Viral Diseases in Transplant and Immunocompromised Patients

For the last few years, the number of immunocompromised individuals is growing fast, due to more intensive antitumor therapy, transplantations and the concomitant immunosuppressive therapy, and the HIV epidemic, as well. Immunosuppressed patients very often are affected with nosocomial infections in hospitals, and with infections in the society. The defense from viral diseases depends mainly on the immune system. When there is immune deficiency, the illness is taking severely longer and has complicated outcome. Usually immunocompromised individuals have one or more defects in the defensive mechanisms and leading cause of death is infection.The viruses taking part in this process are Epstein Barr virus (EBV), Cytomegalovius (CMV), Herpes simplex viruses (HSV1, HSV2), Varicella zoster virus (VZV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Human Polyomaviruses (BKV, JC). Many viruses (HIV, CMV, EBV) are depressing the immune resistance and are leading to co-infections with other microbial agents. Some viruses (HSV1/2, HPV, CMV, EBV, BKV, JC) are at latent condition in the infected persons for life. They become activated when decline in the immunity occurs, leading to serious illnesses. For this reason, accurate screening and prompt and precise diagnosis can be performed to prevent exacerbation of diseases and provide appropriate treatment

Hepatitis D virus in Bulgaria: virology, epidemiology and pathogenesis in chronic HBV carriers with liver dysfunction

Inroduction: Hepatitis D (HDV) is the most interesting and unique among animal viruses. It causes viral hepatitis D only in individuals already infected with HBV (hepatitis B). This dual    infection leads to the most aggressive hepatic dysfunction of all human viral hepatitis.Aim: This study was made to outline the hepatitis D virus among patients with chronic liver disorders in northeastern Bulgaria, in the sight of virus epidemiology, pathogenicity and viral genotype.Materials and methods: This is a retrospective study conducted between 2013-2019 at St. Marina University Hospital,Varna, Bulgaria. We have analyzed 418 serum samples from 391 patients with chronic liver disease using ELISA, PCR and HDV sequencing and genotyping.Results and Discussion: From 391 patients with chronic liver abnormalities, 16.6% (95% CI: 15.9% - 23.8%, n = 65) had an etiological association with HDV in ELISA. We found HDV RNA positive results in 63 out of all 65 anti-HDV Ab (antibody) positive patients (96.9%). Twenty-four of them, or 38.1% (95% CI: 26.1% - 51.2%, n = 24), were on antiviral HBV/HDV therapy. For five of them, or 20.8% (95% CI: 7.1% - 42.2%, n = 5), HDV genotype I was found.Conclusion: HDV infection has still many mysteries to discover - in terms of pathogenesis, clinical outcome in chronic HBV/HDV-infected individuals, as well as genotype variations and their role in avoiding immune elimination of the virus. All these unanswered questions pose a great challenge to the scientific thought and efforts of humankind to reduce and gradually eliminate viral hepatitis D

THE ROLE OF anti-EBNA1 IgG DETERMINATION IN EBV DIAGNOSTICS

Purpose: In Bulgaria, the diagnosis of Epstein-Barr virus (EBV) infection is performed via ELISA testing of IgM and IgG against viral capsid antigen (anti-VCA IgM and anti-VCA IgG). With the current study, we try to answer is there any benefit of determination of IgG against the nuclear antigen of EBV (anti-EBNA-1 IgG) in the laboratory practice. Material/Methods: The prospective study included 82 serum/plasma samples tested for anti-VCA IgМ, anti-VCA IgG, anti-EBNA1 IgG and anti-VCA IgG avidity in ELISA (Euroimmun, Luebeck, Germany). Quantitative variables were reported as mean, and standard deviation (mean±SD) and the qualitative variables were reported as a number and a relative proportion (%). Results: Anti-EBNA1 IgG positive patients were 74.4% (95% CI:63.6% - 83.4%) of all tested individuals. Their mean age was significantly higher (30.5;SD±20.5)of this of patients without anti-EBNA1 IgG (14.5; SD±14.1) (p < 0.05).The first group of patients (with infectious mononucleosis, anti-VCA Ig M negative) had the highest number of anti-EBNA1 IgG negative results. Negative for аnti-EBNA 1 IgG were 12% of patients with Hodgkin's lymphoma. Conclusion: Determination of anti-EBNA1 IgG together with anti-VCA should be considered in the initial serological testing in EBV diagnostics. As different immune responses against the EBNA1 antigen exist, clinicians should interpret the results carefully with regard to the clinical symptoms, the immune status and the laboratory markers. We found anti-EBNA1 IgG ELISA tests exceptionally useful to distinguish primary and past infections in anti-VCA IgM(+)/anti-VCA IgG (+) patients

USE OF IMMUNOBLOT IgM IN PATIENTS WITH SEROLOGICAL AND CLINICAL EVIDENCE OF PRIMARY EBV INFECTION AND REACTIVATION

Purpose: Anti-VCA IgM is a marker for establishing primary infection with Epstein-Barr Virus (EBV), it usually appears in combination with anti-VCA IgG. It has been shown that there is a risk of non-specific IgM reactivity due to cross-reactions, interference with rheumatoid factor or autoantibodies. These antibodies may also occur during reactivation. In these cases, Immunoblot based tests may be useful to confirm the ELISA result. We compared the results of anti-VCA IgM in ELISA and Immunoblot IgM in patients with evidence of primary EBV infection (infectious mononucleosis, IM) and/or reactivation/reinfection. Materials/Methods: We examined 32 serum samples with commercial immunoblot (Euroline Anti-EBV Profile 2 (IgM), Euroimmun, Germany). Samples were tested primarily for anti-VCA IgM/IgG in ELISA. Patients with IM were 11, and those with probable reactivation/reinfection - 21. Results: We found positive results at 31.3% (95% CI: 16.1% -50.0 %, n = 10) of all subjects. Patients with IM and isolated anti-VCA IgM in ELISA (81.8%) were negative in Immunoblot IgM. Positive in Immunoblot IgM was 38.1% (n = 8) of the patients with suspected reactivation. We confirmed a primary infection in three of them due to the low avidity of anti-VCA IgG and missing anti-EBNA1 IgG. In five of the patients, the presence of anti-VCA IgM may be interpreted as reactivation/reinfection. Conclusion: Patients with IM and isolated anti-VCA IgM models in ELISA were not confirmed in the Immunoblot test. Approximately 43% of patients of possible reactivation was also negative in the test

OCCULT HEPATITIS B VIRUS INFECTION AMONG PATIENTS WITH LIVER DYSFUNCTION IN VARNA, BULGARIA

Background: Occult hepatitis B infection (OBI) is a challenge in virology and a clinically relevant topic. The present study assessed the presence of HBV-DNA in serum samples of HBsAg negative, patients with data of liver dysfunction, positive for anti- HBc total Ab with or without anti-Hbs Ab. Purpose: The goal of this study was to evaluate the prevalence of occult hepatitis B in Varna region, among patients with chronic liver dysfunction. Materials and methods: The investigation was conducted among 79 people, predominantly patients at Gastroenterology Clinic in the University Hospital St. ”Marina”, Varna, Bulgaria. Quantitative determination of HBV DNA was performed in the National Reference Laboratory for Hepatitis viruses at the National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria. Results: From 79 investigated patients with liver dysfunction16 (20. 25%), were considered as occult HBV carriers. Fourteen of them (17.72%) were positive for HBV DNA with very low values, below 200 IU/ml. Two of the cases (2.53%) were with serum levels comparable to those usually detected in the different phases of serologically evident (overt) infection and are considered as “false” OBI. Conclusions: Our data showed that OBI is more widespread than expected and can be identified as a significant risk factor for the presence of more severe liver damages and an important oncogenic factor for developing cirrhosis and hepatocellular carcinoma

Role of anti-EA-(D) IgM and anti-EA-(D) IgG tests in patients with primary EBV infection, lymphomas and immunosuppression

Purpose: The EA (early antigen) is expressed during the lytic phase of the EBV life cycle, together with VCA (viral capsid antigen) and MA (membrane antigen). Antibodies to EA (D) IgG occur in the course of primary infection, but not in all patients. The titers increase in the first 3-4 weeks and usually last about 3-4 months. Their presence is also associated with reactivation of the infection due to impaired immune control of the viral replication. The aim of this study was to compare the primary immune response against the major antigens (VCA) and EA (D) in patients with clinically proven primary infection and to define the antibody response to the EA (D) antigen as a marker for reactivation in patients at risk. Materials/Methods: We examined 86 persons with lymphomas, incl. Hodgkin's lymphoma and non-Hodgkin's lymphoma, immunosuppressed patients, mainly with AML (acute myeloid leukemia) and primary infection (infectious mononucleosis, IM) patients. We used an indirect ELISA for anti-EA (D) IgM/IgG and anti-VCA IgM/IgG (Euroimmun, Germany). Results: Patients with аnti-EA(D) IgM were 29.1% (95% CI:19.8% - 39.9%, n=25) while patients with аnti-EA(D) IgG were 23.3% (95% CI:14.8%-33.6%, n=20) (p>0.05). As expected, younger individuals with IM diagnosis predominated among the positive patients. We found isolated аnti-EA(D) IgM in four persons (with lymphoma and immunosuppression) and isolated аnti-EA(D) IgG in five patients. Conclusion: The routine diagnostic tests used to detect antibodies to VCA have a much better diagnostic value in defining a primary infection. Use of antibodies against EA (D) in case of isolated anti-VCA IgM and anti-VCA IgG needs further evaluation. Use of anti-EA (D) IgG as a reactivation marker should be compared with Real-time PCR results

DISTRIBUTION OF EPSTEIN - BARR VIRUS AMONG WOMEN OF REPRODUCTIVE AGE AND CHILDREN UP TO 1 YEAR IN THE VARNA REGION

Purpose: The Epstein-Barr virus (EBV) is widespread in the human population and is the major cause of infectious mononucleosis. Also, the virus is associated with the development of Hodgkin's and non-Hodgkin's lymphomas and nasopharyngeal carcinoma. The evidence of its role in neonatal pathology is contradictory and not well known. The aim of this study is to evaluate the EBV serostatus of women of reproductive age in the Varna region (2010-2016) to determine the risk of intrauterine and early postnatal EBV infection. Materials/Methods: We analyzed the results of a total of 1126 women of reproductive age and 360 children up to 1 year tested for anti-VCA IgM (viral capsid antigen) and anti-VCA IgG. An indirect ELISA of Euroimmun – Germany was used. Results: The proportion of positive anti-VCA IgG women in the reproductive age (76.8%; 95% CI: 74.2 %-79.3%) correlates with that of children up to 6 months - 68.0% (95% CI: 62.1% -73.6%), Pearson's = 8.395, p = 0.004. Conclusion: We found high anti-VCA IgG seropositivity among women of reproductive age, which reduces the risk of infection during pregnancy and intrauterine infection of the fetus, respectively. The presence of seronegative women (around 6.0%) and of women with serological evidence of primary infection or reactivation (17%) assumes a group of babies at risk of early infection. Despite the little evidence of virus involvement in neonatal pathology, contamination should be considered and sought after excluding the most common infectious agents

INVESTIGATION OF IMMUNOSUPPRESSED PATIENTS FOR THE PRESENCE OF EBV DNA IN REAL TIME PCR

Epstein-Barr virus (EBV) reactivates during immunosuppression (IS) and immune deficiency. The introduction of stem cell transplantation and the development of transplantology require compliance with criteria for assessing the risk of reactivation of latent viral infections, including EBV. There are no published EBVDNA findings in Bulgaria for such patient groups, Aim: The aim of this study is to assess the possible benefit of EBV PCR testing in immunosuppressed individuals. Materials and Methods: We investigated 50 immunosuppressed patients – 28 with various haematological diseases, 17 after kidney transplantationand5 patients with autologous stem cell transplantation (HSCT). Patients were first tested in an indirect ELISA to detect anti-VCA IgM/IgG (Euroimmun, Luebeck, Germany) and then in quantitative PCR (Sacace Biotechnologies S.r.l., Como, Italy). Results: We found ЕВV DNAin 14.0% (95% CI:5.8% - 26.7%, n=7) of all tested patients. The Real-time PCR results were in the range 100-500 copies/ml at the lower limit of the 500 copies/ml test positivity. The highest is the proportion of patients with haematological diseases (21.4%), predominantly with AML. Conclusion: We found a relatively small proportion of IS patients with detectable EBV DNA. For HSC-transplanted patients, we anticipate probable reactivation or reinfection in one patient, who was anti-VCA IgG positive in the primary study

Seroepidemiological and Molecular Genetic Studies of Cytomegalovirus Infection in Risk Group Patients /// Сероепидемиологични и молекулярно-генетични проучвания на цитомегаловирусната инфекция при рискови групи

[EN] Definition of the seroepidemiological status of the population in North-eastern Bulgaria and establishment of the relative share and age-related spread of CMV infection in general population and in risk groups; defining diagnostic options for proving reactivation of CMV infection in risk patients-pregnant women, new-borns and immunocompromised patients.[BG] Целта на изследването е дефиниране на сероепидемиологичния статус на населението в Североизточна България и установяване относителния дял и възрастово свързаното разпространение на CMV инфекцията за общата популация и по рискови групи. Дефиниране диагностичните възможности за доказване реактивация на инфекцията при бременни жени, новородени деца и имунонекомпетентни пациенти