/NTM was able to correctly identify 99.6% of the samples, with 100% sensitivity and 0.99 accuracy. The individual values of specificity, sensitivity, and accuracy, obtained for each BiDz sensor, satisfied the recommendations for new diagnostic methods, with sensitivity of 100%, specificity and accuracy ranging from 98% to 100% and from 0.98 to 1.0, respectively. The limit of detection of BiDz sensors ranged from 12 genome copies (Mtb-BiDz) to 2,110 genome copies (Mkan-BiDz). The BiDz-TB/NTM platform would be able to generate results rapidly, allowing the implementation of the appropriate therapeutic regimen and, consequently, the reduction of morbidity and mortality of patients.IMPORTANCEThis article describes the development and evaluation of a new molecular platform for accurate, sensitive, and specific detection and identification of Mycobacterium tuberculosis and other mycobacteria of clinical importance. Based on BiDz sensor technology, this assay prototype is amenable to implementation at the point of care. Our data demonstrate the feasibility of combining the species specificity of BiDz sensors with the sensitivity afforded by asymmetric PCR amplification of target sequences. Preclinical validation of this assay on a large panel of clinical samples supports the further development of this diagnostic tool for the molecular detection of pathogenic mycobacteria.

Genotyping methods and their contributions to the study of tuberculosis dynamic in Latin America.

INTRODUCTION: Mycobacterium tuberculosis genotyping has impacted evolutionary studies worldwide. Nonetheless, its application and the knowledge generated depend on the genetic marker evaluated and the detection technologies that have evolved over the years. Here we describe the timeline of main genotypic methods related to M. tuberculosis in Latin America and the main findings obtained. METHODOLOGY: Systematic searches through the PubMed database were performed from 1993 to May 2021. A total of 345 articles met the inclusion criteria and were selected. RESULTS: Spacer oligonucleotide typing (spoligotyping) was the most widely used method in Latin America, with decreasing use in parallel with increasing use of mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) and whole genome sequencing (WGS). Among the countries, Brazil, Mexico, and Argentina had the most publications, and a considerable part of the articles were in collaboration with Latin American or non-Latin American institutions; a small proportion of studies needed partnerships to perform the genotypic methods. The genotypic methods allowed the identification of M. tuberculosis genotypes with greater capacity for clonal expansion and revealed the predominance of the Euro-American lineage in Latin America. There was a notable presence of the Beijing family in Peru and Colombia. CONCLUSIONS: The data obtained demonstrated the importance of expanding collaborative networks of tuberculosis (TB) research groups to countries with low productivity in this area, the commitment of the few Latin American countries to advance TB research, as well as the inestimable value of building a Latin America database, considering ease of population mobility between countries.

Development of a Novel Lipid-Based Nanosystem Functionalized with WGA for Enhanced Intracellular Drug Delivery.

Despite a considerable number of new antibiotics under going clinical trials, treatment of intracellular pathogens still represents a major pharmaceutical challenge. The use of lipid nanocarriers provides several advantages such as protection from compound degradation, increased bioavailability, and controlled and targeted drug release. Wheat germ agglutinin (WGA) is known to have its receptors on the alveolar epithelium and increase phagocytosis. The present study aimed to produce nanostructured lipid carriers with novel glycosylated amphiphilic employed to attach WGA on the surface of the nanocarriers to improve intracellular drug delivery. High-pressure homogenization was employed to prepare the lipid nanocarriers. In vitro, high-content analysis and flow cytometry assay was employed to study the increased uptake by macrophages when the nanocarriers were grafted with WGA. A lipid nanocarrier with surface-functionalized WGA protein (~200 nm, PDI > 0.3) was successfully produced and characterized. The system was loaded with a lipophilic model compound (quercetin; QU), demonstrating the ability to encapsulate a high amount of compound and release it in a controlled manner. The nanocarrier surface functionalization with the WGA protein increased the phagocytosis by macrophages. The system proposed here has characteristics to be further explored to treat intracellular pathogens.

Intramacrophage potential of a tetrahydropyridine: A promising compound in combating Mycobacterium tuberculosis.

Due to several obstacles in treating tuberculosis (TB), the search for new therapeutic alternatives remains a global priority. The nitrogenous heterocyclic compounds are promising in searching for new anti-Mycobacterium tuberculosis molecules, and our previous results highlight the potential of tetrahydropyridines. After exploring the antimycobacterial potential and putative mechanism of action of a tetrahydropyridine derivative (NUNL02), we seek to measure the oxidative stress caused by NUNL02 inside the extracellular replicating M. tuberculosis since it could be the reason for the NUNL02 bactericidal effect against replicating and starved M. tuberculosis; and to evaluate the anti-M. tuberculosis activity of NUNL02 against the intracellular bacillus (even combined with an anti-TB drug) to explore the potential of this tetrahydropyridine as a promising adjuvant for TB therapy. Briefly, we assessed the activity of NUNL02 against the H37Rv strain and evaluated the combination of NUNL02 and rifampicin (RIF), at previously defined subinhibitory concentrations, against intramacrophage M. tuberculosis. NUNL02, in addition to promote the oxidative stress inside the extracellular replicating M. tuberculosis as a possible indirect mechanism of action, also presented bactericidal potential as promising as RIF against intracellular bacilli. Thus, our findings reinforce NUNL02 as a promising scaffold for the development of new options for TB.

Chemical Profile and Antimicrobial Activity of the Marine Diatom Chaetoceros muelleri.

Microalgae, due to its rapid growth, low nutritional requirements, and versatility of adaptation to different environmental conditions, has aroused the biotechnological interest, synthesizing novel molecules with antioxidant, anticoagulant, anti-inflammatory, antitumor, and antimicrobial activities. In this sense, we carried out the bioprospection of Chaetoceros muelleri, a marine diatom employed in aquaculture, as a candidate to the development of new drugs for the treatment of bacterial infections. The chemical profile of extracts in different solvents (hexane, chloroform, methylene chloride, ethyl acetate, methanol, and acetone) were analyzed by 1 H-NMR. The hexane extract was the most active against all bacteria species tested, including Mycobacterium tuberculosis, with a minimum inhibitory concentration of 100 µg/ml. Contrarily, the methanol extract was inactive against all tested microorganisms and, in addition, was the only one with IC50 >800 µg/mL, showing no cytotoxicity in VERO cell lines. All other extracts showed antibacterial potential and IC50 values varying between 267.58 and 142.47 µg/ml. The fact that C. muelleri is a microalga easily grown on bioreactors on a large scale may promote its biotechnological use, especially as scaffolds for the development of new compounds against bacterial species of clinical and public health interest.

2,2,2-trifluoro-1-(1,4,5,6-tetrahydropyridin-3-yl)ethanone derivative as efflux pump inhibitor in Mycobacterium tuberculosis.

Although the administration of combined therapy is efficient to tuberculosis (TB) treatment caused by susceptible Mycobacterium tuberculosis strains, to overcome the multidrug resistance is still a challenge. Some studies have reported evidence about tetrahydropyridines as a putative efflux pump inhibitor, including in mycobacteria, being a promising strategy against M. tuberculosis. Thus, we investigated the biological potential of 2,2,2-trifluoro-1-(1,4,5,6-tetrahydropyridin-3-yl)ethanone derivative (NUNL02) against two strains of M. tuberculosis. NUNL02 was able to increase the susceptibility of the multidrug resistant strain to the anti-TB drugs, resulting in synergism with rifampicin. Still, we assume that this compound plays a role in the efflux mechanism in M. tuberculosis, besides, to be able to kill the bacillus under the deprivation of essential nutrients. Thus, our findings highlight NUNL02 as a promising prototype to develop a new adjuvant for TB treatment, mainly as EPI.

A highly rifampicin resistant Mycobacterium tuberculosis strain emerging in Southern Brazil.

Here we described phenotypical, molecular and epidemiological features of a highly rifampicin-resistant Mycobacterium tuberculosis strain emerging in Southern Brazil, that carries an uncommon insertion of 12 nucleotides at the codon 435 in the rpoB gene. Employing a whole-genome sequencing-based study on drug-resistant Mycobacterium tuberculosis strains, we identified this emergent strain in 16 (9.19%) from 174 rifampicin-resistant clinical strains, all of them belonging to LAM RD115 sublineage. Nine of these 16 strains were available to minimum inhibitory concentration determination and for all of them was found a high rifampicin-resistance level (≥to 32 mg/L). This high resistance level could be explained by structural changes into the RIF binding site of RNA polymerase caused by the insertions, and consequent low-affinity interaction with rifampicin complex confirmed through protein modeling and molecular docking simulations. Epidemiological investigation showed that most of the individuals (56.25%) infected by the studied strains were prison inmate individuals or that spent some time in prison. The phylogenomic approach revealed that strains carrying on insertion belonged to same genomic cluster, evidencing a communal transmission chain involving inmate individuals and community. We stress the importance of tuberculosis genomic surveillance and introduction of measures to interrupt Mycobacterium tuberculosis transmission chain in this region.

Streptococcus agalactiae colonization and screening approach in high-risk pregnant women in southern Brazil.

INTRODUCTION: Considering that Group B Streptococcus (GBS) persists as an important cause of neonatal morbidity and mortality, the objective of this study was to evaluate the frequency of maternal colonization by GBS, comparing the culture by the Granada broth with the GeneXpert real-time PCR diagnostic methods and the impact of chemoprophylaxis in high-risk pregnant women. METHODOLOGY: A prospective cohort of 110 pregnant women hospitalized for gestational complications was formed and recruited following interview and collection of rectovaginal swabs. RESULTS: The frequency of maternal colonization was 28.2% and statistically associated with Capurro> 37 weeks (p = 0.030) and neonatal infection (p = 0.008). Chemoprophylaxis was offered to 80% of those colonized. Among the pregnant women treated, a fivefold reduction in the rate of prematurity and rate of neonatal infection was observed. The sensitivity was 76.6% and 86.6% in culture and PCR, respectively, with an optimal index of agreement between the methods (K = 0.877). Grenade culture was considered an easy and low-cost method, while GeneXpert presented higher cost and error rate of 10%. However, 23.3% of the pregnant women were diagnosed exclusively by GeneXpert and the results were obtained in two hours. CONCLUSIONS: This study showed a significant prevalence of maternal colonization for GBS and that both culture and molecular methods had peculiarities that allow different applicability, with the culture being feasible for antenatal screening and in the hospital for high-risk pregnant women with no sign of imminent delivery and GeneXpert being prioritized for situations of preterm birth.

Molecular drug susceptibility testing and strain typing of tuberculosis by DNA hybridization.

In Mycobacterium tuberculosis (Mtb) the detection of single nucleotide polymorphisms (SNPs) is of high importance both for diagnostics, since drug resistance is primarily caused by the acquisition of SNPs in multiple drug targets, and for epidemiological studies in which strain typing is performed by SNP identification. To provide the necessary coverage of clinically relevant resistance profiles and strain types, nucleic acid-based measurement techniques must be able to detect a large number of potential SNPs. Since the Mtb problem is pressing in many resource-poor countries, requiring low-cost point-of-care biosensors, this is a non-trivial technological challenge. This paper presents a proof-of-concept in which we chose simple DNA-DNA hybridization as a sensing principle since this can be transferred to existing low-cost hardware platforms, and we pushed the multiplex boundaries of it. With a custom designed probe set and a physicochemical-driven data analysis it was possible to simultaneously detect the presence of SNPs associated with first- and second-line drug resistance and Mtb strain typing. We have demonstrated its use for the identification of drug resistance and strain type from a panel of phylogenetically diverse clinical strains. Furthermore, reliable detection of the presence of a minority population (<5%) of drug-resistant Mtb was possible.

In vitro and in silico analysis of the efficiency of tetrahydropyridines as drug efflux inhibitors in Escherichia coli.

The objectives of this study were to evaluate tetrahydropyridine derivatives as efflux inhibitors and to understand the mechanism of action of the compounds by in silico studies. Minimum inhibitory concentration (MIC) determination, fluorometric methods and docking simulations were performed. The compounds NUNL02, NUNL09 and NUNL10 inhibited efflux, and NUNL02 is very likely a substrate of the transporter protein AcrB. Docking studies suggested that the mechanism of action could be by competition with substrate for binding sites and protein residues. We showed for the first time the potential of tetrahydropyridines as efflux inhibitors and highlighted compound NUNL02 as an AcrB-specific inhibitor. Docking studies suggested that competition is the putative mechanism of action of these compounds.

Evaluation of the Speed-Oligo Mycobacteria assay for the identification of nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) causing human infectious disease have become increasingly common. Rapid and accurate identification to the species level is, therefore, critical. The Speed-Oligo Mycobacteria assay is an oligochromatographic method that was made available recently for the identification and differentiation of mycobacteria. The present study aimed to evaluate the performance of the Speed-Oligo Mycobacteria assay for the identification of NTM. We examined a total of 62 strains (9 type strains, 19 reference strains and 34 clinical isolates) belonging to 13 different species (Mycobacterium intracellulare, M. fortuitum, M. gordonae, M. kansasii, M. marinum, M. peregrinum, M. scrofulaceum, M. abscessus, M. bovis BCG, M. chelonae, M. avium, M. malmoense and M. xenopi). The Speed-Oligo Mycobacteria assay was performed according to the manufacturer's instructions. Discrepant results between Speed-Oligo Mycobacteria and the original identification were reassessed by the Speed-Oligo Mycobacteria assay and resolved by the GenoType Mycobacterium CM assay and by sequencing of 16S rRNA and protein-encoding genes. We found 93.5 % (58/62) concordance for the identification of NTM as compared with the original identification. Three strains were erroneously identified by Speed-Oligo Mycobacteria: one M. kansasii strain was identified as Mycobacterium tuberculosis complex, and one M. chelonae strain and one M. peregrinum strain were both identified as Mycobacterium abscessus. Moreover, one M. chelonae strain was not identified by Speed-Oligo Mycobacteria since it did not react with any species-specific probe. For these strains, sequencing of the genes hsp65, 16S rRNA and rpoB and the GenoType Mycobacterium CM assay were performed. The Speed-Oligo Mycobacteria assay can be a useful tool for the rapid and easy identification of the most common NTM. If applied in clinical practice it could reduce diagnostic delays and contribute to correct clinical and better management of infections caused by NTM.

Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs.

Tuberculosis (TB) remains one of the leading public health problems worldwide. Declared as a global emergency in 1993 by the WHO, its control is hampered by the emergence of multidrug resistance (MDR), defined as resistance to at least rifampicin and isoniazid, two key drugs in the treatment of the disease. More recently, severe forms of drug resistance such as extensively drug-resistant (XDR) TB have been described. After the discovery of several drugs with anti-TB activity, multidrug therapy became fundamental for control of the disease. Major advances in molecular biology and the availability of new information generated after sequencing the genome of Mycobacterium tuberculosis increased our knowledge of the mechanisms of resistance to the main anti-TB drugs. Better knowledge of the mechanisms of drug resistance in TB and the molecular mechanisms involved will help us to improve current techniques for rapid detection and will also stimulate the exploration of new targets for drug activity and drug development. This article presents an updated review of the mechanisms and molecular basis of drug resistance in M. tuberculosis. It also comments on the several gaps in our current knowledge of the molecular mechanisms of drug resistance to the main classical and new anti-TB drugs and briefly discusses some implications of the development of drug resistance and fitness, transmission and pathogenicity of M. tuberculosis.

Antimycobacterial activity of usnic acid against resistant and susceptible strains of Mycobacterium tuberculosis and non-tuberculous mycobacteria.

Tuberculosis remains a serious public health problem, with nine million cases being reported annually. Treatment with antibiotics is the most effective mechanism to control this disease, although the increase in cases with resistant strains, co-infection with HIV, and the long duration of treatment has established the need to develop new drugs. Here we show the activity of usnic acid against susceptible and resistant Mycobacterium tuberculosis strains and against nontuberculous mycobacteria. Further, we did not identify any contribution of efflux in innate resistance to usnic acid.

Clonal diversity of M. tuberculosis isolated in a sea port city in Brazil.

Genotyping tools have been widely used to study the occurrence of outbreaks and to identify the patterns of transmission of Mycobacterium tuberculosis strains. The clonal diversity of 65 clinical isolates of M. tuberculosis was determined by PCR methods. The Double Repeat Element method (DRE-PCR) and spoligotyping identified 45 and 26 distinct patterns respectively. Among these, LAM (38%) was the most frequent lineage, followed by Haarlem (31%) and T (20%). Five orphan patterns were not present in the SITVIT database. Mycobacterial interspersed repetitive units (MIRU) using 12 loci revealed 46 distinct patterns. MIRU loci 10, 23, 26 and 40 had the highest discriminatory power. The high genetic diversity found among M. tuberculosis isolates in this study suggests a high level of recent TB transmission, indicating an endemic mode of TB transmission and a putative importation of new TB genotypes. In addition, the high diversity among the isolates could indicate early detection of the infection in patients and an efficient rate of cure.