Compassionate mesenchymal stem cell treatment in a severe COVID-19 patient: a case report.

COVID-19 presentations range from cold-like symptoms to severe symptoms with the development of acute respiratory distress syndrome (ARDS). We report on a severe COVID-19 patient who was mechanically ventilated and who developed ARDS and bacterial infection. Because of rapid clinical deterioration and the exhaustion of other treatment options, the family and attending physicians requested a compassionate use of adult allogeneic bone marrow-derived mesenchymal stem cells (MSC) in addition to commonly used immunosuppressive, antiviral, and supportive therapy. The clinical course is discussed thoroughly, with a special emphasis on the safety and effect of MSC therapy. Compassionate MSC treatment, given in three rounds, affected ARDS regression. The patient was discharged from the intensive care unit after 31 days and from hospital after 49 days in a good general condition. MSC treatment was not associated with any side effects and was well tolerated in a three-week period; therefore, it should be studied in larger trials and considered for compassionate use.

Case of Acute Disseminated Encephalomyelitis Associated with Cytomegalovirus Reactivation in an Immunocompromised Systemic Lupus Erythematosus Patient.

We present a case of an immunocompromised systemic lupus erythematosus female patient admitted to our hospital for general impairment, monoparesis, and temporary cognitive disability. The case represented a significant diagnostic and therapeutic challenge primarily because of a wide range of differential diagnosis options (CNS lupus, ischemic cerebrovascular disease, viral meningoencephalitis, progressive multifocal leukoencephalopathy, limbic encephalitis, and acute disseminated encephalomyelitis-ADEM). Brain MRI findings were compatible with ADEM, and microbiological tests showed a cytomegalovirus infection (CMV) which is rarely associated with ADEM despite the increasing number of immunocompromised patients prone to symptomatic CMV reactivation. Our patient was treated with intravenous methylprednisolone, immunoglobulin (IVIG), along with antiviral therapy resulting in a favorable therapeutic effect. In conclusion, only a few described ADEM cases have been associated with CMV, and none of them, to the best of our knowledge, in an immunocompromised patient. In this case, a multidisciplinary approach and broad diagnostic considerations were decisive for successful treatment and outcome.

A Comprehensive Analysis of Renal and Endothelium Dysfunction Markers Fourteen Years after Hemorrhagic Fever with Renal Syndrome Contraction.

While the pathology of acute hemorrhagic fever with renal syndrome (HFRS) has been widely researched, details on the chronic HFRS sequelae remain mainly unexplored. In this study, we analyzed the clinical and laboratory characteristics of 30 convalescent HFRS patients 14 years after the disease contraction, mainly emphasizing several endothelial dysfunction parameters. Convalescent HFRS patients exhibited significantly higher serum levels of erythrocyte sedimentation rate, von Willebrand factor, uric acid, C-reactive protein and immunoglobulin A when compared to healthy individuals. Furthermore, 24 h urine analyses revealed significantly lower sodium and potassium urine levels, as well as significantly higher proteinuria, microalbumin levels and ß2-microglobulin levels when compared to healthy individuals. First morning urine analysis revealed significantly higher levels of hematuria in convalescent HFRS patients. None of the additional analyzed endothelium dysfunction markers were significantly different in post-HFRS patients and healthy individuals, including serum and urine P-selectin, E-selectin, soluble intercellular adhesion molecule 1, vascular intercellular adhesion molecule 1 (sVCAM-1) and vascular endothelial growth factor (VEGF). However, binary logistic regression revealed a weak association of serum sVCAM-1 and urine VEGF levels with HFRS contraction. Generally, our findings suggest mild chronic inflammation and renal dysfunction levels in convalescent HFRS patients 14 years after the disease contraction.

Blood markers of endothelial dysfunction and their correlation to cerebrovascular reactivity in patients with chronic hepatitis C infection.

Although liver cirrhosis and hepatocellular carcinoma are major consequences of hepatitis C (HCV), there has been an increasing number of studies examining extrahepatic manifestations, especially those caused by systemic chronic inflammation and metabolic complications that might predispose HCV patients to atherosclerosis and ischemic cerebrovascular disease (CVD). The aim of our study was to assess E-selectin, VCAM-1, ICAM-1 and VEGF-A serum levels in patients with chronic HCV infection and to correlate them with cerebrovascular reactivity. A blood sample was taken from eighteen patients with chronic hepatitis C infection and from the same number of healthy blood donors in the control group. The aim was to analyse markers of endothelial dysfunction and to correlate them with cerebrovascular reactivity expressed as breath-holding index (BHI) determined using transcranial color Doppler. The obtained results revealed significant differences between the groups in all endothelial markers except for the E selectin. While the ICAM-1 and sVCAM-1 were significantly increased in the hepatitis group, VEGF-A was significantly decreased. A significant reduction of 0.5 (95% CI 0.2, 0.8) in the mean BHI was found in the hepatitis group (mean BHI 0.64) compared to controls (mean BHI 1.10). No significant association between the BHI and any of the endothelial markers was found in the control group, while in the hepatitis group, the scatter plot of ICAM-1 vs BHI suggested that the association might be present. In conclusion, the results of this study confirm an association between a chronic HCV infection and altered cerebrovascular reactivity as well as higher levels of markers of endothelial activation (ICAM-1, VCAM-1) as possible indicators of an increased CVD risk.

Clinical, Virological, and Immunological Findings in Patients with Toscana Neuroinvasive Disease in Croatia: Report of Three Cases.

Toscana virus (TOSV) is an arthropod-borne virus, transmitted to humans by phlebotomine sandflies. Although the majority of infections are asymptomatic, neuroinvasive disease may occur. We report three cases of neuroinvasive TOSV infection detected in Croatia. Two patients aged 21 and 54 years presented with meningitis, while a 22-year old patient presented with meningoencephalitis and right-sided brachial plexitis. Cerebrospinal fluid (CSF), serum, and urine samples were collected and tested for neuroinvasive arboviruses: tick-borne encephalitis, West Nile, Usutu, TOSV, Tahyna, and Bhanja virus. In addition, CSF and serum samples were tested for the anti-viral cytokine response. High titers of TOSV IgM (1000-3200) and IgG (3200-10,000) antibodies in serum samples confirmed TOSV infection. Antibodies to other phleboviruses (sandfly fever Sicilian/Naples/Cyprus virus) were negative. CSF samples showed high concentrations of interleukin 6 (IL-6; range 162.32-2683.90 pg/mL), interferon gamma (IFN-γ; range 110.12-1568.07 pg/mL), and IL-10 (range 28.08-858.91 pg/mL), while significantly lower cytokine production was observed in serum. Two patients recovered fully. The patient with a brachial plexitis improved significantly at discharge. The presented cases highlight the need of increasing awareness of a TOSV as a possible cause of aseptic meningitis/meningoencephalitis during summer months. Association of TOSV and brachial plexitis with long-term sequelae detected in one patient indicates the possibility of more severe disease, even in young patients.

Evaluation of cerebrovascular reactivity in chronic hepatitis C patients using transcranial color Doppler.

Hepatitis C viral (HCV) infection is associated with systemic inflammation and metabolic complications that might predispose patients to atherosclerosis, including cerebrovascular atherosclerosis. The aim of this study was to assess cerebrovascular reactivity in patients with chronic hepatitis C. Seventeen patients with chronic hepatitis C infection, as well as 11 healthy blood donors in the control group, were assessed for cerebrovascular reactivity according to the well-established breath-holding test that uses the transcranial color Doppler for measurement of blood flow velocity. Results obtained during the breath-holding revealed significantly lower average peak systolic (AvPS start, P = 0.018), end-diastolic (AvED start, P = 0.031) and mean velocity values at the very beginning of the breath-holding procedure (AvmeanV start, P = 0.02), as well as a lower mean peak systolic velocity at the end of the breath-holding test (AvPS max, P = 0.02) in the hepatitis C group. Vascular reactivity values, calculated as the breath-holding index, were also significantly lower (P = 0.045) in the hepatitis C group. In conclusion, the results of this study suggest an association between chronic HCV infection and altered cerebrovascular reactivity which may ultimately have an unfavorable effect on cerebrovascular hemodynamics and lead to increased risk of cerebrovascular diseases.

Distribution of rotavirus genotypes in three Croatian regions among children ≤5 years of age (2012-2014).

OBJECTIVES: Rotavirus is the major cause of severe diarrhea in young children worldwide. In countries like Croatia, where rotavirus vaccine has not been introduced in the national immunization program, prospective surveillance is necessary to establish the diversity of rotavirus strains. The aim of this study was to describe the prevalence and geographical distribution of rotavirus strains in Croatia and to detect the possible emergence of novel strains. METHODS: The study was conducted among children ≤5 years of age with acute gastroenteritis at three hospitals located in different geographical regions of Croatia, during the years 2012 to 2014. Rotavirus was detected in stools using an immunochromatographic assay and then sent for further molecular analysis. RESULTS: Genotyping of 822 rotaviruses showed that the predominant circulating strain was G1P[8] (61.9%), followed by G2P[4] (19.5%), G1P[4] (3.9%), and G3P[8] (2.9%). A high prevalence of reassortants among common human rotavirus genotypes was detected (7.7%). Possible zoonotic reassortants were found, including G8 and G6 strains. The latter is described for the first time in Croatia. CONCLUSIONS: This study represents pre-vaccination data that are important for decisions regarding immunization strategies in Croatia. The high prevalence of 'common' rotavirus strains circulating in Croatia may advocate for rotavirus vaccine introduction, but further surveillance is necessary to monitor the possible emergence of novel genotypes.

Hla-Cw7 allele as predictor of favorable therapeutic response to interferon-alpha in patients with chronic hepatitis C.

AIM: To evaluate the association between human leukocyte antigens (HLA) class I and therapeutic response to interferon-alpha in Croatian patients with chronic hepatitis C. METHODS: HLA-A, -B, and -C genotyping was performed in 55 patients with sustained virological response and in 57 patients without sustained virological response to interferon-alpha therapy. Patients were treated in the period from 1998-2001 with interferon-alpha at a dose of 3 million units three times a week. Patients who became negative for hepatitis C virus RNA after 12 weeks of therapy completed 48 weeks of therapy. RESULTS: There was no association between therapeutic outcome and frequency of HLA-A, as well as of HLA-B alleles. HLA-Cw7 was significantly more frequent in patients with than those without sustained virological response (27.0% vs 6.7%; P=0.011). CONCLUSION: In Croatian patients with chronic hepatitis C, HLA-Cw7 is the predictor of sustained virological response to interferon-alpha therapy.

[Haemophilus parainfluenzae--a rare cause of endocarditis]. / Haemophilus parainfluenzae--rijedak uzrocnik endokarditisa.

Haemophilus parainfluenzae endocarditis is a rare acute or subacute disease. It is usually associated with dental and surgical procedures in the oral cavity. In a 23-year-old athlete admitted to Department of Infectious Diseases, Split University Hospital, the diagnosis of infective endocarditis was established based on Duke's criteria. The patient was not exposed to risky medical procedures nor he had a predisposing heart disease. The course of illness was characterized by peripheral embolizations (splinter hemorrhages and petechiae) and vegetation on the dorsal mitral valve. After seven days of incubation, blood cultures revealed Haemophilus parainfluenzae. The patient was treated intravenously with amoxicillin-clavulanic acid plus gentamicin for four weeks. Peroral therapy was continued with amoxicillin-clavulanic acid alone for two weeks. By the end of treatment, heart ultrasound showed disappearance of endocardial vegetation. Echocardiographic and clinical examinations performed at 3, 6 and 12 months of therapy showed no pathological aberrations.

Venomous snakebites in children in southern Croatia.

This retrospective study represents observation of 160 children and adolescents aged up to 18 years that experienced venomous snakebites in southern Croatia and were treated in the Clinical Department of Infectious Diseases in the University Hospital Centre Split from 1979 to 2013. The main purpose of this research was to determine the epidemiological characteristics, clinical presentation, local and general complications, and received treatment. Most bites occurred during warm months, from early May to late August (80%), mostly in May and June. Upper limb bites were more frequent (59%) than lower limb bites (40%). Out of the total number of poisoned children, 24% developed local, and 25% general complications. The most common local complications were haemorrhagic blisters that occurred in 20% children, followed by compartment syndrome presented in 7.5% patients. The most dominated general complication was cranial nerve paresis or paralysis, which was identified in 11.2% patients, whereas shock symptoms were registrated in 7% children. According to severity of poisoning, 9.4% children had minor, 35% mild, 30.6% moderate, and 24.4% had severe clinical manifestation of envenomation. Only one (0.6%) child passed away because of snakebite directly on the neck. All patients received antivenom produced by the Institute of Immunology in Zagreb, tetanus prophylaxis as well, and almost all of them received antibiotics, and a great majority of them also received corticosteroids and antihistamines. Neighter anaphylactic reaction nor serum disease were noticed in our patients after administrating antivenom. A total of 26% children underwent surgical interventions, and incision of haemorrhagic blister was the most common applied surgical treatment, which was preformed in 15.6% patients, while fasciotomy was done in 7.5% subjects. All of our surgically treated patients recovered successfully.

A child with Bartonella henselae osteomyelitis of the right humerus.

We present a case of a 22-month-old child with swollen upper part of the right arm and osteolytic lesion of the right humerus, which resembled a neoplastic process. Epidemiological history revealed no scratch marks on the skin or cutaneous papule or pustule. Presumptive diagnosis of hematogenous osteomyelitis was established, but treatment with fusidic acid was unsuccessful. Histological examination of the bioptic specimen of the soft tissue swelling showed a lymph node morphology, with numerous granulomas with central stellate necrosis. Indirect immunofluorescence assay for Bartonella henselae yielded positive results. New treatment included 15 days of trimetoprime and sulfamethoxazole, followed by azithromycin for 5 days. Four months later, swelling resolved and osteolytic lesion almost completely healed with formation of surrounding sclerosis. In conclusion, cat-scratch disease without positive epidemiological history and primary cutaneous papule or pustule may be a serious diagnostic problem, but can be solved by serological and histological examination.

[Is permanent renal function damage possible after hemorrhagic fever with renal syndrome?]. / Je li moguce trajno ostecenje bubrezne funkcije nakon preboljele hemoragijske vrucice s bubreznim sindromom?

AIM: To investigate the possibility of permanent renal function impairment and other organ lesions following hemorrhagic fever with renal syndrome (HFRS). METHODS: Data on 30/37 patients infected with HFRS, treated at the Department of Infectious Diseases, Split University Hospital, in 1995 were retrospectively analyzed. The data were collected three to six years following the appearance of HFRS. In 1998, 30/37 patients presented for control checkup, when their history data were collected, along with physical examination, hematology and biochemistry tests, and urinalysis. Creatinine clearance and sodium, potassium, chlorine, phosphorus, beta 2-microglobulin and N-acetyl-beta-D glucosaminase in 24-h urine were determined. In native urine, erythrocyturia was observed, with 10 erythrocytes per field were considered pathologic result. During the 1998-2001 period, renal scintigraphy by means of technetium labeled diethylene triaminopentacetic acid (99mTC DTPA) was performed in 13/30 patients. RESULTS: Of subjective discomforts, 29/30 (96.7%) patients reported lumbar pain. Elevated blood pressure was found in 9/30 (30.0%), erythrocyturia in 4/30 (13.3%) and hepatic lesion in 4/30 (13.3%) patients. Decreased creatinine clearance values (< 1.2 ml/s) were found in 4 and increased values (> 2.35 ml/s) in 10 patients. Increased sodium in 24-h urine was recorded in 10/23 and increased beta 2-microglobulin in 6/23 (26%) patients. Proteinuria exceeding 150 mg/day was detected in 11/23 (47.8%) patients. Scintigraphy of the kidneys demonstrated reduced glomerular filtration (< 100 ml/min/1.72 m2) in 3/13 patients. Prolonged mean times (> 5 minutes) of radiopharmaceutical passage through the renal parenchymae were found in 7/13 (53.8%) patients. DISCUSSION: Studies performed in 30 patients three years after they had recovered from HFRS revealed changes suggesting a mild to moderate impairment of the renal function. Hypertension found in 9/30 patients was a significant finding, considering the fact that all subjects were soldiers, thus having undergone through examinations to prove them completely healthy prior to joining army. Hypertension results were consistent with those reported from the USA. Although erythrocyturia points to urinary tract damage, its glomerular or postglomerular origin was not examined. Decreased creatinine clearance found in 4/23 patients suggested functional renal impairment. Increased natriuresis found in 10/23 patients implied tubular damage, i.e. reduced ability of tubular cells for sodium reabsorption from primary urine. Non-selective albuminuria detected in 11/23 patients indicated permanent lesion of the glomerular basal membrane. Increased beta 2-microglobulin found in 6/23 patients indicated that the lysosomal enzyme level was elevated only in the acute stage of the disease, but may have been an indicator of permanent lesion. No description of post-HFRS scintigraphic lesion of the kidneys was found in the literature. A decreased value of glomerular filtration, found in three patients, and especially the prolonged mean time of glomerular micropharmaceutical passage in 7/13 (53.8%) patients may have suggested glomerular damage. However, the possible reason may have also been a reduced passage of glomerular filtrate through the damaged lower parts of the nephrons. Transaminase increase during the acute stage of HFRS suggested the possible liver infection, maybe even hantavirus replication in hepatocytes. Even though biopsy confirmed the histologic picture of chronic hepatitis in one patient, the question remains whether it could have been caused by hantavirus. CONCLUSION: Studies performed in 30 patients with a history of HFRS revealed renal function impairment, along with hypertension and damage to the liver parenchyma in some patients. The results obtained showed that the HFRS infection in Croatia may have entailed chronic sequels. To confirm this hypothesis, additional studies including a control group of hantavirus negative persons are needed.

Campylobacter fetus infection presenting with bacteremia and cellulitis in a 72-year-old man with an implanted pacemaker: a case report.

INTRODUCTION: Campylobacter is an important causative agent of intestinal infections in humans. Bacteremia is detected in less than 1% of patients, mainly in immunocompromised patients and in extreme age groups. Cellulitis is a relatively common manifestation of Campylobacter infection, but concomitant bacteremia is a rare event. Infections of the pacemaker area are caused primarily by staphylococci, followed by fungi, streptococci and Gram-negative rods. To the best of our knowledge, this is the first case report of pacemaker pocket infection and bacteremia caused by Campylobacter fetus. CASE PRESENTATION: A 72-year-old Croatian Caucasian man with myelodysplasia, impaired fasting glucose levels and a recently implanted permanent pacemaker was admitted to hospital after six days of fever, development of red swelling of the pacemaker pocket area and worsening of his general condition. No antibiotic therapy was introduced in the outpatient setting. He denied any recent gastrointestinal disturbances. With the exception of an elevated leukocyte count, erythrocyte sedimentation rate, and C-reactive protein and blood glucose levels, other laboratory findings were normal. Treatment with vancomycin plus netilmicin was introduced, and a surgical incision with drainage of the pacemaker pocket was performed. The entire pacemaker system was removed and a new one re-implanted after 14 days of antibiotic therapy. Transesophageal echocardiography showed no pathological findings. Three subsequent blood cultures obtained on admission as well as swab culture of the incised pacemaker area revealed Campylobacter fetus; stool and pacemaker lead cultures were negative. According to the microbiological results, antibiotic therapy was changed to ciprofloxacin plus netilmicin. A clinical examination and the results of a laboratory analysis performed after two weeks of therapy were within normal limits. CONCLUSION: Myelodysplasia, impaired fasting glucose levels and older age could be contributing factors for the development of bacteremic Campylobacter fetus cellulitis. Emergent surgical and antibiotic treatment are mandatory and provide the optimal outcome for such types of pacemaker pocket infection.

Chronic fatigue syndrome after Q fever.

BACKGROUND: Q fever is a common and acute but rare chronic zoonosis caused by Coxiella burnetii. Its acute form manifests as atypical pneumonia, flu-like syndrome, or hepatitis. Some authors observed symptoms of chronic fatigue in a small number of patients after the acute phase of Q fever; in many cases serological assay confirmed the activity of Coxiella burnetii infection. The effect of antibiotic therapy on post-Q-fever fatigue syndrome has not been studied in south-east Europe thus far. CASE REPORTS: Three patients are presented with post-Q-fever fatigue syndrome. All fulfilled the CDC criteria for chronic fatigue syndrome. IgA antibodies to phase I of the growth cycle of Coxiella burnetii were positive in two patients and negative in one. Two patients were treated with doxycycline for two weeks in the acute phase of illness and one with a combination of erythromycin and gentamycin. After 4-12 months they developed post-Q-fever fatigue syndrome and were treated with intracellular active antibiotics (fluoroquinolones and tetracycline) for 3-12 months. Efficacy of the treatment was observed in two patients, but in one patient the results were not encouraging. CONCLUSIONS: These results suggest the possibility of the involvement of Coxiella burnetii infection in the evolution of chronic fatigue syndrome. This is the first report on post-Q-fever fatigue syndrome in Mediterranean countries. Evidence of IgA antibodies to phase I of the growth cycle of Coxiella burnetii is not a prerequisite for establishing a diagnosis of CFS. The recommendation of antibiotic treatment in post-Q-fever fatigue syndrome requires further investigation.

Clinical and epidemiological features of hospitalized acute Q fever cases from Split-Dalmatia County (Croatia), 1985-2002.

BACKGROUND: Q fever shows a wide diversity of clinical manifestation. Q fever is endemic in northern Croatia, but the epidemiological and clinical characteristics of this disease in various ecological areas of southern Croatia are unclear. MATERIAL/METHODS: From January 1985 to December 2002, acute Q fever cases hospitalized at Split University Hospital were analyzed. Acute Q fever was defined as fever (>38 degrees C) with clinical findings in lung and/or liver verified by serologic testing with Coxiella burnetii phase II antigen. RESULTS: During the period of observation, 155 acute Q fever cases were hospitalized. The mean incidence of acute Q fever in the study region was 0.20/100,000/year (95%CI:0-0.78) in the coastal area and 4.64/100,000/year (95%CI:0.44-8.85) in the non-coastal areas, with a male predominance (chi2=60.0; p=0.0000) and a mean male to female ratio of 4.2:1. People of essentially all ages (4-76 years) were affected, the highest rate of infection being recorded in 20- to 49-year-old age groups. In contrast to adults, girls were more frequently affected than boys (2:1). No case of acute Q fever was recorded on any of the nearby islands. Clinically, acute Q fever most commonly presented with both pneumonia and hepatitis (60.0%), followed by pneumonia (25.8%), hepatitis (9.0%), and nonspecific febrile illnesses (5.2%). CONCLUSIONS: C. burnetii is endemic in rural, coastal, and non-coastal areas of southern Croatia and is associated with stock breeding. In these areas, Q fever occurs sporadically and epidemically. Males 20-49 years of age were the prevalent cases.

Dinara - new natural focus of hemorrhagic fever with renal syndrome in Croatia.

AIM: To investigate the characteristics and determine risk factors for hantanvirus infection in natural focus of hemorrhagic fever with renal syndrome (HFRS) on the Dinara Mountain, where outbreak of disease emerged among Croatian soldiers in 1995, and to describe the features of HFRS acquired on the Dinara mountain and determine the scale of the largest HFRS epidemic so far in Croatia. METHODS: During 1996, small mammals were captured in the region of Dinara Mountain where infected Croatian soldiers had sojourned. By taxonomic classification of 42 captured small mammals, three species were determined: 23 yellow-necked mouse, 9 wood mouse, and 5 bank vole. Hantavirus antigen was determined in the lungs of the captured animals by means of direct immunofluorescence assay. The most important features of HFRS were retrospectively determined in 37 soldiers with HFRS treated in the Department for Infectious Diseases of the Split University Hospital. The degree of inapparent exposure to infection was determined by indirect immunofluorescence in 103 soldiers sojourning in this region of natural focus with no apparent signs of HFRS. Epidemiological questionnaire included 50 soldiers with negative serum antibodies, as well as 33 available out of total 37 soldiers with HFRS. Chi-square test was used to determine risk factors. RESULTS: Hantavirus was found in the lungs of 5/42 (12%) captured animals. Mild form of the disease, with few hemorrhagic symptoms and pronounced renal insufficiency, was present in 19/37 patients. The epidemiological questionnaire determined the following risk factors for hantanvirus infection in this focus: service in artillery corps (p=0.040), sleep in wooden barracks (p=0.004), station in forest biotope (p=0.037), usage of natural camouflage (p=0.024), smoking (p=0.010), and the presence of rodents in the place of housing (p<0.001). CONCLUSION: A new natural focus of HFRS in Croatia, and the first one in Dalmatia, was defined by seroepidemiologic, mamologic, and virologic analysis. The risk factors for infection in the new focus have been identified. Our patients suffered from a mild form of HFRS, which predominates in south-eastern Europe, without lethal outcome.

First isolation of Rickettsia conorii from human blood in Croatia.

AIM: To detect and isolate rickettsial strains from blood samples of patients with presumptive diagnosis of Mediterranean spotted fever (MSF) in the coastal region of south Croatia, and to compare the results with routine serology. METHODS: A "suicide" polymerase chain reaction (PCR), and a shell vial culture were done on samples of ethylenediamine tetra-acetic acid (EDTA) and citrate-anticoagulated blood samples. Indirect immunofluorescence was performed on sera collected from 17 patients clinically diagnosed with MSF during summer in three consecutive years, from 1998 to 2000. RESULTS: The primers used in PCR amplified the expected part of the rickettsia genomic DNA and Rickettsia conorii grew from the shell vial-cultured blood of a single patient. In 13 patients, the diagnosis was confirmed serologically by paired sera, whereas in 4 patients the diagnosis remained presumptive, since no paired sera were available. Analyzing sequences of the ompA and citrate synthase gene, respectively, derived from the shell vial isolate, a 100% similarity with Rickettsia conorii, strain Seven (Malish), was found. CONCLUSION: To the best of our knowledge, this is the first isolation of Rickettsia conorii from a human sample in Croatia, and the first proof of a causative agent of MSF in the country. Beside PCR-based methods and isolation, correct diagnosis of MSF could be still routinely reached by serology.