Tuberculosis in Ukrainian War Refugees and Migrants in the Czech Republic and Slovakia: A Molecular Epidemiological Study.

BACKGROUND: The war in Ukraine has led to significant migration to neighboring countries, raising public health concerns. Notable tuberculosis (TB) incidence rates in Ukraine emphasize the immediate requirement to prioritize approaches that interrupt the spread and prevent new infections. METHODS: We conducted a prospective genomic surveillance study to assess migration's impact on TB epidemiology in the Czech Republic and Slovakia. Mycobacterium tuberculosis isolates from Ukrainian war refugees and migrants, collected from September 2021 to December 2022 were analyzed alongside 1574 isolates obtained from Ukraine, the Czech Republic, and Slovakia. RESULTS: Our study revealed alarming results, with historically the highest number of Ukrainian tuberculosis patients detected in the host countries. The increasing number of cases of multidrug-resistant TB, significantly linked with Beijing lineage 2.2.1 (p < 0.0001), also presents substantial obstacles to control endeavors. The genomic analysis identified the three highly related genomic clusters, indicating the recent TB transmission among migrant populations. The largest clusters comprised war refugees diagnosed in the Czech Republic, TB patients from various regions of Ukraine, and incarcerated individuals diagnosed with pulmonary TB specialized facility in the Kharkiv region, Ukraine, pointing to a national transmission sequence that has persisted for over 14 years. CONCLUSIONS: The data showed that most infections were likely the result of reactivation of latent disease or exposure to TB before migration rather than recent transmission occurring within the host country. However, close monitoring, appropriate treatment, careful surveillance, and social support are crucial in mitigating future risks, though there is currently no evidence of local transmission in EU countries.

Resistance patterns and transmission of mono- and polyresistant TB: clinical impact of WGS.

Objectives: Rapidly diagnosing drug-resistant TB is crucial for improving treatment and transmission control. WGS is becoming increasingly accessible and has added value to the diagnosis and treatment of TB. The aim of the study was to perform WGS to determine the rate of false-positive results of phenotypic drug susceptibility testing (pDST) and characterize the molecular mechanisms of resistance and transmission of mono- and polyresistant Mycobacterium (M.) tuberculosis. Methods: WGS was performed on 53 monoresistant and 25 polyresistant M. tuberculosis isolates characterized by pDST. Sequencing data were bioinformatically processed to infer mutations encoding resistance and determine the origin of resistance and phylogenetic relationship between isolates studied. Results: The data showed the variable sensitivity and specificity of WGS in comparison with pDST as the gold standard: isoniazid 92.7% and 92.3%; streptomycin 41.9% and 100.0%; pyrazinamide 15% and 94.8%; and ethambutol 75.0% and 98.6%, respectively. We found novel mutations encoding resistance to streptomycin (in gidB) and pyrazinamide (in kefB). Most isolates belonged to lineage 4 (80.1%) and the overall clustering rate was 11.5%. We observed lineage-specific gene variations encoding resistance to streptomycin and pyrazinamide. Conclusions: This study highlights the clinical potential of WGS in ruling out false-positive drug resistance following phenotypic or genetic drug testing, and recommend this technology together with the WHO catalogue in designing an optimal individualized treatment regimen and preventing the development of MDR TB. Our results suggest that resistance is primarily developed through spontaneous mutations or selective pressure.

Whole genome sequencing of multidrug-resistant Mycobacterium tuberculosis isolates collected in the Czech Republic, 2005-2020.

The emergence and spread of resistant tuberculosis (TB) pose a threat to public health, so it is necessary to diagnose the drug-resistant forms in a clinically short time frame and closely monitor their transmission. In this study, we carried out a first whole genome sequencing (WGS)-based analysis of multidrug resistant (MDR) M. tuberculosis strains to explore the phylogenetic lineages diversity, drug resistance mechanisms, and ongoing transmission chains within the country. In total, 65 isolates phenotypically resistant to at least rifampicin and isoniazid collected in the Czech Republic in 2005-2020 were enrolled for further analysis. The agreement of the results obtained by WGS with phenotypic drug susceptibility testing (pDST) in the determination of resistance to isoniazid, rifampicin, pyrazinamide, streptomycin, second-line injectables and fluoroquinolones was more than 80%. Phylogenetic analysis of WGS data revealed that the majority of MDR M. tuberculosis isolates were the Beijing lineage 2.2.1 (n = 46/65; 70.8%), while the remaining strains belonged to Euro-American lineage. Cluster analysis with a predefined cut-off distance of less than 12 single nucleotide polymorphisms between isolates showed 19 isolates in 6 clusters (clustering rate 29.2%), located mainly in the region of the capital city of Prague. This study highlights the utility of WGS as a high-resolution approach in the diagnosis, characterization of resistance patterns, and molecular-epidemiological analysis of resistant TB in the country.

Cryptosporidium scrofarum n. sp. (Apicomplexa: Cryptosporidiidae) in domestic pigs (Sus scrofa).

We describe the morphological, biological, and molecular characteristics of Cryptosporidium pig genotype II and propose the species name Cryptosporidium scrofarum n. sp. to reflect its prevalence in adult pigs worldwide. Oocysts of C. scrofarum are morphologically indistinguishable from C. parvum, measuring 4.81-5.96 µm (mean=5.16)×4.23-5.29 µm (mean=4.83) with a length to width ratio of 1.07±0.06 (n=400). Oocysts of C. scrofarum obtained from a naturally infected pig were infectious for 8-week-old pigs but not 4-week-old pigs. The prepatent period in 8-week-old Cryptosporidium-naive pigs was 4-6 days and the patent period was longer than 30 days. The infection intensity of C. scrofarum in pigs was generally low, in the range 250-4000 oocysts per gram of feces. Infected pigs showed no clinical signs of cryptosporidiosis and no pathology was detected. Cryptosporidium scrofarum was not infectious for adult SCID mice, adult BALB/c mice, Mongolian gerbils (Meriones unguiculatus), southern multimammate mice (Mastomys coucha), yellow-necked mice (Apodemus flavicollis), or guinea pigs (Cavia porcellus). Phylogenetic analyses based on small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. scrofarum is genetically distinct from all known Cryptosporidium species.