Unraveling tuberculosis patient cluster transmission chains: integrating WGS-based network with clinical and epidemiological insights.

Background: Tuberculosis remains a global health threat, and the World Health Organization reports a limited reduction in disease incidence rates, including both new and relapse cases. Therefore, studies targeting tuberculosis transmission chains and recurrent episodes are crucial for developing the most effective control measures. Herein, multiple tuberculosis clusters were retrospectively investigated by integrating patients' epidemiological and clinical information with median-joining networks recreated based on whole genome sequencing (WGS) data of Mycobacterium tuberculosis isolates. Methods: Epidemiologically linked tuberculosis patient clusters were identified during the source case investigation for pediatric tuberculosis patients. Only M. tuberculosis isolate DNA samples with previously determined spoligotypes identical within clusters were subjected to WGS and further median-joining network recreation. Relevant clinical and epidemiological data were obtained from patient medical records. Results: We investigated 18 clusters comprising 100 active tuberculosis patients 29 of whom were children at the time of diagnosis; nine patients experienced recurrent episodes. M. tuberculosis isolates of studied clusters belonged to Lineages 2 (sub-lineage 2.2.1) and 4 (sub-lineages 4.3.3, 4.1.2.1, 4.8, and 4.2.1), while sub-lineage 4.3.3 (LAM) was the most abundant. Isolates of six clusters were drug-resistant. Within clusters, the maximum genetic distance between closely related isolates was only 5-11 single nucleotide variants (SNVs). Recreated median-joining networks, integrated with patients' diagnoses, specimen collection dates, sputum smear microscopy, and epidemiological investigation results indicated transmission directions within clusters and long periods of latent infection. It also facilitated the identification of potential infection sources for pediatric patients and recurrent active tuberculosis episodes refuting the reactivation possibility despite the small genetic distance of ≤5 SNVs between isolates. However, unidentified active tuberculosis cases within the cluster, the variable mycobacterial mutation rate in dormant and active states, and low M. tuberculosis genetic variability inferred precise transmission chain delineation. In some cases, heterozygous SNVs with an allelic frequency of 10-73% proved valuable in identifying direct transmission events. Conclusion: The complex approach of integrating tuberculosis cluster WGS-data-based median-joining networks with relevant epidemiological and clinical data proved valuable in delineating epidemiologically linked patient transmission chains and deciphering causes of recurrent tuberculosis episodes within clusters.

Effect of NAT2, GSTM1 and CYP2E1 genetic polymorphisms on plasma concentration of isoniazid and its metabolites in patients with tuberculosis, and the assessment of exposure-response relationships.

Objectives: Isoniazid is a key drug in the chemotherapy of tuberculosis (TB), however, interindividual variability in pharmacokinetic parameters and drug plasma levels may affect drug responses including drug induced hepatotoxicity. The current study investigated the relationships between isoniazid exposure and isoniazid metabolism-related genetic factors in the context of occurrence of drug induced hepatotoxicity and TB treatment outcomes. Methods: Demographic characteristics and clinical information were collected in a prospective TB cohort study in Latvia (N = 34). Time to sputum culture conversion (tSCC) was used as a treatment response marker. Blood plasma concentrations of isoniazid (INH) and its metabolites acetylisoniazid (AcINH) and isonicotinic acid (INA) were determined at three time points (pre-dose (0 h), 2 h and 6 h after drug intake) using liquid chromatography-tandem mass spectrometry. Genetic variations of three key INH-metabolizing enzymes (NAT2, CYP2E1, and GSTM1) were investigated by application PCR- and Next-generation sequencing-based methods. Depending on variables, group comparisons were performed by Student's t-test, one-way ANOVA, Mann-Whitney-Wilcoxon, and Kruskal-Wallis tests. Pearson correlation coefficient was calculated for the pairs of normally distributed variables; model with rank transformations were used for non-normally distributed variables. Time-to-event analysis was performed to analyze the tSCC data. The cumulative probability of tSCC was obtained using Kaplan-Meier estimators. Cox proportional hazards models were fitted to estimate hazard rate ratios of successful tSCC. Results: High TB treatment success rate (94.1%) was achieved despite the variability in INH exposure. Clinical and demographic factors were not associated with either tSCC, hepatotoxicity, or INH pharmacokinetics parameters. Correlations between plasma concentrations of INH and its metabolites were NAT2 phenotype-dependent, while GSTM1 genetic variants did not showed any effects. CYP2E1*6 (T > A) allelic variant was associated with INH pharmacokinetic parameters. Decreased level of AcINH was associated with hepatotoxicity, while decreased values of INA/INH and AcINH/INH were associated with month two sputum culture positivity. Conclusion: Our findings suggest that CYP2E1, but not GSTM1, significantly affects the INH pharmacokinetics along with NAT2. AcINH plasma level could serve as a biomarker for INH-related hepatotoxicity, and the inclusion of INH metabolite screening in TB therapeutic drug monitoring could be beneficial in clinical studies for determination of optimal dosing strategies.

Exploring Variability in Rifampicin Plasma Exposure and Development of Anti-Tuberculosis Drug-Induced Liver Injury among Patients with Pulmonary Tuberculosis from the Pharmacogenetic Perspective.

Genetic polymorphisms can exert a considerable impact on drug pharmacokinetics (PK) and the development of adverse drug reactions (ADR). However, the effect of genetic polymorphisms on the anti-tuberculosis (anti-TB) drug, and particularly rifampicin (RIF), exposure or anti-TB drug-induced liver injury (DILI) remains uncertain. Here, we evaluated the relationship between single nucleotide polymorphisms (SNPs) detected in the RIF pharmacogenes (AADAC, SLCO1B1, SLCO1B3, ABCB1, and NR1I2) and RIF PK parameters, as well as anti-TB treatment-associated DILI. In total, the study enrolled 46 patients with drug-susceptible pulmonary TB. The RIF plasma concentration was measured using the LC-MS/MS method in the blood samples collected pre-dose and 2 and 6 h post-dose, whilst the DILI status was established using the results from blood biochemical analysis performed before and 10-12 days after treatment onset. The genotyping was conducted using a targeted NGS approach. After adjustment for confounders, the patients carrying the rs3732357 GA/AA genotype of the NR1I2 gene were found to have significantly lower RIF plasma AUC0-6 h in comparison to those with GG genotype, while the difference in RIF plasma Cmax was insignificant. None of the analyzed SNPs was related to DILI. Hence, we are the first to report NR1I2 intronic SNP rs3732357 as the genetic component of variability in RIF exposure. Regarding anti-TB treatment-associated DILI, the other preexisting factors promoting this ADR should be considered.

Comparative Microbiome Analysis of Three Epidemiologically Important Tick Species in Latvia.

(1) Background: Amplicon-based 16S rRNA profiling is widely used to study whole communities of prokaryotes in many niches. Here, we comparatively examined the microbial composition of three tick species, Ixodes ricinus, Ixodes persulcatus and Dermacentor reticulatus, which were field-collected in Latvia. (2) Methods: Tick DNA samples were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). (3) Results: The results showed significant differences in microbial species diversity and composition by tick species and life stage. A close similarity between microbiomes of I. ricinus and I. persulcatus ticks was observed, while the D. reticulatus microbiome composition appeared to be more distinct. Significant differences in alpha and beta microbial diversity were observed between Ixodes tick life stages and sexes, with lower taxa richness indexes obtained for female ticks. The Francisella genus was closely associated with D. reticulatus ticks, while endosymbionts Candidatus Midichlorii and Candidatus Lariskella were associated with I. ricinus and I. persulcatus females, respectively. In I. ricinus females, the endosymbiont load negatively correlated with the presence of the Rickettsia genus. (4) Conclusions: The results of this study revealed important associations between ticks and their microbial community and highlighted the microbiome features of three tick species in Latvia.

Assessment of Amikacin- and Capreomycin-Related Adverse Drug Reactions in Patients with Multidrug-Resistant Tuberculosis and Exploring the Role of Genetic Factors.

Following the introduction of all-oral treatment regimens for patients with drug-resistant tuberculosis (TB), second-line injectable drug applications have been reduced in the last few years. However, they are still important for anti-TB therapy. This study aims to analyze the occurrence of amikacin- and capreomycin-related adverse drug reactions (ADR) in patients with multidrug-resistant tuberculosis (MDR-TB) and evaluate the role of multiple patient-, disease-, and therapy-related factors on the frequency of the observed adverse events. In addition, the possible role of genetic risk factors was studied by full-length mitochondrial DNA sequencing. Toward this aim, we retrospectively evaluated 47 patients with MDR-TB who received amikacin and/or capreomycin. In total, 16 (34.0%) patients developed ototoxicity and 13 (27.7%) developed nephrotoxicity, including 3 (6.4%) patients who experienced both adverse events. Ototoxicity development was more common in patients who received amikacin. No other factors showed a significant impact. Nephrotoxicity was likely associated with previous renal health impairment. Full mitochondrial genome sequencing did not reveal any specific ADR-associated variants, and results showed no differences in adverse event occurrence for any specific variants, mutation count, or mitochondrial haplogroup. The absence of the previously reported ototoxicity-related mtDNA variants in our patients with ototoxicity and nephrotoxicity highlighted the complex nature of the ADR occurrence.