[Frequency of drug resistance and immune escape mutations in the hepatitis B virus genome detected in pregnant women in the Republic of Guinea].

The aim of the work is to assess the prevalence of hepatitis B virus drug resistance mutations and immune escape mutations in pregnant women in the Republic of Guinea. MATERIALS AND METHODS: Blood plasma samples obtained from 480 pregnant women from different regions of the Republic of Guinea with laboratory-confirmed viral hepatitis B were studied. Nucleotide sequences for genotype identification and mutation detection were obtained using nested-PCR followed by Sanger sequencing, based on overlapping pairs of primers spanning the complete genome of the virus. RESULTS AND DISCUSSION: In the examined group, the viral genotype E was the most prevalent (92.92%) compared with subgenotypes A1 (1.67%), A3 (1.46%), D1 (0.63%), D2 (1.04%) and D3 (2.29%). Among the examined HBV-infected pregnant women, 188 (39.17%) had undetectable HBsAg. Drug resistance mutations were detected in 33 individuals, which amounted to 6.88%. The following mutations were found: S78T (27.27%), L80I (24.24%), S202I (15.15%), M204I/V (42.42%). The presence of polymorphic variants not described as drug resistant has also been shown in positions associated with the development of drug resistance to tenofovir, lamivudine, telbivudine and entecavir (L80F, S202I, M204R). When analyzing the MHR and the region of a determinant, mutations were detected in 318 (66.25%) of pregnant women. In 172 of them, which amounted to 54.09%, multiple mutations were found. The amino acid substitutions in 13 positions associated with HBsAg-negative hepatitis B and/or potentially affecting HBsAg antigenicity were identified. CONCLUSION: The high prevalence of immune escape and drug resistance mutations potentially associated with false-negative result of HBsAg screening, prophylaxis failure, and virological failure of therapy that has been identified among treatment naive pregnant women imposes a serious problem.

[The prevalence clinically significant virus mutations among patients with chronic viral hepatitis B.]

The prevalence of clinically significant virus mutations in patients with chronic viral hepatitis B from the Kyrgyz Republic was analyzed. Blood plasma samples of 64 patients with verified chronic viral hepatitis B obtained from Kyrgyzstan indigenous people were used in the work. Asymmetric PCR was carried out with extended oligonucleotides and the first reaction amplification product was further used in a new PCR with one of the nested pairs overlapping primers that flanked the entire HBV genome together, followed by sequencing. Based on the phylogenetic analysis of 64 HBV isolates obtained from patients from the Kyrgyz Republic, it was shown that only the genotype D virus was present in the examined group, the HBV subgenotype D1 (68.75%) prevailed compared with the HBV subgenotype D2 (18.75%) and subgenotype D3 (12.5%). For all subgenotypes, several independent infection sources are obvious, subclusters that include isolates from Kyrgyzstan, Kazakhstan and Uzbekistan are distinguished, as well as subclusters that include isolates only from Kyrgyzstan, which are less similar to isolates previously deposited in the international database, which probably indicates an independent HBV homologous evolution in the region. Clinically significant mutations were identified in 26.5% of patients. Including 12.5% with escape mutations that prevent the virus detection and / or allow the virus to replicate despite the vaccine (122K, 128V, 133I, 134N). Another 12.5% of the isolates are characterized by mutations that are independently associated with the liver cirrhosis and hepatocellular carcinoma development, including 21, 24, 27 nucleotides deletions in the Pre-S2 region and the S11F mutation in the PreCore region. In one case, unusual 236S and 250P mutations were found in the positions described as drug resistance sites of the P region associated with the resistance development to adefovir, tenofovir, and entecavir. The hepatitis B virus genetic structure analysis, early virus mutations detection in patients with chronic hepatitis B virus can help to choose the right vaccination strategy, antiviral and immunosuppressive therapy, as well as predict the clinical course and disease progression.

[Antimicrobial resistance and clinical significant resistance mechanisms of Salmonella isolated in 2014-2018 in St.Petersburg, Russia.]

The article presents the results of antimicrobial resistance monitoring of Salmonella isolated from children and adults with diarrhea in St. Petersburg in 2014-2018. In 746 isolates of 42 serovars more than 90,0% belonged to three: S. enteritidis (79,6%), S. typhimurium (6,8%) and S. infantis (3,8%). The antimicrobial susceptibility testing (according the EUCAST) to 7 classes of antimicrobials revealed the resistance in 78,6% of Salmonella. Low-level quinolone resistance (MIC of ciprofloxacin 0,12-0,5 mg/l) was detected in 63,3% isolates (S. enteritidis -71,0%, S. typhimurium - 15,7%, S. infantis - 89,3%) and was due to five kinds of single nucleotide substitutions in gyrA: Asp87Tyr - 36,1% of studied isolates (only S. infantis); Ser83Phe - 22,2% (only S. enteritidis); Asp87Asn - 19,4% (S. enteritidis, S. typhimurium, S. hadar, S. newport); Ser83Tyr -11,1% (S. enteritidis and S. infantis) and Asp87Gly - 8,3% (only S. enteritidis). Only in one S. kentucky isolate with high-level fluoroquinolone resistance (MIC of ciprofloxacin > 8,0 mg/l) two substitutions (Ser83Phe and Asp87Asn) were detected. Two Salmonella isolates (S. typhimurium and S. corvallis) had plasmid-mediated quinolone resistance (qnrS). Extended-spectrum cephalosporin resistance was found in 6 Salmonella serovars (1,6%). The bla-genes were detected: of genetic group CTX-M1 - in 10 isolates (serovars S. typhimurium, S. enteritidis, S. abony, S. coeln and S. virchow), CTX-M2 - in 2 S. typhimurium isolates, CTX-M9 - in three S. enteritidis isolates. In one S. typhimurium CTX-M1 and CTX-M2 were detected. The gene of CMY-2 (molecular class C cephalosporinase) was revealed in two isolates (S. newport and S. enteritidis). Our study showed that Salmonella (the main bacterial pathogen of acute diarrhea in children and adults) isolated in Saint-Petersburg had antimicrobial resistance to drugs of choice for salmonellosis treatment.

[Hepatitis B virus identification in a blood plasma at a low viral load.]

To analyze the method for detecting HBV DNA in peripheral blood at low viral load and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of blood and liver tissue biopsy material were used from 128 patients living in the Russian Federation and the Republic of Uzbekistan without CHB and with CHB confirmed detection of circle covalently closed HBV DNA in hepatocytes. Plasma viral load was measured using the «AmpliSens® HBV-Monitor-FL¼ kit. HBV at low viral load was detected by nested PCR. Analytical sensitivity was checked by step dilution. According to our method, at the first stage, an asymmetric PCR is carried out using extended oligonucleotide primers with different melting points, complementary to the hepatitis B different genotypes genomes greatest similarity region. To increase the sensitivity, a second PCR is performed using the first reaction amplification product and internal primers. The sensitivity of the method for DNA extraction from 100 µl of plasma was 5 IU / ml, specificity 100%. Since, in spite of the HBV genotypes characteristic geographical distribution, the detection of "alien" genovariants for certain territories is becoming more frequent, we tested the method in geographically remote but active international relations with the Russian Federation regions with a high frequency of hepatotropic viruses. The developed method for detecting HBV DNA in blood plasma at low viral load based on PCR technology allows the various HBV gene variants identification and genotyping, both characteristic and rare in the Russian Federation, circulating in other world regions. The method can be used to detect HBV in risk groups, in a population, as well as when screening blood donors in order to ensure the blood transfusions safety.