Discordance Between Phenotypic and WGS-Based Drug Susceptibility Testing Results for Some Anti-Tuberculosis Drugs: A Snapshot Study of Paired Mycobacterium tuberculosis Isolates with Small Genetic Distance.

Background: Current tuberculosis treatment regimens primarily rely on phenotypic drug susceptibility testing and rapid molecular assays. Although whole-genome sequencing (WGS) offers a promising alternative, disagreements between phenotypic and molecular testing methods remain. In this retrospective study, we compared the phenotypic and WGS-predicted drug resistance profiles of paired Mycobacterium tuberculosis isolates with small genetic distances (≤10 single nucleotide variants) obtained from patients with longitudinal single-episode or recurrent tuberculosis. Additionally, we investigated the distribution of drug-resistance-conferring variants among the identified M. tuberculosis genotypes. Methods: Paired M. tuberculosis isolates from 46 patients with pulmonary tuberculosis (2002-2019) were analyzed. Spoligotyping was performed for all the isolates. WGS data were processed using TB-Profiler software to genotype the strains and detect variants in M. tuberculosis genes associated with drug resistance. The significance of these variants was evaluated using the M. tuberculosis variant catalog developed by the World Health Organization. Phenotypic drug susceptibility test results were obtained from patients' medical records. Results: Among the 46 isolate pairs, 25 (54.3%) harbored drug-resistance-associated variants, with 20 demonstrating identical WGS-predicted drug resistance profiles. Drug-resistant isolate pairs belonged to Lineages 2 and 4, with the most common sub-lineages being 2.2.1 (SIT1 and SIT190 spoligotypes), and 4.3.3 (SIT42). Agreement between phenotypic and WGS-based drug susceptibility testing was highest (>90%) for rifampicin, isoniazid, ethambutol, fluoroquinolones, streptomycin, and amikacin when calculated for M. tuberculosis isolates or isolate pairs. In most discordant cases, isolate pairs harbored variants that could cause low- or moderate-level resistance or were previously associated with variable minimum inhibitory concentrations. Notably, such discrepancies mostly occurred in one isolate from the pair. In addition, differences in resistance-related variant distributions among M. tuberculosis genotypes were observed for most of the analyzed drugs. Conclusion: The simultaneous performance of phenotypic and WGS-based drug susceptibility testing creates the most accurate drug resistance profile for M. tuberculosis isolates and eliminates important limitations of each method.

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.

Understanding the high-risk human papillomavirus prevalence and associated factors in the European country with a high incidence of cervical cancer.

BACKGROUND: High-risk human papillomavirus (HR-HPV) is a known cause of cervical cancer (CC). Latvia has a high incidence of CC compared with the average incidence in the European Union. This study aims to fill the data gap on the HR-HPV burden in Latvia, providing information on its prevalence and associated factors. METHODS: The cross-sectional study was conducted from February 2021 to April 2022. Participants 25-70 years old visiting a general practitioner (general population) or those referred to a colposcopy clinic with changes in their cervical cytology (colposcopy population) collected vaginal self-sample and completed a paper-based questionnaire. Samples were analyzed with Cobas 6800 System (Roche) for HPV16, HPV18 and other HR-HPV (HPV31/33/35/39/45/51/52/56/58/59/66/68). Descriptive statistics for categorical variables were performed. The Chi-square test was used to determine for the statistical significance of differences in the proportions of the dependent variable between subgroups of the independent variable. Univariate and multivariate binary logistic regression were used to identify factors associated with positive HR-HPV status. Results were considered statistically significant at P < 0.05. RESULTS: A total of 1274 participants provided a valid sample. The prevalence of any HR-HPV infection was 66.8% in the colposcopy group and 11.0% in the general population. Factors associated with positive HR-HPV status were marital status single/divorced/widowed (vs. married/cohabiting) [adjusted OR (aOR) 2.6; P = 0.003], higher number of lifetime sex partners [aOR 5.1 (P < 0.001) and 4.0 (P = 0.001)] for six or more and three to five partners in the general population; in the colposcopy group, the statistical significance remained only for Latvian ethnicity (vs. other) (aOR 1.8; P = 0.008) and current smoking (vs. never) (aOR 1.9; P = 0.01). CONCLUSION: We documented a comparison to European Union HR-HPV infection burden in Latvia. Any HR-HPV positivity was significantly associated with sexual and other health behavior.