Online Service with Automated Interpretation of Sequencing Data and Prediction of Pyrazinamide Resistance in Mycobacterium tuberculosis.

Pyrazinamide plays an important role in the treatment of tuberculosis. However, the microbiological test for pyrazinamide resistance is more complex and less reliable than testing of susceptibility to other anti-tuberculosis drugs due to the need to grow the pathogen at pH 5.5. Identification of mutations that cause resistance to anti-tuberculosis drugs can replace microbiological methods. Mutations in the pncA gene are responsible for the main mechanism of the resistance to pyrazinamide and are found in more than 90% of resistant strains. However, the genetic method for determining drug susceptibility is very complex, because mutations leading to pyrazinamide resistance are diverse and scattered throughout the gene. We have developed a software package for automatic data interpretation and prediction of the resistance to pyrazinamide based on Sanger sequencing results. The effectiveness of detection of pyrazinamide resistance in 16 clinical samples was compared using the BACTEC MGIT 960 automated system and pncA gene Sanger sequencing with automated analysis of the results. A significant advantage of the developed method over a single microbiological study was shown, due to greater reliability of the results irrespective of the purity of isolates.

A Method for Evaluation of the Level of Circulating Mitochondrial DNA by ND1 and ND2 Genes.

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.

SNP rs657152 Is Not Associated with the Level of Viral Load in COVID-19 or the Probability of Disease in the Population of Caucasians in Eastern Siberia.

Cross-replicating associations with rs657152 at the 9q34.2c locus and rs11385942 at the 3p21.31 locus found in patients with severe COVID-19 in the Caucasian population require the study of the discovered phenomenon in various populations, including as an independent biological marker. Primers and TaqMan probes for PCR discrimination of the A and C alleles in single nucleotide polymorphism (SNP) rs657152 have been developed. The polymorphism of the rs657152 A/C locus was determined in 129 patients with COVID-19 and in a control group of 466 healthy individuals. There were no significant differences in the frequency of distribution of the A and C alleles, 0.47/0.53 and 0.45/0.55, between patients and healthy subjects, respectively. Also, no differences were found in the distribution of alleles in patients with a high viral load in the smear (Ct in the range of 16-25) in comparison with an average and low viral load (Ct in the range of 26-40).

[The detection of highly-transmissible genotypes of agent in clinical samples for prognosis of unfavorable course of tuberculosis.]

The bio-information search was carried out and the design of primers and TaqMan probes was developed to detect DNA of agent of tuberculosis subtypes CC1 and CC2-W148 of Beijing genotype and also Ural genotype in various clinical material (phlegm, spinal fluid, pleural fluid, etc.) using real-time polymerase chain reaction technique. The 180 clinical samples from 143 patients with tuberculosis of lungs were used to carry out an approval of sensitivity and specificity of the developed tests concerning studies at the genetic analyzer GeneXpert. The sensitivity of tests on CC1, CC2-W148 и Ural relating to polymerase chain reaction of analyzer Gene Expert made up to 91%, 106% and 81% correspondingly. In all cases, the specificity made up to 100%. In parallel studies the samples with DNA of СС2-W148 genotype were more often positive on mutation of resistance to Rifampicin-Rif (+) according the results of GeneXpert (χ² = 27,1; p < 0,01) related to other genotypes. At the same time, detection of СС2-W148 strain in patient was more often accompanied by discrepancy of results: GeneXpert - Rif (+) and resistance to Rifampicin in bacteriological study under ultimate validation of multiple medicinal resistance of tuberculosis (χ² = 5,1; p < 0,05). The analysis was applied to negative effect of combination of allele-336G of CD209 gene of patient with genotype of tuberculosis mycobacterium Beijing detected previously (Ogarkov et al., 2012). The significant prevalence was observed related to widespread medicinal resistance (χ² =4,3; p < 0,05) in patients with allele-336G of CD209 gene in combination with CC2-W148 clone in comparison with other combinations in patients. The obtained results testify a possibility of application of genetic typing of tuberculosis agent and patient for early diagnosis of development of various complications of tuberculosis at the stages of primary examination of primarily detected patients with tuberculosis.

[Dilated cardiomyopathy as a cause of ischemic stroke]. / Dilatatsionnaya kardiomiopatiya kak prichina ishemicheskogo insul'ta.

Dilated cardiomyopathy (DCMP) is a disease of the myocardium characterized by the dilatation of heart cavities with the development of systolic dysfunction but without a decrease in the thickness of the myocardium. DCMP is a frequent cause of cardioembolic syndrome, in particular cardioembolic ischemic stroke (CES). A case of a patient with DCMP after CES is presented.

Draft genome sequence of Mycobacterium tuberculosis strain B9741 of Beijing B0/W lineage from HIV positive patient from Siberia.

We report a draft genome sequence of Mycobacterium tuberculosis strain B9741 belonging to Beijing B0/W lineage isolated from a HIV patient from Siberia, Russia. This clinical isolate showed MDR phenotype and resistance to isoniazid, rifampin, streptomycin and pyrazinamide. We analyzed SNPs associated with virulence and resistance. The draft genome sequence and annotation have been deposited at GenBank under the accession NZ_LVJJ00000000.

[Correlation between Haemostatic and Vegetate Nervous System Parameters under Examination Stress].

The work is aimed to an assessment of the correlation between of autonomic nervous system tone and the level of reactivity of the students' cardiovascular system and hemostasis system under examination stress. It was found that the students under exam stress are characterized with high values of respiration rate and with lower ones of tidal volume. The value of respiratory minute volume decreases in male students due to the bronchoconstrictor effects of stress at the tracheobronchial tree. This finding confirms a statistically significant reduction in the flow of small, medium and large bronchi, indicating an increased parasympathetic nervous system tone. A statistically significant increase in the power of fast waves as a result of determining heart rate variability in students of both hender during the exam also testifies the activation of the vagus nerve and can be used as a marker of exam stress. While exam stress, trombocrite value decreases and only femail students show a statistically significant reduction in the platelet number and increase of their volume. The tendency to increased thrombotic events under exam stress is demonstarated, being reached statistically significant differences in the case of female students--an increase in the initial thrombosis rate. In the case of mail students, an increase of prothrombin time value is testified under exam stress.

Undertreated HIV and drug-resistant tuberculosis at a referral hospital in Irkutsk, Siberia.

SETTING: A referral hospital for tuberculosis (TB) in Irkutsk, the Russian Federation. OBJECTIVE: To describe disease characteristics, treatment and hospital outcomes of TB-HIV (human immunodeficiency virus). DESIGN: Observational cohort of HIV-infected patients admitted for anti-tuberculosis treatment over 6 months. RESULTS: A total of 98 patients were enrolled with a median CD4 count of 147 cells/mm(3) and viral load of 205 943 copies/ml. Among patients with drug susceptibility testing (DST) results, 29 (64%) were multidrug-resistant (MDR), including 12 without previous anti-tuberculosis treatment. Nineteen patients were on antiretroviral therapy (ART) at admission, and 10 (13% ART-naïve) were started during hospitalization. Barriers to timely ART initiation included death, in-patient treatment interruption, and patient refusal. Of 96 evaluable patients, 21 (22%) died, 14 (15%) interrupted treatment, and 10 (10%) showed no microbiological or radiographic improvement. Patients with a cavitary chest X-ray (aOR 7.4, 95%CI 2.3-23.7, P = 0.001) or central nervous system disease (aOR 6.5, 95%CI 1.2-36.1, P = 0.03) were more likely to have one of these poor outcomes. CONCLUSION: High rates of MDR-TB, treatment interruption and death were found in an HIV-infected population hospitalized in Irkutsk. There are opportunities for integration of HIV and TB services to overcome barriers to timely ART initiation, increase the use of anti-tuberculosis regimens informed by second-line DST, and strengthen out-patient diagnosis and treatment networks.

Administrative interventions associated with increased initiation on antiretroviral therapy in Irkutsk, Siberia.

A bundle of initiatives to integrate human immunodeficiency virus (HIV) and tuberculosis (TB) services was assessed for the impact on antiretroviral therapy (ART) initiation at a TB referral hospital in Irkutsk, Russian Federation, from February 2014 to December 2015. The ART initiation rates in 166 ART-naïve patients undergoing anti-tuberculosis treatment (34.1% with multidrug or extensively drug-resistant TB) increased significantly from 14 (17%) pre-intervention to 44 (54%) post-intervention (P < 0.001). A survey of TB hospital staff identified administrative prioritisation as the most important initiative for increasing ART initiation.

Un ensemble d'initiatives visant à intégrer les services relatifs au virus de l'immunodéficience humaine (VIH) et à la tuberculose (TB) a été évalué en termes d'impact sur la mise en route du traitement antirétroviral (TAR) dans un hôpital de référence de la TB à Irkoutsk, Fédération de Russie, entre février 2014 et décembre 2015. Les taux de mise en route du TAR chez 166 patients n'en ayant jamais reçu et traités pour TB (34,1% avec une TB multi-résistante ou ultra-résistante) ont significativement augmenté de seulement 14 (17%) avant l'intervention à 44 (54%) après l'intervention (P < 0,001). Une enquête auprès du personnel de la TB a identifié la priorisation administrative comme l'initiative la plus importante dans l'augmentation de l'initiation du TAR.

Se evaluó un conjunto de iniciativas encaminadas a integrar los servicios de atención de la infección por el virus de la inmunodeficiencia humana (VIH) y la tuberculosis (TB), con el objeto de determinar la repercusión de la integración sobre el comienzo del tratamiento antirretrovírico (TAR) en el hospital de referencia de la TB de Irkutsk, en la Federación de Rusia, de febrero del 2014 a diciembre del 2015. La tasa de iniciación del TAR en 166 pacientes que nunca lo habían recibido y que estaban en curso de tratamiento antituberculoso (34,1% con TB multirresistente o extremadamente multirresistente) aumentó de manera significativa de solo 14 pacientes antes de la intervención (17%) a 44 pacientes después de la misma (54%; P < 0,001). Al interrogar al personal encargado de la TB en este hospital de referencia, se puso en evidencia que la priorización administrativa del TAR constituía la iniciativa de más había influido en el incremento de su utilización.

GENETIC DIVERSITY OF THE MYCOBACTERIUM TUBERCULOSIS ISOLATES IN THE REPUBLIC SAKHA (YAKUTIA), RUSSIA.

The population structure of the M. tuberculosis in Yakutia was estimated by the MIRU-VNTR method of 24 loci genotyping. 199 strains from 199 patients with pulmonary tuberculosis were tested. The greatest number of the strains (34.2%, 68/199) belonged to the genotype Beijing. The significant predominance (X² = 15.5; p < 0.001) of the multidrug and extensively drug-resistance (MDR/XDR) among the isolates of Beijing genotype was revealed in subtype CC2/ W148 - 9.5% (19/199). Strains of the genotype S (15.6%, 31/199) were the second most common genotype after Beijing. The majority of S-strains had an identical profile 233325153325141344222372. S genotype strains also significantly more frequently carried the MDR/ XDR (X² = 59.8;p<0.001) among non-Beijing isolates. The genotype strain Ural ranks the third in the prevalence - 10.0% (20/199). The strains belonging to the family LAM (8.5%, 17/199) had considerable genetic heterogeneity. A great genetic diversity was also found in minor genotypes T and Haarlem. A phylogenetic reconstruction of the epidemic spread of the S-genotype and subtype CC2/W148 of the Beijing genotype in Yakutia was performed with estimation of the probable time of origin in the scale proposed by Merker M. et al. (2015). It was shown that the strains of the subtype CC2/W148 had been formed from four distinct phylogenetic sublines in recent historical period (XX century). It was estimated that phylogenetic relationships accounted for 30 MIRU-VNTR profiles of S-strains from Yakutia and 31 reference S-profiles from Europe and Canada. The profiles of the S-genotype from Yakutia form a phylogenetically compact group, indicating that all evolutionary history of these strains happened in the Sakha Republic. The time of the ancestral S-genotype spreading in Yakutia was estimated to be in the range from 300 to 600 years.

[Identification of ubiquitous and endemic Mycobacterium tuberculosis genotypes in the Republic of Buryatia].

The MIRU-12 and MIRU-24 methods were used for genotyping and estimation of M. tuberculosis population structure in Buryatia. It was studied in 283 strains isolated from individuals from the regional TB dispensary and medical clinics of the penitentiary system of the republic. It was found that the prevalence of pandemic genotypes Beijing MIT17 and MIT16 and also subtype Beijing MIT642 (24.5% - 46/188) in the studied area were dominant. The isolates of the Beijing MIT642 were common among all groups of patients with pulmonary tuberculosis with the tendency to formation of MDR and chronic course of the disease.

[Prognostic value of parameters of cerebral hemodynamics in patients with carotid ischemic stroke].

We studied the dynamics of neurological deficit and parameters of the blood flow through middle cerebral arteries in 97 patients with acute ischemic stroke (IS). Absolute and relative parameters of the blood flow were compared to the severity of neurological deficit in the 1st, 5-7th and 21-23rd days. There was a strong correlation between the asymmetry in blood flow velocity in the first day of II and NIHSS scores in 21-23rd days (r= -0,96; p<0,01). The less pronounced asymmetry in the first day of IS predicted the better recovery of damaged functions in patients with IS.

[The early diagnostic of somatic inflammatory diseases in patients with acute ischemic stroke].

We examined 29 patients with acute ischemic stroke. Measurement of plasma nitrite and N-nitroso-compounds (NO2- +RNNO) to test for the presence of acute inflammatory process allows to timely and definitely determine its onset before the development of clinical symptoms and to operatively control the treatment, the latter being particularly important due to the restricted communication ability of neurologic patients.

[The state of acid-alkaline balance and oxygen-transport function of blood in patients with acute carotid ischemic stroke].

The gas composition, acid-alkaline state of arterial and venous blood and oxygen-transport function in carotid ischemic stroke was studied in 97 patients admitted to a hospital in the first 24h after stroke. Measurements were made at admission and after 5-7 days and 21-23 days. The relative hyperoxia, which reached maximal values to the first day, was found in patients in the acute stage of ischemic stroke. The increase in partial pressure of CO2 (pCO2) and relative acidosis seen to 5-7 days represent the compensatory reaction and lead to the increase in affinity of hemoglobin to oxygen thus improving the tissue oxygenation. We found the inverse correlations between the parameters of oxygen delivery (OD) and oxygen consumption (OC) in the first day and the severity of neurological deficit assessed with the NIHSS in the 21-23 days (r = -0.42; p < 0.01 and r = -0.55; p < 0.01 for OD and OC, respectively), i.e., the decrease in oxygen delivery and consumption corresponded to the greater severity of the stroke course.