QuantiFERON TB and TB Plus tests in Slovenia between 2008 and 2019

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.Background: The QuantiFERON TB (QFT TB) test is primarily intended to detect latent (LTBI) tuberculosis (TB) infections. This work is based on a retrospective analysis of QFT TB test results in Slovenia between 2008 and 2019. It also draws attention to the influence of pre-analytical factors on a test result, on reasons that lead to indeterminate results, and on the sensitivity of the test in patients with active tuberculosis (TB). Methods: Between 2008 and 2019, we performed the QFT TB test in Laboratory for Mycobacteria of the University Clinic Golnik on 29,352 blood samples of patients from various health care institutions in Slovenia. A retrospective analysis was performed on QFT TB test results. Results: The proportion of positive QFT TB test results is gradually decreasing. In 2008, the proportion was 20.8% and in 2019, it declined to 10.7%, meaning that it has decreased by 48.6% in the last 12 years. The biggest problem are indeterminate results (on average 4.3% per year), as they are unpredictive to a physician. The sensitivity of the QFT TB test in patients with active TB is 82.9%, which is comparable to the data from abroad. Conclusion: The QFT TB test is highly sensitive to pre-analytical factors and the clinical condition of subjects therefore it is crucial that we follow instructions regarding blood taking and dispatching. The proportion of positive QFT TB test results has been rapidly declining over the years, which is a consequence of efforts to limit the spread of active TB in our country

Next-generation sequencing to characterize pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries

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

Mycobacteria in aquarium fish

Mycobacteria potentially pathogenic for humans are commonly present in aquarium fish sold in Slovenian pet shops. Mycobacterium marinum is the most important causative agent of skin mycobacteriosis in humans. The purpose of the present study was to evaluate, by means of a questionnaire, aquarium hobbyists’ and pet shop salespersons’ awareness of the health risks related to aquarium fish handling. A total of 198 participants took part in the study, 76.3% of these were aquarium hobbyists, and 23.7% were pet shop salespersons with up to 15 years of fish handling experience. About one third (35.8%) of all participants recognized that fish may be a source of infection for humans and that fish may contract tuberculosis. Fewer (24.4%) were aware of the fact that fish tuberculosis is a zoonotic disease. The vast majority of respondents were unfamiliar with the clinical manifestations of either fish tuberculosis or cutaneous mycobacteriosis in humans. Despite the generally acknowledged belief that aquarium water may pose a risk to human health, the respondents’ aquarium handling practices were surprising as the majority never used waterproof gloves. Several differences were revealed between the two groups of respondents. Pet shop salespersons were better educated on fish handling, and more were aware of the health risks linked to aquarium fish than aquarium hobbyists. The latter often perceived pet shop salespersons as relevant sources of information. However, overall awareness of the zoonotic potential of fish mycobacteria proved to be fairly low. Therefore, more effort should be made to increase the awareness of the role of mycobacteria in infections associated with exposure to aquarium fish.Za ljudi potencialno patogene vrste mikobakterij so v Sloveniji splošno prisotne pri akvarijskih ribah, ki so naprodaj v trgovinah z domačimi ljubljenčki, med drugimi tudi vrsta Mycobacterium marinum, ki je najpomembnejša povzročiteljica kožne mikobakterioze pri ljudeh. Namen raziskave je bil, s pomočjo vprašalnika, oceniti zavedanje ljubiteljskih akvaristov in prodajalcev v trgovinah z akvarističnim programom o zdravstvenih tveganjih, povezanih z rokovanjem z ribami. V raziskavi je sodelovalo 198 oseb, 76,3 % ljubiteljskih akvaristov in 23,7 % prodajalcev v trgovinah, ki so v povprečju imeli do 15 let izkušenj pri rokovanju z akvarijskimi ribami. Približno tretjina (35,8 %) sodelujočih se je strinjala s trditvijo, da so ribe lahko vir okužbe za ljudi in da ribe lahko zbolijo za tuberkulozo. V manjšem deležu (24,4 %) so se sodelujoči strinjali s trditvijo, da je ribja tuberkuloza zoonoza. Velika večina anketirancev ni poznala kliničnih znakov ribje tuberkuloze in kožne mikobakterioze pri ljudeh. Kljub splošno izraženemu prepričanju, da lahko voda iz akvarija predstavlja tveganje za zdravje ljudi, je bila splošna praksa rokovanja z ribami presenetljiva, saj velika večina sodelujočih pri rokovanju z ribami nikoli ne uporablja vodoodpornih rokavic. Med obema skupinama anketirancev smo odkrili več razlik. Prodajalci v trgovinah so bolj poučeni o rokovanju z ribami in bolj ozaveščeni o zdravstvenih tveganjih povezanih z akvarijskimi ribami, kot ljubiteljski akvaristi. Slednji prodajalce dojemajo kot zaupanja vreden vir informacij. V splošnem je ozaveščenost o zoonotskem potencialu mikobakterij pri akvarijskih ribah precej slaba, zato bi morali nameniti več pozornosti ozaveščanju o vlogi mikobakterij pri okužbah, povezanih z akvarijskimi ribami

Next-generation sequencing of drug resistant Mycobacterium tuberculosis clinical isolates in low-incidence countries

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

Subspecies-specific sequence detection for differentiation of Mycobacterium abscessus complex

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

Genomic insights into the Mycobacterium kansasii complex : an update

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