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.

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.

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.

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.

The objective: to evaluate changes in detection frequency of Mycobacterium tuberculosis of the main phylogenetic lines with various genetic determinants of resistance to rifampicin, isoniazid and fluoroquinolones in 1998–2003 and 2016–2021. Subjects and Methods. 965 mycobacterial cultures were studied, those cultures were isolated from pulmonary tuberculosis patients in 1998–2003 and 2016–2021. The spoligotypes of isolated Mycobacterium tuberculosis cultures and presence of gene mutations associated with resistance to isoniazid, rifampicin, and fluoroquinolones were determined. Results. In 2016–2021 versus 1998–2003, the incidence of Beijing subline increased from 50,72% to 64,60%. Within Euro-American line, the detection frequency of subline LAM9 decreased (from 40,00% to 14,81%), while the detection frequency of subline T1 increased (from 27,11% to 41,36%). Mycobacterium tuberculosis with MDR genotype rpoB531_Ser->Leu + katG315_Ser-> Thr [1] was selected. Mycobacterium tuberculosis with genotypic resistance to fluoroquinolones was detected only in cultures of 2016–2021, and Mycobacterium tuberculosis with preXDR genotype was significantly more common in Beijing subline compared to Euro-American line (21,67% vs. 3,73%, p <0,05).

Introduction. Mixed mycobacterial infections are an urgent problem for phthisiology and pulmonology, which has not been studied enough. The aim of the study was to assess the frequency of detection of mixed populations of mycobacteria in patients with tuberculosis and mycobacteriosis and to determine the species composition of these populations. Materials and methods. Clinical isolates of mycobacteria isolated from 5531 patients who were treated or who sought advice from the FSBI «TSNIIT» were studied. The culture was isolated in the BACTEC MGIT 960 automatic crop growth registration system, species identification was carried out by molecular genetic methods using the GenoType Mycobacterium CM/AS kit (Hain Livescience, Germany) and/or a set based on multiplex PCR in real time. The conclusion about the presence of a mixed population was made if a mixture of two species of mycobacteria was isolated during the entire observation period or the species structure of the population changed dynamically. Descriptive statistics were used to evaluate the results of the study, the chi-squared criterion was used to determine significant differences between groups; differences were considered statistically significant at p < 0.05. The entire analysis was carried out using Microsoft Excel (Microsoft, USA). Results. The frequency of detection of mixed mycobacterial infection by culture method in patients of phthisiological institutions for the period 2011-2023 was 1.16%. The most common combinations of species were M. tuberculosis + M. avium and M. avium + M. intracellulare.

The objective : to perform intraspecific differentiation of M. abscessus strains isolated from patients with pulmonary lesions using molecular genetic tests. Results . Intraspecific differentiation of 164 cultures of M. abscessus from 114 patients without cystic fibrosis showed that the subspecies M. abscessus subsp. dominated in the analysis set (90 of 114 patients; 78,95%), followed by M . abscessus subsp. massiliense (24/114; 21,05%). In 54 examined cystic fibrosis patients excreting non-tuberculous mycobacteria, M. abscessus was most often detected (41 patients out of 54; 75,93%). Intraspecific identification of the obtained M. abscessus isolates demonstrated the prevalence of the subspecies M. abscessus subsp. abscessus (29/41; 70,73%) over M. abscessus subsp. massiliense (11/41; 26,83%). In one case, a rare M. abscessus subsp. bolletii (1/41; 2,44%) was detected.

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.

В статье представлен опыт работы отдела микробиологии ФГБНУ «ЦНИИТ» в условиях пандемии COVID-19. Предложен оригинальный подход, основанный на новейших разработках в области ускоренной диагностики туберкулеза (ТБ), микобактериоза и новой коронавирусной инфекции COVID-19, который позволяет в течение нескольких часов от момента поступления в лабораторию диагностического материала получить результат тестирования на соответствующие заболевания. Обновленный алгоритм позволяет оптимизировать диагностику и повысить эффективность исследований, необходимых в учреждениях фтизиатрического профиля в текущей ситуации.

Исследование проведено с целью оценки достоверности положительных результатов полимеразной цепной реакции в режиме реального времени (ПЦР-РВ) для диагностики туберкулеза (ТБ) при регистрации сигнала флуоресценции после 35-го цикла амплификации. Установлено, что среди 1168 лиц, обследованных с диагностической целью методом ПЦР-РВ и имевших положительный результат данного теста, 28,4% случаев составили лица, у которых ДНК M. tuberculosis выявили из мокроты на поздних циклах амплификации. В дальнейшем у этой группы лиц диагноз ТБ был подтвержден в 80,1% случаев. Аналогичная картина наблюдалась и для других видов диагностического материала. При исследовании промывных вод бронхов положительный результат ПЦР-РВ был получен у 47 лиц, из них у 24 (51,1%) – на поздних циклах амплификации, при этом подтверждение диагноза ТБ в данной группе составило 75%. При исследовании плеврального экссудата у 22 лиц из 84 (26,2%) положительный результат ПЦР-РВ был получен на поздних циклах амплификации, диагноз ТБ был подтвержден в 86,4% случаев. При исследовании ликвора у 5 лиц из 18 (27,8%) ДНК M. tuberculosis определили на поздних циклах, при этом у всех 5 лиц (100% случаев) в дальнейшем был диагностирован ТБ.

Приведен обзор методик, применяемых для идентификации микроорганизмов, принадлежащих к микобактериям туберкулезного комплекса и нетуберкулезных микобактерий. Описаны технологические особенности применяемых методов идентификации. Предложен порядок действий исполнителя для решения задачи по идентификации микобактерий.

Проведен краткий обзор основных этапов применения технологии автоматизации лабораторных исследований во фтизиатрии. Изложена методика определения чувствительности микобактерий к противотуберкулезным препаратам методом пропорций на жидких питательных средах: от приготовления рабочих растворов противотуберкулезных препаратов до подготовки пробирок, посева культур и проведения внутрилабораторного контроля качества.

Проведен краткий обзор основных понятий, связанных с лекарственной чувствительностью микобактерий и фенотипических методов ее тестирования. Изложена методика определения чувствительности микобактерий к противотуберкулезным препаратам (ПТП) методом пропорций на плотных питательных средах.

На большом клиническом материале проведена сравнительная оценка эффективности использования в Республике Марий Эл различных методов выявления микобактерий (МБ). Сопоставлены результаты исследований, выполненных различными микробиологическими методами, для выявления МБ у пациентов с подозрением на туберкулез (ТБ) в учреждениях нетуберкулезного профиля и для диагностики ТБ в специализированной бактериологической лаборатории противотуберкулезного учреждения. Показано, что в настоящее время для повышения эффективности диагностики ТБ существует необходимость в параллельном использовании одновременно комплекса различных микробиологических методов.

Проведен краткий обзор основных понятий, связанных с лекарственной чувствительностью микобактерий и фенотипических методов ее тестирования. Изложена методика определения чувствительности микобактерий к противотуберкулезным препаратам методом абсолютных концентраций на плотной яичной питательной среде Левенштейна–Йенсена.