The COVID-19 pandemic and heightened perception of the risk of emerging viral infections have boosted the efforts to better understand the virome or complete repertoire of viruses in health and disease, with a focus on infectious respiratory diseases. Next-generation sequencing (NGS) is widely used to study microorganisms, allowing the elucidation of bacteria and viruses inhabiting different body systems and identifying new pathogens. However, NGS studies suffer from a lack of standardization, in particular, due to various methodological approaches and no single format for processing the results. Here, we review the main methodological approaches and key stages for studies of the human virome, with an emphasis on virome changes during acute respiratory viral infection, with applications for clinical diagnostics and epidemiologic analyses.

Diagnosis of acute respiratory infections (ARIs) is challenging due to the broad diversity of potential microbial causes. We used metagenomic next-generation sequencing (mNGS) to analyze the nasopharyngeal virome of ARI patients, who had undergone testing with a clinical multiplex PCR panel (Amplisens ARVI-screen-FRT). We collected nasopharyngeal swabs from 49 outpatient adults, 32 of whom had ARI symptoms and were PCR-positive, and 4 asymptomatic controls in Kazakhstan during Spring 2021. We assessed the biodiversity of the mNGS-derived virome and concordance with PCR results. PCR identified common ARI viruses in 65% of the symptomatic cases. mNGS revealed viral taxa consisting of human, non-human eukaryotic and bacteriophage groups, comprising 15, 11 and 28 genera, respectively. Notable ARI-associated human viruses included rhinovirus (16.3%), betaherpesvirus 7 (14.3%) and Epstein-Barr virus (8.16%). The primary phage hosts were Streptococcus spp. (32.7%), Pseudomonas aeruginosa (24.5%) and Burkholderia spp. (20.4%). In total, 47% of ARIs were linked solely to bacterial pathogens, a third to viral-bacterial co-infections, and less than 10% to only viral infections by mNGS. PCR showed low concordance with mNGS, except for rhinovirus. These results underscore the importance of broad diagnostic methods and question the effectiveness of commonly used PCR panels in ARI diagnosis.

Fire blight of fruit crops is one of the most dangerous diseases for apple trees and other plants of the Rosaceae family, and in Kazakhstan, it is subject to quarantine. To study the spread of fire blight, a phytopathological evaluation of 59 apple varieties of domestic and foreign breeds was carried out in various regions of the south and southeast of Kazakhstan while also considering climatic conditions. The susceptibility of an apple tree to fire blight is influenced by the climatic conditions prevailing in a particular fruit region of Kazakhstan. Samples were collected from various varieties of apple trees with fire blight symptoms for molecular genetic analysis. The phytopathological evaluation and results of the PCR analysis made it possible to identify the causative agent of the disease and its spread to apple varieties in the main fruit regions of Kazakhstan. A molecular study of the resistance to the fire blight pathogen was carried out using the most effective molecular markers. A set of 10 (FBE-1_Y320; FBE-2_Y192; FBE-2_Y495; FBE-2_Y551; FB-MR5-K35; FB-MRS-R240; FB-MR5-R249; FB-MR5-rp16k15_M106; RLP1a; and RLP1b) SNPs was selected, including SNPs reported to be associated with three trait loci, as well as the two markers AE10-375 and GE-8019. Interestingly, the SNP analysis revealed that for all ten markers linked to fire blight resistance, the genotypes of all 59 apple cultivars were identical. No differences in the presence or absence of these markers were observed among the studied varieties. The 26 apple varieties of domestic and foreign breeds most resistant to fire blight were identified in the molecular analysis using the markers AE-375 and GE-8019. Among the studied 59 apple varieties, 23 varieties were identified using the AE-375 marker and 7 varieties with the GE-8019 marker. Samuret, Honeycrisp, Pinova, and Red Topaz were found to be resistant using markers AE-375 and GE-8019. The most promising apple varieties for further breeding for resistance to fire blight programs were selected.

Background/Objectives: Acinetobacter baumannii is an increasingly significant nosocomial pathogen causing severe infections globally. The emergence of multidrug-resistant A. baumannii strains has raised concerns about the efficacy of current treatment options. This study aimed to investigate the molecular epidemiology and antimicrobial resistance patterns of A. baumannii isolates from Kazakhstan. Methods: We collected nine A. baumannii isolates in 2022–2023 in Karaganda, Kazakhstan, which were then subjected to whole-genome sequencing (WGS) using the IonTorrent platform for genome characterization. Multilocus sequence typing (MLST) was used to classify the isolates into distinct clonal complexes. In addition, antibiotic susceptibility testing was conducted using the standard methods for a range of antibiotics commonly used against A. baumannii. Results: Our results revealed a high degree of genomic diversity among isolates from Kazakhstan, with multiple distinct classes identified: ST78 (n = 4, 44.4%), ST15 (n = 2, 22.2%), ST2 (n = 2, 22.2%), and ST193 (n = 1, 11%). MLST analysis showed that ST78^Pas/1104^Oxf (harboring blaOXA-72 and blaOXA-90 genes) were prevalent among the multidrug-resistant isolates. Based on the results of MLST, KL, and OCL, the analyzed isolates were assigned to specific international clones: IC2—ST2(Pas)-KL2/168-OCL1, IC4—ST15(Pas)-KL9-OCL7, and IC6—ST78(Pas)-KL49-OCL1. Notably, these isolates exhibited resistance to multiple antibiotics including meropenem, imipenem, gentamicin, amikacin, and ciprofloxacin. Conclusions: This study highlighted the complex molecular epidemiology of A. baumannii in Kazakhstan over a two-year period, underscoring the need for targeted surveillance strategies to monitor antimicrobial resistance patterns. The emergence and dissemination of multidrug-resistant strains within this timeframe emphasizes the importance of whole-genome sequencing as a diagnostic tool and underscores the challenges posed by these infections.

GCT is characterized by specific biochemical markers expression, such as human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP), which are the main tools in the diagnosis and monitoring of GCT treatment. They are expressed in 15-20% of cases of seminoma and in 60-80% of cases of non-seminoma. MicroRNA profiling allows to identify a number of microRNAs that are superior to classical serum tumor markers in the diagnosis of primary tumors, as well as in subsequent monitoring and prediction of recurrence. We analyzed the expression of 9 microRNAs (microRNA clusters 302/367 and 371-373, microRNA375) in the blood serum of 20 children with extracranial GCT at different stages of therapy and showed their usefulness and informativeness in early detection of events. Taking into consideration the high sensitivity and specificity, serum microRNAs 367,371,372,373,302d are of great interest for clinical use in malignant GCT. Significant expression of miR 375-3p was not detected either in malignant GCT or in teratomas.

Background: Respiratory infections are a leading cause of morbidity and mortality worldwide, with a significant proportion thought to have a viral aetiology. Traditional diagnostic approaches often rely on targeted assays for “common” respiratory pathogens, leaving a substantial fraction of infections unidentified. Here, we used metagenomic next generation sequencing (mNGS) to characterize the nasopharyngeal virome associated with acute respiratory infection (ARI), with and without a positive PCR test result for a panel of common respiratory viruses. Methods: Nasopharyngeal swabs from symptomatic outpatients (n=49), of whom 32 tested positive by a multiplex viral PCR, and asymptomatic controls (n=4) were characterized by mNGS. The virome taxa were stratified into human, non-human eukaryotic host, and bacteriophage sub-groups. We used a phage host classification as a proxy to establish bacterial taxa present in the nasopharynx. We then compared the virome biodiversity and presence of pathogens with known respiratory effects across the participant sub-groups. Results: The nasopharyngeal virome exhibited similar diversity across the PCR-positive and - negative subsets. Among the top ARI-associated human viruses were enterovirus (16.3%, human rhinovirus, HRV-A), roseolovirus (14.3%, human betaherpesvirus 7, HBV-7) and lymphocryptovirus (8.16%, Epstein-Barr virus, EBV). The top three ARI-associated phage hosts were Streptococcus spp (32.7%), Pseudomonas aeruginosa (24.5%) and Burkholderia spp. (20.4%). The virome of both asymptomatic and symptomatic subjects was also abundant in the Staphylococcus (60.4%) and Propionibacterium (Cutibacterium) acnes bacteriophages (90.6%). The PCR and mNGS results were relatively concordant for human rhinovirus (HRV), but not for other PCR panel targets, including human parainfluenza (HPIV), adenovirus (HAdV), bocavirus (BoV) and seasonal coronavirus (HCoV). Conclusions: mNGS revealed a high diversity of pathogens that could be cause to respiratory symptomatology, either as a single infection or a co-infection between viral and bacterial species. The clinical significance of the mNGS versus multiplex PCR findings warrants further investigation.