T-Lymphocyte Subset Absolute Counts in the Peripheral Blood of Mediterranean Spotted Fever Patients: Relations to Disease Severity.

INTRODUCTION: Mediterranean spotted fever (MSF) in Bulgaria is caused by Rickettsia conorii conorii. AIM: This study aims at investigating the absolute counts of T-lymphocyte subsets in the peripheral blood of patients with MSF in order to establish relationships with disease severity. MATERIALS AND METHODS: The absolute counts of T-lymphocyte subsets were tested in the blood of 62 patients in the acute stage of MSF. They were assigned into three age and sex matched groups, based on the severity of disease - with mild, moderate or severe forms. Controls were 32 age and sex matched healthy individuals. The diagnosis was confirmed by an immunofluorescence assay. Immunophenotyping was performed using Epics XL-MCL Coulter, USA flow-cytometer. RESULTS: The absolute counts of immune competent (CD3+) cells, as well as the counts of helper/inducer (CD3+ CD4+) and suppressor/ cytotoxic (CD3+ CD8+) T-cell subsets decreased in parallel with disease severity. Naïve (CD4+ CD45RA+) and activated memory (CD4+ CD45RO+) T-cell subsets were reduced, particularly in severe MSF. Taken as a whole, the counts of activated (CD3+ HLA-DR+) and that of presenting accessory (CD28+) or stimulatory (CD38+) molecules Т-cell subsets was increased, but in the first two subsets the trend from mild to severe forms of the disease was descending. CONCLUSION: Reduced T-lymphocyte subset counts are likely related to trans-migration into perivascular inflammatory foci. The increased number of T-lymphocytes bearing activation molecules reflects a mobilization of the cell-mediated immune response. An important issue of this study is the possible prognostic value of T-cell subsets counting, predicting the evolution of a clinical condition to clinical forms, according to the disease severity.

Levels of certain endothelial biomarkers during the acute phase and convalescence in patients with different severity of Mediterranean spotted fever.

INTRODUCTION: Mediterranean spotted fever (MSF) is a tick-borne disease caused by Rickettsia conorii subspp. conorii. It is transmitted by the bite of the tick Rhipicephalus sanguineus. Modified by the rickettsial invasion, the micro-vascular endothelium acquires an activated inflammatory phenotype and initiates secretion of cytokines and expression of cell adhesion molecules (CAMs) and chemoattractans. AIM: This study aims at investigating the alterations in the soluble cellular adhesion molecules (sCAMs) and chemokine MCP-1 levels in patients with MSF of varying severity in the acute and convalescence stage in order to assess their diagnostic and prognostic value. MATERIALS AND METHODS: The soluble forms of cellular adhesion molecules (sCAMs)--sE-selectin and sP-selectin, the intercellular (sICAM-1) and vascular (sVCAM-1) adhesion molecules as well as the monocyte chemoattractant protein-1 (MCP-1) were studied in the sera of 80 patients with MSF. The presence of MSF was confirmed serologically by indirect fluorescence assay (IFA). In order to study disease dynamics, serum samples from 80 patients were drawn on day 1 following the onset of rash; in 60 patients (part of the surveyed 80) a second sample was taken in the convalescence period--14 days post hospital discharge. The investigation was focused on mild, moderate and severe forms of MSF. Enzyme linked immune-sorbent assay was used for sCAMs determination (Quantikine IVD colorimetric RESULTS: Overall, in the acute stage, patients presented with increased levels of sE-selectin, sICAM-1, sVCAM-1 and MCP-1, whereas sP-selectin level was decreased. The levels of sE-selectin, sICAM-1 and sVCAM-1 were significantly elevated in mild, moderate and severe forms of the disease with sE-selectin level exhibiting a plateau tendency and sICAM and sVCAM levels demonstrating an upward trend from mild towards severe MSF forms. MCP-1 level was elevated only in severe MSF. In all forms of MSF, in the convalescence period, sICAM-1, sVCAM-1 and MCP-1 concentration returned to reference levels whereas sE-selectin level persisted elevated. In the convalescence stage, sP- selectin concentration also showed an upward tendency, which in severe forms of MSF slightly exceeded the level in controls. sP-selectin levels correlated directly with platelet count, whereas sICAM-1 and sVCAM-1 levels showed a reverse correlation, sE-selectin, sICAM-1 and MCP-1 levels directly correlated with aminotransferase activity (ALT and/or AST). CONCLUSION: The soluble forms of CAMs reflect the endothelial inflammatory potential. There is evidence that endothelium activation is more potent in severe forms of MSF. Assessment of the endothelial response in the course of the disease is an important predictor of the outcome, the choice of therapeutic approach and disease prognosis.

Some epidemiological features of the Mediterranean spotted fever re-emerging in Bulgaria.

INTRODUCTION: The Mediterranean spotted fever (MSF), caused by Rickettsia conorii conorii strain Malish, is transmitted by the brown dog tick Rhipicephalus sanguineus. In Bulgaria, cases of MSF occurred in two epidemic waves, the first in 1948-1970, (after there were no report of new cases more than for 20 years) and the second started in 1993 and is still going on. The AIM of the study was to investigate the epidemiological characteristics of the re-emerging MSF in Plovdiv city and its suburbs, which is the largest endemic region in the country. MATERIALS AND METHODS: The MSF patients treated between 1993 and 2011 were 1254. MSF was confirmed by immunofluorescent assay (IFA) in the Reference Rickettsioses Laboratory. Descriptive and analytic epidemiological methods were used to determine the routes and ways of infection, the epidemiological locus, seasonality, patients' age, gender and social structure, and the clinical severity of the cases. RESULTS: We established that MSF spread in the region in two distinct phases: from 1993 to 2003 during which the disease was increasingly spreading and the second phase taking place from 2004 till 2011 during which the disease was gradually decreasing. The incidence is between 0.13 and 25.62, mean 10.91 per 100 000 populations (11.88 and 9.56 per 100 000 populations for phases I and II, respectively); between 1.14% and 6.25% of the infected people died (mean 2.07%, 3.46% and 1.25% for phases I and II, respectively). The age distribution of patients shows predominance of 40-59-year-olds (31.66%), reaching a peak in patients older than 60 years (37.5%). Children and adolescents under 19 years are less affected (18.12%), while the least affected are the young adults between 20-39 (12.7%). Urban population is almost twice as affected as rural population regardless of the gender. The disease has summer seasonality, peaking in August. Eschar (tache noire) was found in 77.91% of the patients. Despite the decline and predominance of mild forms (43.12%), the re-emerging MSF still presents with lots of severe forms (11.45%) and malignant forms (8.54%), which makes almost one fifth of all patients to be at serious risk of getting ill with a severe disease with complications and possible fatal outcome. CONCLUSION: Almost two decades after it re-emerged in Bulgaria, MSF is still potentially active despite the decreasing incidence and mortality rate. During the phase of decline, the re-emerging MSF kept the basic epidemiologic characteristics it had in the first phase of rapid increase. The epidemiologic characteristics of MSF in the region are in support of its prevention and control.

Mediterranean spotted fever--classification by disease course and criteria for determining the disease severity.

INTRODUCTION: Mediterranean spotted fever (MSF) in Bulgaria is caused by Ricketsia conorii conorii with a major vector the dog tick, Rhipicephalus sanguineus. The first cases of re-emerging MSF were reported in this country in the early 1990s after some 20 years of absence and then registered an annual increase until 2001-2003 after which the disease prevalence declined. MSF still poses a serious health problem in the country as severe, complicated cases with lethal outcome occur. The aim of this paper was to classify the forms of MSF according to the course of the disease process and to devise criteria for the disease severity in order to enable comparison of clinical manifestations of the disease at different stages of spreading, in different age groups, and between endemic and non-endemic regions in this country and abroad. PATIENTS AND METHODS: The study was carried out in a comparative aspect during the first phase of increase (1993-2003) with incidence of 11.88 per 100000 population and during the second phase of decline (2004-2011) with incidence of 9.56 per 100000 population. The disease was etiologically confirmed in 883 hospitalized patients by the positive antibody response to the specific antigen--Ricketsia conorii conorii by means of the immunofluorescence assay (IFA). The criteria we used for the classification of the forms of MSF included: 1. Typicality: forms having the most characteristic features of the MSF - eschar, fever, papular/maculopapular rash on the trunk and extremities, including hands and feet. 2. Manifestation: forms represented by all or some of the typical symptoms, giving sufficient grounds for preliminary diagnosis. 3. DURATION: fulminant, acute and protracted forms. The criteria for severity differentiate between mild, moderate, severe or malignant forms, and include clinical and laboratory parameters as shown in the present study. RESULTS: Classification of the forms according to MSF course defines them in order of severity, typicality, manifestation, duration of symptoms, complications and age characteristics. According to the accepted criteria for severity and with respect to the studied I and II phase of the disease the mild forms are 41.16%-35.62% (p > 0.05), moderate forms are 32.79%-43.11% (p < 0.01), severe forms are 16.03%-11.37% (p = 0.05), malignant forms are 6.56%-8.68% (p > 0.05), and mortality is 3.46%-1.19% (p < 0.05). The mean age was significantly higher for patients with severe forms of MSF (58.59 +/- 4.32 yrs) compared with those with moderate (46.10 +/- 3.71 yrs, p < 0.05) and mild forms (42.05 +/- 3.50 yrs, p < 0.01). For children up to 14 years old mild forms are more common than in adults over 65 (p < 0.0001). Among children up to 14 years old there were no lethal outcomes, while mortality rate in the patients older than 65 was as much as 10%. All this indicates that MSF runs a milder course in children and a severe, complicated course in the elderly. CONCLUSION: The criteria for MSF severity we have selected are based on our own experience and the experience of other authors. They are based on the reaction of human organism to the pathogenic agent and can be used during the different phases of emergence and development of rikettsial diseases, regardless of their geographic distribution. Unified use of these criteria would eliminate the differences in the data reported by different researchers regarding the disease development and severity.

Dynamics in serum cytokine responses during acute and convalescent stages of Mediterranean spotted fever.

UNLABELLED: Mediterranean spotted fever (MSF) is a re-emerging rickettsiosis in Bulgaria after 20 years of absence (1972-1992), and it has since been affecting many people annually in the endemic regions of the country. The role of cytokines in MSF is still in the focus of research due to their complex participation in the immune pathogenesis of the disease. AIM: To study the changes in the serum cytokine concentrations in MSF patients. PATIENDS AND METHODS: Eighty patients with MSF and 20 healthy controls were enrolled in the study. The pro-inflammatory and immunoregulatory cytokines IL-1beta, TNF-a, IL-6, IL-8, IL-12, IFN-gamma, IL-2, and IL-10 were studied in the burst of disease, at clinical recovery stage, and two weeks later. The disease etiology was verified by indirect IFA in the Referral Rickettsiosis Laboratory. The cytokine levels were determined by ELISA (BioSource Europe S.A). RESULTS: In the disease flare up patients showed a manifold increase in the activity of IL-1beta (p < 0.01), TNF-alpha (p < 0.001), IL-6 (p < 0.001), and IL-8 (p < 0.001) compared with the controls. Significant elevation in IFN-gamma and IL-12 values (p < 0.001) was also found. The increase in the immunoregulatory IL-10 also reached statistical significance (p < 0.001), while the rise in IL-2 did not (p > 0.05). Followed in dynamics, only IL-1beta and IL-6 measured up the control levels at the time of clinical recovery. Two weeks later, in the early convalescence IL-12 and TNF-alpha further diminished but did not normalize their values. CONCLUSION: Our findings show that MSF is characterized by a Th1 cytokine profile. The patient's immune system responds by proinflammatory and immunoregulatory cytokine production that accompanies the rickettsial vasculitis and contributes to the healing process. The latter is probably not fully achieved in the early convalescent period, according to our data concerning some pro-inflammatory cytokines' elevation at this period.

Immune response and immunologic memory in medical personnel vaccinated with hepatitis B vaccine.

UNLABELLED: The occupation-related nature of Hepatitis B viral infection in medical personnel has been well documented in a lot of studies. The only reliable way of prevention of this infection is immunisation with hepatitis B vaccine. AIM: To follow-up the primary immune response after immunisation with recombinant vaccine and its duration in adult immunocompetent subjects. MATERIALS AND METHODS: One hundred sixty-five health-care workers working at St. George University Hospital, Plovdiv in 2009/2010 were included in the study and allocated to two groups. Group 1 (N1 = 70) was followed up for the primary immune response after immunization; group 2 (N2 = 95) was with documented immunization in 1998/1999 (n = 81) and in 1994/1995 (n = 14). Tests based on ELISA for quantitative determination of anti-HBs in mIU/ml were used. The measurement were performed at the National Reference Laboratory of Viral Hepatitis at the NCIPD, Sofia. Descriptive statistics, non-parametric and parametric tests, qualitative correlation were used to analyse data. RESULTS: Group 1 mean age was 40.3 +/- 2.6 years; anti-HBs concentration of > or = 10 mIU/ ml was found in 92.8%. No association between the immune response and the commonly involved factors such as gender, age, overweight, smoking, etc., was found. In group 2, anti-HBs concentration of > or = 10 mIU/ml was found in 77.9%: it was in 75.3% in those immunized 10 years before, and in 92.9% in those immunized 15 years before (t = 0.24, p > 0.05). A booster dose of the vaccine was received by 15/21 subjects from group 2 (those immunized 10 years before that) with anti-HBs < 10 mIU/ml. After the booster, 9/15 produced anti-HBs in protective concentrations (anamnestic immune response). The actual level of seroprotection among the immunized more than 10 years ago was 92%. CONCLUSION: This study and the documentation of the primary postvaccinal immunity in high-risk medical personnel will help specify if additional hepatitis B vaccine shots are needed.

HIV/AIDS-associated Kaposi's sarcoma with multiple skin-mucosal disseminations following ultraviolet (puva) photochemotherapy.

UNLABELLED: HIV/AIDS infection in Bulgaria has spread over about 1200 registered patients and it is supposed that the number of the undetected cases is four times higher. Kaposi's sarcoma is rarely observed in our country and no cutaneous-mucosal dissemination is reported for the time being. AIM: The aim of the study is to present a case of disseminated Kaposi's sarcoma in a HIV/ AIDS patient who underwent Psoralen--UVA radiation treatment (PUVA) for total alopecia. METHODS: HIV was proved through ELISA and Western blot (InnoLia HIV I/II Score). PCR method (COBAS-Amplicor HIV-1 MT, 1,5) was used to determine viral load (VL). Monitoring was realized by flow-cytometric phenotype analysis of the immune cells. Biopsy of a skin lesion was performed for histomorphological analysis. Computed axial tomography (CAT) of the visceral organs was also applied. RESULTS: The patient's face, chest, back and upper extremities are covered by more than 50 typical for Kaposi's sarcoma skin tumors and several isolated lesions are found in the oral cavity mucosa. The histological results show dilated vascular spaces with large endothelial cells and spindle-like tumor cells in irregularly formed fascicles. Monitoring of the immune cells and the viral load before and after the application of highly active antiretroviral therapy (HAART) showed CD4+ T cell number = 0.147 x 10(9)/l and VL = 216 000 copies HIV-RNA/ml plasma when the disorder was first detected. A very good effect appeared 4 months after the HAART start: the mucous membrane lesions disappeared and the skin tumors decreased by number and dimensions. In the same time the CD4+ T cell number increased up to 0.255 x 10(9)/l and VL values decreased < 400 c/ml. CONCLUSION: Disseminated form of Kaposi's sarcoma can be provoked by additional immunosuppressive factors like the implementation of PUVA therapy. Early initiation of HAART improves the process and prevents visceral dissemination.