Using experience during the COVID-19 pandemic to ensure the quality of PCR studies.

The issues of laboratory diagnostics have been relevant since the first days of the SARS-CoV-2 pandemic and play a key role in the fight against the spread of a new coronavirus infection. A direct method for the etiological diagnosis of the causative agent of COVID-19 is the detection of SARS-CoV-2 RNA using the nucleic acid amplification method. In the context of a pandemic and the mass appeal of patients for medical care, the issues of ensuring the quality of ongoing molecular biological studies at all stages (preanalytical, analytical, postanalytical) become the most relevant. the results and timing of the study not only affect the diagnosis and treatment tactics of a particular patient, but are also the basis for the introduction of anti-epidemic measures, the adoption of organizational measures. The study is to summarize the experience in creating an effective and reliable system for managing the quality of molecular biological research in a pandemic using the example of a federal budgetary healthcare institution. The experience of the laboratory of a federal healthcare institution in the context of the COVID-19 pandemic was analyzed, errors were analyzed at the preanalytical, analytical and postanalytical stages of PCR studies with the identification of quality criteria, the impact on which significantly leads to quality improvement. The quality control system for PCR studies is based on the development of regulatory documents and instructions for the patient and laboratory staff, registration of all documents in a single information system with access to information from all structural divisions, with the possibility of uploading data to the patient's personal account on the institution's website. Quality indicators of all stages of PCR studies and measures that significantly affect the quality of laboratory studies were identified; Measures have been identified to reduce the turnaround time of a PCR test: distribution of biomaterial flows, optimization of operators' work, purchase of additional equipment, a patient feedback system, and an infection control information system. The obtained results make it possible to create a reliable quality control system, minimize the risk of obtaining erroneous research results, optimizing the work of the clinical diagnostic laboratory and increasing its productivity.

[PCR research as an auxiliary method for diagnostics of periphresitical infection after endostreditision of joints (clinical case).]

Periprosthetic infection (PPI) after arthroplasty of large joints is the third (among the main causes of unsatisfactory results of surgical treatment) a serious threat to the health of patients. The «gold standard¼ for the diagnosis of PPI is the bacteriological examination of samples of periprosthetic tissues and synovial fluid. In 10-30% of cases, it is impossible to isolate microorganisms, which is explained by the difficulty of cultivation and taking antibiotics before sampling. The purpose of study is to demonstrate the diagnostic value of PCR diagnostics for identifying the genetic material of an infectious pathogen of a culture-negative periprosthetic infection. Material of the study is a description of a clinical case of a culture-negative periprosthetic infection that caused a second two-stage revision of the hip joint prosthesis In the first episode of PPI that occurred 3 years after hip replacement, a microbiological examination of the puncture of the trochanteric zone of the operated joint revealed a massive increase in methicillin-resistant Staphylococcus epidermidis (MRSE). A two-stage revision joint replacement was performed. 5 years after the revision, the patient was hospitalized with clinical and radiological signs of PPI, while examining the puncture of the joint revealed characteristic PPI cytosis. Microbiological examination of punctate and intraoperative aspirate at the first stage of the repeated two-stage revision endoprosthesis replacement did not reveal aerobic and anaerobic microorganisms. In PCR studies, the DNA of methicillin-sensitive Staphylococcus aureus (MSSA) was detected in washouts from the removed components of the endoprosthesis; no resistance marker (mecA gene) was found. Given the concomitant oncological disease, this result determined the appointment of pathogenetic antibiotic therapy, the effectiveness of which was confirmed after 8 weeks at the II stage of revision. The PCR study of joint and trochanteric punctures (before surgery), flushing from the removed spacer components (after ultrasound treatment) and intraoperative aspirate from the joint did not reveal Staphylococcus aureus DNA and resistance marker (mecA gene). In some cases of periprosthetic infection, traumatologists and orthopedists deal with culturally negative results of a microbiological study of the patient's biomaterial and swabs from the components of endoprostheses in the presence of clinical manifestations of PPI, confirmed by laboratory diagnostics and X-ray examination. According to the literature, such clinical situations are observed in 10-30% of cases and are caused by previous antibiotic therapy in the early stages of an infectious complication. After surgical treatment of PPI for the selection of adequate antibiotic therapy, such patients need to at least indirectly determine the type of infection pathogen, which is achieved by the use of additional diagnostic methods, such as a PRC study. In the case described by us, after a course of antibiotic therapy, prescribed according to the results of the first PCR study, the patient's body does not contain DNA traces of the desired infectious agent. Thus, the repeated PCR not only confirmed the accuracy of the initial diagnosis of the source of infection, but also further illustrated the success of the rehabilitation of the periprosthetic infection using a correctly selected antibacterial drug at the previous stage of the study. The use of the PCR method made it possible to diagnose the pathogen and prescribe adequate antibiotic therapy for culture-negative periprosthetic infection.

Spectrum of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies.

Distribution of cancer-predisposing mutations demonstrates significant interethnic variations. This study aimed to evaluate patterns of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies. APC gene defects were identified in 26/38 (68%) subjects with colon polyposis; 8/26 (31%) APC mutations were associated with 2 known mutational hotspots (p.E1309Dfs*4 [n = 5] and p.Q1062fs* [n = 3]), while 6/26 (23%) mutations were novel (p.K73Nfs*6, p.S254Hfs*12, p.S1072Kfs*9, p.E1547Kfs*11, p.L1564X and p.C1263Wfs*22). Biallelic mutations in MUTYH gene were detected in 3/12 (25%) remaining subjects with polyposis and in 6/90 (6.7%) patients with colorectal cancer (CRC) carrying KRAS p.G12C substitution, but not in 231 early-onset CRC cases negative for KRAS p.G12C allele. In addition to known European founder alleles p.Y179C and p.G396D, this study revealed a recurrent character of MUTYH p.R245H germ-line mutation. Besides that, 3 novel pathogenic MUTYH alleles (p.L111P, p.R245S and p.Q293X) were found. Targeted next-generation sequencing of 7 APC/MUTYH mutation-negative DNA samples identified novel potentially pathogenic POLD1 variant (p.L460R) in 1 patient and known low-penetrant cancer-associated allele CHEK2 p.I157T in 3 patients. The analysis of 1120 healthy subjects revealed 15 heterozygous carriers of recurrent MUTYH mutations, thus the expected incidence of MUTYH-associated polyposis in Russia is likely to be 1:23 000.

The nucleoid protein Dps binds genomic DNA of Escherichia coli in a non-random manner.

Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions accumulating them in the form of Fe2O3 within its protein cavity, interacts with DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis from some genes via competition with RNA polymerase or, vice versa, competing with inhibitors to activate transcription.

Hereditary breast-ovarian cancer syndrome in Russia.

Hereditary breast-ovarian cancer syndrome contributes to as much as 5-7% of breast cancer (BC) and 10-15% of ovarian cancer (OC) incidence. Mutations in the "canonical" genesBRCA1andBRCA2occur in 20-30% of affected pedigrees. In addition toBRCA1andBRCA2 mutations, germ-line lesions in theCHEK2,NBS1, andPALB2genes also contribute to familial BC clustering. The epidemiology of hereditary breast-ovarian cancer in Russia has some specific features. The impact of the "founder" effect is surprisingly remarkable: a single mutation,BRCA15382insC, accounts for the vast majority ofBRCA1defects across the country. In addition, there are two other recurrentBRCA1alleles:BRCA14153delA andBRCA1185delAG. BesidesBRCA1, in Russia breast cancer is often caused by germ-line alterations in theCHEK2andNBS1genes. In contrast toBRCA1andBRCA2, theCHEK2andNBS1heterozygosity does not significantly increase the OC risk. Several Russian breast cancer clinics recently started to investigate the efficacy of cisplatin in the therapy ofBRCA1-related cancers; initial results show a unique sensitivity ofBRCA1-associated tumours to this compound.