DETECTION AND DIFFERENTIATION OF NON-TUBERCULOUS MYCOBACTERIA AND M. TUBERCULOSIS COMPLEX BY REAL TIME PCR

Goal of the study: to define the design of primers and probes specific to DNA of non-tuberculous mycobacteria and evaluate their diagnostic value in case of simultaneous detection of non-tuberculous mycobacteria and M. tuberculosis complex by real time PCR.Materials and methods. Primer 3, Primer BLAST, Ugene Uni Pro were used to design primers and probes. Preliminary assessment of specificity and sensitivity of detection of non-tuberculous mycobacteria DNA was performed on cultures belonging to 18 types of non-tuberculous mycobacteria, 16 strains of M. tuberculosis complex and 14 types of microorganisms being none Mycobacterum. Analytic sensitivity was tested on 284 cultures of non-tuberculous mycobacteria and diagnostic sensitivity was tested on 124 sputum samples. The kit ofM-Sorb-Tub-Avtomat (ZAO Sintol) was used for DNA isolation. Cultures were subcultured on the liquid medium of Middlebrook 7H9 in Bactec MGIT 960. Cultures were identified with the use of standard microbiological techniques. Analysis of DNA isolated from cultures was performed by the reagent kit of GenoTypeCM/AS (Hain Lifescience, Germany).Results. 100% specificity and sensitivity of PCR was demonstrated in mycobacterial cultures and 100% specificity and 69-70% sensitivity was demonstrated in diagnostic material analysis

Microbiological diagnosis of coincident mycobacterial infection in cystic Fibrosis

Diseases caused by mycobacteria, leprosy and tuberculosis, are widely known for many centuries. However, interest in mycobacteriosis, as an infectious disease of human organs caused by other mycobacteria, was only apparent from the late 50s of the 20th century. Over the years of observation, the number of cases of mycobacteriosis in various regions of the world has increased significantly, including in patients with cystic fibrosis. A key sign of comorbidity in cystic fibrosis is a chronic microbial infection that determines the severity of the course and the prognosis of the disease. In order to identify and identify mycobacteria isolated from patients with cystic fibrosis from 2011 to 2018, sputum was examined from 160 patients with cystic fibrosis and suspected mycobacterial infection. Mycobacteria were secreted in 17 (10.6%) patients with cystic fibrosis, of which non-tuberculous mycobacteria predominated. Thirteen patients were infected with M. abscessus (76.4%), two with M.avium (11.7%), one with M.xenopi (5.8%), one with M. tuberculosis (5.8%). Determination of drug sensitivity of mycobacteria showed that the culture of M.tuberculosis was sensitive to all antituberculosis drugs, and strains of nontuberculous mycobacteria had a different spectrum of drug susceptibility to drugs used for the therapy of mycobacteriosis. Identification of infectious agents will help ensure the timely initiation of adequate treatment, proper monitoring and prevention of the spread of non-tuberculosis mycobacteria among patients with cystic fibrosis.Заболевания, вызываемые микобактериями, лепра и туберкулез, широко известны на протяжении многих столетий. Однако интерес к микобактериозу, как инфекционному поражению органов человека, вызываемому другими микобактериями, проявился только с конца 50-х годов ХХ века. За годы наблюдений число случаев заболевания микобактериозом в различных регионах мира значительно увеличилось, в том числе и у больных муковисцидозом. ключевой признак коморбидности при муковисцидозе - хроническая микробная инфекция, которая определяет тяжесть течения и прогноз заболевания. С целью выявления и идентификация микобактерий, выделенных от больных муковисцидозом за период с 2011 по 2018 год, была исследована мокрота от 160 пациентов с муковисцидозом и подозрением на микобактериальную инфекцию. У 17 (10,6%) пациентов с муковисцидозом были выделены микобактерии, из них преобладали нетуберкулезные микобактерии. Тринадцать пациентов были инфицированы M.abscessus(76,4%), два - M.avium (11,7%), один - M.xenopi (5,8%), один - M.tuberculosis (5,8%). определение лекарственной чувствительности микобактерий показало, что, культура M.tuberculosis была чувствительна ко всем противотуберкулезным препаратам, а штаммы нетуберкулезных микобактерий имели различный спектр лекарственной чувствительности к препаратам, применяемым для терапии микобактериозов. Идентификация возбудителей инфекции будет способствовать обеспечению своевременного начала адекватного лечения, надлежащего мониторинга и профилактики распространения нетуберкулезных микобактерий среди больных кистозным фиброзом (муковисцидозом)

Выявление и дифференциация нетуберкулезных микобактерий и микобактерий туберкулезного комплекса методом пцр в режиме реального времени

Goal of the study: to define the design of primers and probes specific to DNA of non-tuberculous mycobacteria and evaluate their diagnostic value in case of simultaneous detection of non-tuberculous mycobacteria and M. tuberculosis complex by real time PCR.Materials and methods. Primer 3, Primer BLAST, Ugene Uni Pro were used to design primers and probes. Preliminary assessment of specificity and sensitivity of detection of non-tuberculous mycobacteria DNA was performed on cultures belonging to 18 types of non-tuberculous mycobacteria, 16 strains of M. tuberculosis complex and 14 types of microorganisms being none Mycobacterum. Analytic sensitivity was tested on 284 cultures of non-tuberculous mycobacteria and diagnostic sensitivity was tested on 124 sputum samples. The kit ofM-Sorb-Tub-Avtomat (ZAO Sintol) was used for DNA isolation. Cultures were subcultured on the liquid medium of Middlebrook 7H9 in Bactec MGIT 960. Cultures were identified with the use of standard microbiological techniques. Analysis of DNA isolated from cultures was performed by the reagent kit of GenoTypeCM/AS (Hain Lifescience, Germany).Results. 100% specificity and sensitivity of PCR was demonstrated in mycobacterial cultures and 100% specificity and 69-70% sensitivity was demonstrated in diagnostic material analysis.Цель исследования: определить дизайн праймеров и зондов, специфичных к ДНК нетуберкулезных микобактерий (НТМБ), и оценить их диагностическую значимость при одновременном выявлении НТМБ и M. tuberculosis complex (МБТК) методом ПЦР в режиме реального времени.Материалы и методы. Дизайн праймеров и зондов осуществляли с использованием ПО Primer 3, Primer BLAST, Ugene Uni Pro. Предварительную оценку специфичности и чувствительности выявления ДНК НТМБ проводили на культурах, принадлежащих к 18 видам НТМБ, 16 штаммам МБТК и 14 видам микроорганизмов, не относящихся к роду Mycobacterum. Аналитическую чувствительность оценивали на 284 культурах НТМБ, диагностическую чувствительность - на 124 образцах мокроты. Выделение ДНК проводили набором «М-Сорб-Туб-Автомат» (ЗАО «Синтол»). Культуры подвергали субкультивированию на жидкой среде Middlebrook 7H9 в системе Bactec MGIT 960. Идентификацию культур проводили с использованием стандартных микробиологических методов. Анализ ДНК, выделенной из культур, выполняли c помощью набора реагентов GenoTypeCM/AS (Hain Lifescience, Германия).Результаты. Показаны 100%-ные специфичность и чувствительность ПЦР при работе с культурами микобактерий и 100%-ная специфичность и 69,70%-ная чувствительность при анализе диагностического материала

Mutations in the Mycobacterium tuberculosis genome associated with genotypic MDR: dominant variants in the current (2011-2018) population of Russian strains and meta-analysis of world data

The mutations associated with resistance to rifampicin and isoniazid studied in the genome of M.tuberculosis isolated from 3,144 patients with pulmonary tuberculosis from the clinical and consulting departments of the Central TB Research Institute for the period 2011 - May 2018. The obtained results compared with the spectrum of mutations described for the worldwide population of resistant M.tuberculosis according to TBDReaMDB. The uniqueness of the world population of M.tuberculosis was shown on the specific genetic markers of resistance. The prevalence of MTB strains with the combination of mutations leading to a high level of resistance to rifampicin (rpoB 531 Ser-> Leu) and isoniazid (katG 315 Ser-> Thr(1)) were shown for Russian population of M.tuberculosis. These mutations did not adversely affect the transmissibility of the pathogen. The prevalence of these mutant variants was higher than in other regions of the world. The obtained results allow to make a conclusion about the unfavorable epidemiological situation related to the spread of MDR tuberculosis in the Russian Federation.Изучены мутации, ассоциированные с устойчивостью к рифампицину и изониазиду, в геноме M.tuberculosis, выделенных от 3144 больных туберкулезом легких из клинических и консультационного отделений ЦНИИТ, за период 2011 - май 2018 г.г. Проведено сравнение полученных результатов со спектром мутаций, описанных для общемировой популяции резистентных M.tuberculosis по данным TBDReaMDB. Показана уникальность мировых популяций M.tuberculosis по специфическим генетическим маркерам резистентности. для российской популяции штаммов M.tuberculosis выявлено доминирование штаммов с сочетанием мутаций, приводящих к высокому уровню резистентности к рифампицину (rpoB 531 Ser->Leu) и изониазиду (katG 315 Ser->Thr(1)), не оказывающих отрицательного влияния на трансмиссивность возбудителя. Распространенность этих мутантных вариантов была выше, чем в других регионах мира. Полученные результаты позволяют сделать вывод о неблагоприятной эпидемиологической ситуации, связанной с распространением МЛУ туберкулеза в РФ

Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia

BACKGROUND Patients with elevated triglyceride levels are at increased risk for ischemic events. Icosapent ethyl, a highly purified eicosapentaenoic acid ethyl ester, lowers triglyceride levels, but data are needed to determine its effects on ischemic events. METHODS We performed a multicenter, randomized, double-blind, placebo-controlled trial involving patients with established cardiovascular disease or with diabetes and other risk factors, who had been receiving statin therapy and who had a fasting triglyceride level of 135 to 499 mg per deciliter (1.52 to 5.63 mmol per liter) and a low-density lipoprotein cholesterol level of 41 to 100 mg per deciliter (1.06 to 2.59 mmol per liter). The patients were randomly assigned to receive 2 g of icosapent ethyl twice daily (total daily dose, 4 g) or placebo. The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina. The key secondary end point was a composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke. RESULTS A total of 8179 patients were enrolled (70.7% for secondary prevention of cardiovascular events) and were followed for a median of 4.9 years. A primary end-point event occurred in 17.2% of the patients in the icosapent ethyl group, as compared with 22.0% of the patients in the placebo group (hazard ratio, 0.75; 95% confidence interval [CI], 0.68 to 0.83; P<0.001); the corresponding rates of the key secondary end point were 11.2% and 14.8% (hazard ratio, 0.74; 95% CI, 0.65 to 0.83; P<0.001). The rates of additional ischemic end points, as assessed according to a prespecified hierarchical schema, were significantly lower in the icosapent ethyl group than in the placebo group, including the rate of cardiovascular death (4.3% vs. 5.2%; hazard ratio, 0.80; 95% CI, 0.66 to 0.98; P=0.03). A larger percentage of patients in the icosapent ethyl group than in the placebo group were hospitalized for atrial fibrillation or flutter (3.1% vs. 2.1%, P=0.004). Serious bleeding events occurred in 2.7% of the patients in the icosapent ethyl group and in 2.1% in the placebo group (P=0.06). CONCLUSIONS Among patients with elevated triglyceride levels despite the use of statins, the risk of ischemic events, including cardiovascular death, was significantly lower among those who received 2 g of icosapent ethyl twice daily than among those who received placebo. (Funded by Amarin Pharma; REDUCE-IT ClinicalTrials.gov number, NCT01492361

Diagnosis of Bactec samples by immunoglobulins of mouse hyperimmune sera obtained against modified antigens of the cell wall of Mycobacterium tuberculosis

The objective: using double-site enzyme immunoassay (ELISA) to evaluate the specificity of the antigens of digestive and chemically modified cell walls (CW) of M. tuberculosis.Subjects and methods. Hyperimmune sera of mice were obtained against modified CW antigens; immunoglobulins of different subclasses were isolated from them. With their help, 152 Bactec cultures with mycobacterial and non-mycobacterial growth from patients with lung diseases were tested by ELISA.Results. When CW was treated with proteinase K (prK), the protein content decreased by 10 times, and upon hydrolysis of NaOH, by more than 30 times. In immunoblotting, there was a narrowing of the spectrum of recognized antigens by the sera of hyperimmune mice (compared with whole CW), which indicated a decrease in their immunogenicity. Modification of WC of M. tuberculosis disavows 54 kDa antigen, causing a strong IgG1 subclass response.Diagnostic efficacy in ELISA with Bactec cultures increases with the use of immunoglobulins obtained against antigens treated with proteinase K – 79.14% (Pr.A) and 86.68% (Pr.G), when compared with immunoglobulins against the original drug – 70.69% (Pr.A) and 69.11% (Pr.G). Specificity increases significantly when using IgG1 antibodies after immunization with CW treated with prK (71.92% versus 25.93% in the initial preparation). Thus, new antigens of M. tuberculosis were identified, new antibody preparations for diagnosis in microbiological cultures were created against them

IN VITRO ACTION OF THE DRUG CANDIDATE OF PBTZ169, HYDROCHLORIDE ACTION IN RESPECT OF CLINICAL STRAINS OF MYCOBACTERIUM TUBERCULOSIS WITH EXTENSIVE DRUG RESISTANCE

Goal of the study: to study the specific in vitro action of PBTZ169, hydrochloride, in respect of clinical strains of M. tuberculosis with extensive drug resistance (XDR).Materials and methods. Strains of M. tuberculosis isolated from 20 XDR tuberculosis patients (10 strains from HIV positive patients and 10 strains from HIV negative patients) were used in the study, their susceptibility to 10 anti-tuberculosis first and second lines drugs plus linezolid was tested by Bactec MGIT 960. Bacteriostatic and bactericidal action of PBTZ169, hydrochloride, was defined as per the growth speed of M. tuberculosis strains in Bactec MGIT 960 compared to the growth of strains on the medium free from drugs and the medium containing control drugs (INH 0.1 мcg/ml, RIF 1 мcg/ml, AMK 1 мcg/ml and LFX 1.5 мcg/ml).Results. The used clinical strains of Mycobacterium tuberculosis were resistant to 7-10 anti-tuberculosis drugs, of them 3 were resistant to linezolid. When testing bacteriostatic action it was found that PBTZ169, hydrochloride, in the concentration of 0.037 mcg/ml fully suppressed growth of all 10 strains isolated from HIV negative patients and of the majority of strains isolated from HIV positive tuberculosis patients (7 out of 10). In the same concentration PBTZ169HCl demonstrated bactericidal action in respect of all strains isolated from HIV negative tuberculosis patients and 4 out of 10 strains isolated from HIV positive tuberculosis patients. Forthe remaining 6 out of 10 strains from HIV positive tuberculosis patients minimum bactericide concentration made 0.111 mcg/ml

X-RAY MORPHOLOGICAL SEMIOTICS OF NON-TUBERCULOUS MYCOBACTERIAL PULMONARY DISEASE

Objective: to evaluate the X-ray radiological features of nontuberculous mycobacterial pulmonary disease (NTMPD) versus morphological findings.Material and methods. The investigation enrolled 37 patients, in whom the radiographic signs of dissemination were determined and various types of NTMPD were identified. The investigation was conducted on a Siemens Somatom Emotion 16 multislice computed tomography (MSCT) scanner using a high-resolution algorithm (Quick Time Virtual Reality). To clarify the activity of pathological changes in the thoracic organs, 16 (43.2%) patients underwent a radionuclide study with 99mTc-technetrile on a Nucline Spirit gamma camera in planar and single photon emission computed tomography modes.The diagnosis was verified by sputum smear microscopy and clinical laboratory and bronchologic examinations: bronchoalveolar lavage in 11 (29.7%) patients, various types of bronchial biopsies in 17 (46.0%), morphological examinations, and videoassisted thoracoscopic surgery for pulmonary resection in 9 (24.3%).Results. The dissemination foci in mycobacterial diseases were characterized by their location in the lung parenchyma with vascular and bronchial involvement and reactive changes in the pulmonary pleurae and intrathoracic lymph nodes (ITLN). In 92.7% of cases, the detected foci were predominantly centrilobular with endobronchial localization. Their contours were mixed with clear and fuzzy outlines in 98.7% of cases. In 70.3% of cases, the foci were asymmetrically localized mainly in the subpleural areas of the lung and 12.3% of cases were accompanied by reactive involvement of the visceral pleura.CT study revealed a tree-in-bud sign in 96.7% of cases, frosted glass in 10.2%, and mosaic perfusion in 13.2%. A more than 10-mm increase in ITLN was found in 11.7% of cases.In a number of cases, it was difficult to speak about the activity of the pathological process in the lung and ITLN, as shown by MSCT. In this case, a lung radionuclide study with 99mTc-technetrile was carried out in the planar mode. The degree of tracer accumulation, localization, and extent were analyzed in the planar mode.Conclusion. Thus, the CT typical signs of NTMPD are the asymmetric location of its foci with an endobronchial extension, peribronchovascular localization of foci; the presence of a CT tree-in-bud sign; and the slight involvement of the pulmonary pleurae in the process. 99mTs-technetril radionuclide study has established that the most active inflammatory process is located in the lung and the tracer accumulates in the pathologically altered lymph nodes