Diagnostic PCR Research Articles

Efficacy of peri-implantitis prevention with needle-containing mouthwash

Insufficient motivation of patients determines low hygienic indicators of the mouth in persons with dental implants, which determines the search for new personal hygiene products. The possibility of tweeter substances, in particular, the production of the company «Solagift» (Tomsk), are of interest. Preliminary microbiological studies have shown the obvious bacteriostatic effect of a number of needle-containing substances on periodontopathogens in cell culture, including «Bio-efficient cellular juice of Siberian fir».
 The objective of the study was the clinical testing of the mouth rinses containing «Bio-efficient cellular juice of the Siberian fir».
 Within 18 months, the condition of the perioplantate tissues has been traced in 54 patients who used an oral roster during an individual oral hygiene 1:5 «Bio -efficient cellular juice of Siberian fir» or «Stomatophyt». A comparison of the effectiveness of inflammation prevention was carried out quarterly before the professional hygiene of the mouth using hygienic and periodontal indicators, the detection of mucositis and periymplatitis, and PCR diagnostics of periympetate spaces. A survey of patients with an assessment of rinses was carried out.
 The results of the study showed the advantages of the rinseum with the «Bio-efficient cellular juice of the Siberian fir» in hygienic and periodontal indices, the detection of periodontalopatogens in both patients with implants with an initially healthy periodontal and against the background of inflammatory periodontal diseases. Mucosite was detected less often when using a needle -containing rinseum, periiompetlet was recorded singlely in comparison groups. The subjective assessment of the implications of the needle-containing roster of the mouth is characterized by a higher assessment in comparison with the rinseum based on the «Stomatophyt» on the prevention of the development of inflammation and odor from the mouth.

Species composition and insecticide resistance in malaria vectors in Ellibou, southern Côte d’Ivoire and first finding of Anopheles arabiensis in Côte d’Ivoire

BackgroundKnowing the species composition and insecticide resistance status of the target vector population is important to guide malaria vector control. The aim of this study was to characterize the malaria vector population in terms of species composition, insecticide susceptibility status and potential underlying resistance mechanisms in Ellibou, southern Côte d’Ivoire.MethodsA 1-year longitudinal entomological survey was conducted using light traps and pyrethroid spray catches to sample adult mosquitoes in combination with larval sampling. The susceptibility status of Anopheles gambiae sensu lato (s.l.) to bendiocarb, deltamethrin, DDT and malathion was assessed using the World Health Organization insecticide susceptibility test. Additionally, An. gambiae specimens were screened for knockdown (kdr) and acetylcholineesterase (ace1) target site resistance alleles, and the expression levels of eight metabolic resistance genes, including seven cytochrome P450 monooxygenases (P450s) and one glutathione S-transferase (GST), measured with reverse transcription quantitative real-time polymerase chain reaction (qPCR).ResultsOverall, 2383 adult mosquitoes from 12 different taxa were collected with Culex quinquefasciatus and An. gambiae being the predominant taxa. Molecular identification of An. gambiae s.l. revealed the presence of Anopheles arabiensis, Anopheles coluzzii, An. gambiae sensu stricto (s.s.) and Anopheles coluzzii/An. gambiae s.s. hybrids. Anopheles gambiae mosquitoes were resistant to all insecticides except malathion. PCR diagnostics revealed the presence of ace1-G280S and the kdr L995F, L995S and N1570Y target-site mutations. Additionally, several genes were upregulated, including five P450s (i.e., CYP6P3, CYP6M2, CYP9K1, CYP6Z1, CYP6P1) and GSTE2.ConclusionThis is the first documented presence of An. arabiensis in Côte d’Ivoire. Its detection – together with a recent finding further north of the country – confirms its existence in the country, which is an early warning sign, as An. arabiensis shows a different biology than the currently documented malaria vectors. Because the local An. gambiae population was still susceptible to malathion, upregulation of P450s, conferring insecticide resistance to pyrethroids, together with the presence of ace1, suggest negative cross-resistance. Therefore, organophosphates could be an alternative insecticide class for indoor residual spraying in the Ellibou area, while additional tools against the outdoor biting An. arabiensis will have to be considered.

Prospective Analysis of Antibody Diagnostic Tests and TTS1 Real-Time PCR for Diagnosis of Melioidosis in Areas Where It Is Endemic.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.

Nosocomial outbreaks of aseptic meningitis: A public health concern

Nosocomial outbreaks of aseptic meningitis: A public health concern

Development of a Real-Time PCR Assay for Detection of Macrolide Resistance Mutations in Mycoplasma genitalium and Its Application for Epidemiological Surveillance in Russia.

A real-time PCR assay based on probe fluorescence quenching by a primer was developed and validated for detection of macrolide resistance (MR)-associated mutations in the 23S rRNA gene of Mycoplasma genitalium. The assay involves identification of any nucleotide substitutions at positions 2058, 2059, and 2611 of 23S rRNA (Escherichia coli numbering) by the probe-based melting curve analysis immediately after amplification and was capable of detecting target mutations in clinical specimens and spiked samples with 92% sensitivity and 100% specificity. We applied this new assay to assess the prevalence of MR-associated mutations in 949 nonduplicate urogenital samples positive for M. genitalium by routine diagnostic PCR, which were collected from symptomatic patients in five cities in the European part of Russia during the period 2009-2019. Forty-three (4.92%) samples revealed the presence of MR mutations, and no trend toward an increase in resistance prevalence was observed over the 10-year period of the study. The most commonly detected mutations were A2058G (26/43; 60.47%) and A2059G (13/43; 30.23%), while other mutations were rare: A2058T (3/43; 6.98%) and C2611T (1/43; 2.33%). The data obtained underline the need for regular epidemiological monitoring to ensure effective patient management, rational use of antibiotics, and prevention of further spread of resistance.

Мавпяча віспа (mpox) - нова проблема глобального масштабу для людства

Purpose - to draw attention to an infection that was little known, but has now become a global problem for society; to familiarize readers with the peculiarities of the 2022 monkeypox outbreak and to increase the level of alertness of doctors to this disease. Monkeypox is a global problem because the disease is spreading rapidly, covering 111 countries. Three cases were diagnosed in Ukraine. It is predominantly a self-limited infection, but there are severe and deadly complications. The lethality of this disease ranges from 0% to 11%. The course of the disease is more severe in children and people with reduced immunity. Vertical transmission of the virus from mother to child is possible, resulting in congenital monkeypox. Monkeypox is a zoonotic disease and its natural reservoir is not exactly known, but rodents are most likely to act. In most cases, person-to-person transmission of the virus occurs through close skin to skin contact, often during sexual intercourse. At the beginning of the outbreak 98% of cases of disease were was diagnosed in homo- and bisexuals. Airborne transmission is also possible. Infection is possible through close contact with infectious skin lesions. Clinically, the initial period resembles influenza, but lymphadenopathy is characteristic, which is considered a pathognomonic symptom of mpox. The rash is similar to that of chickenpox, but with more prevalent location on palms and soles than in chickenpox. In the presence of a vesicular rash in a patient, it is necessary to exclude monkeypox. PCR diagnostics of the virus in samples of vesicles or crusts has the greatest diagnostic value. Hygienic skin care is important. Antiviral drugs (tecovirimat, brincidofovir) are recommended only in severe cases. To reduce the spread of infection, international rules apply as for other infections, such as COVID-19. The monkeypox virus vaccine is recommended primarily for groups at risk of infection, including medical personnel who may come into contact with the patient or samples for laboratory testing. Being aware for this infection, following international health regulations, it is possible to prevent the further spread of monkeypox. No conflict of interests was declared by the authors.

Detection and Identification of Mycoplasmopsis agassizii in Captive Tortoises with Different Clinical Signs in Italy

Mycoplasmopsis agassizii causes the Upper Respiratory Tract Disease (URTD) in tortoises. The severity of the disease usually ranges from mild to severe respiratory signs. Animals can recover, die, or become asymptomatic carriers and are source of infection for other tortoises. This study describes (i) the clinical history and the results obtained in ten years of diagnostic PCR activity for detecting M. agassizii in different species of captive tortoises in Italy, and (ii) the phylogenetic analysis of the 16S rRNA gene sequences of M. agassizii. A total of 26.0% out of 169 samples resulted positive by PCR and 32 out of 75 (42.7%) animals with symptoms were positive. Sequences ob-tained from the PCR products were conserved, differed from the sequence of the M. agassizii type strain PS6, and were identical to many M. agassizii sequences deposited in databases. In particular, the sequences were identical or very similar to sequences obtained previously from tortoises in It-aly. Since samples collected from different anatomical sites resulted positive, it is suggested that pools of conjunctival, nasal and oral swabs are tested for diagnostic purpose in both symptomatic and asymptomatic animals.

Molecular forms of the Indian Anopheles subpictus complex differ in their susceptibility to insecticides and the presence of knockdown resistance (kdr) mutations in the voltage-gated sodium channel.

To investigate the differential insecticide-susceptibility of two molecular forms of Anopheles subpictus complex (A and B) against DDT and pyrethroids, the occurrence of knockdown resistance (kdr) mutations in these forms, and the association of kdr mutations with insecticide resistance. Insecticide susceptibility tests of An. subpictus s.l., collected from coastal and inland areas of mainland India, were performed against DDT, permethrin and deltamethrin using the WHO standard insecticide susceptibility test kit. The mosquitoes were characterized for molecular forms using a diagnostic PCR developed in this study. Representative samples of An. subpictus molecular forms A and B were sequenced for a genomic region encompassing the IIS4-5 linker to the IIS6 segments of the voltage-gated sodium channel to identify kdr mutations. A common PIRA-PCR was developed for identifying L1014F-kdr mutation and used for genotyping in both molecular forms of An. subpictus. Molecular form A of An. subpictus was resistant to all three insecticides, i.e., DDT, Permethrin and deltamethrin, whereas Form B was categorized as 'possibly resistant' to these insecticides. Significantly higher mortalities in WHO insecticide susceptibility tests were recorded in Form B compared to Form A in sympatric populations. Molecular characterization of the IIS4-5 linker to IIS-6 segments of the voltage-gated sodium channel revealed the presence of two alternative nucleotide transversions at L1014 residue in Form A, both leading to the same amino acid change, i.e., Leu-to-Phe; however, such mutations could not be observed in Form B. PIRA-PCR-based kdr-genotyping of field populations revealed high frequencies of L1014F-kdr mutations in Form A and the absence of this mutation in Form B. The proportion of L1014F mutation was significantly higher in resistant mosquitoes following insecticide-bioassay with DDT (p<0.0001), permethrin (p<0.001) and deltamethrin (p<0.01) as compared to their susceptible counterparts. Significant differences in insecticide susceptibility were found between two molecular forms of An. subpictus complex in sympatric populations. The L1014F-kdr mutation was observed in Form A only, which was found to be associated with DDT, permethrin and deltamethrin resistance.

The current situation on bovine leukemia in Dagestan and methods of its diagnosis

The article presents the current situation on bovine leukemia in the municipalities of Dagestan and a comparative analysis of serological and molecular-genetic methods of diagnosis. In municipal districts, urban districts and areas of animal husbandry for the first half of 2022, the number of animals examined by immune diffusion 531111 identified 2871, infected with the leukemia virus, which is 0,5 % from the number diagnosed. The degree of infection of livestock with bovine leukemia virus in rural areas varied from 0,01 to 5,2 %. The average coverage rate of serological studies of animals on leukemia – 57,2 %. Hematological studies of seropositive animals were not carried out during the analyzed period. 79 new leukemia-affected points have been registered, 95 unhealthy points have been transferred since 2021, only 3 points have been rehabilitated. As on 01.07.2022, 171 leukemia-affected points are registered, including 38 in agricultural enterprises, 4 of them in breeding farms, 27 farms and 106 farms. A comparative analysis of the data on the carrier of antibodies and DNA provirus showed that ELISA revealed 7 animals more carriers of antibodies than in AGID, PCR study revealed 14 and 7 animals more carriers of DNA provirus than carriers of antibodies in AGID and ELISA, respectively. To maximize the detection of seropositive BLV of animals, the integrated use of AGID, ELISA and PCR methods as a promising system in anti-leukemia measures is proposed. The introduction of ELISA and PCR diagnostics into the veterinary practice of the Dagestan Republic will make it possible to determine virus transmission in animals from the first day after birth (ELISA) and from the 20th day of the calf's life (PCR), as well as to increase the effectiveness and shorten the duration of anti-leukemia measures the final stages of the recovery of agricultural enterprises from leukemia infection.

Изучение генетической вариабельности выделенных изолятов вируса лейкоза крупного рогатого скота в Белгородской области

Abstract. The purpose of the research Was to study genetic diversity of bovine leukemia virus isolates obtained in the Belgorod Region using restriction fragment length polymorphism method (RFLP). Scientific novelty. Bovine leukosis is one of the most common chronic infectious diseases of cattle in many countries of the world, which causes significant economic losses in the livestock industry. The typing of bovine leukemia virus (BLV), the study of its genetic structure, the evaluation of the mutation vector and a more detailed disclosure of the biological properties of the pathogen represent fundamental and applied value. Methods. The object of the research was 3-4-year-old cows infected with leukemia virus (n = 10), identified by serological methods in disadvantaged dairy farms. The immunodiffusion (ID) test, hematologic studies, PCR, genotyping, statistical processing of obtained data were conducted. Results. Conducted hematological studies determined the stage of the leukemic process in each animal. The target env fragment of the BLV gene (444 bp) was amplificated by 2-stage nested PCR, and this region was genotyped for all studied leukemia virus isolates using the restriction fragment length polymorphism (RFLP) method. In the course of the work, specific regions of the BLV env (gp51) gene, 970 bp long, were also obtained. We have given a primary assessment of the genetic diversity of BLV with the establishment of a genetic group (Belgian genotype according to RFLP). In the course of the work, specific regions of the env gp51 BLV gene, 444 bp long, were obtained. These fragments will be used for further DNA sequencing followed by phylogenetic analysis and determination of amino acid changes in the structure of the surface glycoprotein (gp51) of the bovine leukemia virus. Monitoring studies of BLV genotypes and the study of antigenic changes in the pathogen will allow timely development of the latest means of controlling and restricting the spread of bovine leukosis and improvement of diagnostic serological and PCR test systems.

EFFECT OF SYNBIOTIC ON INTESTINAL HISTOMORPHOMETRY AND GROWTH RATE IN QUAILS, EXPERIMENTALLY INFECTED WITH FIELD STRAIN OF SALMONELLA GALLINARUM

This study was planned to investigate the effect of synbiotic on intestinalhistomorphometry and the growth rate of quails experimentally infected with a field strain ofSalmonella gallinarum. Day old Japanese quails (120), with an average body weight of 6.09±1.1 g,were randomly assigned into 4 groups A, B, C, and D. Quails of groups A and B were given synbioticon a daily and weekly basis respectively, along with challenge while group C was negative control andgroup D was the positive control group. A total of 60 organ samples of the intestine were collectedfrom apparently healthy and freshly dead quails respectively to isolate the field strain of Salmonellagallinarum. Bacterial isolation and molecular identification were performed in accordance withlaboratory diagnostic culture techniques and PCR. Results from the current study indicate thatnonantibiotic feed additives such as synbiotics boosted the gut histomorphometric parametersincluding the villus height, villus width, and crypt depth under the challenge of Salmonella gallinarum.There was a significant increase in all these parameters due to synbiotic feeding except for the negativecontrol group which showed the lowest values. On day 21, DD achieved a maximum villus height of636.88 ± 65.93b µm in the duodenal mucosa, whereas, maximum villus width of 182.01 ± 15.40c µmin duodenal mucosa was achieved by group AA. Maximum villus height of 276.89 ± 21.16b µm andcrypt depth of 26.66 ± 1.15a µm in jejunum mucosa were recorded in group AA. Statistical results byusing the technique of one-way ANOVAs indicated that there was a significant increase inmorphmetric parameters of duodenal and jejunum mucosa in the groups fed synbiotic as compared tothe negative control. Hence results illustrate that there is an overall increase in histological parametersof duodenal and jejunum mucosa in the groups fed synbiotic as compared with the negative controlgroup. This study supports the beneficial effects of synbiotic on intestinal health and the growth rate ofJapanese quails by improving FCR.

Evaluation of the World Health Organization Global Invasive Bacterial Vaccine-Preventable Disease (IB-VPD) Surveillance Network's Laboratory External Quality Assessment Programme, 2014-2019.

Introduction. In 2009, the World Health Organization (WHO) established the Global Invasive Bacterial Vaccine Preventable Disease (IB-VPD) Surveillance Network (GISN) to monitor the global burden and aetiology of bacterial meningitis, pneumonia and sepsis caused by Haemophilus influenzae (Hi), Neisseria meningitidis (Nm) and Streptococcus pneumoniae (Sp).Hypothesis/Gap Statement. The GISN established an external quality assessment (EQA) programme for the characterization of Hi, Nm and Sp by culture and diagnostic PCR.Aim. To assess the performance of sentinel site laboratories (SSLs), national laboratories (NLs) and regional reference laboratories (RRLs) between 2014 and 2019 in the EQA programme.Methodology. Test samples consisted of bacterial smears for Gram-staining, viable isolates for identification and serotyping or serogrouping (ST/SG), plus simulated cerebrospinal fluid (CSF) samples for species detection and ST/SG by PCR. SSLs and NLs were only required to analyse the slides for Gram staining and identify the species of the live isolates. RRLs, and any SLs and NLs that had the additional laboratory capacity, were also required to ST/SG the viable isolates and analyse the simulated CSF samples.Results. Across the period, 69-112 SS/NL labs and eight or nine RRLs participated in the EQA exercise. Most participants correctly identified Nm and Sp in Gram-stained smears but were less successful with Hi and other species. SSLs/NLs identified the Hi, Nm and Sp cultures well and also submitted up to 56 % of Hi, 62 % of Nm and 33 % of Sp optional ST/SG results each year. There was an increasing trend in the proportion of correct results submitted over the 6 years for Nm and Sp. Some SSLs/NLs also performed the optional detection and ST/SG of the three organisms by PCR in simulated CSF from 2015 onwards; 89-100 % of the CSF samples were correctly identified and 76-93 % of Hi-, 90-100 % of Nm- and 75-100 % of Sp-positive samples were also correctly ST/SG across the distributions. The RRLs performed all parts of the EQA to a very high standard, with very few errors across all aspects of the EQA.Conclusion. The EQA has been an important tool in maintaining high standards of laboratory testing and building of laboratory capacity in the GISN.

Diagnosis of avian metapneumovirus infection using multiplex PCR

Avian metapneumovirus infection causes significant economic damage to poultry farming, which consists of losses from a decrease in safety and productivity, as well as the cost of health and preventive measures.The causative agent of the disease is avian metapneumovirus family Paramyxoviridae subfamily Pneumovirinae genus Metapneumovirus. The viral genome consists of 8 genes encoding 9 proteins: N-nucleoprotein, P-phosphoprotein, Mmatrix protein, F-fusion protein, M2 (M2-1,M2-2) - elongation/transcription factor, SH-(small) hydrophobic protein, Gglycoprotein, L-(large) polymerase. The virus lacks non-structural NS1 and NS2 proteins that counteract interferon.Based on the nucleotide seguence of the variable glycoprotein G gene, avian metapneumovirus was classified into four 4 subtypes of MPV are officially known, although recent publications have reported of two new pneumoviruses, GuMPV and AMPV PAR-05 isolated from the seagull and parrot in North America.The diversity of pathogen subtypes and differences in virulence properties of metapneumovirus create difficulties both in the prevention of this disease and in its diagnosis.The complexity of isolating the metapneumovirus from the test material is due to the short period of persistence of the virus in the organs of birds. It is possible to isolate the RNA of the causative agent of MPVI by the molecular biological method within 17-19 days after infection. The aim of our work was development and testing of primers and probes for the simultaneous detection and differentiation of avian metapneumovirus subtypes A and B by PCR with real-time fluorescence detection, with high specificity.Using vaccine strains 8544 and VC-03, the conditions for PCR were selected according to the composition of the PCR mixture and the annealing temperature of primers and probes. During PCR, we recorded the fluorescence signal through the FAM channel for cDNA obtained from the 8544 vaccine strain, and through the HEX channel for the VC-03 vaccine strain. The specificity of the developed primers and probes was also tested in studies with heterogeneous viruses such as Newcastle disease, classical and variant strains of chicken infectious bronchitis virus, and with bird DNA.The studies conducted to determine the specificity of the primers selected by us for multiplex PCR diagnostics make it possible to simultaneously specifically detect subtypes A and B of the causative agent of MPVI in birds in real time using different fluorescence channels, while other non-target respiratory pathogens were not observed.

Pathogenicity and Virulence of Flavobacterium columnare Isolated from Farmed African Catfish, Clarias gariepinus (Burchell 1822)

Columnaris disease caused by the multi-variant Flavobacterium columnare is known to be responsible for severe mortality in wild and cultured freshwater fish species worldwide. Incidence of conditions presenting with clinical signs typical of Columnaris disease has been reported by catfish farmers in Nigeria. There is, therefore, the need to ascertain the pathogenicity and virulence of F. columnare isolates from disease conditions, as a prelude to molecular characterization of genetic variants of the bacterium in Nigeria. Four groups of 10 African catfish, Clarias gariepinus weighing 32 – 40 g were experimentally infected with 106 CFU/L of different isolates of F. columnare, identified using diagnostic PCR. Similar number of fish in a fifth group were not infected (Control). All Experimental treatments were replicated once. Percentage morbidity and mortality were observed and compared between experimental groups using descriptive statistics, while cellular structures of normal, discoloured and necrotic skin of affected fish were also studied. All F. columnare isolates used in the experimental infection were observed to be pathogenic, causing clinical signs and mortality in infected fish. Lesions observed were external, with characteristic dermatitis typically referred to as saddle-back lesion being prominent. There is gross variation in morbidity and mortality among the different isolates, with the highest being 80% (for both) and the least, 55% and 40%, respectively. There was vacuolation in the epidermal cells and reduction in melanocytes in the discoloured skin but complete erosion of the epidermis in severe dermatitis. There is the likelihood that some of the F. columnare isolates used in this study differ genetically, indicating the need for the identification of the different genetic variants available in Nigeria.

ДИАГНОСТИКА БАКТЕРИАЛЬНЫХ РЕСПИРАТОРНЫХ ИНФЕКЦИЙ У СВИНЕЙ С ПОМОЩЬЮ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИЕЙ С ГИБРИДИЗАЦИОННО-ФЛУОРЕСЦЕНТНОЙ ДЕТЕКЦИЕЙ В РЕЖИМЕ РЕАЛЬНОГО ВРЕМЕНИ

Indications for laboratory testing for the purpose of etiological diagnosis of bacterial respiratory infections were the presence in pigs of an acute disease with local symptoms of respiratory tract damage. The aim of the study was to use PCR with hybridization-fluorescence detection in real time to detect bacterial pathogens of respiratory infections of pigs Mycoplasma hyopneumoniae, Streptococcus pneumoniae, Pasterella multocida, Staphylococcus aureus. We used PCR kits "Vet-factor" (Russia) and LLC NPF "Litech" (Russia). The materials for laboratory studies were: blood, flushes from the nasal mucosa, pieces of parenchymal organs, lymph nodes, trachea and tonsils from sows and corpses of piglets aged from one to four months. The following equipment was used to set up the reactions: The Real-time CFX Connect amplifier (Bio-Rad Laboratories, USA). The results of the PCR analysis were taken into account by the presence or absence of the intersection of the fluorescence curve with the threshold line set at the appropriate level (which corresponds to the presence or absence of the threshold cycle value "Ct" for the sample under study). The results obtained during laboratory diagnostic studies of the material from pigs received by the Kropotkin regional Laboratory over the past 4 years (from 2019 to 2022) indicate a high frequency of detection of bacterial pathogens of respiratory infections in pigs (from 10 to 72%). PCR diagnostics with real-time hybridization-fluorescence detection is optimal for rapid detection of Mycoplasma hyopneumoniae, Streptococcus pneumoniae, Pasterella multocida, Staphylococcus aureus in pigs.

Видовой состав микрофлоры матки и вымени у коров при острых послеродовых эндометритах и маститах

The polyetiology of postpartum mastitis and endometritis makes it difficult to develop and use specific drugs. Therefore, there is a need for a more detailed study of the microbiological composition of the udder microflora, which will make it possible to correctly and appropriately select antimicrobial drugs. The main properties of microorganisms isolated from postpartum mastitis and endometritis in cows were studied. Of 166 samples from the exudate of the uterus and udder of cows, 32 (19,28 %) cultures of Escherichia coli (E. coli) were isolated, while in 14 cultures (43,75 %) hemolytic activity was confirmed, and 17 cultures (53,12 %) were highly pathogenic for white mice. In 35 samples (21,08 % of the total number of cultures), bacteria of the genus Klebsiella were isolated, active hemolysis was observed in 12 (34,28 %) samples, 16 (45,71 %) cultures were pathogenic for white mice. Bacteria of the genus Proteus were isolated in 24 samples (14,46 %) of the total number of cultures, hemolytic activity was detected in 11 (45,83 %) cultures, pathogenicity for laboratory animals was determined in 9 cultures (37,50 %). The analysis of isolated microorganisms from the contents of the uterus and udder from cows showed that the species composition of the microflora in postpartum mastitis and endometritis is practically the same, which gives us the opportunity to assume that pathogenic microorganisms can be transferred from the focus of inflammation in the mammary gland by lymphogenous or hematogenous way to the uterus, and back. With an increase in the number of postpartum mastitis and endometritis in cows in large livestock complexes with an intensive housing system, it is advisable to use modern PCR diagnostics. Real-time PCR has significant advantages over traditional methods, as well as higher sensitivity and specificity.

Опыт использования синдромальных подходов к диагностике респираторных инфекций

Irrational or excessive use of antibiotics, the absence or insufficient use of rapid diagnostics tests at the point of care ("POC"), as well as insufficient infection control are key factors in the development of multi- and pan-resistance to antibiotics. The article presents the clinical experience of using the syndromic approach Patients and methods. The work was carried out on the basis of the Morozovskaya State children's hospital from February to June 2022 in accordance with the standards of medical care for patients with acute respiratory infections using multiplex PCR test (mPCR) – BioFire FilmArray respiratory panel (BioFire RP). The study included inpatient children (n = 40) with symptoms of acute respiratory infections and respiratory failure (DN) of different severity. Results. The analysis of the nosological structure showed that acute obstructive bronchitis/acute bronchitis (J20.9) was found in 47.5%, pneumonia (J18.9) in 20% of children, acute bronchiolitis (J21.9) at 7.5%, other viral lesions of the respiratory tract (J00-J06) – 25%. The standard PCR ARVI panel identified pathogens in 45% (n = 18), the BioFire RP panel in 55% (n = 22) of patients (p = 0.503). An important indicator is the speed of obtaining results. The median time from the moment of taking the smear to the moment of transmitting the result to the physician was 2.56 hours (CI: 1.91–3.62) when examined using the BioFire RP panel compared with 28.4 hours (CI: 9.64–33) when using standard PCR diagnostics for respiratory viruses (p &lt; 0.001). Conclusion. Methods of rapid molecular diagnostics can reduce the time of hospitalization of patients. In the study, the duration of hospitalization is 1 day shorter compared to the analysis of retrospective indicators of hospitalization terms. This is due to the ability to quickly identify the agent of respiratory infection, which in turn affects the tactics of treatment, optimizes the appointment of antibiotics, which affects to the duration of hospitalization. Key words: respiratory infection, BioFire, PCR, virus, rapid diagnosis, etiotropic therapy

Mass screening and treatment (MSaT) for identifying and treating asymptomatic cases of malaria-malaria elimination demonstration project (MEDP), Mandla, Madhya Pradesh

BackgroundMass screening and treatment (MSaT) aims at reducing the spread of malaria in communities by identifying and treating infected persons regardless of the symptoms. This study was conducted to identify and treat asymptomatic cases using MSaT approaches in the community.MethodsThree rounds of MSaT using cluster combination approaches were carried out during September 2018 to December 2019 to identify and treat asymptomatic malaria cases in the community. All individuals who were present in the household were screened using RDT irrespective of malaria related symptoms. Simultaneously thick and thin blood smear and blood spot were collected for further analysis using microscopy and diagnostic PCR done in a subset of the samples.ResultsLogistic regression analysis revealed that asymptomatic malaria cases significantly less among the older age groups compared with < 5 years children (OR ranged between 0.52 and 0.61; p < 0.05), lowest in cluster 4 (OR = 0.01; p < 0.0001); during third round of MSaT survey (OR = 0.11; p < 0.0001) and significantly higher in moderate to high endemic areas (OR = 88.30; p < 0.0001).ConclusionOver the three rounds of MSaT, the number of asymptomatic cases were significantly less in the older age groups, and during third round. Similarly, the asymptomatic cases were significantly less in the low endemic area with API < 1 (cluster four). Therefore, the malaria elimination programme may consider the MSaT strategy to identify asymptomatic cases that would be otherwise missed by routine fever based surveillance. This MSaT strategy would help accomplish the malaria elimination goal in an expedited manner.

Development of a specific and sensitive diagnostic PCR for rapid molecular authentication of the medicinal plant Portulaca oleracea

Adulteration by Bacopa monnieri (BM) in Portulaca oleracea (PO) plants frequently occurs; it decreases the efficacy of traditional Chinese medicine (TCM) and leads to fraud in the herbal marketplace. In this study, a diagnostic PCR assay was established for the rapid authentication of PO and BM in the herbal market. The sequence divergences in internal transcribed spacer 2 (ITS2) between PO and its adulterant species were used to design diagnostic PCR primers. The specific designed primer sets were evaluated and show that the diagnostic PCR assay can be used to verify the authenticity of PO and BM. The detection limits of the primer set for PO and BM identification were 10 pg and 1 pg, respectively. The reactivity of diagnostic PCR was 0.1% PO genomic DNA and 0.01% BM genomic DNA in the test sample during DNA amplification. In addition, multiplex PCR (mPCR) for PO and BM identification was also established. The samples were more susceptible to the effect of steaming in authentication by singleplex PCR and mPCR than boiling and drying treatment. Furthermore, commercial samples from the market were used to demonstrate the applicability of the developed diagnostic PCR for PO authentication and diagnose BM adulteration, and the investigation found that approximately 72.2% (13/18) of PO plants in the herbal market were adulterated. In conclusion, the diagnostic PCR assay was successfully developed and its specificity, sensitivity and reactivity for PO and BM authentication were proven. These developed PCR-based molecular methods can be applied as an identification tool for PO authenticity and can be practically applied for inspection of BM adulteration in the herbal market in the future.

Hepatitis E Virus (HEV) Synopsis: General Aspects and Focus on Bangladesh.

HEV is the most common cause of acute hepatitis globally. This review summarizes the latest knowledge on the epidemiology, clinical characteristics, testing, and treatment of HEV infection. We also focused on Bangladesh to highlight the distinct challenges and the possible remedies. In low-income settings, the virus is mainly transmitted between people by fecal contamination of drinking water causing large outbreaks, and sporadic cases. The disease is usually mild and self-limiting acute hepatitis. Still, pregnant women and their offspring in low-income countries are at particular risk for severe disease, with up to 20% maternal mortality. Despite the high burden of the disease, HEV remains a relatively neglected virus, with detection hampered by costly tests and a lack of suitable treatments. Molecular PCR diagnostics, together with ELISA antibody tests, remain the preferred methods for diagnosis of HEV; however, rapid bedside diagnostics are available and could offer a practical alternative, especially in low-income countries. One vaccine (HEV 239) is only available in China and Pakistan, as efficacy against the other genotypes remains uncertain. The effectiveness trial conducted in Bangladesh might lead the way in gathering more efficacy data and could, together with improved surveillance and raised awareness, dramatically reduce the global burden of HEV.