Fecal microbiota transplantation in the treatment of astrovirus infection in a recipient of an allogeneic hematopoietic stem cell transplant: a clinical case

Background: Secondary immunodeficiency in recipients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the pediatric practice is often accompanied by bacterial and viral infections of the gastrointestinal tract (GIT), resistant to conventional therapy. Fecal microbiota transplantation (FMT) promotes intestinal recolonization and eradication of gastrointestinal symptoms. Clinical case description: A 2.5-year-old patient underwent allo-HSCT from a haploidentical related donor (father) as a part of the treatment of acute myeloid leukemia. A month after the last procedure, diarrhea (up to 10 times a day) and abdominal pain appeared. The astrovirus RNA and Clostridium difficile toxin A were detected in the feces. The FMT was prescribed. After two FMT procedures, the intestinal syndrome leveled out, and the tests for the astrovirus RNA and clostridial toxins were negative. The content of cholic and, in particular, deoxycholic acids, as well as their conjugates with glycine and taurine, in the feces increased; the acetic acid content increased with a simultaneous decrease in the level of propionic acid, which indicates the restoration of the intestinal microbiotas functional potential. Conclusion: FMT contributes to the restoration of the normal intestinal microflora, the elimination of clostridial toxins, enteroinvasive E. coli and astrovirus infection in allo-HSCT recipients, as evidenced by the indicators of the intestinal microbiota activity, and can be used in allo-HSCT recipients with infections refractory to conventional therapy

A fluorescent microspheres-based microfluidic test system for the detection of immunoglobulin G to SARS-CoV-2

Background: The pandemic of the new coronavirus infection, COVID-19, is currently ongoing in the world. Over the years, the pathogen, SARS-CoV-2, has undergone a series of mutational genome changes, which has led to the spread of various genetic variants of the virus. Meanwhile, the methods used to diagnose SARS-CoV-2, to establish the disease stage and to assess the immunity, are nonspecific to SARS-CoV-2 variants and time-consumable. Thus, the development of new methods for diagnosing COVID-19, as well as their implementation in practice, is currently an important direction. In particular, application of systems based on chemically modified fluorescent microspheres (with a multiplex assay for target protein molecules) opens great opportunities. Aim: development of a microfluidic diagnostic test system based on fluorescent microspheres for the specific detection of immunoglobulins G (IgG) to SARS-CoV-2. Methods: A collection of human serum samples was characterized using enzyme-linked immunosorbent assay (ELISA) and commercially available reagent kits. IgG to SARS-CoV-2 in the human serum were detected by the developed immunofluorescent method using microspheres containing the chemically immobilized RBD fragment of the SARS-CoV-2 (Kappa variant) viral S-protein. Results: The level of IgG in the blood serum of recovered volunteers was 9-300 times higher than that in apparently healthy volunteers, according to ELISA (p0.001). Conjugates of fluorescent microspheres with the RBD-fragment of the S-protein, capable of specifically binding IgG from the blood serum, have been obtained. The immune complexes formation was confirmed by the fluorescence microscopy data; the fluorescence intensity of secondary antibodies in the immune complexes formed on the surface of microspheres was proportional to the content of IgG (r 0.963). The test system had a good predictive value (AUC 70.3%). Conclusion: A test system has been developed, based on fluorescent microspheres containing the immobilized RBD fragment of the SARS-CoV-2 S-protein, for the immunofluorescent detection of IgG in the human blood serum. When testing the system on samples with different levels of IgG to SARS-CoV-2, its prognostic value was shown. The obtained results allow us to present the test system as a method to assess the level of immunoglobulins to SARS-CoV-2 in the human blood serum for the implementation in clinical practice. The test system can also be integrated into various microfluidic systems to create chips and devices for the point-of-care diagnostics

Clinical laboratory diagnostics of antibodies to SARS-CoV-2: from a QR code to the reality

Background: The immune response to SARS-CoV-2 includes the production of specific immunoglobulins to protein antigens of SARS-CoV-2. Depending on the type and level of immunoglobulins, it is possible to assess the stage of the disease and evaluate the effectiveness of vaccination. The main approach to the determination of immunoglobulins to SARS-CoV-2 in human biological fluids is enzyme-linked sorbent immunoassay. Its data, in particular, are used to issue an electronic COVID-19 certificate with a QR code. However, the qualitative and quantitative composition of immunoglobulins for a QR code is not officially regulated. Aim: measuring the immunoglobulins level in the human blood serum with different types of immunity to the new coronavirus infection (COVID-19) to select the most informative indicators of protective immunity. Methods: The study included 76 blood serum samples from male and female volunteers (age, 18 to 50 y.o.) in compliance with the ethical standards. The detection of IgA, IgM, IgG (total to different regions of SARS-CoV-2, S-protein IgG and RBD-fragment IgG), IgG avidity, and the level of the SARS-CoV-2 N-antigen was performed by enzyme-linked immunosorbent assay (ELISA) using commercially available reagent kits. Results: The indicators of the level of antibodies (both "protective" IgG and IgA of the initial phase of infection) are most pronounced in persons who have been vaccinated and have had COVID-19, and least pronounced in unvaccinated people. For recovered unvaccinated individuals, the level of total protective antibodies and IgG to the S-protein, including the RBD fragment, is the lowest; the avidity of IgG is lower than that in the other groups, too. The IgG avidity in vaccinated patients is higher than that in recovered ones. It should be noted that there were no differences in the level of both total IgG to SARS-CoV-2, to the S-protein and to the RBD-fragment of the S-protein for recovered and vaccinated individuals. Conclusion: The analysis of COVID-19 immunoglobulins indicates a different profile of the humoral immune response following vaccination and previous infection with COVID-19. To quickly assess the immune response to previous and current COVID-19 infection, as well as to detect the post-vaccination immunity, it is advisable to use the total level of IgG to SARS-CoV-2. For deeper assessment of protective immunity and production of protective antibodies, it is better to evaluate the quantitative content of IgG to the S protein and its RBD fragment. The equal level of IgA in the experimental groups indicates an ongoing interaction with SARS CoV-2 in the population. Thus, the electronic COVID-19 certificate is of little use when it is formed by only one of the indicators without taking into account the rest

Genotyping, Assessment of Virulence and Antibacterial Resistance of the Rostov Strain of Mycobacterium tuberculosis Attributed to the Central Asia Outbreak Clade

The Central Asia Outbreak (CAO) clade is a growing public health problem for Central Asian countries. Members of the clade belong to the narrow branch of the Mycobacterium tuberculosis Beijing genotype and are characterized by multidrug resistance and increased transmissibility. The Rostov strain of M. tuberculosis isolated in Russia and attributed to the CAO clade based on PCR-assay and whole genome sequencing and the laboratory strain H37Rv were selected to evaluate the virulence on C57Bl/6 mice models by intravenous injection. All mice infected with the Rostov strain succumbed to death within a 48-day period, while more than half of the mice infected by the H37Rv strain survived within a 90-day period. Mice weight analysis revealed irreversible and severe depletion of animals infected with the Rostov strain compared to H37Rv. The histological investigation of lung and liver tissues of mice on the 30th day after injection of mycobacterial bacilli showed that the pattern of pathological changes generated by two strains were different. Moreover, bacterial load in the liver and lungs was higher for the Rostov strain infection. In conclusion, our data demonstrate that the drug-resistant Rostov strain exhibits a highly virulent phenotype which can be partly explained by the CAO-specific mutations

Activation of Neutrophils by Mucin–Vaterite Microparticles

Nano- and microparticles enter the body through the respiratory airways and the digestive system, or form as biominerals in the gall bladder, salivary glands, urinary bladder, kidney, or diabetic pancreas. Calcium, magnesium, and phosphate ions can precipitate from biological fluids in the presence of mucin as hybrid nanoparticles. Calcium carbonate nanocrystallites also trap mucin and are assembled into hybrid microparticles. Both mucin and calcium carbonate polymorphs (calcite, aragonite, and vaterite) are known to be components of such biominerals as gallstones which provoke inflammatory reactions. Our study was aimed at evaluation of neutrophil activation by hybrid vaterite–mucin microparticles (CCM). Vaterite microparticles (CC) and CCM were prepared under standard conditions. The diameter of CC and CCM was 3.3 ± 0.8 µm and 5.8 ± 0.7 µm, with ƺ-potentials of −1 ± 1 mV and −7 ± 1 mV, respectively. CC microparticles injured less than 2% of erythrocytes in 2 h at 1.5 mg mL−1, and no hemolysis was detected with CCM; this let us exclude direct damage of cellular membranes by microparticles. Activation of neutrophils was analyzed by luminol- and lucigenin-dependent chemiluminescence (Lum-CL and Luc-CL), by cytokine gene expression (IL-6, IL-8, IL-10) and release (IL-1β, IL-6, IL-8, IL-10, TNF-α), and by light microscopy of stained smears. There was a 10-fold and higher increase in the amplitude of Lum-CL and Luc-CL after stimulation of neutrophils with CCM relative to CC. Adsorption of mucin onto prefabricated CC microparticles also contributed to activation of neutrophil CL, unlike mucin adsorption onto yeast cell walls (zymosan); adsorbed mucin partially suppressed zymosan-stimulated production of oxidants by neutrophils. Preliminary treatment of CCM with 0.1–10 mM NaOCl decreased subsequent activation of Lum-CL and Luc-CL of neutrophils depending on the used NaOCl concentration, presumably because of the surface mucin oxidation. Based on the results of ELISA, incubation of neutrophils with CCM downregulated IL-6 production but upregulated that of IL-8. IL-6 and IL-8 gene expression in neutrophils was not affected by CC or CCM according to RT2-PCR data, which means that post-translational regulation was involved. Light microscopy revealed adhesion of CC and CCM microparticles onto the neutrophils; CCM increased neutrophil aggregation with a tendency to form neutrophil extracellular traps (NETs). We came to the conclusion that the main features of neutrophil reaction to mucin–vaterite hybrid microparticles are increased oxidant production, cell aggregation, and NET-like structure formation, but without significant cytokine release (except for IL-8). This effect of mucin is not anion-specific since particles of powdered kidney stone (mainly calcium oxalate) in the present study or calcium phosphate nanowires in our previous report also activated Lum-CL and Luc-CL response of neutrophils after mucin sorption

Isolation and Characterization of the First <i>Zobellviridae</i> Family Bacteriophage Infecting <i>Klebsiella pneumoniae</i>

In order to address the upcoming crisis in the treatment of Klebsiella pneumoniae infections, caused by an increasing proportion of resistant isolates, new approaches to antimicrobial therapy must be developed. One approach would be to use (bacterio)phages and/or phage derivatives for therapy. In this study, we present a description of the first K. pneumoniae phage from the Zobellviridae family. The vB_KpnP_Klyazma podovirus, which forms translucent halos around the plaques, was isolated from river water. The phage genome is composed of 82 open reading frames, which are divided into two clusters located on opposite strands. Phylogenetic analysis revealed that the phage belongs to the Zobellviridae family, although its identity with the closest member of this family was not higher than 5%. The bacteriophage demonstrated lytic activity against all (n = 11) K. pneumoniae strains with the KL20 capsule type, but only the host strain was lysed effectively. The receptor-binding protein of the phage was identified as a polysaccharide depolymerase with a pectate lyase domain. The recombinant depolymerase protein showed concentration-dependent activity against all strains with the KL20 capsule type. The ability of a recombinant depolymerase to cleave bacterial capsular polysaccharides regardless of a phage’s ability to successfully infect a particular strain holds promise for the possibility of using depolymerases in antimicrobial therapy, even though they only make bacteria sensitive to environmental factors, rather than killing them directly

Unusual Large-Scale Chromosomal Rearrangements in <i>Mycobacterium tuberculosis</i> Beijing B0/W148 Cluster Isolates

<div><p>The <i>Mycobacterium tuberculosis</i> (MTB) Beijing family isolates are geographically widespread, and there are examples of Beijing isolates that are hypervirulent and associated with drug resistance. One-fourth of Beijing genotype isolates found in Russia belong to the B0/W148 group. The aim of the present study was to investigate features of these endemic strains on a genomic level. Four Russian clinical isolates of this group were sequenced, and the data obtained was compared with published sequences of various MTB strain genomes, including genome of strain W-148 of the same B0/W148 group. The comparison of the W-148 and H37Rv genomes revealed two independent inversions of large segments of the chromosome. The same inversions were found in one of the studied strains after deep sequencing using both the fragment and mate-paired libraries. Additionally, inversions were confirmed by RFLP hybridization analysis. The discovered rearrangements were verified by PCR in all four newly sequenced strains in the study and in four additional strains of the same Beijing B0/W148 group. The other 32 MTB strains from different phylogenetic lineages were tested and revealed no inversions. We suggest that the initial largest inversion changed the orientation of the three megabase (Mb) segment of the chromosome, and the second one occurred in the previously inverted region and partly restored the orientation of the 2.1 Mb inner segment of the region. This is another remarkable example of genomic rearrangements in the MTB in addition to the recently published of large-scale duplications. The described cases suggest that large-scale genomic rearrangements in the currently circulating MTB isolates may occur more frequently than previously considered, and we hope that further studies will help to determine the exact mechanism of such events.</p></div

Comparative phylogenetic analysis of strains under study and 12 whole genomes from the NCBI database.

<p>Phylogenetic tree based on all SNPs of genomes was constructed using the Neighbor-Joining algorithm. Evolutionary distances were calculated using p-distance method.</p

Genotyping and drug resistance data of the B0/W148 strains sequenced in this study.

<p>¹ RIF - rifampicin, INH - isoniazid, EMB - ethambutol, STR - streptomycin, PZA - pyrazinamide, ETH - ethionamide, AMI- amikacin, CAPR - capreomycin, OFL – ofloxacin.</p><p>² B0 designation according to Narvskaya <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Narvskaya1" target="_blank">[6]</a>, W148 according to Bifani <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Bifani1" target="_blank">[14]</a>.</p><p>³ SITVITWEB was used for identification of data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Demay1" target="_blank">[15]</a>.</p><p>⁴ 24 – VNTR: s154, s580, s960, s1644, s2059, s2531, s2687, s2996, s3007, s3192, s4348, s802, s2165, s2461, s577, s2163, s4052, s4156, s424, s1955, s2347, s2401, s3171, s3690 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Supply1" target="_blank">[16]</a>.</p