Mini-Multilocus Sequence Typing Scheme for the Global Population of Neisseria gonorrhoeae.

The increasing problem of antimicrobial resistance in N. gonorrhoeae necessitates the development of molecular typing schemes that are suitable for rapid and mass screening. The objective of this study was to design and validate a mini-MLST scheme for N. gonorrhoeae based on global pathogen population data. Using sequences of seven housekeeping genes of 21,402 isolates with known MLSTs from the PubMLST database, we identified eighteen informative polymorphisms and obtained mini-MLST nucleotide profiles to predict MLSTs of isolates. We proposed a new MLST grouping system for N. gonorrhoeae based on mini-MLST profiles. Phylogenetic analysis revealed that MLST genogroups are a stable characteristic of the N. gonorrhoeae global population. The proposed grouping system has been shown to bring together isolates with similar antimicrobial susceptibility, as demonstrated by the characteristics of major genogroups. Established MLST prediction algorithms based on nucleotide profiles are now publicly available. The mini-MLST scheme was evaluated using a MLST detection/prediction method based on the original hydrogel DNA microarray. The results confirmed a high predictive ability up to the MLST genogroup. The proposed holistic approach to gonococcal population analysis can be used for the continuous surveillance of known and emerging resistant N. gonorrhoeae isolates.

MIR27A Gene Polymorphism Modifies the Effect of Common DPYD Gene Variants on Severe Toxicity in Patients with Gastrointestinal Tumors Treated with Fluoropyrimidine-Based Anticancer Therapy.

To reduce severe fluoropyrimidine-related toxicity, pharmacogenetic guidelines recommend a dose reduction for carriers of four high-risk variants in the DPYD gene (*2A, *13, c.2846A>T, HapB3). The polymorphism in the MIR27A gene has been shown to enhance the predictive value of these variants. Our study aimed to explore whether rs895819 in the MIR27A gene modifies the effect of five common DPYD variants: c.1129-5923C>G (rs75017182, HapB3), c.2194G>A (rs1801160, *6), c.1601G>A (rs1801158, *4), c.496A>G (rs2297595), and c.85T>C (rs1801265, *9A). The study included 370 Caucasian patients with gastrointestinal tumors who received fluoropyrimidine-containing chemotherapy. Genotyping was performed using high-resolution melting analysis. The DPYD*6 allele was associated with overall severe toxicity and neutropenia with an increased risk particularly pronounced in patients carrying the MIR27A variant. All carriers of DPYD*6 exhibited an association with asthenia regardless of their MIR27A status. The increased risk of neutropenia in patients with c.496G was only evident in those co-carrying the MIR27A variant. DPYD*4 was also significantly linked to neutropenia risk in co-carriers of the MIR27A variant. Thus, we have demonstrated the predictive value of the *6, *4, and c.496G alleles of the DPYD gene, considering the modifying effect of the MIR27A polymorphism.

Evaluation of the Polygenic Risk Score for Alzheimer's Disease in Russian Patients with Dementia Using a Low-Density Hydrogel Oligonucleotide Microarray.

The polygenic risk score (PRS), together with the ɛ4 allele of the APOE gene (APOE-ɛ4), has shown high potential for Alzheimer's disease (AD) risk prediction. The aim of this study was to validate the model of polygenic risk in Russian patients with dementia. A microarray-based assay was developed to identify 21 markers of polygenic risk and ɛ alleles of the APOE gene. This case-control study included 348 dementia patients and 519 cognitively normal volunteers. Cerebrospinal fluid (CSF) amyloid-ß (Aß) and tau protein levels were assessed in 57 dementia patients. PRS and APOE-ɛ4 were significant genetic risk factors for dementia. Adjusted for APOE-ɛ4, individuals with PRS corresponding to the fourth quartile had an increased risk of dementia compared to the first quartile (OR 1.85; p-value 0.002). The area under the curve (AUC) was 0.559 for the PRS model only, and the inclusion of APOE-ɛ4 improved the AUC to 0.604. PRS was positively correlated with tTau and pTau181 and inversely correlated with Aß42/Aß40 ratio. Carriers of APOE-ɛ4 had higher levels of tTau and pTau181 and lower levels of Aß42 and Aß42/Aß40. The developed assay can be part of a strategy for assessing individuals for AD risk, with the purpose of assisting primary preventive interventions.

Alteration of Blood Immune Biomarkers in MCI Patients with Different APOE Genotypes after Cognitive Training: A 1 Year Follow-Up Cohort Study.

Many studies aim to detect the early phase of dementia. One of the major ways to achieve this is to identify corresponding biomarkers, particularly immune blood biomarkers. The objective of this study was to identify such biomarkers in patients with mild cognitive impairment (MCI) in an experiment that included cognitive training. A group of patients with MCI diagnoses over the age of 65 participated in the study (n = 136). Measurements of cognitive functions (using the Mini-Mental State Examination scale and Montreal Cognitive Assessment) and determination of 27 serum biomarkers were performed twice: on the first visit and on the second visit, one year after the cognitive training. APOE genotypes were also determined. Concentrations of EGF (F = 17; p = 0.00007), Eotaxin (F = 7.17; p = 0.008), GRO (F = 13.42; p = 0.0004), IL-8 (F = 8.16; p = 0.005), MCP-1 (F = 13.46; p = 0.0001) and MDC (F = 5.93; p = 0.016) increased after the cognitive training in MCI patients. All these parameters except IL-8 demonstrated a weak correlation with other immune parameters and were poorly represented in the principal component analysis. Differences in concentrations of IP-10, FGF-2, TGFa and VEGF in patients with MCI were associated with APOE genotype. Therefore, the study identified several immune blood biomarkers that could potentially be associated with changes in cognitive function.

Genomic Diversity and Chromosomal Rearrangements in Neisseria gonorrhoeae and Neisseria meningitidis.

Chromosomal rearrangements in N. gonorrhoeae and N. meningitidis were studied with the determination of mobile elements and their role in rearrangements. The results of whole-genome sequencing and de novo genome assembly for 50 N. gonorrhoeae isolates collected in Russia were compared with 96 genomes of N. gonorrhoeae and 138 genomes of N. meningitidis from the databases. Rearrangement events with the determination of the coordinates of syntenic blocks were analyzed using the SibeliaZ software v.1.2.5, the minimum number of events that allow one genome to pass into another was calculated using the DCJ-indel model using the UniMoG program v.1.0. Population-level analysis revealed a stronger correlation between changes in the gene order and phylogenetic proximity for N. meningitidis in contrast to N. gonorrhoeae. Mobile elements were identified, including Correa elements; Spencer-Smith elements (in N. gonorrhoeae); Neisserial intergenic mosaic elements; IS elements of IS5, IS30, IS110, IS1595 groups; Nf1-Nf3 prophages; NgoФ1-NgoФ9 prophages; and Mu-like prophages Pnm1, Pnm2, MuMenB (in N. meningitidis). More than 44% of the observed rearrangements most likely occurred with the participation of mobile elements, including prophages. No differences were found between the Russian and global N. gonorrhoeae population both in terms of rearrangement events and in the number of transposable elements in genomes.

Prognosis of Alzheimer's Disease Using Quantitative Mass Spectrometry of Human Blood Plasma Proteins and Machine Learning.

Early recognition of the risk of Alzheimer's disease (AD) onset is a global challenge that requires the development of reliable and affordable screening methods for wide-scale application. Proteomic studies of blood plasma are of particular relevance; however, the currently proposed differentiating markers are poorly consistent. The targeted quantitative multiple reaction monitoring (MRM) assay of the reported candidate biomarkers (CBs) can contribute to the creation of a consistent marker panel. An MRM-MS analysis of 149 nondepleted EDTA-plasma samples (MHRC, Russia) of patients with AD (n = 47), mild cognitive impairment (MCI, n = 36), vascular dementia (n = 8), frontotemporal dementia (n = 15), and an elderly control group (n = 43) was performed using the BAK 125 kit (MRM Proteomics Inc., Canada). Statistical analysis revealed a significant decrease in the levels of afamin, apolipoprotein E, biotinidase, and serum paraoxonase/arylesterase 1 associated with AD. Different training algorithms for machine learning were performed to identify the protein panels and build corresponding classifiers for the AD prognosis. Machine learning revealed 31 proteins that are important for AD differentiation and mostly include reported earlier CBs. The best-performing classifiers reached 80% accuracy, 79.4% sensitivity and 83.6% specificity and were able to assess the risk of developing AD over the next 3 years for patients with MCI. Overall, this study demonstrates the high potential of the MRM approach combined with machine learning to confirm the significance of previously identified CBs and to propose consistent protein marker panels.

Prediction of ceftriaxone MIC in Neisseria gonorrhoeae using DNA microarray technology and regression analysis.

BACKGROUND: Decreased susceptibility of Neisseria gonorrhoeae to extended-spectrum cephalosporins is a major concern. Elucidation of the phenotypic and genetic characteristics of such isolates is a priority task. METHODS: We developed a method for predicting the N. gonorrhoeae ceftriaxone susceptibility level (MICcro) by identifying genetic determinants of resistance using low-density hydrogel microarrays and a regression equation. A training dataset, containing 5631 isolates from the Pathogenwatch database and 181 isolates obtained in the Russian Federation during 2018-19, was used to build a regression model. The regression equation was tested on 14 WHO reference strains. Ceftriaxone resistance determinants for the 448 evaluated clinical isolates collected in Russia were identified using microarray analysis, and MICcro values were calculated using the regression equation and compared with those measured by the serial dilution method. RESULTS: The regression equation for calculating MICcro values included 20 chromosomal resistance determinants. The greatest contributions to the increase in MICcro were shown to be PBP2: Ala-501→Pro, Ala-311→Val, Gly-545→Ser substitutions, Asp(345-346) insertion; and PorB: Gly-120→Arg substitution. The substitutions PBP2: Ala-501→Thr/Val, PorB: Gly-120→Asn/Asp/Lys and PBP1: Leu-421→Pro had weaker effects. For 94.4% of the isolates in the evaluation set, the predicted MICcro was within one doubling dilution of the experimentally determined MICcro. No ceftriaxone-resistant isolates were identified in the analysed samples from Russia, and no interpretative errors were detected in the MICcro calculations. CONCLUSIONS: The developed strategy for predicting ceftriaxone MIC can be used for the continuous surveillance of known and emerging resistant N. gonorrhoeae isolates.

Multiplex Autoantibody Detection in Patients with Autoimmune Polyglandular Syndromes.

The diagnosis of autoimmune polyglandular syndrome (APS) types 1/2 is difficult due to their rarity and nonspecific clinical manifestations. APS-1 development can be identified with assays for autoantibodies against cytokines, and APS-2 development with organ-specific antibodies. In this study, a microarray-based multiplex assay was proposed for simultaneous detection of both organ-specific (anti-21-OH, anti-GAD-65, anti-IA2, anti-ICA, anti-TG, and anti-TPO) and APS-1-specific (anti-IFN-ω, anti-IFN-α-2a, and anti-IL-22) autoantibodies. Herein, 206 serum samples from adult patients with APS-1, APS-2, isolated autoimmune endocrine pathologies or non-autoimmune endocrine pathologies and from healthy donors were analyzed. The prevalence of autoantibodies differed among the groups of healthy donors and patients with non-, mono- and multi-endocrine diseases. APS-1 patients were characterized by the presence of at least two specific autoantibodies (specificity 99.5%, sensitivity 100%). Furthermore, in 16 of the 18 patients, the APS-1 assay revealed triple positivity for autoantibodies against IFN-ω, IFN-α-2a and IL-22 (specificity 100%, sensitivity 88.9%). No anti-cytokine autoantibodies were found in the group of patients with non-APS-1 polyendocrine autoimmunity. The accuracy of the microarray-based assay compared to ELISA for organ-specific autoantibodies was 88.8-97.6%. This multiplex assay can be part of the strategy for diagnosing and predicting the development of APS.

Summary and Trends of the Russian Gonococcal Antimicrobial Surveillance Programme, 2005 to 2016.

The Russian Gonococcal Antimicrobial Surveillance Programme (RU-GASP) was established in 2004 and operated continuously during the years from 2005 to 2016. The aims of this study were to summarize the RU-GASP results over this 12-year period and evaluate the trends in Neisseria gonorrhoeae antimicrobial resistance in Russia. In total, 5,038 verified N. gonorrhoeae isolates from 40 participating regions were tested for susceptibility to six antimicrobials via an agar dilution method. DNA loci involved in antimicrobial resistance were identified via minisequencing or DNA microarray techniques. From 2005 to 2016, increasing susceptibility to penicillin G (from 22.6% to 63.0%), tetracycline (from 34.8% to 53.0%), and ciprofloxacin (from 50.6% to 68.6%) was observed, but resistance to these drugs remained high. The proportions of isolates nonsusceptible to azithromycin and spectinomycin peaked in 2011 and decreased thereafter. Of the isolates, only 6 and 23 were identified as nonsusceptible to ceftriaxone according to the CLSI definitions and EUCAST breakpoint (0.57% of the total population), respectively. Comparison of N. gonorrhoeae antimicrobial resistance genetic determinants in 2005 versus those in 2016 showed a significant decrease in the number of isolates carrying chromosomal mutations. The proportion of isolates with wild-type genotypes increased from 11.7% in 2005 to 30.3% in 2016. Thus, the RU-GASP can be considered a successful gonorrhea surveillance program, and the current state of N. gonorrhoeae antimicrobial resistance in Russia is less serious than that in other WHO GASP regions.

Examination of bedaquiline- and linezolid-resistant Mycobacterium tuberculosis isolates from the Moscow region.

Objectives: To study the isolates with acquired resistance to bedaquiline and linezolid that were obtained from patients enrolled in a clinical study of a novel therapy regimen for drug-resistant TB in Moscow, Russia. Methods: Linezolid resistance was detected using MGIT 960 with a critical concentration of 1 mg/L. The MIC of bedaquiline was determined using the proportion method. To identify genetic determinants of resistance, sequencing of the mmpR ( Rv0678 ), atpE , atpC , pepQ , Rv1979c , rrl , rplC and rplD loci was performed. Results: A total of 85 isolates from 27 patients with acquired resistance to linezolid and reduced susceptibility to bedaquiline (MIC ≥0.06 mg/L) were tested. Most mutations associated with a high MIC of bedaquiline were found in the mmpR gene. We identified for the first time two patients whose clinical isolates had substitutions D28N and A63V in AtpE, which had previously been found only in in vitro -selected strains. Several patients had isolates with elevated MICs of bedaquiline prior to treatment; four of them also bore mutations in mmpR , indicating the presence of some hidden factors in bedaquiline resistance acquisition. The C154R substitution in ribosomal protein L3 was the most frequent in the linezolid-resistant strains. Mutations in the 23S rRNA gene (g2294a and g2814t) associated with linezolid resistance were also found in two isolates. Heteroresistance was identified in ∼40% of samples, which reflects the complex nature of resistance acquisition. Conclusions: The introduction of novel drugs into treatment must be accompanied by continuous phenotypic susceptibility testing and the analysis of genetic determinants of resistance.

Simultaneous drug resistance detection and genotyping of Mycobacterium tuberculosis using a low-density hydrogel microarray.

BACKGROUND: Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs. METHODS: We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray. RESULTS: The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype. CONCLUSIONS: The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex.

Molecular epidemiology of drug-resistant Neisseria gonorrhoeae in Russia (Current Status, 2015).

BACKGROUND: The widespread distribution of Neisseria gonorrhoeae strains that are resistant to previously used and clinically implemented antibiotics is a significant global public health problem. In line with WHO standards, the national Gonococcal Antimicrobial Surveillance Programme (RU-GASP) has been in existence in Russia since 2004; herein, the current status (2015) is described, including associations between N. gonorrhoeae antimicrobial susceptibility, primary genetic resistance determinants and specific strain sequence types. METHODS: A total of 124 N. gonorrhoeae strains obtained from 9 regions in Russia in 2015 were examined using N. gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST), an antimicrobial susceptibility test according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria and an oligonucleotide microarray for the identification of mutations in the penA, ponA, rpsJ, gyrA and parC genes responsible for penicillin G, tetracycline, and fluoroquinolone resistance. Genogroup (G) isolates were evaluated based on their porB and tbpB sequence types (STs). RESULTS: NG-MAST analysis showed a diversified population of N. gonorrhoeae in Russia with 58 sequence types, 35 of which were described for the first time. The STs 807, 1544, 1993, 5714, 9476 and 12531, which were typical for some Russian Federation regions and several countries of the former Soviet Union, were represented by five or more isolates. The internationally widespread ST 1407 was represented by a single strain in the present study. Division into genogroups facilitated an exploration of the associations between N. gonorrhoeae sequence type, antimicrobial resistance spectra and genetic resistance determinant contents. Preliminarily susceptible (G-807, G-12531) and resistant (G-5714, G-9476) genogroups were revealed. The variability in the most frequently observed STs and genogroups in each participating region indicated geographically restricted antimicrobial susceptibility in N. gonorrhoeae populations. CONCLUSIONS: Resistance or intermediate susceptibility to previously recommended antimicrobials, such as penicillin G (60.5 %), ciprofloxacin (41.1 %) and tetracycline (25 %), is common in the N. gonorrhoeae population. Based on previous reports and current data, ceftriaxone and spectinomycin should be recommended for first-line empiric antimicrobial monotherapy for gonorrhoea in Russia.

Evaluation of a low-density hydrogel microarray technique for mycobacterial species identification.

In addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of the gyrB gene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when using gyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.

Rotary-based platform with disposable fluidic modules for automated isolation of nucleic acids.

We describe the development and evaluation of a rotary-based platform with multiple disposable fluidic modules for simultaneous automatic nucleic acid (NA) isolation from up to 24 biological samples. The procedure is performed inside insulated individual disposable modules, which minimizes both the risk of infection of personnel and laboratory cross-contamination. Each module is a segment of a circular cylinder containing a leak-proof inlet port for sample input, reservoirs with lyophilized chemicals and solvents, fluidic channels, stoppers, valves, a waste reservoir and an outlet port equipped with the standard micro test tube for NA collection. The entire platform, apart from the rotor that accommodates 24 modules, consists of functional elements that provide spinning of the rotor, reagent mixing, pressure delivery, and heating of reaction mixtures. The transfer of the reaction mixtures inside the modules is performed either with rotation of the rotor or with excessive air pressure applied to the module's reservoirs. The entire process takes less than 40 min, starting from the sample loading to the recovery of the purified NA, and it allows NA isolation both from bacterial cells and viral particles. The feasibility and reproducibility of the developed platform was demonstrated by the NA isolation from suspensions of Bacillus thuringiensis and Mycobacterium tuberculosis cells within a concentration range of 10(8) to 10(2) cells/ml. Isolation of NAs from blood plasma samples with varying concentration of hepatitis B and C viruses from 10(7) to 10(2) particles/ml were also successful. The purity and integrity of the extracted NAs were both reliable for performing quantitative PCR.

Mutations in Mismatch Repair Genes and Microsatellite Instability Status in Pancreatic Cancer.

Patients with pancreatic cancer (PC) showing mismatch repair (MMR) deficiency may benefit from immunotherapy. Microsatellite instability (MSI) is a hallmark of MMR deficiency (MMR-D). Here, we estimated the prevalence of MSI in PC, investigated germline and somatic mutations in the three MMR genes (MLH1, MSH2, and MSH6), and assessed the relationship between MMR genes mutations and MSI status in PC. Clinical specimens from PC patients were analyzed using targeted next-generation sequencing, including paired normal and tumor specimens from 155 patients, tumor-only specimens from 86 patients, and normal-only specimens from 379 patients. The MSI status of 235 PCs was assessed via PCR. Pathogenic/likely pathogenic (P/LP) germline variants in the MMR genes were identified in 1.1% of patients, while somatic variants were found in 2.6% of patients. No MSI-H tumors were detected. One patient carried two variants (P (VAF = 0.57) and LP (VAF = 0.25)) simultaneously; however, their germline/somatic status remains unknown due to the investigation focusing solely on the tumor and MSI analysis was not performed for this patient. MSI is rare in PC, even in tumors with MMR genes mutations. Our findings underscore the importance of assessing tumor MMR-D status in PC patients with confirmed Lynch syndrome when deciding whether to prescribe immunotherapy.

Dynamics of Cognitive Impairment in MCI Patients over a Three-Year Period: The Informative Role of Blood Biomarkers, Neuroimaging, and Genetic Factors.

Given the high growth rates of cognitive decline among the elderly population and the lack of effective etiological treatments, early diagnosis of cognitive impairment progression is an imperative task for modern science and medicine. It is of particular interest to identify predictors of an unfavorable subsequent course of cognitive disorders, specifically, rapid progression. Our study assessed the informative role of various risk factors on the dynamics of cognitive impairment among mild cognitive impairment (MCI) patients. The study included patients with MCI (N = 338) who underwent neuropsychological assessment, magnetic resonance imaging (MRI) examination, blood sampling for general and biochemical analysis, APOE genotyping, and polygenic risk score (PRS) evaluation. The APOE ε4/ε4 genotype was found to be associated with a diminished overall cognitive scores initial assessment and negative cognitive dynamics. No associations were found between cognitive changes and the PRS. The progression of cognitive impairment was associated with the width of the third ventricle and hematological parameters, specifically, hematocrit and erythrocyte levels. The absence of significant associations between the dynamics of cognitive decline and PRS over three years can be attributed to the provided suitable medical care for the prevention of cognitive impairment. Adding other risk factors and their inclusion in panels assessing the risk of progression of cognitive impairment should be considered.

Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays.

BACKGROUND: The steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) requires rapid and reliable methods to identify resistant strains. The current molecular methods to detect MTB resistance to second-line drugs either do not cover an extended spectrum of mutations to be identified or are not easily implemented in clinical laboratories. A rapid molecular technique for the detection of resistance to second-line drugs in M. tuberculosis has been developed using hybridisation analysis on microarrays. METHODS: The method allows the identification of mutations within the gyrA and gyrB genes responsible for fluoroquinolones resistance and mutations within the rrs gene and the eis promoter region associated with the resistance to injectable aminoglycosides and a cyclic peptide, capreomycin. The method was tested on 65 M. tuberculosis clinical isolates with different resistance spectra that were characterised by their resistance to ofloxacin, levofloxacin, moxifloxacin, kanamycin and capreomycin. Also, a total of 61 clinical specimens of various origin (e.g., sputum, bronchioalveolar lavage) were tested. RESULTS: The sensitivity and specificity of the method in the detection of resistance to fluoroquinolones were 98% and 100%, respectively, 97% and 94% for kanamycin, and 100% and 94% for capreomycin. The analytical sensitivity of the method was approximately 300 genome copies per assay. The diagnostic sensitivity of the assay ranging from 67% to 100%, depending on the smear grade, and the method is preferable for analysis of smear-positive specimens. CONCLUSIONS: The combined use of the developed microarray test and the previously described microarray-based test for the detection of rifampin and isoniazid resistance allows the simultaneous identification of the causative agents of MDR and XDR and the detection of their resistance profiles in a single day.

Gonococcal Genetic Island in the Global Neisseria gonorrhoeae Population: A Model of Genetic Diversity and Association with Resistance to Antimicrobials.

The aim of this work was to study the genetic diversity of the gonococcal genetic island (GGI) responsible for the type IV secretion system (T4SS) and the association of a functionally active GGI with antimicrobial resistance. An analysis of the GGI in a sample of 14,763 genomes of N. gonorrhoeae isolates from the Pathogenwatch database collected in 1996-2019 from 68 countries was performed. A model of GGI's genetic diversity that divides the global gonococcal population into fifty-one GGI clusters and three GGI superclusters based on the allele type of the traG gene and substitutions of the atlA and ych genes for eppA and ych1 has been proposed, reflecting differences among isolates in the T4SS functionality. The NG-MAST and MLST typing schemes (with accuracies of 91% and 83%, respectively) allowed the determination of both the presence of a GGI and the GGI cluster and, correspondingly, the structure of the GGI and the ability to secrete DNA. A statistically significant difference in the proportion of N. gonorrhoeae isolates resistant to ciprofloxacin, cefixime, tetracycline, and penicillin was found when comparing populations with a functional and a non-functional GGI. The presence of a functional GGI did not affect the proportion of azithromycin-resistant isolates.

Azithromycin Susceptibility Testing and Molecular Investigation of Neisseria gonorrhoeae Isolates Collected in Russia, 2020-2021.

The aim of this work was to study the resistance to macrolides (azithromycin) in the modern Russian population of N. gonorrhoeae with the analysis of genetic resistance determinants. Azithromycin is not used to treat gonococcal infection in Russia. However, among 162 isolates collected in 2020-2021, 22 isolates (13.6%) were phenotypically resistant to azithromycin. Mutations in 23S rRNA genes were found only in two isolates; erm and mefA genes were absent. Azithromycin resistance was shown to be predominantly associated with mutations in the mtrR and mtrD genes of the MtrCDE efflux pump and their mosaic alleles which may have formed due to a horizontal transfer from N. meningitidis. A total of 30 types of mtrR alleles and 10 types of mtrD alleles were identified including mosaic variants. Matching between the mtrR and mtrD alleles was revealed to indicate the cooperative molecular evolution of these genes. A link between the mtrR and mtrD alleles and NG-MAST types was found only for NG-MAST 228 and 807, typical of N. gonorrhoeae in Russia. The high level of resistance to azithromycin in Russia may be related to the spread of multiple transferable resistance to antimicrobials regardless of their use in the treatment of gonococcal infection.

Emergence of Azithromycin-Resistant Neisseria gonorrhoeae Isolates Belonging to the NG-MAST Genogroup 12302 in Russia.

The goal of this work was to determine the factors affecting the emergence of azithromycin-resistant Neisseria gonorrhoeae isolates in Russia, where azithromycin was never recommended for the treatment of gonococcal infections. Clinical N. gonorrhoeae isolates collected in 2018-2021 (428 isolates) were analyzed. No azithromycin-resistant isolates were found in 2018-2019, but in 2020-2021, a significant increase in the ratio of azithromycin-resistant isolates was observed: 16.8% and 9.3%, respectively. A hydrogel DNA microarray was developed for the analysis of resistance determinants: mutations in the genes encoding the mtrCDE efflux system and in all four copies of the 23S rRNA gene (position 2611). A majority of the azithromycin-resistant Russian isolates belonged to the NG-MAST G12302 genogroup, and the resistance was associated with the presence of a mosaic structure of the mtrR gene promoter region with the -35 delA deletion, an Ala86Thr mutation in the mtrR gene, and a mosaic structure of the mtrD gene. A comparative phylogenetic study of modern Russian and European N. gonorrhoeae populations allowed us to conclude that the emergence of azithromycin resistance in Russia in 2020 was the result of the appearance and spread of European N. gonorrhoeae strains belonging to the G12302 genogroup due to possible cross-border transfer.