[Clinical and diagnostic value of including PCR blood test in the traditional algorithm for identifying causative agents of infective endocarditis: a cohort study of 124 patients].

BACKGROUND: If infective endocarditis (IE) is suspected, the determination of the etiology is of fundamental importance for the verification of the disease and the appointment of effective therapy. Microbiological diagnostic features are important, but they often need to be supplemented by culture-independent studies of pathological agents. AIM: To investigate of the diagnostic advantage and value of quantitative analysis of molecular biological methods (polymerase chain reaction - PCR, sequencing) in addition to microbiological examination of whole venous blood in IE. MATERIALS AND METHODS: We examined 124 patients with suspected or significant IE (DUKE 2015) hospitalized in the Vinogradov City Clinical Hospital (2015-2021). All patients underwent parallel microbiological (cultural) and molecular biological (PCR or PCR followed by sequencing) examination of venous whole blood samples. RESULTS: The introduction of an early parallel PCR study into the algorithm for the etiological diagnosis of IE made it possible to obtain an additional advantage in 43/124 (34.7%) patients, which made it possible to exclude unreliable results in the determination of CoNS skin commensals and pathogens atypical for IE or contamination and identify the true pathogens, and also for the first time to isolate the etiopathogenetic pathogen with a negative microbiological study. It was shown that in IE associated with CoNS, the association with the disease was confirmed by PCR in 21.4% (3/14) and refuted in 71.4% (10/14). The coincidence of the results of microbiological and PCR studies of blood samples was obtained only in 35/95 (36.8%). Positive results of PCR analysis of blood of biological material with negative results of culture were obtained in 22/51 (43.1%), of which 2/22 (9.0%) were able to confirm the presence of Bartonella spp DNA. The presented complex algorithm made it possible to significantly increase the possibility of intravital identification of the pathogen in the blood from 58.9 to 76.6%. IE with unknown etiology was present in 29/124 (23.4%) patients. A parallel PCR study allowed timely correction of antibiotic therapy in 43/124 (34.7%) patients. CONCLUSION: Expansion of indications for the use of PCR studies, primarily whole venous blood samples, is justified, not only in IE with negative results of microbiological examination, but also as a control method for the reliability of the results of traditional (cultural) diagnostic methods.

[Infective Endocarditis with Unknown Etiology: Possibilities of Conquering and Role of Microbiologistics].

Current infectious endocarditis (IE) is characterized by changes in its etiological and epidemiological profiles associated with increased incidence of IE of undetermined etiology. This requires a search for ways to enhance the effectivity of diagnosis. Microbiologistics along with high-tech methods becomes decisively important for identifying the pathogen by studying cultures of blood and tissues from the affected heart valve. This determines timely diagnosis and treatment to be introduced to medical practice as a component of personalized medicine. The article focuses on the validity and features of microbiological (cultural), immunochemical, and molecular biological [MALDI-TOF MS (matrix-activated laser desorption/ionization with time-of-flight mass spectrometry), polymerase chain reaction, sequencing] studies.

[Infective endocarditis: Importance of molecular biological techniques in etiological diagnosis]. / Infektsionnyi endokardit: znachenie molekulyarno-biologicheskikh metodov v etiologicheskoi diagnostike.

AIM: To investigate the specific features of conventional bacteriological methods and current molecular biological techniques for the etiological diagnosis of infective endocarditis (IE). SUBJECTS AND METHODS: Examinations were made in 53 patients treated at City Clinical Hospital Sixty-Four, Moscow Healthcare Department, in 2012-2015 who underwent simultaneous bacteriological and molecular biological (polymerase chain reaction (PCR) or PCR with further sequencing) examinations of blood or resected cardiac valve tissues. RESULTS: The investigation included 53 patients (31 men; median age, 62 years) with IE (Duke 2009); its primary form was observed in 32 (60.4%) patients. Blood bacteriological tests and PCR assays were positive in 28 (52.8%) and 34 (64.2%) patients, respectively. There were concordant results in 21 of the 28 positive blood culture cases and discordant results in 7 (25%); at the same time 3 cases showed a compete discordance in the detected causative agents (the growth of Enterococcus spp. was revealed by bacteriological examination and that of Staphylococcus spp., Streptococcus spp., and Escherichia coli by DNA PCR) and a pathogen could not be identified by DNA PCR in 4 patients who had positive blood bacteriological results. The positive PCR results for cocci and fungi were obtained in 10 of the 25 (47.2%) examinees with culture-negative IE. Rare causative agents were not revealed. The tissues obtained from 8 resected damaged heart valves displayed a wider spectrum of pathogens than did blood samples, which was associated with the formation of bacterial films. CONCLUSION: The etiological agent of IE was revealed in venous blood by bacteriological examination in 52.8% of the examinees, by PCR in 64.2%, and by either in 71.7%. There were concordant and discordant results in 67.9 and 32.1% of the patients, respectively; among whom 18.9% were found to have pathogen DNA revealed by PCR in culture-negative IE.

[The validation of kit of reagents for quantitative detection of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode].

The validation of kit of reagents destined to detection and quantitative evaluation of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode was implemented. The comparison was made against international WHO standard--The first WHO international standard for human cytomegalovirus to implement measures the kit of reagents "AmpliSens CMV-screen/monitor-FL" and standard sample of enterprise DNA HCMV (The central research institute of epidemiology of Rospotrebnadzor) was applied. The fivefold dilution of international WHO standard and standard sample of enterprise were carried out in concentrations of DNA HCMV from 106 to 102. The arrangement of polymerase chain reaction and analysis of results were implemented using programed amplifier with system of detection of fluorescent signal in real-time mode "Rotor-Gene Q" ("Qiagen", Germany). In the total of three series of experiments, all stages of polymerase chain reaction study included, the coefficient of translation of quantitative evaluation of DNA HCMV from copy/ml to ME/ml equal to 0.6 was introduced for this kit of reagents.

[The development and testing of reagents kit for detection and qualitative evaluation of DNA of methicillin sensitive and methicillin resistant Staphylococcus aureus and also methicillin resistant coagulase negative Staphylococcus spp. applying technique of polymerase chain reaction in "real time" mode].

The reagents kit is developed to identify and quantitatively detect DNA of methicillin sensitive and methicillin resistant Staphylococcus aureus, methicillin resistant coagulase negative Staphylococcus spp. in biological material using technique of polymerase chain reaction with hybridizational fluorescent detection and having higher analytical and diagnostic characteristics. The application of the given reagents kit makes it possible to optimize the epidemiologic monitoring of propagation of methicillin resistant strains of Staphylococcus spp. Significantly decreasing duration and laboriousness of study.

[Use of immunological and molecular biological methods to diagnose cerebral toxoplasmosis in HIV infection].

Cerebral toxoplasmosis is one of the leading causes of neurologic diseases with high mortality rates in patients with HIV infection. Invasion was difficult to diagnose for a number of objective reasons. The objective of the investigation was to determine the clinical sensitivity of different laboratory techniques as both a single study and their various combinations to verify the diagnosis of cerebral toxoplasmosis in HIV-infected patients. Blood and cerebrospinal fluid were tested in 51 patients with Stage 4B HIV infection (AIDS) with the verified diagnosis of cerebral toxoplasmosis. Separate determination of specific antibodies of IgG, IgM, IgA and toxoplasma DNA in the blood and cerebrospinal fluid was shown to have an insufficient clinical sensitivity (37.3-68.6%). The benefits of various combinations of immunological and molecular biological assays enhancing the diagnostic efficiency up to 76.5-96.1% are demonstrated.

[Toxoplasmosis in HIV infection: invasion reactivation criteria].

Contemporary representation of toxoplasmosis reactivation criteria in HIV infection is generalized. Significance of the issue is justified: toxoplasmosis is a leading neurological pathology in AIDS with a high lethality percentage due to complexity of clinical confirmation and difficulties of laboratory confirmation of the start of reactivation. Clinical, instrumental, immunologic, molecular genetic invasion reactivation criteria are discussed in the article and analysis of their effectiveness is performed; their most feasible combinations are justified. Further system analysis of the cerebral toxoplasmosis reactivation criteria specified in the article in combination with search of new pathogen dissemination markers will allow to obtain important information that has both fundamental interest and important practical significance.

[Detection of rubella virus RNA in clinical material by real time polymerase chain reaction method].

AIM: Development of a reagent kit for detection of rubella virus RNA in clinical material by PCR-RT. MATERIALS AND METHODS: During development and determination of analytical specificity and sensitivity DNA and RNA of 33 different microorganisms including 4 rubella strains were used. Comparison of analytical sensitivity of virological and molecular-biological methods was performed by using rubella virus strains Wistar RA 27/3, M-33, "Orlov", Judith. Evaluation of diagnostic informativity of rubella virus RNAisolation in various clinical material by PCR-RT method was performed in comparison with determination of virus specific serum antibodies by enzyme immunoassay. RESULTS: A reagent kit for the detection of rubella virus RNA in clinical material by PCR-RT was developed. Analytical specificity was 100%, analytical sensitivity - 400 virus RNA copies per ml. Analytical sensitivity of the developed technique exceeds analytical sensitivity of the Vero E6 cell culture infection method in studies of rubella virus strains Wistar RA 27/3 and "Orlov" by 11g and 31g, and for M-33 and Judith strains is analogous. Diagnostic specificity is 100%. Diagnostic specificity for testing samples obtained within 5 days of rash onset: for peripheral blood sera - 20.9%, saliva - 92.5%, nasopharyngeal swabs - 70.1%, saliva and nasopharyngeal swabs - 97%. Positive and negative predictive values of the results were shown depending on the type of clinical material tested. CONCLUSION: Application of reagent kit will allow to increase rubella diagnostics effectiveness at the early stages of infectious process development, timely and qualitatively perform differential diagnostics of exanthema diseases, support tactics of anti-epidemic regime.

[Prevalence of human papillomavirus (Papillomaviridae; Human papillomavirus) of high carcinogenic risk based on the results of screening of three anatomical loci in men stratified by sexual behavior and HIV status].

INTRODUCTION: Human papillomavirus (HPV) of high carcinogenic risk (HCR), in addition to being the etiological agent of cervical cancer, also contribute to development of cancer of the anus, vagina, penis, vulva and oropharyngeal cancer. In this connection, further study of the biological properties of this agent and its prevalence in different populations is an urgent task.The aim of the study was to examine the prevalence of HCR HPV in three anatomical loci in men stratified by HIV (human immunodeficiency virus) infection status (negative, HIV+/positive/HIV-) as well as by sexual behavior: men who have sex with men (MSM), heterosexual men (HM). MATERIAL AND METHODS: The study included 256 men from Moscow and Moscow region: 73 МSМ/HIV+, 66 МSМ/ HIV-, 58 HM/HIV+, and 59 HM/HIV-. All men were tested for 14 HCR genotypes of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). Smears were taken from three anatomical loci: urethra, anus, oropharynx. Testing was preformed using real-time polymerase chain reaction assay (PCR-RT). RESULTS: The highest prevalence of HCR HPV detection, regardless of the locus, was recorded for МSМ/HIV+ (82.2%), and the lowest for HM/HIV (20.3%). The highest detection of HCR HPV in scrapings of epithelial cells from anus was recorded for МSМ/HIV+ (79.5%). The highest incidence of this pathogen in oropharynx also was registered for МSМ/HIV+ (13.7%). The highest incidence of HCR HPV in scrapings of epithelial cells from urethra was recorded for HM/HIV+ (24%). The prevalence of HCR HPV among men was found to differ markedly depending on the anatomical locus, HIV status and sexual behavior. DISCUSSION: For the first time, there were obtained data on the prevalence of HCR HPV in men with different patterns of sexual behavior and HIV status in the Moscow region. CONCLUSION: Screening for HCR HPV in male population based on the identification of 14 genotypes of the virus in three anatomical loci (urethra, oropharynx, anus) by PCR-RT will provide the information necessary to improve the system of epidemiological monitoring and proper planning of preventive measures among men with any risk factors for HPV persistence (presence of HIV infection and/or belonging to the MSM group). HPV screening algorithm development is required for men considering their HIV status and sexual behavior. We recommend testing for 14 HCR HPV genotypes in three loci (urethra, anus, oropharynx).

[Cytomegalovirus infection in infants with perinatal damage of the central nervous system].

Ninety children, aged from 7 days to 12 months, with perinatal damage of the central nervous system (CNS) were virologically tested. The persistence of cytomegalovirus (CMV) DNA in the blood, urine and oropharyngeal swabs was identified in 18.9% of children. In 14.4% of children, CMV DNA was found only in oropharyngeal swabs and urine samples. In some cases, the severity of neurologic symptoms and absence of positive changes in the restoration treatment could be caused by the activity of CMV infection, blood CMV DNA and the increase in blood-brain barrier permeability. The authors recommend performance of a comprehensive immunological and molecular-biological survey in patients with suspected CMV infection. In case of positive reactions, a specific antiviral treatment should be started. Tree clinical cases are reported.