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1.
Testa QuantiFERON TB in TB Plus v Sloveniji v obdobju 2008–2019
Urška Skamen, Eva Sodja, Marija Žolnir-Dovč, 2022, izvirni znanstveni članek

Povzetek: Izhodišča: Test QuantiFERON TB (QFT TB) je prvenstveno namenjen za odkrivanje latentne (LTBI) okužbe z bacili tuberkuloze (TB). Članek obsega retrospektivno analizo rezultatov testa QFT TB v Sloveniji med letoma 2008 in 2019, opozarja na vplive predanalitskih dejavnikov na rezultate testa ter govori o razlogih za nastanek nejasnih rezultatov in o občutljivosti testa pri bolnikih z aktivno obliko TB. Metode: V obdobju 2008–2019 smo v Laboratoriju za mikobakterije Klinike Golnik s testom QFT TB testirali 29.352 vzorcev krvi bolnikov iz različnih zdravstvenih ustanov v Sloveniji. Na rezultatih testa QFT TB smo izvedli retrospektivno analizo. Rezultati: Delež pozitivnih rezultatov testa QFT TB se z leti postopno znižuje. V letu 2008 je znašal 20,8 %, v letu 2019 pa 10,7 %, kar pomeni, da je v zadnjih dvanajstih letih upadel za 48,6 %. Največjo težavo predstavljajo nejasni rezultati (letno povprečno 4,3 %), saj so za zdravnika nepovedni. Občutljivost testa QFT TB pri bolnikih z aktivno obliko TB je 82,9 %, kar je primerljivo s podatki iz tujine. Zaključek: Pomembno je, da pri odvzemu in pošiljanju krvi dosledno sledimo navodilom, saj je test QFT TB zelo občutljiv na predanalitske dejavnike in klinično stanje preiskovancev. Delež pozitivnih rezultatov testa QFT TB z leti pospešeno upada, kar je povezano z napori za omejitev širjenja TB in upadom primerov aktivne TB v naši državi.
Ključne besede: tuberkuloza - 2008-2019, Mycobacterium tuberculosis - 2008-2019, latentna tuberkuloza - 2008-2019, Slovenija, test QuantiFERON TB, predanalitski dejavniki, nejasen rezultat
Objavljeno v DiRROS: 29.08.2022; Ogledov: 528; Prenosov: 170
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2.
Next-generation sequencing to characterize pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries
Eva Sodja, Simon Koren, Nataša Toplak, Sara Truden, Marija Žolnir-Dovč, 2021, izvirni znanstveni članek

Povzetek: Objectives. Next-generation sequencing (NGS) provide a comprehensive analysis of the genetic alterations that are most commonly linked with pyrazinamide (PZA) resistance. There are no studies reporting molecular background of PZA resistance in TB isolates from Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance, pncA, using Ion Torrent technology in comparison to phenotypic BACTEC MGIT 960 DST in clinical TB isolates from two countries of the Balkan Peninsula. Methods. Between 1996 and 2017, we retrospectively selected 61 TB isolates. To identify gene variants related to drug resistance in genomic DNA extracted from TB isolates, AmpliSeq libraries were generated automatically using the AmpliSeq™ Kit for Chef DL8 and the Ion AmpliSeq TB Research Panel. Result.s Of all 61 TB isolates included, 56 TB were phenotypically resistant to any antibiotic. Among them, 38/56 (67.9%) TB isolates were phenotypically resistant to pyrazinamide and pncA mutations were detected in 33/38 cases (86.8%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS to conventional BACTEC MGIT 960 DST revealed very strong agreement (90.2%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS. Conclusions. Detection of PZA resistance using NGS seems to be a valuable tool for surveillance of TB drug resistance also in the Balkan Peninsula, with great potential to provide useful information at least one weak earlier than is possible with phenotypic DST.
Ključne besede: tuberculosis, Mycobacterium tuberculosis, high-throughput nucleotide sequencing, pyrazinamide, microbial sensitivity tests, next-generation sequencing, drug susceptibility testing, Slovenia, Republic of North Macedonia
Objavljeno v DiRROS: 10.01.2022; Ogledov: 857; Prenosov: 524
.pdf Celotno besedilo (1,53 MB)
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3.
Subspecies-specific sequence detection for differentiation of Mycobacterium abscessus complex
Alina Minias, Lidia Żukowska, Jakub Lach, Tomasz Jagielski, Dominik Strapagiel, Su-Young Kim, Won-Jung Koh, Heather Adam, Ruth Bittner, Sara Truden, Marija Žolnir-Dovč, Jarosław Dziadek, 2020, izvirni znanstveni članek

Povzetek: Mycobacterium abscessus complex (MABC) is a taxonomic group of rapidly growing, nontuberculous mycobacteria that are found as etiologic agents of various types of infections. They are considered as emerging human pathogens. MABC consists of 3 subspecies - M. abscessus subsp. bolletti, M. abscessus subsp. massiliense and M. abscessus subsp. abscessus. Here we present a novel method for subspecies differentiation of M. abscessus named Subspecies-Specific Sequence Detection (SSSD). This method is based on the presence of signature sequences present within the genomes of each subspecies of MABC. We tested this method against a virtual database of 1505 genome sequences of MABC. Further, we detected signature sequences of MABC in 45 microbiological samples through DNA hybridization. SSSD showed high levels of sensitivity and specificity for differentiation of subspecies of MABC, comparable to those obtained by rpoB sequence typing.
Ključne besede: Mycobacterium abscessus complex, nontuberculous mycobacteria, diagnosis
Objavljeno v DiRROS: 23.11.2020; Ogledov: 11999; Prenosov: 979
.pdf Celotno besedilo (1,23 MB)
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5.
Genomic insights into the Mycobacterium kansasii complex : an update
Tomasz Jagielski, Paulina Borówka, Zofia Bakuła, Jakub Lach, Błażej Marciniak, Anna Brzostek, Jarosław Dziadek, Mikołaj Dziurzyński, Lian Pennings, Jakobus van Ingen, Marija Žolnir-Dovč, Dominik Strapagiel, 2020, izvirni znanstveni članek

Povzetek: Only very recently, has it been proposed that the hitherto existing Mycobacteriumkansasii subtypes (I–VI) should be elevated, each, to a species rank. Consequently, the former M. kansasii subtypes have been denominated as Mycobacterium kansasii (former type I), Mycobacterium persicum (II), Mycobacterium pseudokansasii (III), Mycobacterium innocens (V), and Mycobacterium attenuatum (VI). The present work extends the recently published findings by using a three-pronged computational strategy, based on the alignment fraction-average nucleotide identity, genome-to-genome distance, and core-genome phylogeny, yet essentially independent and much larger sample, and thus delivers a more refined and complete picture of the M. kansasii complex. Furthermore, five canonical taxonomic markers were used, i.e., 16S rRNA, hsp65, rpoB, and tuf genes, as well as the 16S-23S rRNA intergenic spacer region (ITS). The three major methods produced highly concordant results, corroborating the view that each M. kansasii subtype does represent a distinct species. This work not only consolidates the position of five of the currently erected species, but also provides a description of the sixth one, i.e., Mycobacterium ostraviense sp. nov. to replace the former subtype IV. By showing a close genetic relatedness, amonophyletic origin, and overlapping phenotypes, our findings support the recognition of the M. kansasii complex (MKC), accommodating all M. kansasii-derived species and Mycobacterium gastri. None of the most commonly used taxonomic markers was shown to accurately distinguish all the MKC species. Likewise, no species-specific phenotypic characteristics were found allowing for species differentiation within the complex, except the non-photochromogenicity of M. gastri. To distinguish, most reliably, between the MKC species, and between M. kansasii and M. persicum in particular, whole-genome-based approaches should be applied. In the absence of clear differences in the distribution of the virulence-associated region of difference 1 genes among the M. kansasii-derived species, the pathogenic potential of each of these species can only be speculatively assessed based on their prevalence among the clinically relevant population. Large-scale molecular epidemiological studies are needed to provide a better understanding of the clinical significance and pathobiology of the MKC species. The results of the in vitro drug susceptibility profiling emphasize the priority of rifampicin administration in the treatment of MKC-induced infections, while undermining the use of ethambutol, due to a high resistance to this drug.
Ključne besede: Mycobacteriumkansasii complex, Mycobacteriumostraviense sp. nov., non-tuberculous mycobacteria (NTM), whole genome sequencing, taxonomy
Objavljeno v DiRROS: 31.07.2020; Ogledov: 1846; Prenosov: 1317
.pdf Celotno besedilo (10,22 MB)
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6.
Next-generation sequencing of drug resistant Mycobacterium tuberculosis clinical isolates in low-incidence countries
Eva Sodja, Nataša Toplak, Simon Koren, Minka Kovač, Sara Truden, Biljana Ilievska Poposka, Marija Žolnir-Dovč, 2019, izvirni znanstveni članek

Povzetek: Drug resistant tuberculosis (TB), especially multidrug (MDR) and extensively drug-resistant (XDR) TB, is still a serious problem in global TB control. Slovenia and North Macedonia are low-incidence countries with TB incidence rates of 5.4 and 10.4 in 2017, respectively. In both countries, the percentage of drug resistant TB is very low with sporadic cases of MDR-TB. However, global burden of drug-resistant TB continues to increase imposing huge impact on public health systems and strongly stimulating the detection of gene variants related with drug resistance in TB. Next-generation sequencing (NGS) can provide comprehensive analysis of gene variants linked to drug resistance in Mycobacterium tuberculosis. Therefore, the aim of our study was to examine the feasibility of a full-length gene analysis for the drug resistance related genes (inhA, katG, rpoB, embB) using Ion Torrent technology and to compare the NGS results with those obtained from conventional phenotypic drug susceptibility testing (DST) in TB isolates. Between 1996 and 2017, we retrospectively selected 56 TB strains from our National mycobacterial culture collection. Of those, 33 TB isolates from Slovenian patients were isolated from various clinical samples and subjected to phenotypic DST testing in Laboratory for Mycobacteria (University Clinic Golnik, Slovenia). The remaining 23 TB isolates were isolated from Macedonian patients and sent to our laboratory for assistance in phenotypic DST testing. TB strains included were either mono-, poly- or multidrug resistant. For control purposes, we also randomly selected five TB strains susceptible to first-line anti-TB drugs. High concordance between genetic (Ion Torrent technology) and standard phenotypic DST testing for isoniazid, rifampicin and ethambutol was observed, with percent of agreement of 77%, 93.4% and 93.3%, sensitivities of 68.2%, 100% and 100%, and specificities of 100%, 80% and 88.2%, respectively. In conclusion, the genotypic DST using Ion Torrent semiconductor NGS successfully predicted drug resistance with significant shortening of time needed to obtain the resistance profiles from several weeks to just a few days.
Ključne besede: drug resistant tuberculosis, next-generation sequencing, low-incidence countries, phenotypic drug susceptibility testing
Objavljeno v DiRROS: 24.07.2020; Ogledov: 1820; Prenosov: 1024
.pdf Celotno besedilo (144,02 KB)
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