Molecular Methods For Diagnosis Research Articles

СЛУЧАЙ ИЗ ПРАКТИКИ: ЭПИЗОДИЧЕСКАЯ ПАРОКСИЗМАЛЬНАЯ МОЗЖЕЧКОВАЯ АТАКСИЯ 2 ТИПА

Instability walking is a fairly common complaint made by patients who come to be examined by a neurologist in polyclinics and hospitals. From a medical point of view, this symptom is included in the concept of "ataxia". Neurologists distinguish four types of ataxia depending on the localization of the pathological process: cortical, cerebellar, vestibular, and sensitive. There are often combinations of different types of ataxia in the same patient, for example, cortical-vestibular, sensitive-cerebellar. In addition to establishing the variant of the atactic syndrome, there is an urgent question of verifying the etiological factor that led to this pathology. Pathological heredity becomes the least obvious. In this article, we draw attention to a similar case from practice in order to form the alertness of doctors regarding hereditary coordination disorders. Episodic ataxia is a group of rare hereditary diseases of the nervous system, the main clinical manifestation of which are paroxysms of cerebellar ataxia of varying duration and frequency. The most studied from a genetic and clinical point of view are episodic cerebellar ataxia of the first and second types. The authors present a clinical case of episodic paroxysmal cerebellar ataxia type 2 in a 22-year-old patient. The diagnosis of the disease was based on a typical clinical picture and confirmed by a molecular genetic study that revealed a mutation in the CACNA1A gene. The article discusses aspects of the pathogenesis of the main forms of episodic paroxysmal ataxia, age of onset, frequency and duration of seizures, features of the clinical picture, neurological status. This clinical observation draws the attention of practitioners to the possibilities of modern methods of molecular genetic diagnosis and treatment of these conditions.

Drug-Resistant Tuberculosis: Development Mechanisms and Methods of Molecular Genetic Diagnosis

The widespread occurrence of drug-resistant tuberculosis is an important public health challenge. To better understand this phenomenon, the article summarizes current ideas on the development of mechanisms of Mycobacterium tuberculosis resistance to antituberculosis drugs. Special attention is paid to the mechanism of acquired resistance based on mutations in the genes encoding antituberculosis drug targets or enzymes that translate pro-drug into its active form; the effect of these mutations on fitness of the pathogen is in the focus of the article. It emphasizes the leading role of molecular genetic methods for diagnosing M. tuberculosis drug resistance and importance of these methods for preventing the expansion of the pathogen’s resistance range and the spread of resistant clones in the population. A comparison of sequencing and PCR-based methods capacities led to a conclusion that at the current stage of technological development it is reasonable to use each of these approaches for specific purposes: domestic PCR-based tests — for diagnosis, and sequencing — for basic research of M. tuberculosis evolution and epidemiological monitoring. Promising areas of M. tuberculosis resistance research were proposed to develop new approaches for diagnosis and treatment of drug-resistant tuberculosis and to provide effective personalized therapy.

Comparison of immunological and molecular methods for laboratory diagnosis of ocular toxoplasmosis in blood, serum and tears in Brazil.

Ocular toxoplasmosis (OT) is caused by protozoan T. gondii. Ophthalmological examination is considered the gold standard for OT diagnosis, and laboratory tests are used for diagnostic confirmation. However, these tests can present different results, which change depending on their basis, on sample type and on patients' clinical alteration. Thus, the aim of the present study is to assess immunodiagnostic and molecular techniques applied in blood, serum and tear fluid to diagnose T. gondii infection in patients seen at an Ophthalmology Clinic. In total, 160 patients were included in the study, 40 of them had OT with active lesions (G1); 40 had OT with healed lesions (G2), 40 had non-toxoplasmic uveitis (G3) and 40 had no ocular alterations (G4). Serum samples were subjected to Immunoenzymatic Assay (ELISA) and to Indirect Immunofluorescence Reaction (IFAT) to search for anti-T. gondii IgM and IgG. Tear fluid samples were analyzed through ELISA for IgA research. All blood and tear fluid samples were subjected to conventional polymerase chain reaction (cPCR) and in a Nested PCR model for T. gondii DNA amplification with targets B1, GRA7 and REP 529. IgG and IgM anti-T. gondii was detected in serum samples from 106 and 15 patients, respectively, when combining ELISA and IFAT results. Anti-T.gondii IgA antibodies were detected in 9.2% of the tear material. Nested PCR with GRA7 target showed higher positivity in blood samples (24.4%); Nested PCR with B1 target showed a higher frequency of positivity in tears (15%). Biological samples of patients with active lesions showed the highest positivity frequencies in all immunodiagnostic assays, as well as in most PCR models. The present results highlighted the need of associating techniques with different fundamentals to confirm OT diagnosis. Furthermore, further tear fluid analyses should be performed to validate this biological material as lesser invasive alternative for the more accurate OT diagnosis.

Ustekinumab as first line therapeutic choice of biological therapy in bionaive patient affected with ulcerative colitis and intestinal reactivation of cytomegalovirus infection

Ulcerative colitis is a chronic autoimmune disease characterized by inflammation of colonic mucous layer. Patients affected with ulcerative colitis are often subjected to immunosuppressive therapy that makes them susceptible to opportunistic infections. This article presents clinical case of moderate-to-severe ulcerative colitis, complicated by cytomegalovirus infection. We demonstrated the necessity of the inclusion of cytomegalovirus infection into the differential diagnostic algorithm of ulcerative colitis relapse using molecular methods of diagnosis. Also, we justified the choice of specific antiviral therapy and first line biological therapy in a bionaive steroid-dependent, refractory to thiopurines patient. The choice of ustekinumab as therapeutic armamentarium showed to be safe and effective in attainment and maintenance of persistent clinical, laboratory and endoscopic remission in a patient affected with ulcerative colitis with intestinal reactivation of cytomegalovirus infection.

Can Multiplex SYBR Green Real-Time PCR Assay Serve as a Detection and Quantification Method Comparable to the TaqMan Method for SARS-CoV-2 Diagnosis?

The reopening of schools, business, and social sectors during the COVID-19 pandemic has caused a current increase in the number of COVID-19 cases and clusters all over the globe. While the COVID-19 pandemic is far from over, the reopening and resumption of all economic sectors are essential to recovering the world economy. Health experts all over the world have determined that the real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) method is the gold standard for diagnosing COVID-19 infections due to the test’s high sensitivity and specificity. During the past 3 years when WHO declared the COVID-19 pandemic, the cost of laboratory diagnosis of COVID-19 using a robust RT-qPCR assay is still considerably expensive, especially for low and middle-income countries. Therefore, numerous studies have reported optimized SYBR green methods which are more economical than the qPCR probe assay. Continuous diagnostic testing is vital to mitigate the spread of COVID-19. However, there is a question as to whether SYBR Green may serve as an excellent detection and quantification method for molecular diagnosis to perform SARS-CoV-2 screening. This review summarizes the numerous studies using SYBR Green RT-PCR to detect SARS-CoV-2. The reliability of SYBR Green qPCR assays for determining gene expression based on their performance is justified and the quality is comparable to the TaqMan method.

Work-Up and Treatment Strategies for Individuals with PIK3CA-Related Disorders: A Consensus of Experts from the Scientific Committee of the Italian Macrodactyly and PROS Association.

PIK3CA-related disorders encompass many rare and ultra-rare conditions caused by somatic genetic variants that hyperactivate the PI3K-AKT-mTOR signaling pathway, which is essential for cell cycle control. PIK3CA-related disorders include PIK3CA-related overgrowth spectrum (PROS), PIK3CA-related vascular malformations and PIK3CA-related non-vascular lesions. Phenotypes are extremely heterogeneous and overlapping. Therefore, diagnosis and management frequently involve various health specialists. Given the rarity of these disorders and the limited number of centers offering optimal care, the Scientific Committee of the Italian Macrodactyly and PROS Association has proposed a revision of the most recent recommendations for the diagnosis, molecular testing, clinical management, follow-up, and treatment strategies. These recommendations give insight on molecular diagnosis, eligible samples, preferable sequencing, and validation methods and management of negative results. The purpose of this paper is to promote collaboration between health care centers and clinicians with a joint shared approach. Finally, we suggest the direction of present and future research studies, including new systemic target therapies, which are currently under evaluation in several clinical trials, such as specific inhibitors that can be employed to downregulate the signaling pathway.

Phenotype versus genotype discordant rifampicin susceptibility testing in tuberculosis: implications for a diagnostic accuracy.

An accurate diagnosis of drug resistance in clinical isolates is an important step for better treatment outcomes. The current study observed a higher discordance rate of rifampicin resistance on Mycobacteria Growth Indicator Tube (MGIT) drug susceptibility testing (DST) than Lowenstein-Jenson (LJ) DST when compared with the rpoB sequencing. We detected a few novel mutations and their combination in rifampicin resistance isolates that were missed by MGIT DST and may be useful for the better management of tuberculosis (TB) treatment outcomes. Few novel deletions in clinical isolates necessitate the importance of rpoB sequencing in large data sets in geographic-specific locations, especially high-burden countries. We explored the discordance rate on MGIT and LJ, which is important for the clinical management of rifampicin resistance to avoid the mistreatment of drug-resistant TB. Furthermore, MGIT-sensitive isolates may be subjected to molecular methods of diagnosis for further confirmation and treatment options.

Comparison of laboratory methods for diagnosis of human visceral leishmaniasis in the Center-West Region of São Paulo State, Brazil

Visceral leishmaniasis (VL) is a neglected disease and represents an important public health problem. The present study aimed to compare the conventional laboratory methods with the molecular method for VL diagnosis in human blood samples. Direct parasitological test of bone marrow aspirate (direct parasitological test), rapid immunochromatographic test (RIT), indirect immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR) were used to evaluate 26 human samples with clinical suspicion of VL. The positivity rates obtained for direct parasitological test, RIT, IFAT and PCR were 80.8%, 80.8%, 73.1% and 84.6%, respectively. PCR had greater positivity and agreement with the other methodologies. Therefore, the use of PCR in the diagnostic routine should be encouraged to complement the serological results.

Identification of Etiological Agents of Pertussis-Like Syndrome in Children Younger Than 5 Years Old Hospitalized in Southwestern Iran

Background: Despite the global vaccination program, there are many new cases of pertussis in different societies annually. Objectives: This study aimed to investigate the prevalence of some microorganisms associated with pertussis-like syndrome and compare the clinical presentations between Bordetella pertussis and pertussis-like syndrome in children. Methods: Children younger than 5 years old suspected of pertussis-like syndrome were admitted to a hospital in Ahvaz, Iran, and examined from July 2018 to July 2019. Nasopharyngeal samples were evaluated using molecular methods. The studied microorganisms were the following: Bordetella pertussis, B. parapertussis, Mycoplasma pneumoniae, Chlamydophila pneumoniae, adenovirus, respiratory syncytial virus, and parainfluenza virus type III. Results: Forty-five children were enrolled. Bordetella pertussis was detected in 15 cases (33.3%), respiratory syncytial virus in 14 (31.1%), C. pneumoniae in 3 (6.7%), and parainfluenza virus type III in 3 (6.7%). The collected samples were negative in terms of M. pneumoniae, adenovirus, and B. parapertussis. In the case of paroxysmal cough, the clinical symptoms were significantly different between pertussis and pertussis-like groups. Conclusions: The results indicated that children with pertussis-like syndrome are commonly infected with B. pertussis and respiratory syncytial virus, so more attention should be paid to this issue. The study also demonstrated the importance of molecular diagnosis methods, along with diagnosis based on clinical symptoms, in children suspected of pertussis-like syndrome.

Genetic variation of sequencing 18srDNA gene of Trichophyton mentagrophytes isolates

Background. Trichophyton species are considered the most frequent causative and considerable agents in infection concerns. Objective. The existing training designed to conduct the sequencing analysis of nucleotides of ITS1-5.8S-ITS2 for Trichophyton mentagrophytes. Methods. The isolation and identification of pathogenic fungi, Trichophyton mentagrophytes from clinical specimens was done based on the standard morphological and molecular methods. The genomic DNA of fungal isolates were extracted and purified to amplify with primers of 18S rRNA gene for detection and sequencing the nitrogenous bases to define the genetic variation among clinical isolates of Trichophyton mentagrophytes in compared with strains recorded in NCBI GenBank. Results. Eight isolates of Trichophyton mentagrophytes were isolated and identified from clinical specimen of dermatophytosis. The DNA sequencing analysis showed the presence of some genetic variation in nitrogenous bases between the local isolates and that recorded in locus of world NCBI GenBank. Multiple Alignment sequence using NCBI BLAST revealed the recoding a new mutant isolate among the local isolates based on DNA homology percent. Conclusion. The present results proved the successful the use of molecular methods in diagnosis the fungal isolates of Trichophyton mentagrophytes especially the sequencing analysis to find the genetically bases of these isolates.

A-294 Duplicate Testing of Samples Suspected of SARS-CoV-2 Infection Could Prevent Misdiagnosis of Covid-19

Abstract Background There is no doubt that the whole world has been shaken by the SARS-CoV-2 pandemic, which has affected both global health and the global economy. At the same time the importance of molecular methods for the accurate diagnosis of respiratory infections has been reinforced together with the need of testing methodologies that minimize the risk of misleading results. During the last three years of the pandemic, SARS-CoV-2 exhibited a tremendous adaptation capability through numerous mutations, leading to the appearance of the well-known Variants of Concern (VOCs) responsible of increased transmission rates. Therefore, the development of molecular methods capable of detecting all variants has become a challenge. To overcome this, potential genetic targets with low mutation rates have been identified, including ORF1ab, N gene, S gene and E gene. While Real-time PCR based detection assays of viral genomes are very accurate, testing naso/oropharyngeal samples using singlet analysis does not guarantee the error free detection of true positive results for SARS-CoV-2 infections. Furthermore, it has been documented that the probability of a false-negative testing results is influenced by a number of variables . Therefore, testing protocol adaptations, such as replicate testing, has been shown to efficiently reduce the probability of missing SARS-CoV-2 positive cases. The pilot study presented here investigates the outcome of testing 10 014 nasopharyngeal samples collected from patients with a suspected SARS-CoV-2 infection using two separate detection kits. In this study the error rate of single vs duplicate testing is discussed. Methods 10 014 nasopharyngeal swabs, collected from three different U.K. hospital trusts and Kent based care homes, were tested at the North Kent Pathology Service (NKPS). Each swab collected was placed in sterile transport media and stored at 4°C prior to viral genome isolation using the HigherPurity™ Viral RNA extraction kit. All collected specimens were processed within 24 h from collection. Total RNA isolated was immediately analyzed using two commercial CE-IVD kits: Vitassay qPCR SARS-CoV-2 and Primerdesign™ LTD, Coronavirus (COVID-19) Genesig Real-Time PCR assay. All samples were tested in duplicate. Results Following the testing and subsequent analysis, 491 samples (4.9%) were detected positive for SARS-CoV-2 using both assay kits. Of these, 302 samples (61.5%) were reported as duplicate positive (on both replicate 1 and 2) whereas 189 samples (38.5%) were detected positive on either replicate 1 or 2. In particular, 85 (17.3%) were detected positive on replicate 1 and 104 (21.2%) were detected positive on replicate 2. Conclusions Approximately 19% of tested individuals were detected as SARS-CoV-2 positive based on a positive Real-time PCR result in one of the two reactions. This percentage represents the potential SARS-CoV-2 infections that can be missed when a single reaction Real-time PCR testing is employed for infection detection in the wider population, and equates to 940 individuals out of 100 000 potentially being falsely confirmed as SARS-CoV-2 negative The authors believe that in the presence of fast transmitting variants this is a significant number of false negative tests and should be taken into consideration when testing protocols are developed and disease modelling is employed.

Identification of a Novel ARSA Gene Mutation Through High-Throughput Molecular Diagnosis Method in Two Girls with Late Infantile Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare leukoencephalopathy caused by pathogenic mutations in the ARSA gene. It manifests as severe motor symptoms, mental problems, and sometimes, seizures. We aimed to investigate the phenotypic manifestations and genetic causes of MLD in an Iranian family. We present the case of a 3-year-old girl who presented with hypotonia, muscular atrophy, and seizures. Neurological and neuromuscular examinations were performed to evaluate clinical characteristics. Whole exome sequencing (WES) was used to detect disease-causing variants. In silico analysis was performed to predict the pathogenicity of this variant. GROMACS software was utilized for molecular dynamic simulation (MDS). Neurological studies revealed marked slowing of motor conduction velocities and an increased motor unit action potential duration. Brain MRI scan revealed white matter abnormalities. By applying WES, we identified a novel homozygous missense variant (NM_000487.6, c.938G > C, p.R313P) in ARSA. Direct sequencing identified this homozygous variant in her asymptomatic younger sister, whereas both parents carried a heterozygous variant. This mutation has not been reported in genetic databases or in literature. In silico analysis predicted that any variation in this DNA position would cause disease, as it is highly conserved. The c.938G > C variant was classified as a pathogenic variant according to ACMG/AMP guidelines. MDS analysis indicated that c.938G > C had a significant impact on both the structure and stabilization of ARSA, ultimately resulting in impaired protein function. The identification of this variant expands the spectrum of ARSA gene mutations associated with MLD and highlights the importance of genetic testing for the diagnosis of MLD.

Breast cancer: genetic personal risk factors: A review

Determination of cancer risk factors allow us to develop diagnostics tests that improved identification and reduced the rate of mortality of most frequent cancer diseases including breast cancer, prostate cancer, gastrointestinal tumors. Today individual risk of breast cancer considers personal genetics, medical history of patient, lifestyle, and a number of additional factors. Calculation of the first mathematical models for breast cancer risk assessment included anthropometric data, hormonal status, and family history of cancer. The discovery of BRCA1 and BRCA2 genes role in the development of breast cancer and the accumulation of data from population studies contributed to the introduction of the genetic component into mathematical models. The trend of the last decade is the integration of the polygenic component into the scheme for calculating the individual risk of breast cancer. In this review, we have analyzed existing models, assessed their relevance for certain groups of patients, studied the trends in the development of methods for molecular genetic diagnosis of breast cancer and determining the personal risk of developing the disease.

Exome sequencing identifies novel variants associated with non-syndromic hearing loss in the Iranian population.

Autosomal recessive non-syndromic hearing loss (ARNSHL) is a public health concern in the Iranian population, with an incidence of 1 in 166 live births. In the present study, the whole exome sequencing (WES) method was applied to identify the mutation spectrum of NSHL patients negative for GJB2 gene mutations. First, using ARMS PCR followed by Sanger sequencing of the GJB2 gene, 63.15% of mutations in patients with NSHL were identified. Among the identified mutations in GJB2:p.Val43Met and p.Gly21Arg were novel. The remaining patients were subjected to WES, which identified novel mutations including MYO15A:p.Gly39LeufsTer188, ADGRV1:p.Ser5918ValfsTer23, MYO7A: c.5856+2T>c (splicing mutation), FGF3:p.Ser156Cys. The present study emphasized the application of WES as an effective method for molecular diagnosis of NSHL patients negative for GJB2 gene mutations in the Iranian population.

Kaedah Diagnostik Semasa dan Penggunaan Ujian Titik Penjagaan Pantas (POC) Bagi Mendiagnos Hiperkolesterolemia Famili (FH)

Familial hypercholesterolemia (FH) is an autosomal dominant inherited genetic disease characterized by increased concentrations of low-density lipoprotein (LDL-C) cholesterol in the blood. The risk of premature coronary heart disease in FH patients may increase without early treatment. Advancement in molecular biology techniques has enable early detection and diagnosis of FH. These techniques are cost-effective and have a shorter turnaround time. The current diagnostic tools available for FH diagnosis involving algorithm-based scoring criteria and various molecular diagnosis methods including next-generation sequencing (NGS), Sanger sequencing, Multiplex ligation-dependent probe amplification (MLPA) and DNA hybridisation assay are discussed in this review. However, molecular genetic testing is not widely available due to time-consuming procedures, high cost and requires trained personnel. Thus, this 36 review highlights the use of point of care (POC) testing as an approach to diagnose FH, particularly in countries lacking infrastructure and expertise in this field. Lateral flow testing (LFA) has gained attention as a POC diagnostic tool due to its simplicity, low cost and involved simple procedure and settings. The advantages of LFA made this technique a potential tool in addressing challenges in diagnosing FH, particularly for early diagnosis of family members.

OPTIMIZATION OF MOLECULAR GENETIC DIAGNOSTICS OF PATIENTS WITH ADVANCED NON-SMALL CELL LUNG CANCER BY INTRODUCING ROS1 TESTING IN THE REPUBLIC OF KAZAKHSTAN

Relevance: Currently, molecular diagnosis in NSCLC in Kazakhstan includes detection of EGFR, ALK driver mutations status, and PD-L1-status, but not ROS1, what limits the access of patients with this driver mutation to vital therapy.
 The study aimed to optimize the methods of molecular genetic diagnosis of patients with NSCLC by introducing ROS1 testing in the Republic of Kazakhstan.
 Methods: The biopsy and surgical material of non-small cell lung cancer (NSCLC) fixed in 10% buffered formalin was studied. After the initial morphological diagnosis of adenocarcinoma, EGFR, and ALK mutation status determination, EGFR, and ALK-negative tumor assays were sent for further determination of ROS1 mutation status. First, we performed immunohistochemistry (IHC) using the Ventana BenchMark Ultra platform using the ROS1 antibody (SP283) and the OptiView DAB Detection Kit imaging system. After that, samples with positive and doubtful IHC results were sent for RT-PCR (reverse transcriptase polymerase chain reaction) to confirm the ROS1 mutation status.
 Results: A total of 99 tumor samples from patients with EGFR-negative and ALK-negative lung adenocarcinoma were studied by IHC from January 01 till September 30, 2022. The results of IHC staining were assessed as: 0 (negative) – 59 samples, 1+ (negative) – 25 samples, 2+ (doubtful) – 12 samples, 3+ (positive) – 3 samples. Cases with ≥70% immunostaining were considered positive. Samples with an IHC stain score of 2+ (doubtful), 3+ (positive), and a few samples of 1+ were sent for confirmation by PCR.
 Overall, 22 samples were tested using RT-PCR, and results were considered as follows:1 (4%) – positive, 13 (59%) – negative, 8 (37%) -– invalid.
 Conclusion: A large proportion of positive and questionable results were obtained when determining ROS1 mutation status using IHC, and a large proportion of invalid results during subsequent RT-PCR testing. Choosing methods for nationwide ROS1 implementation, one should evaluate the economics of the methods to be implemented and compare them with a standard validated FISH method.

OPTIMIZATION OF MOLECULAR GENETIC DIAGNOSTICS OF PATIENTS WITH ADVANCED NON-SMALL CELL LUNG CANCER BY INTRODUCING ROS1 TESTING IN THE REPUBLIC OF KAZAKHSTAN

Relevance: Currently, molecular diagnosis in NSCLC in Kazakhstan includes detection of EGFR, ALK driver mutations status, and PD-L1-status, but not ROS1, what limits the access of patients with this driver mutation to vital therapy.
 The study aimed to optimize the methods of molecular genetic diagnosis of patients with NSCLC by introducing ROS1 testing in the Republic of Kazakhstan.
 Methods: The biopsy and surgical material of non-small cell lung cancer (NSCLC) fixed in 10% buffered formalin was studied. After the initial morphological diagnosis of adenocarcinoma, EGFR, and ALK mutation status determination, EGFR, and ALK-negative tumor assays were sent for further determination of ROS1 mutation status. First, we performed immunohistochemistry (IHC) using the Ventana BenchMark Ultra platform using the ROS1 antibody (SP283) and the OptiView DAB Detection Kit imaging system. After that, samples with positive and doubtful IHC results were sent for RT-PCR (reverse transcriptase polymerase chain reaction) to confirm the ROS1 mutation status.
 Results: A total of 99 tumor samples from patients with EGFR-negative and ALK-negative lung adenocarcinoma were studied by IHC from January 01 till September 30, 2022. The results of IHC staining were assessed as: 0 (negative) – 59 samples, 1+ (negative) – 25 samples, 2+ (doubtful) – 12 samples, 3+ (positive) – 3 samples. Cases with ≥70% immunostaining were considered positive. Samples with an IHC stain score of 2+ (doubtful), 3+ (positive), and a few samples of 1+ were sent for confirmation by PCR.
 Overall, 22 samples were tested using RT-PCR, and results were considered as follows:1 (4%) – positive, 13 (59%) – negative, 8 (37%) -– invalid.
 Conclusion: A large proportion of positive and questionable results were obtained when determining ROS1 mutation status using IHC, and a large proportion of invalid results during subsequent RT-PCR testing. Choosing methods for nationwide ROS1 implementation, one should evaluate the economics of the methods to be implemented and compare them with a standard validated FISH method.

Non-invasive methods of molecular diagnosis, clinical monitoring and approaches to the personalized therapy of diffuse midline glioma

The purpose of the study was to summarize and analyze modern data about non-invasive methods of molecular diagnosis and approaches to the personalized therapy of diffuse midline glioma (DMG). Material and Methods. The search and analysis of publications was carried out using Google Scholar, Pubmed, Elsevier, Web of Science, Elibrary systems. The review includes publications published from 2011 to 2022. Of the 102 articles found, 59 were used to write the review. Results. In this review, we discuss the spectrum of somatic driver mutations present in DMG tumor cells and their relationship with the sensitivity of tumor cells to certain types of therapy - a pharmacogenetic approach to the selection of individual treatments (targeted therapy). We provide examples of new methods of targeted therapy for DMG, which are currently at the stage of preclinical laboratory development. Also, we discuss examples of the use of 3D cell cultures for the development of targeted therapies, including the use of perfusion systems. The review describes the methods of analysis of liquid biopsy, which allow the detection of tumor-specific biomarkers in the non-invasive diagnosis of DMG, including a number of methods that have not yet been tested in the clinic. The following is a list of tumor-specific biomarkers for diagnosing, monitoring, and selecting targeted therapy for DMG. Finally, we discuss the possibility of implementing these methods in the clinic and present the results of several clinical trials. Conclusion. In oncology, new methods of molecular genetics, such as analysis of liquid biopsy, allow diagnosis and monitoring of treatment in cases where classical methods that require tissue sampling are not applicable (for example, the analysis of genetically heterogeneous tumors and tumors of surgically inaccessible localization). These tumors include DMG, a primary brain tumor most common in children. The available data confirm the relevance of the search for new specific tumor biomarkers, as well as targets for targeted therapy of the paediatric-type diffuse gliomas.

Pyrococcus furiosus Argonaute with isothermal amplification for fast and ultra-sensitive diagnosis of acute hepatopancreatic necrosis disease in shrimp

The acute hepatopancreas necrosis disease (AHPND) caused by Vibrio species containing the virulence Photorhabdus insecticidal-related genes, PirAvp and PirBvp, is a high-mortality-rate disease in shrimp farming with strong transmission, quick onset, and severe symptoms. It is vital to control the disease transmission with frequent screening and early diagnosis; hence, developing fast diagnostic methods for AHPND with point-of-care testing (POCT) capacity is particularly important. In this study, we have established a method for fast and ultra-sensitive diagnosis of AHPND by combining a nuclease, Pyrococcus furiosus Argonaute (PfAgo), with recombinase polymerase amplification (RPA). Through systematic screening and optimizations, a strategy of using two guide DNA (gDNA) molecules to program PfAgo is realized for precisely tailored nuclease cleavage. The fabricated procedure can be completed in 75 min with a sensitivity of one copy per reaction, which is better than all the currently available molecular diagnosis methods for AHPND. The procedure requires only simple device support, which is suitable for POCT. This study exhibits the advantages of the PfAgo cleavage system concerning flexibility and sensitivity. The established RPA-PfAgo method is valuable for the prevention of AHPND in shrimp farms, presenting a good example of utilizing PfAgo for highly sensitive molecular diagnosis.

Diagnostic methods for peritoneal molecular residual disease in gastric cancer

Peritoneal metastasis of gastric cancer serving as the most frequent form of metastasis, is one of the leading causes of death. A portion of surgically treated patients often suffer from small peritoneal residual metastasis, which will lead to recurrence and metastasis of gastric cancer patients after surgery. Given these, the prevention and treatment of peritoneal metastasis of gastric cancer deserves more attention. Molecular residual disease (MRD) refers to the molecular abnormalities of tumor origin that cannot be found by traditional imaging or other laboratory methods after treatment, but can be found by liquid biopsy, representing the possibility of tumor persistence or clinical progress. In recent years, the detection of MRD based on ctDNA has gradually become a research hotspot in the prevention and treatment of peritoneal metastasis. Our team established a new method for MRD molecular diagnosis of gastric cancer, and reviewed the research achievements in this field.