Genotypic Distribution and the Epidemiology of Multidrug Resistant Tuberculosis in Upper Northern Thailand

The epidemiology and genotypes of multidrug-resistant tuberculosis (MDR-TB), a global public health threat, remain limited. The genotypic distribution and factors associated with MDR-TB in upper northern Thailand between 2015 and 2019 were investigated. The DNA sequencing of rpoB, katG, and inhA promoter of 51 multidrug-resistant Mycobacterium tuberculosis isolates revealed nine patterns of the rpoB gene mutation distributed in seven provinces. The S531L mutation was the most common mutation in all provinces. The rpoB mutation in Chiang Rai, Chiang Mai, and Lampang was highly diverse compared to other areas. Here, the mutation profiles that have yet to be reported in northern Thailand (H526P, Q513P, and H526C) were detected in Chiang Rai province. The S315T katG mutation was the most common genotype associated with INH resistance, especially in Chiang Mai and Lampang. Further analysis of data from 110 TB patients (42 MDR-TB and 68 drug-susceptible TB) revealed that p = 0.011) and ≥60 years of age was a significant factor associated with the S315T katG-mutation (OR = 8.867, 95% CI 0.981–80.177, p = 0.047). This study highlighted the necessity for continuous surveillance and risk factor monitoring for effective control of MDR-TB

In Vitro Antimycobacterial Activity of Human Lactoferrin-Derived Peptide, D-hLF 1-11, against Susceptible and Drug-Resistant Mycobacterium tuberculosis and Its Synergistic Effect with Rifampicin

Tuberculosis is a highly contagious disease caused by the Mycobacterium tuberculosis complex (MTBC). Although TB is treatable, multidrug-resistant, extensively drug-resistant, and totally drug-resistant forms of M. tuberculosis have become a new life-threatening concern. New anti-TB drugs that are capable of curing these drug-resistant strains are urgently needed. The purpose of this study is to determine the antimycobacterial activity of D-enantiomer human lactoferricin 1-11 (D-hLF 1-11) against mycobacteria in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide colorimetric assay, resazurin microplate assay, and microscopic observation drug susceptibility assay. Three previously described antimicrobial peptides, protegrin-1, AK 15-6, and melittin, with potent anti-TB activity, were included in this study. The findings suggest that D-hLF 1-11 can inhibit the growth of M. tuberculosis with a minimum inhibitory concentration of 100–200 µg/mL in susceptible, isoniazid (INH)-monoresistant, rifampicin (RF)-monoresistant, and MDR strains. The peptide can also inhibit some nontuberculous mycobacteria and other MTBC in similar concentrations. The antibiofilm activity of D-hLF 1-11 against the biofilm-forming M. abscessus was determined by crystal violet staining, and no significant difference is observed between the treated and untreated biofilm control. The checkerboard assay was subsequently carried out with M. tuberculosis H37Rv and the results indicate that D-hLF 1-11 displays an additive effect when combined with INH and a synergistic effect when combined with RF, with fractional inhibitory concentration indices of 0.730 and 0.312, respectively. The red blood cell hemolytic assay was initially applied for the toxicity determination of D-hLF 1-11, and negligible hemolysis (<1%) was observed, despite a concentration of up to 4 mg/mL being evaluated. Overall, D-hLF 1-11 has potential as a novel antimycobacterial agent for the future treatment of drug-sensitive and drug-resistant M. tuberculosis infections

Nano-Delivery System of Ethanolic Extract of Propolis Targeting <i>Mycobacterium tuberculosis</i> via Aptamer-Modified-Niosomes

Tuberculosis (TB) therapy requires long-course multidrug regimens leading to the emergence of drug-resistant TB and increased public health burden worldwide. As the treatment strategy is more challenging, seeking a potent non-antibiotic agent has been raised. Propolis serve as a natural source of bioactive molecules. It has been evidenced to eliminate various microbial pathogens including Mycobacterium tuberculosis (Mtb). In this study, we fabricated the niosome-based drug delivery platform for ethanolic extract of propolis (EEP) using thin film hydration method with Ag85A aptamer surface modification (Apt-PEGNio/EEP) to target Mtb. Physicochemical characterization of PEGNio/EEP indicated approximately −20 mV of zeta potential, 180 nm of spherical nanoparticles, 80% of entrapment efficiency, and the sustained release profile. The Apt-PEGNio/EEP and PEGNio/EEP showed no difference in these characteristics. The chemical composition in the nanostructure was confirmed by Fourier transform infrared spectrometry. Apt-PEGNio/EEP showed specific binding to Mycobacterium expressing Ag85 membrane-bound protein by confocal laser scanning microscope. It strongly inhibited Mtb in vitro and exhibited non-toxicity on alveolar macrophages. These findings indicate that the Apt-PEGNio/EEP acts as an antimycobacterial nanoparticle and might be a promising innovative targeted treatment. Further application of this smart nano-delivery system will lead to effective TB management

Combined Locked Nucleic Acid Probes and High-Resolution Melting Curve Analysis for Detection of Rifampicin-Resistant Tuberculosis in Northern Thailand

Rifampicin-resistant tuberculosis (RR-TB) has become a major threat globally. This study aims to develop a new assay, RIF-RDp, to enhance the detection of RR-TB based on combined locked nucleic acid (LNA) probes with high-resolution melting curve analysis (HRM). Two new LNA probes were designed to target the class-III and IV mutations of rpoB, H526D, and D516V. LNA probes showed 100% specificity in the detection of mutant targets among characterized and blinded Mycobacterium tuberculosis (Mtb) isolates. The performance of RIF-RDp was evaluated using 110 blinded clinical Mtb isolates in northern Thailand against drug-susceptibility testing (DST), DNA sequencing, and a commercial real-time PCR kit. This assay showed sensitivity and specificity of 94.55% and 98.18% compared to DST, and 96.36% and 100% compared to DNA sequencing. The efficacy of RIF-RDp was comparable to the commercial kit and DNA sequencing. The Cohen&rsquo;s Kappa statistic showed almost perfect agreement between RIF-RDp and the commercial kit (&kappa; = 0.95), and RIF-RDp and DNA sequencing (&kappa; = 0.96). Furthermore, this is the first report of the rare mutation profiles, S531W, and a triple codon deletion (510&ndash;512) in northern Thailand. According to high accuracy, the RIF-RDp assay may render an easy-to-use, low-cost, and promising diagnostics of RR-TB in the future