Abstract

Toxin-producing strains of Clostridioides difficile (C. diff) are the most commonly identified cause of healthcare-associated infection in the elderly. Risk factors include advanced age, hospitalization, prior or concomitant systemic antibacterial therapy, chemotherapy, and gastrointestinal surgery. Patients with unspecified and new-onset diarrhea with ≥3 unformed stools in 24 h are the target population for C. diff infection (CDI) testing. To present data on the risks of laboratory misdiagnosis in managing CDI. Materials. In two general hospitals, we examined 116 clinical stool specimens from hospitalized patients with acute diarrhea suspected of nosocomial or antibiotic-associated diarrhea (AAD) due to C. diff. Enzyme immunoassay (EIA) tests for the detection of C. diff toxins A (cdtA) and B (cdtB) in stool, automated CLIA assay for the detection of C. diff GDH antigen and qualitative determination of cdtA and B in human feces and anaerobic stool culture were applied for CDI laboratory diagnosis. MALDI-TOF (Bruker) was used to identify the presumptive anaerobic bacterial colonies. The following methods were used as confirmatory diagnostics: the LAMP method for the detection of Salmonella spp. and simultaneous detection of C. jejuni and C. coli, an E. coli Typing RT-PCR detection kit (ETEC, EHEC, STEC, EPEC, and EIEC), API 20E and aerobic stool culture methods. Results. A total of 40 toxigenic strains of C. diff were isolated from all 116 tested diarrheal stool samples, of which 38/40 produced toxin B and 2/40 strains were positive for both cdtA and cdtB. Of the stool samples positive for cdtA (6/50) and/or cdtB (44/50) by EIA, 33 were negative for C. diff culture but positive for the following diarrheal agents: Salmonella enterica subsp. arizonae (1/33, LAMP, culture, API 20E); C. jejuni (2/33, LAMP, culture, MALDI TOF); ETEC O142 (1/33), STEC O145 and O138 (2/33, E. coli RT-PCR detection kit, culture); C. perfringens (2/33, anaerobic culture, MALDI TOF); hypermycotic enterotoxigenic K. pneumonia (2/33) and enterotoxigenic P. mirabilis (2/33, culture; PCR encoding LT-toxin). Two of the sixty-six cdtB-positive samples (2/66) showed a similar misdiagnosis when analyzed using the CLIA method. However, the PCR analysis showed that they were cdtB-negative. In contrast, the LAMP method identified a positive result for C. jejuni in one sample, and another was STEC positive (stx1+/stx2+) by RT-PCR. We found an additional discrepancy in the CDI test results: EPEC O86 (RT-PCR eae+) was isolated from a fecal sample positive for GHA enzyme (CLIA) and negative for cdtA and cdtB (CLIA and PCR). However, the culture of C. diff was negative. These findings support the hypothesis that certain human bacterial pathogens that produce enterotoxins other than C. diff, as well as intestinal commensal microorganisms, including Klebsiella sp. and Proteus sp., contribute to false-positive EIA card tests for C. diff toxins A and B, which are the most widely used laboratory tests for CDI. Conclusions. CDI presents a significant challenge to clinical practice in terms of laboratory diagnostic management. It is recommended that toxin-only EIA tests should not be used as the sole diagnostic tool for CDI but should be limited to detecting toxins A and B. Accurate diagnosis of CDI requires a combination of laboratory diagnostic methods on which proper infection management depends.

Abstract

Ticks are important carriers and reservoirs of pathogens, causing a number of diseases in humans and animals, causing significant damage to livestock. The search for and study of bacterial communities associated with ticks living in Kazakhstan are of the greatest relevance.

In this study, we characterized the microbial communities of ticks collected from cattle in eight different regions of Kazakhstan. Unique and common bacterial taxa identified in tick samples were analyzed using 16S rRNA metagenomic sequencing data. The following bacterial taxa were identified by region of Kazakhstan: West Kazakhstan region (WKR) 24 (45.28%), Abay region 21 (39.62%), East Kazakhstan region (EKR) 20 (37.74%), North Kazakhstan region (NKR) 20 (37.74%), Almaty region 18 (33.96%), Zhambyl region 17 (32.08%), Aktobe region 10 (18.87%), and Turkestan region 10 (18.87%).

During the work, various pathogenic bacteria of the families Moraxellaceae, Rhodobacteraceae, Ehrlichiaceae, Burkholderiaceae, Campylobacteraceae, Chlamydiaceae, Enterobacteriaceae, Peptostreptococcaceae, Crocodylidae, Enterobacteriaceae, Enterobacteriaceae, Vibrionaceae, Pseudomonadaceae, Lactobacillaceae, Microbacteriaceae, Mycoplasmataceae, Bacillaceae, Paenibacillaceae, Erwiniaceae, Pseudomonadaceae, Staphylococcaceae, Streptococcaceae were identified in ticks in the studied areas of Kazakhstan in 2024. The most frequently encountered bacteria included Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Campylobacter jejuni, as well as a representative of the genus Rickettsia as a pathogen/endosymbiont.

To assess statistically significant differences in the level of microbiota alpha diversity between the regions, an analysis of variance (ANOVA) was performed. The significance threshold was p<0.05. The significance level: α=0.05. All statistical calculations were performed in Python 3.11 using the scipy.stats libraries. The analysis was performed using the alpha diversity indices: Chao1, Shannon and Pielou. All three indices Chao 1, p-value < 0.0001, Shannon, p-value < 0.0001, Pielou p-value/0.0319 < 0.05, show statistically significant differences between the regions according to the ANOVA test. This means that for all three indices, diversity varies significantly between the regions.

Thus, it can be concluded that microbial communities in different regions are characterized by a similar level of diversity, with small but statistically acceptable variations. These differences can be due to both environmental factors and the specific species composition of ticks in each region.

Abstract

SARS-CoV-2 infection affects the respiratory system but also many tissues and organs that may be adversely compromised. Accordingly, clinical and experimental evidence has assessed virus ability to infect different cell phenotypes, translate viral proteins and promote virus replication. Among them, Envelope (E) viroporin sustains virus replication, promote inflammatory processes and remodelling of host cells. Despite advances on structure and sequence, E-protein specific location and effects in human host cells are still controversial and poorly investigated. Using lentiviral vectors, we established HEK293 and hiPS cell lines stably expressing E-protein. Immunocytochemistry showed E-protein mainly locates within the endoplasmic reticulum (ER), the ERGIC and the Golgi compartments, while only HEK293 cells display some protein staining in cytoplasm periphery suggesting a possible insertion into the plasmalemma. Electrophysiological recordings in HEK293 cells revealed E-protein self-assembles in the plasma membrane to mediate a cation efflux pore that is sensitive to amantadine blockade. Calcium fluorescence imaging in HEK293 and hiPS cells demonstrate E-protein expression induces a marked depletion of thapsigargin-sensitive intracellular calcium stores. The altered calcium homeostasis associates to reduced cell metabolic activity, mitochondrial potential, proliferation rate and ER stress. Trilineage differentiation of hiPS cells indicates E-protein expression preserves cell pluripotency while it selectively impairs mesodermal differentiation. These results unveil a critical role of stable E-viroporin expression that through alteration of ER Ca²⁺ homeostasis, metabolic activity and induction of ER stress affects important human cell function, including the process from pluripotent to mesodermal progenitors, a critical transition process in self-repair and homeostasis of most human tissue and organs.

Abstract

Dengue is the most important mosquito borne disease caused by the dengue virus (DENV), Affecting 390 million world population every year mainly in the tropical and subtropical regions where India is contributing approximately 35% of total cases. DENV has four serotypes (DENV 1 – 4) belonging to the

Flaviviridae family and it has a positive-sense, single-stranded RNA genome of approximately 10.7 Kilobases (Kb). Replication of the DENV genome is by RNA-dependent RNA polymerase (RdRp) encoded by NS5. RdRp of RNA viruses are error-prone due to lack of proof-reading activity and results in continuous accumulation of mutations in the viral genome. Accumulation of mutation leads to genetic and antigenic diversity within and across DENV serotypes which may also contribute to the emergence of new virulent strains. There have been very minimal efforts towards characterizing the replication fitness of clinical isolates of DENV in cell culture and in dengue mouse model. We have isolated DENV from acute dengue patients from 2020-25 and performed whole genome sequencing to understand the genetic and antigenic variation as compared to previously reported dengue virus. Furthermore, we have evaluated the replication fitness of these DENV in cell culture and mouse model. Phylogenetic analysis revealed that DENV1-4 isolates belonged to the genotype I, cosmopolitan, genotype III and genotype I, respectively. Next, we analyzed the percent homology, synonymous and non-synonymous mutations in circulating dengue virus and compared them with already reported Indian and global DENV genome sequences. Variability in the amino acid sequences of both structural and non-structural proteins of predicted B-cell and T-cell epitopes was also analyzed. Furthermore, we show the differences in replication fitness of low passage clinical isolates of DENV in a human monocyte cell line and in AG129 mice. Collectively, our results provide a comprehensive insight into the variation in currently circulating dengue virus in India. Their molecular evolution and replication fitness study would aid the ongoing efforts of antiviral and vaccine development.