V processe formirovaniya ustojchivosti M. tuberculosis k ftorhinolonam v organizme hozyaina mogut odnovremenno sosushchestvovat' puly M. tuberculosis, chuvstvitel'nye k preparatam etoj gruppy, i puly M. tuberculosis s razlichnymi determinantami ustojchivosti. Cel'yu issledovaniya bylo izuchit' osobennosti rosta in vitro shtammov M. tuberculosis, otlichayushchihsya geneticheskimi determinantami ustojchivosti k ftorhinolonam, v usloviyah konkurencii za pitatel'nye veshchestva. Issledovanie provedeno na pyati klinicheskih shtammah M. tuberculosis s mnozhestvennoj lekarstvennoj ustojchivost'yu, razlichayushchihsya strukturoj gyrA. SHtammy kul'tivirovali poparno i individual'no v optimal'nyh usloviyah (sreda Middlebrook 7H9) i v usloviyah mul'tistressa (50% sreda Middlebrook 7H9, 2 mM KNO2, 0,02% H2O2). Vremya eksperimenta sostavilo 21 sutki. CHislo kletok kazhdogo iz sovmestno kul'tiviruemyh shtammov ocenivali po kalibrovochnym krivym zavisimosti porogovogo cikla polimeraznoj cepnoj reakcii po celevomu dlya mutacii kanalu ot koncentracii kletok M. tuberculosis. Po chislu kletok kazhdogo shtamma pri sovmestnom kul'tivirovanii vychislyali velichinu konkurentnogo fitnesa i udel'nuyu skorost' rosta. Bylo ustanovleno, chto shtammy M. tuberculosis s mutaciyami v gyrA ustupali v skorosti rosta shtammu s dikim tipom gyrA, chto bylo osobenno sil'no vyrazheno v usloviyah mul'tistressa. SHtamm s naibolee rasprostranennoj mutaciej gyrA_D94G imel naimen'shuyu skorost' rosta iz vsekh issledovannyh shtammov. Byla vyskazana gipoteza, chto medlennyj rost M. tuberculosis s etoj mutaciej mozhet privodit' k tolerantnosti k protivotuberkuleznym preparatam i v rezul'tate etogo shtamm poluchaet preimushchestvo v usloviyah himioterapii po sravneniyu s drugimi mutantnymi po gyrA variantami.

Given that the adaptive immune response is important for the control and elimination of viral infections that cause disease in humans, it is of utmost importance to assess the adaptive response to SARS-CoV-2. Neutralizing antibodies and CD4 (+) /CD8 (+) T cells contribute to the control of SARS-CoV-2. Tuberculosis remains the leading cause of death among bacterial infections worldwide. Currently, the treatment of tuberculosis is complicated by co-infection with COVID-19. The aim of this work was to investigate the formation of neutralizing antibodies against SARS-CoV-2 and SARS-CoV-2-specific CD4 (+) and CD8 (+) T cells in patients with pulmonary TB. The levels of neutralizing antibodies to SARS-CoV-2 and the number of SARS-CoV-2-specific T cells were assessed at two time points (3 and 6 months after COVID-19) in patients diagnosed with pulmonary tuberculosis (69 people: 33 women and 36 men, aged 18 to 70 years). The control group included patients who had COVID-19 but did not have tuberculosis (35 people: 25 women and 10 men, aged 18 to 70 years). The study showed similar levels of neutralizing antibodies to SARS-CoV-2 in both groups 3 months after COVID-19. Antibody levels decreased in both groups 6 months after COVID-19 compared to 3 months. The antibody level was significantly lower in the TB group ( p = 0.01). The number of SARS-CoV-2-specific T cells was lower in TB patients 6 months after COVID-19 ( p < 0.001) compared to the control group. Thus, TB coinfection reduces the specific immune response against SARS-CoV-2 6 months after COVID-19.

As M. tuberculosis strains develop resistance to fluoroquinolones, pools of M. tuberculosis sensitive to drugs of this group and pools of M. tuberculosis with different resistance determinants can simultaneously coexist in the host organism. The goal of this research was to run an in vitro investigation of growth characteristics of M. tuberculosis strains which have different genetic determinants of resistance to fluoroquinolones, in the setting of competition for nutrients. The research used five clinical strains of multidrug-resistant M. tuberculosis differing in gyrA structure. Strains were cultured in pairs and individually under optimal conditions (Middlebrook 7H9 medium) and under conditions of multistress (50% Middlebrook 7H9 medium, 2 mM KNO2, 0.02% H2O2). The experiment took 21 days. The number of cells of each co-cultured strain was estimated from calibration curves. These curves showed the dependence of the threshold cycle of the polymerase chain reaction — respective to the channel targeted by the mutation — on the concentration of M. tuberculosis cells. The competitive fitness value and specific growth rate were calculated from the number of cells of each strain when co-cultured. M. tuberculosis strains with mutations in gyrA were found to be inferior in growth rate to the wild-type gyrA strain, which was particularly pronounced under multistress conditions. The strain with the most common gyrA_D94G mutation had the lowest growth rate of all strains examined. It has been hypothesised that the slow growth of M. tuberculosis with this mutation may lead to tolerance to anti-tuberculosis drugs, and as a result, the strain gains an advantage under chemotherapy conditions compared to other gyrA mutant variants.

Given the fact, that adaptive immune response is important for control and elimination of viral infections causing human diseases, estimation of adaptive response to SARS-CoV-2 is extremely important. The neutralizing antibodies and CD4+/CD8+ T cells contribute to the SARS-CoV-2 control. Tuberculosis remains the leading cause of mortality among bacterial infections all over the world. Currently, treatment of tuberculosis is complicated by the COVID-19 co-infection. The aim of the study was to investigate the formation of neutralizing antibodies against SARS-CoV-2 and CD4+ and CD8+ T cells specific for SARS-CoV-2 in patients with pulmonary TB. The levels of neutralizing antibodies against SARS-CoV-2 and the amount of T cells specific for SARS-CoV-2 were estimated at two time points (3 and 6 months after COVID-19) in patients diagnosed with pulmonary tuberculosis (69 individuals: 33 females and 36 males aged 18–70 years). Patients without tuberculosis (35 individuals: 25 females and 10 males aged 18–70 years) who had undergone COVID-19 served as the control group. The study showed equal levels of SARS-CoV-2 neutralizing antibodies in both groups 3 months after COVID-19. The levels of antibodies decreased 6 months after COVID-19 compared to the levels reported 3 months after the disease in both groups. The antibody levels were significantly lower in the group of patients with TB (p = 0.01). The amount of SARS-CoV-2 specific T cells was lower in TB patients 6 months after COVID-19 (p < 0.001) compared to the control group. Thus, TB co-infection reduces the specific immune response to SARS-CoV-2 6 months after COVID-19.

The diagnosis of mycobacterial co-infection is one of the pressing public health issues. The study was aimed to determine discriminatory power of multiplex PCR used for species identification when detecting mixed mycobacterial populations. The study involved model samples representing the mixtures of DNA of two mycobacterial species with the ratios of 1 : 1, 1 : 9, 1 : 99, and 1 : 999 and different total DNA concentrations (103 gEq/mL to 106 gEq/mL). The model samples were assessed using the multiplex PCR-based AmpliTube-RV-Differentiation kit (Syntol LLC; Russia). It has been shown that the kit is capable of detecting the mixtures of mycobacterial species with high discriminatory power. The discriminatory power of real-time PCR used for analysis of the mixture of DNA of two mycobacterial species depended on the total DNA content in the sample and varied between 0.1% for high-rate samples (total DNA concentration 106 gEq/mL) and 50% for low-rate samples (total DNA concentration 103 gEq/mL) and corresponded to the amount of DNA of the species in the sample of at least 5 × 102 gEq/mL. When the amount of DNA of each species in the mixture was at least 5 × 102 gEq/mL, the results of PCR test for detection of co-infection did not depend on the mucobacterial species contained in the mixture, which should be taken into account when analyzing PCR results.

Diagnostika mikobakterial'noj koinfekcii — odna iz aktual'nyh problem zdravoohraneniya. Cel'yu issledovaniya bylo opredelit' diskriminiruyushchuyu sposobnost' metoda mul'tipleksnoj PCR vidovoj identifikacii pri vyyavlenii smeshannyh populyacij mikobakterij. Issledovanie vypolneno na model'nyh obrazcah, predstavlyayushchih soboj smes' DNK mikobakterij dvuh vidov v sootnoshenii 1 : 1, 1 : 9, 1 : 99 i 1 : 999 s raznoj summarnoj koncentraciej DNK (ot 103 GE/ml do 106 GE/ml). Model'nye obrazcy issledovali naborom «Amplitub-RV-differenciaciya» («Sintol»; Rossiya), osnovannom na mul'tipleksnoj PCR. Pokazano, chto nabor sposoben vyyavlyat' smesi vidov mikobakterij s vysokoj diskriminiruyushchej sposobnost'yu. Diskriminiruyushchaya sposobnost' metoda PCR v rezhime real'nogo vremeni pri analize smesi DNK dvuh vidov mikobakterij zavisela ot summarnogo soderzhaniya DNK v obrazce i var'irovala ot 0,1% dlya vysokonagruzhennyh obrazcov (summarnaya koncentraciya DNK × 106 GE/ml) do 50% dlya nizkonagruzhennyh obrazcov (summarnaya koncentraciya DNK × 103 GE/ml) i sootvetstvovala kolichestvu DNK vida v smesi ne menee 5 × 102 GE/ml. Pri kolichestve DNK kazhdogo vida v smesi ne menee 5 × 102 GE/ml rezul'tat PCR na vyyavlenie koinfekcii ne zavisel ot vida mikobakterij, vhodyashchih v smes', chto neobhodimo uchityvat' pri analize rezul'tatov PCR.

In order to study the in vitro adaptation of M.tuberculosis strains of various phylogenetic lineages and drug resistance under multistress conditions created by a lack of nutrients, reactive forms of nitrogen and oxygen, 17 strains of M.tuberculosis were studied (10 strains of the East Asian lineage of the Beijing sublineage and 7 strains Euro-American lineage). We studied the culture yield, the state of M.tuberculosis cells in culture and the secretome when cultivating strains under conditions of multistress, nitrosative stress and in optimal conditions. It has been established that M. tuberculosis strains of frequently SITs with a sensitive genotype or with widespread mutations, in contrast to strains with rare mutations and with an expanded spectrum of phenotypic drug resistance, adapt well to stress conditions in vitro. M.tuberculosis strains of the Beijing sublineage adapt to stress at an earlier time than M.tuberculosis of the Euro-American lineage; this was especially evident in the multistress model.

S uchetom togo, chto adaptivnyj immunnyj otvet vazhen dlya kontrolya i ustraneniya virusnyh infekcij, vyzyvayushchih zabolevaniya u lyudej, krajne vazhna ocenka adaptivnogo otveta na SARS-CoV-2. Nejtralizuyushchie antitela i T-limfocity CD4+/CD8+ sposobstvuyut kontrolyu SARS-CoV-2. Tuberkulez do sih por ostaetsya glavnoj prichinoj smerti sredi bakterial'nyh infekcij v mire. Na dannyj moment lechenie tuberkuleza oslozhneno koinfekciej COVID-19. Cel'yu raboty bylo issledovat' obrazovanie nejtralizuyushchih antitel protiv SARS-CoV-2 i specifichnyh dlya SARS-CoV-2 T-kletok CD4+ i CD8+ u pacientov s TB legkih. Urovni nejtralizuyushchih antitel k SARS-CoV-2 i kolichestvo specifichnyh k SARS-CoV-2 T-kletok ocenivali v dvuh vremennyh tochkah (cherez 3 i cherez 6 mesyacev posle perenesennogo COVID-19) u bol'nyh s diagnozom tuberkulez legkih (69 chelovek: 33 zhenshchiny i 36 muzhchin ot 18 do 70 let). V kontrol'nuyu gruppu voshli pacienty, perenesshie COVID-19 bez tuberkuleza (35 chelovek: 25 zhenshchin i 10 muzhchin ot 18 do 70 let). V rezul'tate issledovaniya byli zaregistrirovany odinakovye urovni nejtralizuyushchih antitel k SARS-CoV-2 v obeih gruppah cherez 3 mesyaca posle perenesennogo COVID-19. Urovni antitel snizilis' v dvuh gruppah cherez 6 mesyacev posle COVID-19 po sravneniyu s 3 mesyacami. Uroven' antitel byl dostoverno nizhe v gruppe bol'nyh TB (r = 0,01). Kolichestvo SARS-CoV-2-specifichnyh T-kletok bylo nizhe u bol'nyh TB cherez 6 mesyacev posle perenesennogo COVID-19 (r < 0,001) po sravneniyu s kontrol'noj gruppoj. Takim obrazom, koinfekciya TB snizhaet specificheskij immunnyj otvet protiv SARS-CoV-2 cherez 6 mesyacev posle perenesennogo COVID-19.

Objective. To evaluate endosonographic images (EGI) for different peripheral lung lesions (PPLs) genesis, to identify patterns of malignancy in the differential diagnosis of tuberculosis (TB), mycobacteriosis (NTM), malignancy neoplasm, to compare the main combinations of variants of the echo characteristics of lung lesions visualized during rEBUS navigation with the data of a comprehensive microbiological, cytological and morphological studies. Methods. A cohort retrospective study included 145 patients (81 women (mean age 44.94±0.24 years) and 64 men (mean age 47.91±0.24)), with newly diagnosed PPLs and with negative sputum TB tests, who underwent diagnostic navigational bronchoscopy (rEBUS) and surgical diagnostics, and EGI of PPLs was visualized with rEBUS. Depending on the final verification, were endosonographic images divided into 2 groups: the Neo group (n=63), whose biopsy (during bronchoscopic or surgical diagnosis) verified the malignancy PPls, and the TB+NTM group (n=82) – infectious PPLs caused by mycobacterial infection (73 cases – pulmonary TB, 9 cases – mycobacteriosis). Results. It was established that differences were statistically significant in shape, contour/border, and the presence of inclusions (linear arcs, zones of hypo-anechogenesis) between the comparison groups. If there are echoic features of malignancy PPLs on EGI with rEBUS (irregular shape (found in 60,3%), lobulated shape (in 17,4%), visualized but blurred contour (in 57.2%), fuzzy/poorly visualized contour (in 25.4%), heteroechoic structure, with “linear arcs” (49.2%) with an air bronchogram (in 36,5%)), tissue bronchobiopsy (brush and TBBL) are priority for cytological and histological examination. If there are echoic features of delimited infectious inflammation on EGI of PPLs in rEBUS (round or oval shape (found in 34.1% of cases), definite, closed contour (in 78.0%), homoechoic structure (29,3%), heteroechoic structure with zones of hypo-anechoicity (in 32.9%) with an air bronchogram (in 23.2%)), bronchobiopsy is a priority, allowing to cover the largest area of the lungs (BAL) for microbiological examination (microscopy, PCR-RT, culture) in combination with tissue biopsy (cytological examination). A classification has been created that includes 3 types of EGI to determine rational approaches to bronchobiopsy for the purpose of differential diagnosis of TB, mycobacteriosis and neoplasia. Conclusions. The use of EGI of PPLs assessment during bronchoscopy with rEBUS navigation determines rational approaches to the complex of navigational bronchobiopsies in the differential diagnosis of TB, mycobacteriosis and malignant processes.

Ftorhinolony — osnovnaya gruppa preparatov, primenyaemyh dlya lecheniya tuberkuleza s mnozhestvennoj lekarstvennoj ustojchivost'yu (MLU-TB). Cel'yu issledovaniya bylo ocenit' raznoobrazie mutacij v gene gyrA, a takzhe ustanovit' associaciyu mutacij v gyrA s fenotipicheskoj ustojchivost'yu k levofloksacinu i obshchim profilem lekarstvennoj ustojchivosti vozbuditelya. Issledovanie provedeno na diagnosticheskom materiale ot 2836 bol'nyh tuberkulezom legkih. Dlya opredeleniya mutacij v gyrA ispol'zovali nabory «TB-BIOCHIP-2» ili «Amplitub-FQ-RV». Fenotipicheskuyu lekarstvennuyu chuvstvitel'nost' M. tuberculosis (MBT) opredelyali v sisteme BACTEC MGIT 960. Pokazano, chto u MBT dominirovali mutacii D94G (41,63%; 95%DI: 38,03–45,32%) i A90V (21,32%; 95%DI: 18,44–24,50%), prichem izolyaty s etimi mutaciyami byli polucheny v tom chisle i ot vpervye vyyavlennyh bol'nyh tuberkulezom legkih. Ustanovleno, chto mutaciya D94A ne yavlyalas' strogo associirovannoj s fenotipicheskoj ustojchivost'yu k ftorhinolonam. Ustojchivost' k ftorhinolonam, kak pravilo, byla associirovana s mnozhestvennoj lekarstvennoj ustojchivost'yu (93,52%; 95%DI 91,43–95,12%). V 2,31% (95%DI 1,78–3,00%) sluchaev vyyavlena genotipicheskaya geterorezistentnost' k ftorhinolonam: smeshannye populyacii vklyuchali 2—4 pula MBT s raznoj strukturoj QRDR gyrA. Na osnovanii poluchennyh rezul'tatov mozhno zaklyuchit', chto v sovremennoj populyacii MBT proiskhodit formirovanie ustojchivosti k ftorhinolonam, kak pravilo, na fone uzhe imeyushchejsya MLU. Naibolee perspektivnymi v evolyucionnom plane predstavlyayutsya MBT s mutaciyami v gyrA D94G i A90V.

Fluoroquinolones are the main group of drugs used for treatment of multidrug resistant tuberculosis (MDR-TB). The study was aimed to assess the diversity of mutation in the gyrA gene and to evaluate the association of gyrA mutations with the phenotypic resistance to levofloxacin and the general drug resistance profile of the pathogen. The study involved assessment of diagnostic materials obtained from 2836 patients with pulmonary tuberculosis. TB-BIOCHIP-2 and AmplitubeFQ-RV kits were used for identification of the gyrA mutations. Phenotypic drug susceptibility of M. tuberculosis (MTB) was defined using the BACTEC MGIT 960 test system. It was shown that mutations D94G (41.63%; 95% CI: 38.03–45.32%) and A90V (21.32%; 95% CI: 18.44–24.50%) prevailed in MBT, although some isolates carrying these mutations were obtained from the newly diagnosed patients with pulmonary tuberculosis. It was found that mutation D94A was not strongly associated with the phenotypic resistance to fluoroquinolones. Fluoroquinolone resistance was usually associated with multiple drug resistance (93.52%; 95% CI 91.43–95.12%). In 2.31% (95% CI 1.78–3.00%) of cases, genotypic heteroresistance to fluoroquinolones was detected: mixed populations included 2–4 MTB pools with various structure of the gyrA QRDR. The results obtained lead to the conclusion that resistance to fluoroquinolones that is usually associated with the existing MDR arises in the modern MTB population. MTB carrying gyrA mutations D94G and A90V seems to be the most promising in evolutionary terms.

The lack of simple, rapid diagnostic tests for isoniazid-resistant rifampicin-susceptible tuberculosis infection (Hr-TB) can result in low treatment efficacy and further amplification of drug resistance. Based on the clinical data, this study sought to estimate the prevalence of Hr-TB in the general population and characterize the phenotypic susceptibility and genetic determinants of isoniazid resistance in M. tuberculosis strains. Molecular-genetic and culture-based drug susceptibility tests were performed on M. tuberculosis isolates and M. tuberculosis DNA obtained from the patients with pulmonary TB undergoing treatment at the Central Tuberculosis Research Institute between 2011 and 2018. The tests revealed that Hr-TB accounted for 12% of all TB cases in the studied sample. Hr-TB strains were either resistant to isoniazid only (45%) or had multiple resistance to 2–6 anti-TB agents. Resistance to isoniazid was caused by mutations in the katG gene. Based on the literature analysis and our own observations, we emphasize the importance of developing simple molecular drug susceptibility tests capable of detecting simultaneous resistance to rifampicin and isoniazid and the necessity of their translation into clinical practice.

Отсутствие ускоренной диагностики туберкулеза с устойчивостью возбудителя к изониазиду с сохраненной чувствительностью к рифампицину (ИР-ТБ) может быть причиной низкой эффективности терапии и приводить к амплификации лекарственной резистентности, в том числе к формированию множественной лекарственной устойчивости. Целью работы было определить частоту встречаемости ИР-ТБ в современной популяции, охарактеризовать фенотипическую чувствительность и генетические детерминанты устойчивости к изониазиду представителей этой группы M. tuberculosis на репрезентативном материале. Анализировали результаты определения лекарственной чувствительности, полученные при исследовании молекулярно- генетическими и/или культуральными методами изолятов M. tuberculosis / ДНК M. tuberculosis, выделенных от больных туберкулезом легких из клинических отделений Центрального научно-исследовательского институа туберкулеза за период 2011–2018 гг. Частота ИР-ТБ составила 12% от всех выявленных случаев туберкулеза. M. tuberculosis с ИР были как монорезистентными к изониазиду (45%), так и полирезистентными (устойчивыми к 2–6 противотуберкулезным препаратам), а устойчивость к изониазиду была обусловлена мутациями в гене katG, приводящими к высокому уровню резистентности. На основании анализа литературных данных и собственных наблюдений подчеркивается важность разработки и внедрения новых простых молекулярных тестов для определения устойчивости одновременно к рифампицину и изониазиду.

Sozdaniye protivotuberkuleznykh preparatov prolongirovannogo deystviya krayne perspektivno, tak kak pozvolyayet sokhranit' priverzhennost' bol'nykh k lecheniyu pri dlitel'nykh kursakh terapii, osobenno pri khimioterapii tuberkuleza s mnozhestvennoy lekarstvennoy ustoychivost'yu. Traditsionno ispol'zuyemyy dlya otsenki kinetiki vykhoda preparatov iz polimernykh nositeley metod UF-spektrofotometrii ne podkhodit dlya primeneniya v mnogokomponentnykh pitatel'nykh sredakh dlya kul'tivirovaniya mikobakteriy tuberkuleza. Tsel'yu issledovaniya bylo razrabotat' metod otsenki vysvobozhdeniya protivotuberkuleznykh preparatov iz biorezorbiruyemykh polimernykh nositeley, pozvolyayushchiy provodit' skrining bol'shogo chisla inkapsulirovannykh prolongirovannykh form protivotuberkuleznykh preparatov i otbirat' naiboleye perspektivnyye kompozitsii. Pri izuchenii dinamiki rosta laboratornogo chuvstvitel'nogo shtamma M. tuberculosis H37Rv v prisutstvii serii kontsentratsiy levofloksatsina (ot 0,03 do 0,4 mkg/ml) byla razrabotana model', predstavlyayushchaya soboy dva parallel'no provodimykh opyta, pozvolyayushchikh otsenit' kinetiku vysvobozhdeniya preparata v kul'tural'nuyu sredu. Vse eksperimenty provodili trekhkratno, pri otsenke ispol'zovali metody opisatel'noy statistiki. Rezul'taty, poluchennyye v etoy modeli dlya trekh inkapsulirovannykh form levofloksatsina v biorezorbiruyemykh polimernykh nositelyakh iz polilaktoglikolida (chastitsy 50 i 100 mkm i matriks), pokazali, chto kinetika nakopleniya preparata v srede sushchestvenno zavisit ot vida polimernogo nositelya. Naiboleye perspektiven iz nikh matriks, kotoryy khorosho vklyuchayet v sebya levofloksatsin i dostatochno ravnomerno vysvobozhdayet yego pri inkubatsii v pitatel'noy srede.

We report here the draft genome sequence of Mycobacterium heckeshornense, isolated from the sputum of a patient admitted to a tuberculosis hospital with suspected pulmonary tuberculosis.