Prognosis of Alzheimer's Disease Using Quantitative Mass Spectrometry of Human Blood Plasma Proteins and Machine Learning.

Early recognition of the risk of Alzheimer's disease (AD) onset is a global challenge that requires the development of reliable and affordable screening methods for wide-scale application. Proteomic studies of blood plasma are of particular relevance; however, the currently proposed differentiating markers are poorly consistent. The targeted quantitative multiple reaction monitoring (MRM) assay of the reported candidate biomarkers (CBs) can contribute to the creation of a consistent marker panel. An MRM-MS analysis of 149 nondepleted EDTA-plasma samples (MHRC, Russia) of patients with AD (n = 47), mild cognitive impairment (MCI, n = 36), vascular dementia (n = 8), frontotemporal dementia (n = 15), and an elderly control group (n = 43) was performed using the BAK 125 kit (MRM Proteomics Inc., Canada). Statistical analysis revealed a significant decrease in the levels of afamin, apolipoprotein E, biotinidase, and serum paraoxonase/arylesterase 1 associated with AD. Different training algorithms for machine learning were performed to identify the protein panels and build corresponding classifiers for the AD prognosis. Machine learning revealed 31 proteins that are important for AD differentiation and mostly include reported earlier CBs. The best-performing classifiers reached 80% accuracy, 79.4% sensitivity and 83.6% specificity and were able to assess the risk of developing AD over the next 3 years for patients with MCI. Overall, this study demonstrates the high potential of the MRM approach combined with machine learning to confirm the significance of previously identified CBs and to propose consistent protein marker panels.

Multiplex Autoantibody Detection in Patients with Autoimmune Polyglandular Syndromes.

The diagnosis of autoimmune polyglandular syndrome (APS) types 1/2 is difficult due to their rarity and nonspecific clinical manifestations. APS-1 development can be identified with assays for autoantibodies against cytokines, and APS-2 development with organ-specific antibodies. In this study, a microarray-based multiplex assay was proposed for simultaneous detection of both organ-specific (anti-21-OH, anti-GAD-65, anti-IA2, anti-ICA, anti-TG, and anti-TPO) and APS-1-specific (anti-IFN-ω, anti-IFN-α-2a, and anti-IL-22) autoantibodies. Herein, 206 serum samples from adult patients with APS-1, APS-2, isolated autoimmune endocrine pathologies or non-autoimmune endocrine pathologies and from healthy donors were analyzed. The prevalence of autoantibodies differed among the groups of healthy donors and patients with non-, mono- and multi-endocrine diseases. APS-1 patients were characterized by the presence of at least two specific autoantibodies (specificity 99.5%, sensitivity 100%). Furthermore, in 16 of the 18 patients, the APS-1 assay revealed triple positivity for autoantibodies against IFN-ω, IFN-α-2a and IL-22 (specificity 100%, sensitivity 88.9%). No anti-cytokine autoantibodies were found in the group of patients with non-APS-1 polyendocrine autoimmunity. The accuracy of the microarray-based assay compared to ELISA for organ-specific autoantibodies was 88.8-97.6%. This multiplex assay can be part of the strategy for diagnosing and predicting the development of APS.

Biomarkers of Community-Acquired Pneumonia: A Key to Disease Diagnosis and Management.

Community-acquired pneumonia (CAP) is a dangerous disease caused by a spectrum of bacterial and viral pathogens. The choice of specific therapy and the need for hospitalization or transfer to the intensive care unit are determined by the causative agent and disease severity. The microbiological analysis of sputum largely depends on the quality of the material obtained. The prediction of severity and the duration of therapy are determined individually, and existing prognostic scales are used generally. This review examines the possibilities of using specific serological biomarkers to detect the bacterial or viral aetiology of CAP and to assess disease severity. Particular emphasis is placed on the use of biomarker signatures and the discovery of biomarker candidates for a single multiplex analysis.

Examination of bedaquiline- and linezolid-resistant Mycobacterium tuberculosis isolates from the Moscow region.

Objectives: To study the isolates with acquired resistance to bedaquiline and linezolid that were obtained from patients enrolled in a clinical study of a novel therapy regimen for drug-resistant TB in Moscow, Russia. Methods: Linezolid resistance was detected using MGIT 960 with a critical concentration of 1 mg/L. The MIC of bedaquiline was determined using the proportion method. To identify genetic determinants of resistance, sequencing of the mmpR ( Rv0678 ), atpE , atpC , pepQ , Rv1979c , rrl , rplC and rplD loci was performed. Results: A total of 85 isolates from 27 patients with acquired resistance to linezolid and reduced susceptibility to bedaquiline (MIC ≥0.06 mg/L) were tested. Most mutations associated with a high MIC of bedaquiline were found in the mmpR gene. We identified for the first time two patients whose clinical isolates had substitutions D28N and A63V in AtpE, which had previously been found only in in vitro -selected strains. Several patients had isolates with elevated MICs of bedaquiline prior to treatment; four of them also bore mutations in mmpR , indicating the presence of some hidden factors in bedaquiline resistance acquisition. The C154R substitution in ribosomal protein L3 was the most frequent in the linezolid-resistant strains. Mutations in the 23S rRNA gene (g2294a and g2814t) associated with linezolid resistance were also found in two isolates. Heteroresistance was identified in ∼40% of samples, which reflects the complex nature of resistance acquisition. Conclusions: The introduction of novel drugs into treatment must be accompanied by continuous phenotypic susceptibility testing and the analysis of genetic determinants of resistance.

A Comparison of the Sensititre MycoTB Plate, the Bactec MGIT 960, and a Microarray-Based Molecular Assay for the Detection of Drug Resistance in Clinical Mycobacterium tuberculosis Isolates in Moscow, Russia.

BACKGROUND: The goal of this study was to compare the consistency of three assays for the determination of the drug resistance of Mycobacterium tuberculosis (MTB) strains with various resistance profiles isolated from the Moscow region. METHODS: A total of 144 MTB clinical isolates with a strong bias toward drug resistance were examined using Bactec MGIT 960, Sensititre MycoTB, and a microarray-based molecular assay TB-TEST to detect substitutions in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, and embB genes that are associated with resistance to rifampin, isoniazid, fluoroquinolones, second-line injectable drugs and ethambutol. RESULTS: The average correlation for the identification of resistant and susceptible isolates using the three methods was approximately 94%. An association of mutations detected with variable resistance levels was shown. We propose a change in the breakpoint minimal inhibitory concentration for kanamycin to less than 5 µg/ml in the Sensititre MycoTB system. A pairwise comparison of the minimal inhibitory concentrations (MICs) of two different drugs revealed an increased correlation in the first-line drug group and a partial correlation in the second-line drug group, reflecting the history of the preferential simultaneous use of drugs from these groups. An increased correlation with the MICs was also observed for drugs sharing common resistance mechanisms. CONCLUSIONS: The quantitative measures of phenotypic drug resistance produced by the Sensititre MycoTB and the timely detection of mutations using the TB-TEST assay provide guidance for clinicians for the choice of the appropriate drug regimen.

Simultaneous drug resistance detection and genotyping of Mycobacterium tuberculosis using a low-density hydrogel microarray.

BACKGROUND: Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs. METHODS: We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray. RESULTS: The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype. CONCLUSIONS: The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex.

Evaluation of a low-density hydrogel microarray technique for mycobacterial species identification.

In addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of the gyrB gene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when using gyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.

Spoligotyping of Mycobacterium tuberculosis complex isolates using hydrogel oligonucleotide microarrays.

Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. This circumstance underscores the relevance of population studies of tuberculosis for transmission dynamics control. In this study, we describe a conversion of the spoligotyping of M.tuberculosis complex isolates on a platform of custom designed hydrogel microarrays (biochips). An algorithm of automated data processing and interpretation of hybridization results using online database was proposed. In total, the 445 samples were tested. Initially, 97 samples representing multiple species of M.tuberculosis complex and nontuberculous mycobacteria were used for protocol optimization and cut-off settings. The developed assay was further evaluated on the out-group of the 348 mycobacterial samples. Results showed high concordance with the conventional membrane-based spoligotyping method. Diagnostic sensitivity and diagnostic specificity of the spoligo-biochip assay were 99.1% and 100%, respectively. The analytical sensitivity was determined to be 500 genomic equivalents of mycobacterial DNA. The high sensitivity and specificity, ease of operation procedures, and the automatic processing of measured data make the developed assay a useful tool for the rapid and accurate genotyping of M. tuberculosis.

Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays.

BACKGROUND: The steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) requires rapid and reliable methods to identify resistant strains. The current molecular methods to detect MTB resistance to second-line drugs either do not cover an extended spectrum of mutations to be identified or are not easily implemented in clinical laboratories. A rapid molecular technique for the detection of resistance to second-line drugs in M. tuberculosis has been developed using hybridisation analysis on microarrays. METHODS: The method allows the identification of mutations within the gyrA and gyrB genes responsible for fluoroquinolones resistance and mutations within the rrs gene and the eis promoter region associated with the resistance to injectable aminoglycosides and a cyclic peptide, capreomycin. The method was tested on 65 M. tuberculosis clinical isolates with different resistance spectra that were characterised by their resistance to ofloxacin, levofloxacin, moxifloxacin, kanamycin and capreomycin. Also, a total of 61 clinical specimens of various origin (e.g., sputum, bronchioalveolar lavage) were tested. RESULTS: The sensitivity and specificity of the method in the detection of resistance to fluoroquinolones were 98% and 100%, respectively, 97% and 94% for kanamycin, and 100% and 94% for capreomycin. The analytical sensitivity of the method was approximately 300 genome copies per assay. The diagnostic sensitivity of the assay ranging from 67% to 100%, depending on the smear grade, and the method is preferable for analysis of smear-positive specimens. CONCLUSIONS: The combined use of the developed microarray test and the previously described microarray-based test for the detection of rifampin and isoniazid resistance allows the simultaneous identification of the causative agents of MDR and XDR and the detection of their resistance profiles in a single day.

An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1, HBV, and HCV.

We describe a novel microarray-based approach for simultaneous identification and quantification of human immunodeficiency virus type 1 (HIV-1) and hepatitis B and C viruses (HBV and HCV) in donor plasma specimens. The method is based on multiplex real-time RT-PCR performed within the microarray hydrogel pads. Double-stranded amplification products are simultaneously detected using nonspecific SYBR Green I dye due to the reaction run in separate pads bearing 5'-immobilized specific primers. Both the sensitivity and specificity of the assay, based on 132 blood specimens analyzed, were 100% (56, 26, and 8 specimens were seropositive to HBV HCV and HIV-1, respectively; 22 were positive to both HIV-1 and HCV and 2 positive to all three viruses; 18 samples were pathogen-negative). The dynamic range of the quantitative analysis covered a six-order interval ranging from 100 to 106 genome equivalents per assay. The 95% detection limits were 14 gEq for HIV-1, 10 gEq (1.7 IU) for HBV, and 15 gEq (7.5 IU) for HCV per assay. The proposed approach is considered to be versatile and could be adapted for simultaneous identification and quantification of numerous genetic targets.

Oligonucleotide microchip for subtyping of influenza A virus.

BACKGROUND: Influenza A viruses are classified into subtypes depending on the antigenic properties of their two outer glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Sixteen subtypes of HA and nine of NA are known. Lately, the circulation of some subtypes (H7N7, H5N1) has been closely watched because of the epidemiological threat they present. OBJECTIVES: This study assesses the potential of using gel-based microchip technology for fast and sensitive molecular subtyping of the influenza A virus. METHODS: The method employs a microchip of 3D gel-based elements containing immobilized probes. Segments of the HA and NA genes are amplified using multiplex RT-PCR and then hybridized with the microchip. RESULTS: The developed microchip was validated using a panel of 21 known reference strains of influenza virus. Selected strains represented different HA and NA subtypes derived from avian, swine and human hosts. The whole procedure takes 10 hours and enables one to identify 15 subtypes of HA and two subtypes of NA. Forty-one clinical samples isolated during the poultry fall in Novosibirsk (Russia, 2005) were successfully identified using the proposed technique. The sensitivity and specificity of the method were 76% and 100%, respectively, compared with the 'gold standard' techniques (virus isolation with following characterization by immunoassay). CONCLUSIONS: We conclude that the method of subtyping using gel-based microchips is a promising approach for fast detection and identification of influenza A, which may greatly improve its monitoring.