Demonstration of intracellular microorganisms (Rickettsia spp., Chlamydia pneumoniae, Bartonella spp.) in pathological human aortic valves by PCR.

OBJECTIVES: Rickettsiae, which causes vasculitis, has not been linked to atherosclerotic cardiovascular disease in contrast to Chlamydia pneumoniae whose association with coronary artery disease and with sclerotic heart valves in patients undergoing aortic valve replacement is well established, even if causality is yet to unproven. In the search for any of these infectious agents, 84 pathological and 15 normal aortic heart valves of patients undergoing forensic autopsy were analysed by PCR and DNA-sequencing. METHODS: Two to four pieces of all valves were examined by semi-nested PCR, with primers specific for 16S rDNA, citrate synthase (gltA) and 17 kDa outer membrane protein (OMP) genes. RESULTS: Genetic material from Rickettsia spp. and C. pneumoniae was found in 17 (20.2%) and 22 (26.2%), respectively, of the 84 pathological aortic valves. In 35 (41.7%) of these 84 valves either C. pneumoniae or Rickettsia spp. were detected by PCR and in six cases (7.1%) these two organisms co-existed. In one case with Lambl's excrescences, previously considered as aseptic, presence of rickettsia-like organisms also was demonstrated by light microscopy, immunohistochemistry and sequencing of the amplified PCR product showing 100% homology with the published sequence for R. helvetica. In three of the 15 control valves, genetic material from only C. pneumoniae was detected compared to Rickettsia spp. that was significantly detected only in the pathological valves (Fisher's Exact test, 1-sided p = 0.046). CONCLUSIONS: The findings suggest that Rickettsia spp. also have a role in the pathogenesis of aortic valve disease.

Subacute bartonella infection in Swedish orienteers succumbing to sudden unexpected cardiac death or having malignant arrhythmias.

During the period 1979-92, an increasing number of sudden unexpected cardiac deaths (SUCD) occurred in young, Swedish, male elite orienteers. Myocarditis was the most common diagnosis in the 16 victims, and in 4 cases was also associated with fatty infiltration mimicking arrhythmogenic right ventricular cardiomyopathy (ARVC). Tissues from autopsies of 5 orienteers were tested for Bartonella by PCR targeting the gltA (citrate-synthase) gene. The products were then sequenced. Antibodies to B. henselae, B. quintana and B. elizabethae were measured by indirect fluorescence antibody assay. Bartonella spp. DNA was detected in the hearts of 4 deceased orienteers, and in the lung of a fifth deceased case. The sequences were close to B. quintana in 2 cases and identical to B. henselae in 3. Four of these 5 cases, as well as 2 additional cases of elite orienteers with ARVC, indicated antibodies to Bartonella. It is suggested that Bartonella-induced silent subacute myocarditis, eventually leading to electric instability, caused the increased SUCD rate among the Swedish orienteers. It is further suggested that Bartonella infection may be a major pathogenetic factor in the development of ARVC-like disease. Although the mode of transmission is unknown, both zoonotic/vector-borne and parenteral person-to-person transmission may be involved.

Relationship of Chlamydia pneumoniae infection to severity of human coronary atherosclerosis.

BACKGROUND: Infection with Chlamydia pneumoniae has been postulated to play a pathogenic role in atherosclerosis. We examined the role of infection with C pneumoniae in relation to the extent of coronary atherosclerosis. METHODS AND RESULTS: Coronary atherosclerosis was graded microscopically on a postmortem basis in a blinded fashion in 60 subjects as mild (n=18) or severe (n=42) atherosclerosis. Serum antibodies to C pneumoniae were measured by microimmunofluorescence test. Paraffin-embedded coronary artery specimens were examined for the presence of chlamydia by use of a genus-specific direct immunofluorescence monoclonal antibody. Frozen coronary artery specimens were examined by immunoperoxidase for the presence of C pneumoniae by use of a specific monoclonal antibody RR-402. Direct immunofluorescence was reactive in 86% of cases with severe atherosclerosis but in only 6% of cases with mild atherosclerosis (P<0.01), whereas immunoperoxidase staining was reactive in 80% and 38% of cases with severe and mild atherosclerosis, respectively (P<0. 01). Elevated IgG and IgA levels against C pneumoniae were not different in cases with severe and mild atherosclerosis (61% and 30% for severe atherosclerosis and 67% and 42% for mild atherosclerosis, respectively). CONCLUSIONS: This study supports the hypothesis that intracellular infection with C pneumoniae may relate to the severity of atherosclerosis in some subjects. Serum antibody titers against C pneumoniae do not differentiate between severe and mild atherosclerosis.

Cause and manner of death among users of anabolic androgenic steroids.

Medicolegally investigated deaths among 34 male users of anabolic androgenic steroids (AAS) are described. Nine persons were victims of homicide, 11 had committed suicide, 12 deaths were judged as accidental and 2 as indeterminate. In two cases of accidental poisoning, the levels of pharmaceuticals and illicit drugs were considered too low to be the sole cause of death and AAS was considered part of the lethal polypharmacia. Chronic cardiac changes were observed in 12 cases. In two cases of accidental poisonous deaths, these changes were regarded as contributory cause of death. Homicides, suicides, and poisonings determined accidental or indeterminate in manner were related to impulsive, disinhibited behavior characterized by violent rages, mood swings, and/or uncontrolled drug intake. The observations in the present study indicate an increased risk of violent death from impulsive, aggressive behavior, or depressive symptoms associated with use of AAS. There are also data to support earlier reports of possible lethal cardiovascular complications from use of AAS. Furthermore, a contributing role of AAS in lethal polypharmacia is suggested. Finally, the observations indicate that use of AAS may be the gateway of approach to abuse of other psychotropic drugs.

Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death.

BACKGROUND: Rickettsia helvetica is the only non-imported rickettsia found in Scandinavia. It was first detected in Ixodes ricinus ticks, but has never been linked to human disease. We studied two young Swedish men who died of sudden cardiac failure during exercise, and who showed signs of perimyocarditis similar to those described in rickettsial disease. METHODS: Samples from the heart and other organs were analysed by PCR and DNA sequencing. May-Grünwald-Giemsa, Grocott, and acridine-orange stains were used for histopathological examinations. Staining of R. helvetica grown on shell-vials in vero cells, and the early descriptions of R. rickettsii by H T Ricketts and S B Wohlbach served as controls. Immunohistochemistry was done with Proteus OX-19 rabbit antisera as the primary antibody. The structure of rickettsia-like organisms was investigated by transmission electron microscopy. Serological analyses were carried out by indirect immunofluorescence with R. helvetica as the antigen. FINDINGS: By use of a semi-nested PCR, with primers specific for the 16S rRNA and 17-kDa outer-membrane-protein genes, and sequence analysis of the amplified products, genetic material from R. helvetica was detected in the pericardium and in a lymph node from the pulmonary hilum in case 1, and in a coronary artery and the heart muscle in case 2. A serological response in case 1 revealed an endpoint titre for R. helvetica of 1/320 (1/256 with R. rickettsii as the antigen). Examination of PCR-positive tissue showed chronic interstitial inflammation and the presence of rickettsia-like organisms predominantly located in the endothelium. These organisms reacted with Proteus OX-19 antisera, and their size and form were consistent with rickettsia. Electron microscopy confirmed that the appearance of the organisms was similar to that described for spotted-fever rickettsia. INTERPRETATION: R. helvetica, transmitted by I. ricinus ticks, may be an important pathogen in the aetiology of perimyocarditis, which can result in sudden unexpected cardiac death in young people.

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.

Rickettsia helvetica in Ixodes ricinus ticks in Sweden.

In the present study further characterization of the amplified sequence of the citrate synthase gene of the spotted fever group Rickettsia isolated from Ixodes ricinus ticks in Sweden showed that it has 100% homology with the deposited sequence of the citrate synthase gene of Rickettsia helvetica. The restriction fragment length polymorphism (RFLP) pattern of an amplified 382-bp product of the citrate synthase sequence, defined by primers RpCS877 and RpCS1258, yielded fragments for our isolate that could be visualized as a double band that migrated at approximately 44 bp, another double band at 85 bp, and a single band at nearly 120 bp after digestion with the restriction enzyme AluI. When calculating a theoretical PCR-RFLP pattern of the sequence of the citrate synthase gene of R. helvetica from the known positions where the AluI enzyme cuts, we arrived at the same pattern that was obtained for our isolate, a pattern distinctly different from the previously published PCR-RFLP pattern for R. helvetica. Investigation of 125 living I. ricinus ticks showed a higher prevalence of rickettsial DNA in these ticks than we had found in an earlier study. Rickettsial DNA was detected by amplification of the 16S rRNA gene, for which a seminested primer system consisting of two oligonucleotide primer pairs was used. Of the 125 ticks, some were pooled, giving a total of 82 tick samples, of which 20 were found to be positive for the rickettsial DNA gene investigated. When considering the fact that some of the positive samples were pooled, the minimum possible prevalence in these ticks was 20 of 125 (16%) and the maximum possible prevalence was 46 of 125 (36.8%). These prevalence estimates conform to those of other studies of spotted fever group rickettsiae in hard ticks in Europe.

Criminal multilation of the human body in Sweden--a thirty-year medico-legal and forensic psychiatric study.

During the 30-year period 1961-1990, a total of 22 deaths with criminal multilation/dismemberment of the human body were registered in Sweden. The multilations occurred in time clusters, mostly during the summer and winter periods, and increased during the three decades, with incidence rates of 0.05, 0.1, and 0.125 per million inhabitants and year, respectively. Multilation was noted 6.6 times more often in large urban areas than in the rest of Sweden. Defensive mutilation, in order to get rid of the corpse or make its identity more difficult, was noted in ten instances, aggressive mutilation following outrageous overkilling in four, offensive mutilation (lust murder) in seven, and necromanic multilation in one instance. In the last-mentioned case the cause of death was natural, while all deaths in the first three groups were homicidal, or homicide was strongly suspected. All perpetrators were males, in six instances assisted by other persons. In more than half of the cases the perpetrator's occupation was associated with application of anatomical knowledge, e.g., butcher, physician, veterinary assistant, or hunter. The perpetrators of the defensive and aggressive mutilations were mostly disorganized, i.e., alcoholics or drug users with previous psychiatric contacts and criminal histories, while the lust murderers were mostly organized, with a history of violent crimes (including the "serial killing" type), drug abuse and mental disorders with anxiety and schizophrenia, in that order to a diminishing degree. There were differences in mode of mutilation, depending on whether the mutilation was carried out by a layman, a butcher, or a physician. In only one case was the perpetrator convicted for the mutilation act itself; in the remaining instances the manslaughter, as a more serious crime, assimilated the mutilation. When the mutilation made it impossible to establish the cause of death, the perpetrators, despite strong circumstantial evidence indicating murder, were acquitted.

Violent mass shootings in Sweden from 1960 to 1995: profiles, patterns, and motives.

During the past few decades, violent mass shooting in Sweden has increased rapidly. In the 36 years between 1960 and 1995, fourteen such occasions were recorded, during which 32 people were killed and 57 were wounded. The 14 offenders were men between the ages of 17 and 61 years. In the 20 years from 1960 to 1979, five shootings were committed by five offenders, leaving 10 dead and 13 wounded; in the 16 years between 1980 and 1995, there were nine different shootings committed by nine offenders, with 22 dead and 44 wounded. Seven of the shootings were classified as mass shootings, six as spree shootings, and one as a serial shooting. In all but four of these cases, the firearms used were illegal weapons. The four legal firearms belonged to an unemployed young laborer, an officer, a former United Nations (U.N.) soldier, and a member of the Swedish military volunteer corps. Of those killed, 68.8% were strangers to the offender; among the wounded, the corresponding figure was 89.5%. Profiles of the offenders and of the victims were studied. The psychiatric diagnoses among the offenders and the measures taken to prevent the increase in mass shooting in Sweden are presented.

High incidence of Chlamydia pneumoniae in sclerotic heart valves of patients undergoing aortic valve replacement.

Chlamydia pneumoniae has previously been demonstrated in the atherosclerotic lesions of various arteries, including the coronary arteries, and has been proposed to play a role in the pathogenesis of atherosclerosis. A prospective study of the incidence of C. pneumoniae in the sclerotic valves of patients undergoing aortic valve replacement because of aortic stenosis and in the aortic valves of cases dying of non-cardiac reasons and undergoing forensic autopsy was undertaken. The results were correlated to serological markers of past (IgG) or persistent (IgA) C. pneumoniae infection. C. pneumoniae, as determined by the polymerase chain reaction (PCR), was detected in the aortic valve in 19/39 (49%) patients and in 1/11 (9%) autopsy controls (p = 0.018) and confirmed by electron microscopy in one patient. There was no significant difference in the incidence rate of IgG or IgA antibody positivity between PCR-positive and PCR-negative cardiac patients. These results extend the hypothesis of a pathogenic role of C. pneumoniae in atherosclerosis to include also aortic valve sclerosis.

Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden.

A spotted fever group rickettsia isolated from the common tick, Ixodes ricinus, was genetically characterized by PCR and genomic sequencing. This study was performed with nymphal and adult ticks collected in southern and central Sweden. I. ricinus is the only North European tick species of medical importance which is regularly collected from humans. No species of the genus Rickettsia has previously been found in Scandinavian ticks, nor has any case of domestic rickettsial infection in humans or animals been reported. According to the nucleotide sequencing, the present Rickettsia sp. belongs to the spotted fever group of rickettsiae. Ticks are the most common arthropod reservoirs and vectors of the rickettsiae of this group. Among 748 ticks investigated, 13 (1.7%) were positive for a Rickettsia sp. Borrelia burgdorferi was detected in 52 (7%) of the ticks, a prevalence similar to or somewhat lower than that previously been recorded in other Swedish studies. There was no evidence of ehrlichial or chlamydial DNA in these ticks. The Rickettsia sp. was further characterized by 16S ribosomal DNA (rDNA) sequencing and restriction fragment length polymorphism (RFLP). The 16S rDNA sequencing resulted in a sequence identical to that described for Rickettsia helvetica, but the pattern obtained with RFLP of the citrate synthetase gene diverged from previously known patterns. The rickettsial agent of one tick which was positive by PCR was confirmed by transmission electron microscopy. The morphology of this rickettsia was similar to that of the spotted fever and typhus group rickettsiae. This represents the first documented isolate of a Rickettsia sp. from Swedish ticks.

An increase in sudden unexpected cardiac deaths among young Swedish orienteers during 1979-1992.

BACKGROUND: Sixteen cases of sudden unexpected cardiac death, 15 males and one female, are known to have occurred among young Swedish orienteers from 1979 to 1992, of which seven cases occurred between 1989 and 1992. This is considered to be indicative of an increased death rate. RESULTS: Histopathological evaluation showed myocarditis in a higher than expected proportion of cases. In one such case, which we studied before the sudden unexpected death occurred, the victim had suffered a Chlamydia pneumoniae infection verified by serology, and a nucleotide sequence was found in the heart and lung by means of the polymerase chain reaction (PCR) that hybridized with a probe specific for that organism. Male Swedish orienteers do not, however, seem to have an increased rate of exposure to this agent. No further sudden unexpected deaths among young orienteers have occurred over the past 3.5 years. At the beginning of that period, attempts were made to modify training habits and attitudes.

[Clear connection between gun ownership and suicide. 92 per cent of attempts result in death]. / Klart samband vapeninnehav och självmord 92 procent avlider av skottskadan.

In a retrospective study involving 20 police districts in Sweden, 348 completed or attempted suicides with firearms were found to have occurred during the period, 1976-88. Statistics on civilian firearms ownership were obtained from police authorities. Firearm suicide was found to be significantly correlated to gun ownership, through the 25 per cent increase in gun ownership during the period was not accompanied by a corresponding increase in the overall suicide rate. The head was the primary target in suicide attempts, both with longarms (rifles and shotguns) and with handguns (pistols and revolvers). Handgun injuries were more common among the survivors. Only 4.6 per cent of the victims survived and could be discharged from hospital. Nearly 10 per cent of those using shotguns or rifles used a string, a rod or their toes to reach the trigger; all of them died. Apart from such well-known predisposing factors such as psychiatric disorders or alcohol abuse that were present in 43 per cent of cases, 24 per cent of the victims were found to have had somatic problems instead.

The population study of women in Gothenburg 1980-81--the third phase of a longitudinal study. Comparison between participants and non-participants.

A representative population sample comprising 1,462 women was studied in Gothenburg, Sweden in 1968-69, and a third follow-up study was carried out in 1980-81. The participation rates in the baseline study and during the follow-up studies were high. In 1980-81 women in two new age strata, aged 26 and 38, were added. Women who had moved to or from Gothenburg during the study period were not found to differ from those who were living in Gothenburg during the total study period, while there were a few differences of statistical significance between refusers and participants in 1980-81. The mortality among initial refusers was about doubled compared to that of those who participated in the baseline study.

Serum gamma-glutamyltransferase in a Swedish female population. Age-related reference intervals; morbidity and prognosis in cases with raised catalytic concentration.

We determined the catalytic concentration of gamma-glutamyltransferase in serum from a population sample of 1408 women in seven age strata between 26 and 72 years. The range in healthy individuals for the different age groups was found to increase with age with a maximum of the central 0.95 fractile interval at 58 years (0.17-1.68 microk/l). The serum gamma-glutamyltransferase activity correlated with body mass index, blood pressure and concentrations of blood glucose and serum ferritin, triglycerides and cholesterol. During follow-up of women with gamma-glutamyltransferase activity greater than 1.20 microk/l, no woman developed any disease possibly related to the original finding of raised serum gamma-glutamyltransferase activity, several individuals being apparently healthy. Apparently, the serum gamma-glutamyltransferase assay is an unspecific indicator of several metabolic abnormalities. High values may be found in individuals in whom all commonly done investigations have given results within the health-associated reference interval.

Bone mineral content in normally menstruating women with hyperprolactinaemia.

Decreased bone density has been reported in women with hyperprolactinaemia due to pituitary tumours. We identified a number of seemingly healthy women with hyperprolactinaemia, i.e. a serum prolactin concentration exceeding 500 mU/l (25 micrograms/l) on three occasions, during a study in 1980/1981 of a representative population sample of greater than 1,400 women in seven different age strata (range 26-72 years). We compared vertebral bone mineral content and bone mineral areal content in 5 hyperprolactinaemic normally menstruating 50-year-old women with that of 6 controls matched for age and menstrual status but found no difference. Since the degree of prolactin elevation was similar in our study group to that previously reported for hyperprolactinaemic subjects with pituitary tumours and the time of exposure to raised hormone concentration appears to be of the same magnitude, other hormonal changes than hyperprolactinaemia per se seem to be the cause of low bone mineral content in women with hyperprolactinaemia and amenorrhoea.

Smoking--a risk factor for cardiovascular disease in women? Considerations based on a prospective population study of women in Gothenburg, Sweden.

A longitudinal population study of 1462 women aged 38-60 was carried out in Gothenburg, Sweden during 1968-69. The participants have been followed up for 12 years. The relationships between smoking and cardiovascular disease and between smoking and mortality have been evaluated. No significant increased risk was observed for smoking women concerning the 12-year incidence of myocardial infarction, angina pectoris, electrocardiographic changes suggesting ischaemic heart disease, stroke or death from all causes. Multivariate analysis could not demonstrate an independent effect of the cigarette smoking habit in women on these end-points. In agreement with the results from the Framingham prospective study we could not in our prospective study verify the markedly increased risk of myocardial infarction in smoking women, which has been observed in a number of cross-sectional studies of women and also in prospective studies of men.

Prognosis for women with different symptoms and signs suggesting ischaemic heart disease--a 12-year follow-up. The population study of women in Gothenburg, Sweden.

A longitudinal population study of a total of 1462 women aged 38-60 was carried out in 1968-69 in Gothenburg, Sweden. The women have been re-studied in 1974-75 and 1980-81. The incidences of myocardial infarction during the following 12-year period were: three of 29 women with a history of angina pectoris (10%), four of 23 women with initial ECG changes at rest (17%) and one of 30 with ECG changes during work (3%). In addition, all women of similar age in Gothenburg with myocardial infarction during the years 1968-70 have been followed-up with respect to mortality, and in all there were 47 women who were alive on arrival at hospital. The figures for the 12-year overall mortality in the population study were: three women with angina pectoris (10%), four women with ECG changes at rest (17%) and three women with ECG changes during work (10%) and in the series of women with myocardial infarction 21 of 47 (45%). It seemed that the mortality among women with a history of angina pectoris or ECG changes at rest or during exercise indicating ischaemic heart disease was only slightly increased, if at all, compared to other women in the population, while having had a myocardial infarction significantly increased the mortality risk.

Relationships between the menopause and risk factors for ischaemic heart disease.

There are still contradictory opinions as to whether there is an association between menopausal age and ischaemic heart disease or not. There is, however, no doubt about the existence of a number of relationships between menstrual status and different risk factors for ischaemic heart disease. Thus, smoking will give rise to an earlier menopause, while the menopause will give rise to increased serum cholesterol and serum triglyceride levels but seems to influence arterial blood pressure and body weight in the opposite way. The relationships between the menopause and risk factors for ischaemic heart disease are complex, which may be one reason for the contradictory results when relating menopausal age to the incidence of ischaemic heart disease.