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 fi rst 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 confi rmed by immunofl uorescent 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 fi fth 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 fi rst phase of rapid increase. The epidemiologic characteristics of MSF in the region are in support of its prevention and control.

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.

The study demonstrates the presence of immune memory and protection 5-15 years after the initial course of newborn immunization with recombinant vaccines against hepatitis B.

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ABSTRACT

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-1β, TNF-α, IL-6, IL-8, IL-12, IFN-γ, 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 EL ISA (BioSource Europe S.A). Results: In the disease flare up patients showed a manifold increase in the activity of IL- 1β (р < 0.01), TNF-α (p < 0.001), IL-6 (р < 0.001), and IL-8 (p < 0.001) compared with the controls. Significant elevation in IFN-γ 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 (р > 0.05). Followed in dynamics, only IL-1β 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-α 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.

Objectives: Mediterranean spotted fever (MSF) is a tick-borne endemic disease caused by Rickettsia conorii conorii and transmitted to humans by the brown dog tick Rhipicephalus sanguineus. It is characterized by fever, maculopapular rash and a tick bite skin lesion "tache noire". The disease affects all age groups and usually has mild to moderate course. It has long been considered a benign infection. However, very severe "malignant" forms with fatal outcome strongly influenced by the patients' age were also observed. We aimed to reveal the features of MSF in children by comparing them with some of the disease patterns in adults.Methods: Our prospective study data are based on the monitoring of all inpatients of the Clinic of Infectious Diseases in Plovdiv City and environs, the largest MSF endemic region in Bulgaria. The total number of patients admitted and treated for MSF over a study period of 10 years is 549, the incidence reaching 9.44/100,000. MSF is confirmed by antibody response to a specific antigen measured by indirect immunofluorescence assay (IFA). IgG ≥ 128 and/or IgM > 64 were considered to be indicative of acute infection.Results: Eighty-five (15.48%) patients were children up to 14 years of age and 464 (84.52%) were aged 15-85 years. The skin lesion "tache noire" was present in 73.68% of children, and in 76.93% of the older age groups; symptoms as anorexia, nausea, vomiting, abdominal pain, and diarrhoea were more frequent in children, till general weakness and malaise was prevalent in adults. Children accounted approximately for 70% of the disease mild forms. No lethality was registered in this age group. Even though there were isolated cases of severe and malignant forms, children did not undergo the fatal complications seen in adults.Conclusion: Knowledge of the peculiarities of MSF in children will contribute to children's health protection, timely diagnose and disease prevention.

Abstract:Mediterranean spotted fever (MSF) is widely prevalent in many endemic regions in Bulgaria. The disease is still not quite thoroughly studied as to some aspects of its pathogenesis and especially to issues that concern the crucial signals for apoptosis in the target microvascular endothelial cells. To study the expression of Bcl-2 family proteins and Caspase-3 in the dermal capillary endothelial cells from skin papules and in the eschar (tache noire) epidermal layers of patients with MSF so that we can establish apoptotic processes and the time of their occurrence and deployment. Immunohistochemical reactions for Bcl-2, Bax and Caspase-3 were obtained in slices of punch-biopsies taken from papules of the skin rash and from the eschars of eight patients with MSF. The average intensity of the reactions was compared with that in control punch-biopsy slices from four healthy subjects. MSF was etiologically confirmed in all patients by positive antibody response to a specific antigen, Rickettsia conorii, with indirect immunofluorescent assay performed by the Rickettsial Reference Laboratory. The immune reaction for Bcl-2 was found to be poorly expressed in the capillary endothelial cells of skin papules of patients without any differences from controls. The expression of Bax and Caspase-3 was strongly upregulated in comparison with the controls. The Bcl-2/Bax ratio was significantly decreased. Microvascular endothelial cells of the eschar showed similar changes. While the Bcl-2/Bax ratio decreased in the epidermal layers of the eschar "tache noire", there were no changes in the intensity of the immunoreactivity of Caspase-3 as compared with controls. The upregulation of Bax and Caspase-3 is an indication of ongoing apoptotic processes in the dermal microvascular endothelial cells of MSF patients. The epidermal layers of the eschar showed increased sensitivity to apoptosis, however, executive phase of apoptosis did not occur.