Hemolysis-in-gel (HIG) test for antibodies to influenza A, measles and mumps using liquid nitrogen freezed erythrocytes coupled with the respective viral antigen.

A drawback with the hemolysis-in-gel test is the constant need for fresh erythrocytes which must be treated with virus before incorporation in the gel. This problem was overcome by freezing small droplets containing erythrocytes to which antigen had been attached. The droplets were stored at -70 degrees C or in liquid nitrogen. This modification was applied in detecting antibodies to influenza, measles and mumps viruses and the results were shown to equal those obtained with fresh erythrocytes.

An echocardiographic study comparing male Swedish elite orienteers with other elite endurance athletes.

Between 1979 and 1992, there were 16 known cases of sudden unexpected cardiac death among young Swedish orienteers, whose autopsies showed myocarditis to be a common finding. Therefore, 96 elite orienteers and 47 controls underwent echocardiography, showing left ventricular wall motion abnormalities in 9% of the orienteers compared with 4% in the controls.

Sudden unexpected cardiac deaths among young Swedish orienteers--morphological changes in hearts and other organs.

During the years 1979-1992 an accumulation of sudden unexpected cardiac deaths (SUD) occurred among young Swedish orienteers. A reevaluation of material saved from 16 autopsies was undertaken. Myocarditis was most frequent. It was found in different stages in the majority of cases, indicating subacute or chronic disease with ongoing reparative processes. There were severe morphological changes in all cases. All but one showed a picture of fibrosis and unspecific hypertrophy and/or degenerative changes in myocytes. The hearts were classified into three groups (A-C), based on the morphological picture of the retrieved heart tissue and the macroscopic description. Group A comprised five cases in which areas with active myocarditis combined with areas of healing or healed myocarditis widely distributed in the left ventricle were the only morphological changes found. Group B comprised four cases demonstrating foci of myocarditis in different stages in the left ventricle and changes resembling those found in arrhythmogenic right ventricular dysplasia (ARVD), including degenerative changes with fibrosis and fatty infiltration located in either ventricle. Group C comprised the remaining seven cases. In none of the cases were coronary artery or valvular anomalies present, nor significant coronary sclerosis or changes outside the heart that could cause SUD.

Sequential changes in hematologic and biochemical parameters in African tick bite fever.

OBJECTIVE: To evaluate the sequential changes and to estimate the frequencies of abnormalities in some commonly measured biological variables in patients with African tick bite fever (ATBF), an emerging spotted fever group (SFG) rickettsiosis in international travelers to rural sub-Saharan Africa. METHODS: A study was done of hemoglobin, total leukocyte count, absolute lymphocyte count, blood platelet count and serum levels of C-reactive protein (S-CRP), alanine aminotransferase (S-ALAT), aspartate aminotransferase, lactic dehydrogenase, gamma-glutamyl transferase, alkaline phosphatase, bilirubin, sodium and creatinine during the first two weeks of illness and prior to the institution of antirickettsial therapy in 108 patients with travel-associated ATBF. RESULTS: There were significant falls in mean total leukocyte count, mean absolute lymphocyte count, and mean platelet count, and significant increases in mean S-CRP and S-ALAT. During the first ten days of illness, elevated S-CRP, lymphopenia and elevated S-ALAT were detected in 91.7%, 73.3% and 40.7% of patients, respectively. Most abnormalities were mild. For 55 patients who underwent both S-CRP and absolute lymphocyte count determination, at least one parameter was abnormal in 52 (94.5%) patients. CONCLUSIONS: The sequential changes in many biological parameters during the acute phase of ATBF mimic those reported in other SFG rickettsioses. Mild abnormalities are frequent, with increased S-CRP and lymphopenia being the two most consistent findings.

Effects of methyl mercury on cytokines, inflammation and virus clearance in a common infection (coxsackie B3 myocarditis).

A myocarditic coxsackievirus B3 (CB3) infection in Balb/c mice was used to investigate the effects of 12 weeks of methyl mercury (MeHg) exposure (3.69 mg/g diet) on inflammatory heart lesions, virus in the heart, the cytokine response, i.e. cachectin/TNF-alpha and gamma-interferon (IFN-gamma) levels in plasma, and on disease complications and mortality. This dose of MeHg did not influence mortality in this infection model. The inflammatory and necrotic lesions in the ventricular myocardium 7 days after the inoculation covered 2.2% of the tissue section area in infected control mice. This damage was increased (n.s.) by 50% (to 3.3% of the tissue section area) in MeHg-treated mice. The response pattern of lymphocyte subsets in situ in myocardial inflammatory lesions was corroborated using an immune histological technique. MeHg treatment tended to increase (2.2-fold, n.s.) the number of Mac 2+ cells (macrophages) in the heart muscle in this infection. Plasma levels of both TNF-alpha and IFN-gamma increased on day 3 of the infection in MeHg-treated as well as in non-MeHg-treated mice, but the mean IFN-gamma response was more pronounced in the MeHg-treated mice. On day 7 of the infection, when most animals still showed clinical signs of disease, cytokine levels were back to normal. MeHg-exposure in non-infected mice did not affect cytokine levels. In situ hybridization of virus RNA in myocardial tissue showed remaining virus in those mice who had the lowest plasma IFN-gamma levels. A 20% increased (P < 0.05) lymphoproliferative response to the T cell mitogen Con A was observed as a result of the MeHg treatment. Even heart tissue lesions and virus persistence tended to be influenced by MeHg in a direction compatible with the development of chronic disease.

Trace element changes in the myocardium during coxsackievirus B3 myocarditis in the mouse.

During most infections plasma, concentrations of trace elements change, but it is unclear if this reflects changes in infected target tissues. In coxsackievirus B3 (CB3) infection, the myocardium is a target in both humans and mice. The concentrations of 12 trace elements were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) in the myocardium of sham-inoculated controls and infected A/J mice 4 and 7 d postinoculation. The size of the inflammatory lesion was positively correlated to the virus content of the heart, as estimated by histopathology and in situ hybridization, respectively. Iron, cobalt, vanadium, and selenium showed transient changes, whereas for the other elements, tendencies on d 4 were manifest on d 7. A three-fold increase in calcium on d 7 suggests prestages of calcification, whereas increases in zinc, selenium, and copper may be the result of the accumulation of immune cells. The magnesium decrease may contribute to the increased sensitivity to cardiac arrhythmias in myocarditis.

Trace element distribution in heart tissue sections studied by nuclear microscopy is changed in Coxsackie virus B3 myocarditis in methyl mercury-exposed mice.

Methyl mercury (MeHg) has been shown to change Coxsackie virus type B3 (CB3) myocarditis in a direction compatible with the development of chronic disease. Murine models of CB3 myocarditis closely mimic the pathogenesis in humans. There are also indications that metals, such as mercury, and trace elements may interact and adversely affect viral replication and development of inflammatory lesions. The effects of low-dose MeHg exposure on myocardial trace element distribution, as determined by means of nuclear microscopy, was studied in CB3 myocarditis. Balb/c mice were fed a MeHg-containing diet (3.9 mg/kg diet) for 12 wk prior to infection. Areas of inflammatory lesions in the myocardium were identified by traditional histologic examination, and serial tissue sections in these selected areas were used for immune histology (macrophages), in situ hybridization of virus genomes, and nuclear microscopy of tissue trace element distribution. Areas with no inflammation or virus were compared with areas of ongoing inflammation and viral replication. In the inflammatory lesions of MeHg-exposed mice as compared to nonexposed mice, the myocardial contents of calcium (Ca), manganese (Mn), and iron (Fe) were significantly increased, whereas the zinc (Zn) content was decreased. The increased Ca and decreased Zn contents in the inflamed heart may partly explain a more severe disease in MeHg-exposed individuals. Although not significant in the present study, with a limited number of mice, the inflammatory and necrotic lesions in the ventricular myocardium on d 7 of the infection was increased by 50% (from 2.2% to 3.3% of the tissue section area) in MeHg-exposed mice and, also, there was a tendency of increased persistence of virus with MeHg exposure. No increased MeHg uptake, either in the inflammatory lesions or in the areas of noninflamed heart tissue in infected mice, could be detected. The present results indicate that a "competition" exists between potentially toxic heavy metals from the environment/diet and important trace elements in the body and that a disturbed trace element balance adversely influences the development of pathophysiologic changes in inflammatory heart disease.

Thallium-201 myocardial imaging at rest in male orienteers and other endurance athletes.

During the period 1979 to 1992, 16 sudden unexpected cardiac deaths were known to have occurred in young Swedish orienteers. Autopsy indicated myocarditis to be the most frequent finding, most often combined with extensive myocardial fibrosis. The aim of the present investigation was to explore whether young male orienteers show a higher frequency than other young elite endurance athletes (controls) in the occurrence of Thallium-201 myocardial perfusion defects at rest, suggestive of fibrosis evoked by myocarditis. Thallium-201 perfusion abnormalities at rest were more frequently found in the controls than in the orienteers (26% vs. 12%, p=0.03). Uneven Tl-201 perfusion was associated with left ventricular mass (r=0.32, r=0.24, p<0.01, p=0.02) and body weight (r=0.30, r=0.31, p<0.01, p=0.03) in orienteers and controls, respectively. Echocardiographic left ventricular wall motion abnormalities were found in 11 athletes (9 orienteers and 2 controls) but only two displayed an abnormal Thallium-201 perfusion scan at rest. Perfusion abnormalities at rest did not occur more frequently in the orienteers but were commonly found in both groups of apparently healthy athletes making it futile to discern abnormals from normals. Thallium-201 perfusion aberrations were not associated with left ventricular wall motion abnormalities obtained by echocardiography.

[Three Swedish cases of African tick-bite fever. Can our native Rickettsia species cause disease in humans?]. / Tre svenska fall av "African tick-bite fever". Kan våra inhemska rickettsier ge sjukdom hos människa?

The article consists in a report of three cases of African tick-bite fever in Swedish tourists returning from brief visits to South Africa. The clinical course included eschar, regional lymphadenopathy, fever and, in two cases, maculopapular rash. Two cases were characterised by significant increases in anti-Rickettsia conorii IgG and IgM antibody titres. However, the aetiological agent was assumed to be Rickettsia africae, based on reports by others and the widespread serological cross-reactivity among spotted fever Rickettsia spp. The third case was diagnosed on clinical grounds. During the past ten years, 50 per cent (41/80) of cases diagnosed serologically as rickettsial (R. conorii antigen) spotted fever at the Swedish Institute for Infectious Disease Control were associated with travel to South Africa. Parallels are drawn to the recent finding of R. helvetica in Swedish ticks (Ixodes ricinus), and the possibility of its pathogenicity to humans is discussed, though no such clinical cases have been reported to date.

Special feature for the Olympics: effects of exercise on the immune system: infections and exercise in high-performance athletes.

The elite athlete has a potentially increased sensitivity to respiratory infections, rendering protective measures particularly important. Some other infections that may appear in clusters in the sports setting, such as gastroenteritis, leptospirosis, herpes simplex and viral hepatitis, also require special precautionary attention. Strenuous exercise during ongoing infection and fever may be hazardous and should always be avoided. In addition, early symptoms of infection warrant caution until the nature and severity of the infection become apparent. Because myocarditis may or may not be accompanied by fever, malaise or catarrhal symptoms, athletes should be informed about the symptoms suggestive of this disease. Although sudden unexpected death resulting from myocarditis is rare, exercise should be avoided whenever myocarditis is suspected. Guidelines are suggested for the management and counselling of athletes suffering from infections, including recommendations on when to resume training. Acute febrile infections are associated with decreased performance resulting from muscle wasting, circulatory deregulation and impaired motor coordination, which require variable amounts of time to become normalized once the infection is over.

Infectious and lymphocytic myocarditis: epidemiology and factors relevant to sports medicine.

Myocarditis is a disease entity of permanent actuality and of special concern in the sports setting. This is because the probability of complications or sequelae increases if exercise is imposed. Myocarditis may pass unrecognized by the sufferer or be easily overlooked by the physician, especially in athletes, where electrocardiographic changes often exist normally. Furthermore, although the immune system of the conditioned individual may be more efficient than that of the sedentary individual, very strenuous and frequently repeated or long-lasting exercise may cause immunosuppression and an increased susceptibility to respiratory tract infections (RTI). Several causative agents of RTI can give rise to myocarditis as well. The true incidence of proven infectious myocarditis in society is unknown. Histopathologically defined myocarditis (lymphocytic myocarditis), on the other hand, has been found in about 1% of unselected routine autopsies. In general, the long-term prognosis in myocarditis is favorable but exercise stress may cause long-term sequelae or sudden death. It is thus essential to refrain from strenuous exercise during RTI.

Sequence variation in the ftsZ gene of Bartonella henselae isolates and clinical samples.

In a search for methods for subtyping of Bartonella henselae in clinical samples, we amplified and sequenced a 701-bp region in the 3' end of the ftsZ gene in 15 B. henselae isolates derived from cats and humans in the United States and Europe. The ftsZ sequence variants that were discovered were designated variants Bh ftsZ 1, 2, and 3 and were compared with 16S rRNA genotypes I and II of the same isolates. There was no ftsZ gene variation in the strains of 16S rRNA type I, all of which were Bh ftsZ 1. The type II strains constituted two groups, with nucleotide sequence variation in the ftsZ gene resulting in amino acid substitutions at three positions, one of which was shared by the two groups. One 16S rRNA type II isolate had an ftsZ gene sequence identical to those of the type I strains. Variants Bh ftsZ 1 and 2 were detected in tissue specimens from seven Swedish patients with diagnoses such as chronic multifocal osteomyelitis, cardiomyopathy, and lymphadenopathy. Patients with similar clinical entities displayed either Bh ftsZ variant. The etiological role of B. henselae in these patients was supported by positive Bartonella antibody titers and/or amplification and sequencing of a part of the B. henselae gltA gene. B. henselae ftsZ gene sequence variation may be useful in providing knowledge about the epidemiology of various B. henselae strains in clinical samples, especially when isolation attempts have failed. This report also describes manifestations of atypical Bartonella infections in Sweden.

Immune function in Swedish élite orienteers.

During 1979-1992 an increased frequency of sudden unexpected cardiac death (SUD) occurred among young male Swedish élite orienteers. Subacute-to-chronic myocarditis was found in 12/16 (75%) at autopsy and Chlamydia pneumoniae, or a cross-reacting agent, was suspected on the basis of diagnostic tests performed. Because myocarditis is an infrequent cause of SUD and clusters of SUD are rare, whereas Chlamydia pneumoniae infections are ubiquitous and seldom cause severe myocarditis, 119 top ranked élite orienteers (67 males and 52 females) and 36 highly trained male middle-distance runners and cross-country skiers, serving as controls, underwent immunologic screening in an effort to reveal possible immune dysfunction. Except for two orienteers and one runner/skier who showed genetic C3-deficiency or IgA-deficiency, the results showed no significant differences between the orienteers and controls with respect to immunoglobulin levels, complement activation, lymphocyte subsets, including activated T lymphocytes, and sIL-2r-alpha. IL-1 beta, IL-6, TNF-alpha, and sCD8, tested in the orienteers only, were normal. However, IFN-gamma was significantly higher in controls than in orienteers, who showed normal levels, whereas the orienteers had increased sELAM-1 and sICAM-1 levels. Finally, sIL-2 receptor-alpha was similarly elevated in orienteers and controls. We conclude that, with the tests employed, no immunologic disturbance could be revealed in the orienteers that may potentially have increased their susceptibility to myocarditis and SUD.

In situ detection of enterovirus genomes in mouse myocardial tissue by ribonucleic acid probes.

We have applied a sensitive in situ hybridization method for the detection of coxsackievirus RNA in myocardial tissue. Two radioactive cRNA probes were used: an RNA transcript representing the 5' end of poliovirus 3 which recognizes a highly conserved region among enteroviruses and an RNA transcript of a 1.1 kb fragment from the polymerase gene region of coxsackievirus B3. The reactivity of these probes was tested by dot-blot hybridization against a panel of enteroviruses. Formaldehyde-fixed and paraffin-embedded tissue of experimentally coxsackievirus B3 infected mice was analyzed for the localization of virus RNA. Both the probes gave signals in mouse myocardial cells, disseminated evenly in the heart tissue 7 days postinfection. At this time point, hybridization-positive cells and inflammatory reaction were mainly found in different areas that may be due to the inability of the immune system to recognize the infected cells before cytolysis. The samples were still positive when fixed 52 hours postmortem indicating that virus RNA is in a relatively stable form in the cells.

Effects of the antiviral WIN 54954 and the immune modulator LS 2616 on cachectin/TNF and gamma-interferon responses during viral heart disease.

The effects of the anti picornaviral drug WIN 54954 (5-(5-(2.6-dichloro-4-(4.5-dihydro-2-oxazolyl)phenoxy)pentyl)-3-me thyl- isoxazole) and the immune modulator LS 2616 (Quinoline-3-carboxamide) on plasma cachectin/TNFa and g-interferon (IFN-g) responses were investigated during the clinical course of a myocarditic coxsackievirus B3 (CB3) infection in the mouse. Virus as well as inflammatory and necrotic lesions were found in the hearts on days 4 and 7 post inoculation (p.i.), respectively. This was demonstrated using in situ virus RNA hybridization and immune histological techniques with monoclonal antibodies against lymphocyte subsets. The CB3 infection increased TNFa levels during the first three days of disease. This response was suppressed by WIN 54954 and LS 2616. IFN-g was decreased in infected mice in the late phase of the disease (day 11). Therapy, however, was protective, and WIN 54954 even tended to increase the IFN-g response at day 5, corresponding to the time when viremia peaks. These results indicate that cytokines may serve as prognostic markers in the therapy of infectious diseases and also that WIN 54954 and LS 2616 are both possible candidates for treatment of coxsackievirus infections in man. It is suggested that a combined antiviral and immune stimulatory treatment could be of future value.

Genotypic and serotypic profile in dilated cardiomyopathy.

Eighteen consecutive patients, admitted with a diagnosis of dilated cardiomyopathy (DCM), to the Cardiology Section, Department of Internal Medicine, University Hospital, Uppsala, Sweden were enrolled into the study. All patients suffered signs of cardiac incompensation of variable duration. Patients were defined by conventional clinical investigations including chest X-ray, ultrasound, g-camera, catheterization and endomyocardial biopsy with histological evaluation by a specially trained pathologist. Angiography was performed to exclude ischemic heart disease. Several patients were diagnosed as having a specific reason for the cardiac insufficiency, like pheochromocytoma, SLE, ethylism, ischemic heart disease and hypertrophic cardiomyopathy. In this group all 7/7 had negative serology against Coxsackie B viruses. In the other group of idiopathic CM, no other etiology could be found. Serological analysis in this group showed high IgM titres against Coxsackie viruses in 6/8 patients. EDTA-blood was taken for tissue-typing using DNA probe hybridisation. 6/12 patients had DQB1:4 using the newest nomenclature, vs 17% in the control population. The reversed picture was observed for DQB1:2, occurring in 1/12 patients, vs 19% in the normal population, thus indicating a protective value of this genotype, which to our knowledge has not been described before. The results indicate a dual dependence of (host) genotype and (virus) serotype according to the Doherty-Zinkernagel hypothesis. Thus, it would also be in agreement with the virus-immune hypothesis suggested more than 20 years ago to explain the enigmatic pathogenesis of DCM.

The epidemiology of infectious myocarditis, lymphocytic myocarditis and dilated cardiomyopathy.

Infectious myocarditis is a common condition which often passes unrecognized, and the true incidence is thus unknown. Lymphocytic myocarditis has been recorded in 1.06% of 12,747 unselected routine autopsies performed over a 10-year period. Dilated cardiomyopathy (DCM) has an estimated frequency of 7.5-10% per 100,000 inhabitants per year. Overall, the enteroviruses, and particularly the Coxsackie-B viruses, predominate among viruses as the cause of myocarditis. As new molecular biological techniques have become available, the cytomegaloviruses (CMV) seem to have emerged as a more common cause of myocarditis than was previously recognized. Among the bacterial myocarditides, diphtheric myocarditis has become a serious threat in Russia and adjacent states during the 1990s. Among newly identified bacteria, Borrelia burgdorferi infection is accompanied by cardiac involvement in 1-8% of cases, where myocarditis with conduction disturbances is the most prominent feature. Chlamydia pneumoniae may be associated with myocarditis and sudden unexpected death. In AIDS, myocarditis with variable aetiology occurs in up to 50% of patients, although asymptomatic in most cases. In lymphocytic myocarditis and DCM, enteroviral-specific nucleotide sequences have been detected in about 30% of patients, and CMV-specific nucleotide sequences in 14%. Borrelia burgdorferi may occasionally be implicated in DCM. In this contribution we focus also on sudden unexpected death (SUD) in young athletes, since, in Sweden, an increased frequency of SUD has recently been observed in young orienteers and myocarditis was a common feature.

Serological and epidemiological analysis of the prevalence of Bartonella spp. antibodies in Swedish elite orienteers 1992-93.

The emergence of the popular, physically demanding and highly nature-interactive sport of orienteering was marked in Sweden by an elevated rate of sudden unexpected cardiac deaths in young competitors during the years 1979-92, with a common underlying cause or causes suspected. Subsequently, sera were collected during 1992-93 from the elite segment of orienteers holding a nationally ranked position, and a survey compiling various epidemiological data was performed. In this study, a total of 1136 sera were analyzed by indirect-fluorescent antibody assay for the presence of IgG antibodies against 3 Bartonella spp.: B. henselae, B. elizabethae and B. quintana. In total, 31% (355/1136) were seropositive for at least 1 species of Bartonella, with titers ranging up to 1/512; 350/1136 (31%) had antibodies against B. elizabethae, 34/1136 (3.0%) against B. henselae and 16/1136 (1.4%) against B. quintana. Males and females showed equal rates of 31% seropositisity to Bartonella spp. (males 241/766; females 114/370). In comparison, 322 time-matched sera from healthy blood donors had antibodies to Bartonella spp. in 6.8% of cases (p < 0.001). The observed high prevalence of Bartonella spp. antibodies found in Swedish elite orienteers may be indicative of a connection with risk factors for the development of myocarditis and sudden unexpected cardiac death.

Left ventricular volume changes during supine exercise in young endurance athletes.

AIM: The primary objective of the study was to measure the relative left ventricular volumes and the changes in left ventricular ejection fraction during supine position from rest to exercise in young endurance athletes. The secondary objective was to examine if there were gender differences regarding the volume reply and ejection fraction with exercise. METHOD: Sixty-five (35 female and 30 males) young healthy Swedish orienteers participated in the study. Left ventricular volume and ejection fraction changes between rest and submaximal supine bicycle exercise were measured with radionuclide ventriculography. RESULTS: The mean left ventricular end-diastolic volume increased by 13% (P < 0.001) but there was no change in end-systolic volume. Stroke volume was found to increase by 21% (P < 0.001). Left ventricular ejection fraction increased significantly (>0.04 units) in 54% of the athletes from rest to exercise; 5% of the athletes showed a decrease in ejection fraction. A negative correlation was found between ejection fraction at rest and the difference in ejection fraction from rest to exercise (r = -0.38, P = 0.002). There were no gender differences in the left ventricular volume changes or ejection fraction. CONCLUSION: During submaximal supine exercise, the adjustments in cardiac volumes in endurance athletes were small. There were no gender disparities concerning the left ventricular volume reply during exercise.