Serological and Molecular Detection of Coxiella Burnetii in Clinical Samples from Veterinarians and Cattle Farm Workers from Gabrovo Region, Bulgaria

Coxiella burnetii, which causes Q fever, is a highly infectious agent that is widespread around the world.  During the last decades, the number of cases reported in Bulgaria varied from year to year. The present study aimed to determine the frequency of C. burnetii infection using ELISA and conventional PCR among freelance veterinarians and cattle farm workers in Gabrovo province, Bulgaria. In the period April 2020 to June, 2021 a total of 154 blood samples of target group was tested in the National Reference Laboratory of Cell cultures, rickettsia and oncogenic viruses (NRL CCROV) at NCIPD - Sofia. Diagnosis of C. burnetii was performed by indirect enzyme-linked immunosorbent assay ELISA (anti-Coxiella burnetii ph. II IgG/IgM) and by end-point PCR technique (to detect the sodB gene region of C. burnetii). By indirect ELISA assay of the tested 154 clinical samples, anti-C. burnetii positive ph. II IgM antibodies were registered in 37% of samples. A relatively high percentage are affected in the active age between 50-60 years old. Anti-C. burnetii positive ph. II IgG antibodies were proven at 50% of tested samples. A positive PCR signal for C. burnetii DNA was obtained at 37/154 (20% of samples) and follows the above reported trend of acute infection of active age patients. Around 10% of tested samples were positive for three C. burnetii laboratory markers. We conclude that Q fever is endemic in Bulgaria. More research is necessary in different Bulgarian regions to set the human risk groups, to diagnose acute and chronic Q fever and to determine the economic impact of Q fever in the cattle industry. In the NRL CCROV was developed diagnostic scheme including complex methods to improve early laboratory diagnosis of C. burnetii, allowing taking proper treatment of suspected with Q fever patients

Mini Review: Q Fever (Coxiellosis): Epidemiology, Pathogenesis and Current Laboratory Diagnosis

Q fever is zooantroponozis with global distribution caused by the strictly intracellular bacterium Coxiella burnetii. Causative agent of Q fever is an obligate intracellular parasite, classified in the genus Coxiella, family Coxiellaceae, class Gammaproteobacteria. The importance of the disease was assessed both in terms of human health and the serious economic damage they cause on livestock. Clinical manifestation of Q fever in humans is characterized by a wide variety - from asymptomatic infection to a chronic disease that can be fatal. Several basic methods have been developed to detection of C. burnetii. PCR and C. burnetii genomic sequences in whole blood are a sensitive and safe method of detection, with >90% sensitivity. A four-fold or greater rise of (CF) antibody (phase 2) between the paired sera is also diagnostic approach. Sensitivity of a four-fold rise in titre has been estimated as 73% ÷78% and specificity has been estimated as 90%, respectively. EIA is method with highly sensitive and specific. EIA detect IgM and then IgG antibodies which develop to phase II antigens in 10 to 14 days from symptom onset. IFA tests are of particular value for confirmation of acute infection and for diagnosis of chronic infection with high sensitivity. The technique detected IgG, IgM and IgA immunoglobulin classes. Suitable specimens for C. burnetii detection are blood samples. Although scientific interest in Q fever has always existed, a number of facts concerning the unforeseen nature of the epidemic, various clinical manifestations both in humans and in animals, the opportunities for chronic and other features of infection remain unclear. For this reason, timely and highly sensitive laboratory diagnosis is crucial for the outcome of the disease and subsequent treatment and monitoring

HEALTHCARE WORKERS IN BULGARIA - ARE THEY PROTECTED FROM VACCINE-PREVENTABLE INFECTIONS?

Background: Healthcare workers (HCWs) are at increased risk of exposure to many viral infections, including vaccine preventable diseases (VPDs) such as measles, mumps and rubella (MMR) as compared to non-HCWs. Immunity of HCWs against these viruses is mandatory in a healthcare setting due to possible exposure from patients or colleagues. Aim: To provide an assessment of anti-measles, mumps and rubella IgG seropositivity among Bulgarian HCWs employed in hospitals and regional health inspectorates (RHI), as an indicator of protective immunity against MMR in this risk group.  Materials and Methods: In the current study, 181 HCWs from Infectious Units in regional hospitals in the country, and HCWs from the RHI, involved in the monitoring and surveillance of MMR cases in Bulgaria were screened. Serum specimens from all participants were tested by a commercial indirect enzyme-linked immunosorbent assay (Anti-Measles, Anti-Mumps, Anti-Rubella IgG EIA-Euroimmun®, Germany) for presence of IgG antibodies against measles, mumps and rubella, as an indicator of protective immunity.  Results: The study included 181 HCWs, 25 male and 156 female, aged 22 to 66 years. The average protective seroprevalence for measles, mumps and rubella was 82.9%, 76.2% and 92.3% percent, respectively. The highest share of negative results were obtained for mumps-specific IgG – 23.2% (42/181), followed by measles 16.6% (60/181) and rubella-specific IgG 7.7% (19/181). Regarding the age distribution, the highest number of HCWs non-immune to measles and mumps was found among the 31- 40-year olds, and against mumps – among the 41-50-year-olds. Conclusion: HCWs are at greater risk of contracting infections than the general population because of contact with sick patients or infectious material. Infected healthcare workers can spread nosocomial diseases to vulnerable patients with more severe illness, leading to complications and even death. Therefore, the vaccination status of HCWs must be strictly monitored

EVALUATION OF INTERACTIONS OF SARS-COV-2 STRUCTURAL PROTEINS WITH SPECIFIC ANTIBODIES BY SPR ASSAY

Background: The World Health Organization admitted that the vaccination against Covid 19 limited the deaths, but not the spread of the disease. This requires a method allowing a specific, rapid and accurate diagnosis of the disease. We report a SPR assay that meets the requirements and can be applied no only for SARS Cov-2 diagnosis but as a tool for early diagnosis of otherinfections. Methods: Surface plasmon resonance (SPR) method was used to identify the binding of S/N protein to monoclonal antibodies. N-protein monoclonal antibody (NP mAb), S-protein monoclonal antibody (SP mAb), and receptor bind domain (RBD) antibody were used as recognition molecules. Ligands were deposited by the matrix-assisted laser evaporation (MAPLE) method, which guarantees maximum interaction specificity. Results: We registered S/N protein binding to the corresponding mAbs and S protein to RBD antibody with high sensitivity: the interactions were observed at protein concentration about 130 femtomoles (fM). A very good specificity was observed: the measured S protein binding activity to NP mAb was below the limit of detection (LOD). The same was noticed for N protein binding to SP mAb. Conclusions: The presented SPR assay possesses high sensitivity and selectivity and provides quantitative analysis. This makes it applicable for following the evolution of acute SARS-CoV-2 infection, especially at the early stages of viral replication which can be clinically useful

Dried Blood Spots as a Clinical Samples for Laboratory Diagnosis and Surveillance of Vaccine-Preventable Diseases in Bulgaria

In recent years the dried blood spots (DBS) had new and innovative applications in medicine, neonatology, virology and microbiology. This study aimed to evaluation of the frequency of detection of viral IgM/IgG markers in dried blood spots and introducing an easy-to-implement protocol for serum extraction in measles, mumps and rubella surveillance. The total 204 clinical samples (102 serum samples and 102 dried blood spots) collected from 102 patients were included. All specimens were tested for presence of specific viral markers (IgM and IgG antibodies) by a commercial indirect enzyme-linked immunosorbent assay (ELISA). Of all tested patients, three (3/102, 2.94%, 95% CI: 0 ÷ 6.22) were confirmed for acute measles infection and two (2/102, 1.96%, 95% CI: 0 ÷ 4.65) for mumps. Double positive ELISA-IgM results were found in their serum samples and DBS. No acute rubella infection and rubella IgM marker were detected in both clinical samples. By immunoassay analysis of all 102 patients, measles, mumps and rubella IgG were found in 83/102 (81%, 95% CI: 73.40 ÷ 88.60), 76/102 (75%, 95% CI: 66.60 ÷ 83.40) and 79/102 (77%, 95% CI: 68.83 ÷ 85.17) serum samples.  Comparative results were obtained in the adequately obtained DBS. Viral IgG seroprevalence in DBS were obtained in 79/102 (77%, 95% CI: 68.83 ÷ 85.17) for measles, 69/102 (68%, 95% CI: 58.67 ÷ 77.33) for mumps and 73/102 (72%, 95% CI: 63 ÷ 81) for rubella, respectively. Double negative results for each screened viral markers were proven in six tested patients.The study shown higher extinction value (Ratio and NovaTec units) in DBS compared to serum samples of same persons were calculated. Our studies show over 90% coincidence in combined ELISA assay of viral markers against measles, mumps, and rubella in serum samples and DBS. DBS clinical approach is non-aggressive and more acceptable to the public (including young children, pregnant women, etc.). It has a variety of new and innovative applications in medicine and in particular in the laboratory diagnosis of acute and past (presence of protective immunity) measles, mumps and rubella infection in the phase of elimination

Hofmeister anions effect on the thickness and morphology of polyelectrolyte multilayers for biofunctionalization of cardiovascular stents

The effect of Hofmeister anions on the surface properties of polyelectrolyte multilayers built from hyaluronan and chitosan by layer-by-layer deposition is studied by ellipsometry and atomic force microscopy. The thickness, roughness and morphology of the resulting coatings were found to depend on the type of the anion. Relationship between the surface properties and the biological response of the polyelectrolyte multilayers is established by assessing the degree of protein (albumin) adsorption

Mini Review: Chemical Characterization and Evaluation of Antiviral and Antibacterial Activity of Extracts from Graptopetalum paraguayense E. Walther (Crassulaceae)

Graptopetalum paraguayense E. Walther (GP) is a traditional Chinese herbal medicine belonging to the Crassulaceae family As herbal medicine, GP possesses several biological/pharmacological perspectives. It was found that GP aqueous extracts exhibit anti-inflammatory and hepatoprotective, antioxidant, anti-hypertensive and antihyperglycemic, immunomodulatory, antineoplastic, antiviral and antibacterial activities. The GP contains in the high range levels phenolic compounds and anthocyanins. Despite the intense study of the plant, little data exist regarding its chemical composition. It was found that the GP aqueous extract has no cytotoxic effect on RD and Lep cells. The extract effectively inhibited herpes simplex virus (HSV) replication in dose-dependent manner moderate activity as well as Gram-positive bacterial strains

Immunity to mumps virus in children population in Bulgaria

AbstractMumps is an acute, contagious, viral vaccine-preventable disease caused by mumps virus (MuV). The measles, mumps, rubella (MMR) vaccine that is used in many countries is considered highly effective with decreased MuV incidence, but suboptimal MuV long-term immunity. This study assessed the MuV seropositivity and antibody titre among vaccinated children in Bulgaria to provide evidence for a better understanding of MuV circulation and immunity in Bulgaria. The samples from 734 immunized children (369 females and 365 males) aged 1 to 16, divided into four age groups (≥1, 2–6, 7–11, and 12–16) were tested. Qualitative and quantitative indirect enzyme-linked immunosorbent assay (Anti-Mumps IgG ELISA, Euroimmun, Germany) was performed to determine the mumps IgG antibody levels in sera. Among all participants, protective MuV immunity was identified in 93%. MuV IgG antibody positivity ranged between 87% in the age group between 1 and 2 years and 96% in the age group 12–16 years, but no statistically significant difference was found among age groups. At the same time, there was a statistically significant difference (p = 0.01) between seropositivity in male and female participants, with male participants having an overall seropositivity of 90% and female 95%. Among the antibody-positive samples, the quantitative measurements of median anti-MuV IgG concentrations showed that titres decreased with increasing age. A slightly waning immunity following vaccination was observed, but positivity remained high among vaccinated children over the years. Similar studies show that maintaining high immunity is crucial to prevent mumps outbreaks

Antiviral properties of chalcones and their synthetic derivatives: a mini review

Chalcones (natural or synthetic derivatives) are aromatic ketones possessing a central backbone that form a core for variety important compounds with different substitutions. Recent scientific advances show that chalcones exhibit different bio-medical activities, including antiviral, which is related to the variety substitutions. This review provides general information on the origin, sources, virucidal and direct antiviral properties of chalcones in vitro, as well as a brief overview of the possible application and molecular modes of action of these compounds. The antiviral effect of chalcones probably results from the disruption of the different stage of viral replication cycle, inhibition of viral or cell enzymes, induction of apoptosis and others. Structural requirements for antiviral activities vary according to the mechanisms of action. Based on the published information, it could be considered that synthetic chalcones are very perspective antiviral candidates and deserve further studies for elucidating of their pharmacological potential

Primate Erythroparvovirus 1 Infection in Patients with Hematological Disorders

Primate erythroparvovirus 1, commonly referred to as Parvovirus B19 (B19V), is a DNA virus that normally results in a mild childhood infection called “erythema infectiosum”. Besides respiratory spread, B19V can also be transmitted through transfusions, which may result in persistent anemia in immunodeficient hosts. Dialysis patients often face acute or chronic anemia after infection with B19V. Here, we describe the laboratory investigation of 21 patients with hematological disorders for B19V infections. B19V DNA was detected in 13 (62%) of them, with specific IgM antibodies in three of the DNA positives. All 13 patients received treatment and were laboratory-monitored over a period of one year. In only two patients (a 14-year-old child with a kidney transplantation and a 39-year-old patient with aplastic anemia), markers of recent B19V infection were still detectable in follow-up samples. For four B19V DNA positive samples, short sequences could be obtained, which clustered with genotype 1a reference strains. Our findings suggest that all cases of hematological disorders should be examined for specific B19V antibodies and DNA for accurate diagnosis and appropriate patient management