Application of MALDI-TOF MS and machine learning for the detection of SARS-CoV-2 and non-SARS-CoV-2 respiratory infections.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) could aid the diagnosis of acute respiratory infections (ARIs) owing to its affordability and high-throughput capacity. MALDI-TOF MS has been proposed for use on commonly available respiratory samples, without specialized sample preparation, making this technology especially attractive for implementation in low-resource regions. Here, we assessed the utility of MALDI-TOF MS in differentiating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vs non-COVID acute respiratory infections (NCARIs) in a clinical lab setting in Kazakhstan. Nasopharyngeal swabs were collected from inpatients and outpatients with respiratory symptoms and from asymptomatic controls (ACs) in 2020-2022. PCR was used to differentiate SARS-CoV-2+ and NCARI cases. MALDI-TOF MS spectra were obtained for a total of 252 samples (115 SARS-CoV-2+, 98 NCARIs, and 39 ACs) without specialized sample preparation. In our first sub-analysis, we followed a published protocol for peak preprocessing and machine learning (ML), trained on publicly available spectra from South American SARS-CoV-2+ and NCARI samples. In our second sub-analysis, we trained ML models on a peak intensity matrix representative of both South American (SA) and Kazakhstan (Kaz) samples. Applying the established MALDI-TOF MS pipeline "as is" resulted in a high detection rate for SARS-CoV-2+ samples (91.0%), but low accuracy for NCARIs (48.0%) and ACs (67.0%) by the top-performing random forest model. After re-training of the ML algorithms on the SA-Kaz peak intensity matrix, the accuracy of detection by the top-performing support vector machine with radial basis function kernel model was at 88.0%, 95.0%, and 78% for the Kazakhstan SARS-CoV-2+, NCARI, and AC subjects, respectively, with a SARS-CoV-2 vs rest receiver operating characteristic area under the curve of 0.983 [0.958, 0.987]; a high differentiation accuracy was maintained for the South American SARS-CoV-2 and NCARIs. MALDI-TOF MS/ML is a feasible approach for the differentiation of ARI without specialized sample preparation. The implementation of MALDI-TOF MS/ML in a real clinical lab setting will necessitate continuous optimization to keep up with the rapidly evolving landscape of ARI.IMPORTANCEIn this proof-of-concept study, the authors used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and machine learning (ML) to identify and distinguish acute respiratory infections (ARI) caused by SARS-CoV-2 versus other pathogens in low-resource clinical settings, without the need for specialized sample preparation. The ML models were trained on a varied collection of MALDI-TOF MS spectra from studies conducted in Kazakhstan and South America. Initially, the MALDI-TOF MS/ML pipeline, trained exclusively on South American samples, exhibited diminished effectiveness in recognizing non-SARS-CoV-2 infections from Kazakhstan. Incorporation of spectral signatures from Kazakhstan substantially increased the accuracy of detection. These results underscore the potential of employing MALDI-TOF MS/ML in resource-constrained settings to augment current approaches for detecting and differentiating ARI.

Sputnik-V reactogenicity and immunogenicity in the blood and mucosa: a prospective cohort study.

Sputnik-V (Gam-COVID-Vac) is a heterologous, recombinant adenoviral (rAdv) vector-based, COVID-19 vaccine now used in > 70 countries. Yet there is a shortage of data on this vaccine's performance in diverse populations. Here, we performed a prospective cohort study to assess the reactogenicity and immunologic outcomes of Sputnik-V vaccination in Kazakhstan. COVID-19-free participants (n = 82 at baseline) were followed at day 21 after Sputnik-V dose 1 (rAd5) and dose 2 (rAd26). Self-reported local and systemic adverse events were captured using questionnaires. Blood and nasopharyngeal swabs were collected to perform SARS-CoV-2 diagnostic and immunologic assays. We observed that most of the reported adverse events were mild-to-moderate injection site or systemic reactions, no severe or potentially life-threatening conditions were reported, and dose 1 appeared to be more reactogenic than dose 2. The seroconversion rate was 97% post-dose 1, remaining the same post-dose 2. The proportion of participants with detectable virus neutralization was 83% post-dose 1, increasing to 98% post-dose 2, with the largest relative increase observed in participants without prior COVID-19 exposure. Dose 1 boosted nasal S-IgG and S-IgA, while the boosting effect of dose 2 on mucosal S-IgG, but not S-IgA, was only observed in subjects without prior COVID-19. Systemically, vaccination reduced serum levels of growth regulated oncogene (GRO), which correlated with an elevation in blood platelet count. Overall, Sputnik-V dose 1 elicited both blood and mucosal SARS-CoV-2 immunity, while the immune boosting effect of dose 2 was minimal. Thus, adjustments to the current vaccine dosing regimen are necessary to optimize immunization efficacy and cost-effectiveness. While Sputnik-V reactogenicity is similar to that of other COVID-19 vaccines, the induced alterations to the GRO/platelet axis warrant investigation of the vaccine's effects on systemic immunology.

High SARS-CoV-2 seroprevalence in Karaganda, Kazakhstan before the launch of COVID-19 vaccination.

COVID-19 exposure in Central Asia appears underestimated and SARS-CoV-2 seroprevalence data are urgently needed to inform ongoing vaccination efforts and other strategies to mitigate the regional pandemic. Here, in a pilot serologic study we assessed the prevalence of SARS-CoV-2 antibody-mediated immunity in a multi-ethnic cohort of public university employees in Karaganda, Kazakhstan. Asymptomatic subjects (n = 100) were recruited prior to their first COVID-19 vaccination. Questionnaires were administered to capture a range of demographic and clinical characteristics. Nasopharyngeal swabs were collected for SARS-CoV-2 RT-qPCR testing. Serological assays were performed to detect spike (S)-reactive IgG and IgA and to assess virus neutralization. Pre-pandemic samples were used to validate the assay positivity thresholds. S-IgG and -IgA seropositivity rates among SARS-CoV-2 PCR-negative participants (n = 100) were 42% (95% CI [32.2-52.3]) and 59% (95% CI [48.8-69.0]), respectively, and 64% (95% CI [53.4-73.1]) of the cohort tested positive for at least one of the antibodies. S-IgG titres correlated with virus neutralization activity, detectable in 49% of the tested subset with prior COVID-19 history. Serologically confirmed history of COVID-19 was associated with Kazakh ethnicity, but not with other ethnic minorities present in the cohort, and self-reported history of respiratory illness since March 2020. Overall, SARS-CoV-2 exposure in this cohort was ~15-fold higher compared to the reported all-time national and regional COVID-19 prevalence, consistent with recent studies of excess infection and death in Kazakhstan. Continuous serological surveillance provides important insights into COVID-19 transmission dynamics and may be used to better inform the regional public health response.

Genetic Predictors of the Development of Complications after Coronary Stenting.

Due to the fact that there are scientific discussions about the significance of gene polymorphisms in the risk of developing cardiovascular complications after a percutaneous coronary intervention, it is of interest to evaluate the genetic predictors of the development of cardiovascular events. This study is a molecular genetic study. Association with the genes of biomarkers for inflammation and immune response increases the risk of cardiovascular events: rs1234313 (TNFSF4): (A/G, OR-4.57 (2.35-8.87), p ≤ 0.0001), (A/G-A/A, OR-3.14 (1.75-5.63), p ≤ 0.0001), and (A/G, OR = 4.01 (2.19-7.36), p ≤ 0.0001); rs3184504 (SH2D3); ATXN2: (C/T, OR-2.53 (1.28-5.01), T/T, OR-2.99 (1.13-7.92), p = 0.017)), (C/T-T/T, OR-2.61 (1.35-5.07), p = 0.000), and (OR-1.89 (1.15-3.09), p = 0.009)). According to the lipid metabolism biomarker genes, rs2943634: (A/C OR-2.57 (1.18-5.62), p = 0.013); according to the endothelial biomarker genes, rs2713604: (DNAJB8-AS1; GATA2): (C/T, OR-4.27 (2.35-7.76), p ≤ 0.0001), (C/T-C/C, OR-4.13 (2.31-7.40), p ≤ 0.0001), (OR-4.05 (2.24-7.30), p ≤ 0.0001), and (C/T, OR-3.46 (1.99-6.00), p ≤ 0.0001). The regression analysis found that in the presence of the rs2943634 gene polymorphism, the risk of late cardiovascular events increases by 4.007 times with 95% CI (1.502:10.692), p = 0.006. The genes of biomarkers for the risk of cardiovascular events are rs1234313(TNFSF4), rs3184504 (SH2D3; ATXN2), rs2943634, and rs2713604 (DNAJB8-AS1; GATA2). The only predictor of the development of new cardiovascular events was rs2943634, which belongs to the group of lipid metabolism biomarkers.

Association of four genetic variants with colorectal cancer in Kazakhstan population.

The study was conducted to search for polymorphisms located in the 10th chromosome associated with colorectal adenocarcinoma in representatives of the Kazakhstan population. Study was performed with 282 colorectal cancer (CRC) patients and 159 controls. Genotyping of SNPs was performed by QuantStudio 12K Flex PCR. For four significant SNPs inheritance model analysis was performed. Increasing risk of CRC was noted for rs10795668 in log-additive model (OR = 1.45, 95% CI: 1.05-1.99, p = 0.023); for rs1035209 in log-additive model (OR = 1.79, 95% CI: 1.18-2.72, p = 0.003); for rs11190164 in log-additive model (OR = 1.67, 95% CI: 1.17-2.38, p = 0.004). Decreasing risk of CRC was noted for rs10506868 in log-additive model (OR = 0.56, 95% CI: 0.37-0.85, p = 0.006). We detected SNPs that are associated with CRC risk in the Kazakhstan population.

The Oxidized proteins and activity of the Cl-/HCО3- exchanger in erythrocytes of patients with acute alcohol intoxication.

BACKGROUND: The purpose of this research was to study the morphological properties and the products of oxidative protein modification in erythrocytes of patients with acute alcohol intoxication. Two groups of subjects were analyzed. The first one included 39 patients with acute alcohol intoxication. The second group consisted of 14 healthy subjects. METHODS: In erythrocytes the activity of Cl-/HCО3- exchanger, the reactive protein carbonyl derivatives and membrane-bound hemoglobin concentration were measured. RESULTS: Our results demonstrated strong alteration of the Cl-/HCО3- exchanger activity in erythrocytes of patients with acute alcohol intoxication. A delay in the beginning of hemolysis during incubation of erythrocytes in the ammonium medium was observed. The concentration of protein carbonyls in erythrocytes of patients significantly increased in comparison to the control ones. A decrease in the membrane-bounded hemoglobin was observed as well. CONCLUSIONS: These findings indicate that ethanol toxicity is manifested by alteration of oxidized protein concentration and Cl-/HCО3- exchanger activity in erythrocytes. It is hypothesized that oxidized proteins are implicated in modulation of the erythrocyte cell volume regulation.

Influence of Combined Therapy on Generation of Neutrophil Extracellular Traps in Patients with Cervical Cancer.

BACKGROUND: According to several authors, neutrophil extracellular traps (NETs) play an important role in the mechanisms of cancer development and metastatic processes, which allows them to be considered as a potential new target for the treatment of cancer. AIM: To investigate the presence of extracellular neutrophil traps in the blood of patients with cervical cancer on the background of the combined treatment. MATERIALS AND METHODS: The study was conducted in 28 patients with cervical cancer. Group 1 received only radiation therapy; Groups 2-radiation therapy with ftorafur; Group 3-radiation therapy with cisplatin. To determine the number of spontaneous extracellular neutrophilic traps in the blood of the examined individuals, we used a technique of I.I. Dolgushin and Yu.S. Andreeva. RESULTS: Peripheral blood neutrophils in 53.57% (33.87; 72.49) of cervical cancer patients showed the ability to generate NETs before treatment. The ability to form NETs was observed in neutrophils isolated from 66.67% (9.43; 99.16) patients of the Group 1. After radiation therapy with ftorafur, the ability of blood neutrophils to form NETs was observed in 50% (1.26; 98.74) of cervical cancer patients. After radiotherapy with cisplatin, 37.50% (15.20; 64.57) of patients were found to have NETs formation. CONCLUSION: The ability to form NETs varied greatly after radiotherapy. The addition of chemotherapy drugs to radiation therapy did not increase the percentage of NETs in the blood of patients with cervical cancer but stimulated the appearance of basophil extracellular traps.

The modified proteins in erythrocytes at hemodialysis patients.

The main goal of our research was to study the modified proteins and activity of integral band 3 protein in erythrocytes of patients with end-stage renal failure during hemodialysis. Depending on the starting nosological variant 2 groups were formed. The first group included 27 patients with end-stage renal failurein the outcome of chronic pyelonephritis, the second - 20 patients with end-stage renal failure, developed on a background of chronic glomerulonephritis. The control group consisted of 15 healthy subjects. The patients of 1-th and 2-nd groups were treated with hemodialysis. The concentration of reactive protein carbonyl derivates, membrane-binding hemoglobin were detected in erythrocytes. Activity of band 3 protein was detected following the protocol of I. Mindukshev et al. (2010). Comparison the results obtained was performed using non-parametric Mann-Whitney U-test (for independent variables). Before hemodialysis the significant increasing of concentration of reactive protein carbonyl derivates and membrane-binding hemoglobin in erythrocytes of both group patients in comparison with healthy ones was observed. During hemodialysis the further augmentation of modified proteins in erythrocytes took place. Different changes in the activity of the protein band 3 in erythrocytes of patients during hemodialysis were found. The similar type of protein band 3 activity changing was determined in erythrocytes of 1-th group patients. There was positive pair correlation between concentration of the reactive protein carbonyl derivates and protein band 3 activity (r= 0,31, p<0,05). Two different types of protein band 3 activity changing were determined in erythrocytes of 2-th group patients. Thus, the differences in modified proteins concentration and protein band 3 activity that occurred in the erythrocytes of patients during hemodialysis were established.

Characteristic of oxidative metabolism in patients with chronic kidney disease depending on etiological nosology.

Development of chronic kidney disease (CKD) is accompanied by change in the oxidative metabolism. The aim of the study was to assess the parameters of oxidative stress in erythrocytes and plasma of patients with CKD 1 and 2 stages depending on the etiological nosology. Two groups were formed. 1(st) - patients with chronic glomerulonephritis (CG), CKD 1, 2. 2(nd) - patients with chronic pyelonephritis (CP), CKD 1, 2. The concentration of carbonyl derivatives was determined by the method of Levine R.L. Malondialdehyde (MDA) concentration - by the method of Goncharenko M.S. and Lapytova A.M. Median and quartiles of parameters were calculated. Statistically significant difference of data in the comparison groups were assessed using the Mann-Whitney test. In group of patients with CG there is the tendency to reduce the concentration of carbonyl derivatives in erythrocytes Md=12.861nmol/ml (12.468, 15.177) as compared with patients with CP Md=14.346nmol/ml (14.130, 14.537). Significant differences in this indicator in these groups were not detected, p=0.715. The concentration of MDA in erythrocytes in these groups varied in the opposite way. In patients with CG concentration of MDA in erythrocytes Md=5.513 µmol/ml (4.487, 6.282) was higher than this indicator in patients with CP Md=4.744 µmol/ml (3.974, 5.256), these changes were not significant, p=0.256. The concentrations of carbonyl derivatives and MDA in plasma in the compared groups were almost identical. In patients with CG concentration of carbonyl derivatives in plasma was Md=0.390nmol/ml (0.381, 0.528), the content of MDA Md=0.846 µmol/ml (0.714, 1.005). In patients with CP concentration of carbonyl derivatives in plasma was Md=0.424nmol/ml (0.312, 0.485) (when comparing 2 groups p=0.668), the content of MDA Md=0.820 µmol/ml (0.740, 0.979) (p=0.886). Thus, in patients with CKD 1, 2 depending on the etiological nosology features of oxidative metabolism were not found.

Myeloperoxidase activity and the oxidized proteins in blood neutrophils of patients with pneumonia.

The main purpose of our investigation was to study myeloperoxidase activity and concentration of oxidized proteins in blood neutrophils of patients with ambulant pneumonia and secondary pneumonia which has arisen on a background of chronic obstructive pulmonary disease (COPD). Patients were divided into 2 groups. 17 patients with ambulant pneumonia moderate severity and respiratory insufficiency of grade 2 were included in the 1-st group. 20 COPD patients with secondary pneumonia moderate severity and with respiratory insufficiency of grade 2 were included in the 2-nd group. The control group consisted of 15 healthy subjects. The reactive protein carbonyl derivates, advanced oxidation protein products (AOPP) and myeloperoxidase activity were detected in neutrophils. In neutrophils of 1-st group patients the augmentation of reactive protein carbonyl derivates was observed in comparison with healthy ones. In neutrophils of 2-nd group patients the slight decrease of reactive protein carbonyl derivates was observed in comparison with healthy ones (by 17%). In neutrophils of 2-nd group patients the significant increasing AOPP in comparison with healthy ones (p <0.01) and 1 group patients (p <0.05) was fixed. Myeloperoxidase activity was higher in neutrophils of 1-th group patients in comparison with healthy ones. In neutrophils of 2-nd group patients myeloperoxidase activity was higher in comparison with the same of 1 group patients (by 67%, p <0.05). Our results showed the different direction of oxidized proteins formation neutrophils of patients with primary and secondary pneumonia. Besides that the varied degree of myeloperoxidase activity was fixed. Our results require more detailed understanding because they can reflect peculiar mechanisms of pneumonia development and determine the characteristics of their progression.