Dmitry Gryadunov

DNA microarrays in the clinic: infectious diseases.

We argue that the most-promising area of clinical application of microarrays in the foreseeable future is the diagnostics and monitoring of infectious diseases. Microarrays for the detection and characterization of human pathogens have already found their way into clinical practice in some countries. After discussing the persistent, yet often underestimated, importance of infectious diseases for public health, we consider the technologies that are best suited for the detection and clinical investigation of pathogens. Clinical application of microarray technologies for the detection of mycobacteria, Bacillus anthracis, HIV, hepatitis and influenza viruses, and other major pathogens, as well as the analysis of their drug-resistance patterns, illustrate our main thesis.

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

BackgroundThe 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.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.

Gel-based microarrays in clinical diagnostics in Russia

Immobilization of molecular probes in 3D hydrogel elements provides some essential advantages compared with conventional flat surfaces. In this article, an integrated technology based on the use of low-density microarrays comprised of hemispherical gel elements, developed at the Engelhardt Institute of Molecular Biology (Moscow, Russia) for various applications will be reviewed. The structure of the gel can be adapted for immobilization of virtually any biological molecules in a natural hydrophilic environment. The discrimination between matching and mismatching duplexes of nucleic acids in these conditions is more reliable than on conventional flat surfaces, minimizing the number of elements needed to detect specific sequences. Protein molecules immobilized in hydrogel-based biochips better preserve their biological properties. As described in this article, such biochips were successfully applied for laboratory diagnostics in a wide variety of clinical conditions involving the identification of bacterial and viral pathogens, cancer-related mutations and protein tumor markers.

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.

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.

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.

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.

Gel-Based Microchips: History and Prospects

The review describes the history of formation and development of the microchip technology and its role in the human genome project in Russia. The main accent was done on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous models. The microchips are manufactured by photoinitiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical properties and stability; this allowed one to work with the DNA fragments of up to 500 nt in length, as well as with quite large protein molecules. At present, the gel-based microchips are widely applied to solve different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA–protein interactions. The oligonucleotide microchips are a cheap and reliable diagnostic tool designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; of orthopoxviruses, including the smallpox virus; of the anthrax pathogen; and chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteo-mics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which opens a wide potential for creating biosensors on the basis of microchips.

Drug Resistance Mechanisms in Bacteria Causing Sexually Transmitted Diseases and Associated with Vaginosis

Here, we review sexually transmitted diseases (STDs) caused by pathogenic bacteria and vaginal infections which result from an overgrowth of opportunistic bacterial microflora. First, we describe the STDs, the corresponding pathogens and the antimicrobials used for their treatment. In addition to the well-known diseases caused by single pathogens (i.e., syphilis, gonococcal infections, and chlamydiosis), we consider polymicrobial reproductive tract infections (especially those that are difficult to effectively clinically manage). Then, we summarize the biochemical mechanisms that lead to antimicrobial resistance and the most recent data on the emergence of drug resistance in STD pathogens and bacteria associated with vaginosis. A large amount of research performed in the last 10–15 years has shed light on the enormous diversity of mechanisms of resistance developed by bacteria. A detailed understanding of the mechanisms of antimicrobials action and the emergence of resistance is necessary to modify existing drugs and to develop new ones directed against new targets.

Detection of single-nucleotide polymorphisms in the p53 gene by LDR/RCA in hydrogel microarrays

To find single-nucleotide polymorphisms (SNPs) in the human genome, three modern technologies of molecular genetic analysis were combined: the ligase detection reaction (LDR), rolling circle amplification (RCA), and immobilized microarray of gel elements (IMAGE). SNPs were detected in target DNA by selective ligation of allele-specific nucleotides in microarrays. The ligation product was assayed in microarray gel pads by RCA. Two variants of microarray analysis were compared. One included selective ligation of short oligonu-cleotides immobilized in a microarray with subsequent amplification with a preformed circular probe (a common circle). The probe was especially designed for human genome research. The other variant employed immobilized allele-specific padlock probes, which could be circularized as a result of selective ligation. Codon 72 SNP of the human p53 gene was used as a model. RCA in microarrays proved to be a quantitative assay and, in combination with LDR, allowed efficient discrimination of alleles. The principles and prospects of LDR/RCA in microarrays are discussed.