APMIS 111: 50713, 2003 Printed in Denmark .

All rights reserved

Copyright C APMIS 2003

ISSN 0903-4641

A study on forensic samples of Bartonella spp. antibodies in Swedish intravenous heroin addicts
RAN FRIMAN1 SVENA McGILL,1 JOVAN RAJS,4 EVA HJELM,2 OLLE LINDQUIST3 and GO
1 Infectious Diseases and 2Clinical Bacteriology at the Department of Medical Sciences, 3Forensic Medicine at the Department of Surgical Sciences, Uppsala University Hospital, Uppsala, and 4Forensic Medicine at the Karolinska Institute, Stockholm, Sweden

McGill S, Rajs J, Hjelm E, Lindquist O, Friman G. A study on forensic samples of Bartonella spp. antibodies in Swedish intravenous heroin addicts. APMIS 2003;111:50713. Infection with Bartonella, an emerging bacterial pathogen which often affects immunodecient patients, has been reported in Sweden over the past few years, with a high seroprevalence of B. elizabethae. A high prevalence of antibodies against B. elizabethae has also been found in urban intravenous drug users in the USA. Using immunouorescence, we retrospectively examined serum samples taken at autopsy from 59 Swedish intravenous drug addicts from the Stockholm area for evidence of antibodies against 6 pathogenic strains of Bartonella. The 59 addicts died following heroin injection during the years 19871992 and include 24 individuals (41%) who were additionally HIV-positive. An overall seropositivity rate for Bartonella spp. of 39% (23/59) was found with the following antigenic reactivities: B. elizabethae, 39% (23/59); B. grahamii, 3% (2/59); B. henselae (Houston-1), 14% (8/59); and B. quintana, 3% (2/59). There were no positive reactions for B. henselae (Marseille) or B. vinsonii subsp. vinsonii. The Bartonella-seropositive cases included 11/23 (48%) individuals who were HIVpositive. Subacute to chronic myocarditis was seen in 2/11 microscopically investigated Bartonellaseropositive cases that were HIV-negative and in 1/14 seronegative cases. No cases of endocarditis or other common manifestations of Bartonella infection were found. An overall Bartonella seropositivity of 21% (9/44) was observed in control forensic autopsy samples. Key words: Bartonella spp. antibodies; Swedish intravenous heroin addicts; forensic samples. Gran Friman, Infectious Diseases, Dept of Medical Sciences, Uppsala University Hospital, S-751 85 Uppsala, Sweden. e-mail: goran.friman/medsci.uu.se

The genus Bartonella, which in 1990 comprised only 2 species, has expanded nearly 10-fold in the past decade to currently comprise 17 named species, including numerous subspecies. This group of small gram-negative bacteria has been implicated in the etiology of an expanding range of human syndromes. Several clinical manifestations of Bartonella infection (e.g. cat scratch disease, bacillary angiomatosis, endocarditis, and bacteremia) become apparent or are exacerbated in immunosuppressed individuals, particularly HIV-positive patients (15). The reserReceived September 9, 2002. Accepted March 5, 2003.

voirs, modes of transmission, and risk factors for infection have yet to be completely elucidated for the majority of Bartonella species. Bartonella infections have recently been found in inner-city homeless alcoholic men as a cause of endocarditis (3, 4), and high Bartonella seroprevalences have been reported in other socially underprivileged groups (6), as well as in inner-city intravenous drug (IVD) users in the USA (7, 8). In the Comer et al. studies of 1996 (7) and 2001 (8) high antibody levels of B. elizabethae were found in IVD users in Baltimore, MD, and Central and East Harlem, New York City, with seroprevalences of 33% and 46%, respectively. In Sweden, a parallel high seroposi507

McGILL et al.

tivity for B. elizabethae has been reported in certain groups, including domestic cats (9), clinical samples (10), and elite orienteers suffering from subacute myocarditis (11, 12). In the current study we have collected forensic samples from IVD users in the Stockholm region who died in connection with heroin injection and tested their sera or blood for antibodies to six Bartonella strains: B. elizabethae, B. grahamii, B. henselae, Houston-1 and Marseille strains, B. quintana, and B. vinsonii subsp. vinsonii, the rst ve of which are regarded as pathogenic (3, 4, 1319).

yr). The male-female ratio of the forensic autopsy cases was nearly identical to that of the case subjects, with 37 males and 7 females. Deaths in the controls were violent in 59% (23 males, 3 females) and nonviolent in 41% (14 males, 4 females). Forensic analysis Bodies were kept in refrigerated rooms of the mortuary at 48 C. Autopsies were performed from 0 6 days after death (mean 3.6 days). Typically, the bodies arrived at the mortuary within hours after being discovered. Before autopsy, blood samples were analyzed for HIV antibodies (20). In consideration of the autopsy personnel, complete autopsy was not carried out in such HIV-positive cases where the cause of death was obvious. Heart sections were taken from the anterior wall, interatrial and interventricular septa, the left anterior and posterior ventricular wall, and the left anterior and posterior papillary muscles as previously recommended (21). Inammatory alteration in the myocardium was dened as a process characterized by the presence of inammatory cells (n15/focus) adjacent to necrotic or degenerated myocytes, with or without brosis according to the Dallas classication (22). Myocardial brosis was dened as focal presence of non-inammatory brous tissue in the ventricular myocardium, but not when found only perivascularly or in the apices of the papillary muscles. For toxicological investigation, femoral vein blood was collected from both sides and pooled; in addition, bladder urine was collected, to which potassium uoride was added. Cultivation of Bartonella spp The following bacterial strains were cultivated on 5% debrinated rabbit-blood heart infusion agar (BBL Microbiology Systems, Cockeysville, MD.): B. elizabethae (F9251; ATCC 49927), B. grahamii (ATCC 700132), B. henselae (Houston-1 isolate, ATCC 49882), B. henselae (Marseille isolate, URLLY8), B. quintana (OK 90268), and B. vinsonii subsp. vinsonii (ATCC VR-152). Cultures were incubated for a period of 3 to 4 days. Co-cultivation of Vero cells with Bartonella spp. was subsequently performed in accordance with previously established standards (1). All organisms were inactivated by gamma irradiation (500,000 rads) and stored at 70 C until use. Serologic testing As previously described (12), the serum samples were serologically tested using indirect uorescent antibody assay (IFA) for antibodies against the six Bartonella antigens listed above. Briey, serum or blood samples were diluted in a phosphate-buffered solution (PBS) with 5% skim milk in 2-fold dilutions from 1:32 and placed on antigen-coated 12-well

MATERIALS AND METHODS

Cases The study was based on deaths investigated at the Government Institute of Forensic Medicine in Stockholm, Sweden, during the 6-year period from 1987 to 1992. According to Swedish law, a medicolegal investigation is necessary when individuals die under unclear or suspicious circumstances or as a result of violence, poisoning, substance abuse, or otherwise suddenly without a previously known potentially fatal disease. During these 6 years, 17,699 bodies were examined; of these, 149 deaths were in direct association with heroin injection. The serviced area included Stockholm city and county, as well as Sdermanland and Gotland counties. Information about the deceased individuals was obtained from police reports and was supplemented, when possible, with information from families and friends, as well as from social workers. Blood or serum specimens from 70 of the 149 addicts who died of a heroin overdose were made available to us by the serum bank of the National Institute of Infectious Disease Control, Stockholm, Sweden. Due to hemolysis, 11 samples were in too poor a condition to be assayed and were thus not included in the study. The 59 case samples used in this study represent a subset of heroin abusers from the Stockholm area, the majority of whom were from the city of Stockholm and for whom direct heroin injection was implicated at autopsy as the primary cause of death. The heroin in all cases was injected intravenously. The subjects, 50 males and 9 females, were all European (Caucasian), with a mean age of 32.1 yr (range 2243 yr). Controls Sera from 44 forensic autopsy cases from the Stockholm area with causes of death unrelated to any type of substance abuse served as controls. The cases were autopsied during the same period of time as the IVD users. The mean age was 40.9 yr (range 2055

508

BARTONELLA IN SWEDISH INTRAVENOUS DRUG ADDICTS

slides. After incubation at 35 C for 30 min, slides were washed with PBS, air-dried, and coated with a 1:120 dilution of commercial uorescein isothiocyanate (FITC)-conjugated rabbit anti-human IgG (Dakopatts, Denmark). The slides were incubated for an additional 30 min, washed as before, mounted in glycerol, and analyzed using a uorescence microscope at 60 magnication. A positive reaction at a dilution of 1:64 or greater was considered seropositive for all antigens. Titers are given as reciprocal values of serum endpoint dilutions. Statistics The chi2 test was used for testing observed frequencies.

RESULTS Intravenous drug (IVD) users In total, 24/59 (41%) case samples were HIVpositive. IFA tests resulted in a high overall seropositivity rate against Bartonella spp.; 39% (23/59) had antibodies to at least one of the 6 Bartonella antigens tested. Individual antigenic reactivities are included in Fig. 1. There were no reactions against B. henselae (Marseille strain) or B. vinsonii subsp. vinsonii. The highest seroprevalence was for B. elizabethae, with all of the 23 seropositive serum samples reacting to this antigen. Titer values were also highest for B. elizabethae, ranging up to 1:512 in several cases. Geometric mean titers (GMT) for B. elizabethae, B. grahamii, B. henselae (Houston-1), and B. quintana were 144, 181, 117, and 64, respectively. More than half of the Bartonella-positive sera (14/23; 23.7% of all 59 sera) reacted only to one antigen, namely B. elizabethae.

The mean age of Bartonella-seropositive and -seronegative victims was similar (32.6 and 31.8 yr, respectively). In the seropositive group, however, there were only 2/23 females (9%) and in the seronegative group there were 7/36 females (19%). Nearly half (48%, 11/23) of the Bartonella-seropositive IVD users were HIV-positive, and of the Bartonella-seronegative IVD users, 36% (13/36) were HIV-positive (p0.3). In 11/23 Bartonella-seropositive cases a complete routine autopsy was performed: 10/11 had histopathologic evidence of hepatitis; 2/11 had active myocarditis with concomitant signs of healing (brosis); 1/11 had myocardial brosis only; and none had endocarditis. A complete breakdown of important postmortem clinical characteristics and serology results of the 23 Bartonella spp.-seropositive heroin users is included in Table 1. Respecting Bartonella-seronegative heroin users, 16/36 underwent a complete routine autopsy, while 14 were microscopically examined: all 14 had hepatitis, 1 had active myocarditis, 3 had myocardial brosis, and none had endocarditis. None of the IVD users had peliosis hepatis, typical of bartonellosis or liver cirrhosis. The average concentration of morphine in the femoral vein blood at autopsy in the seropositive IVD users was 0.37 mg morphine/g blood. None had cocaine or alcohol in the blood. The mean body mass index (BMI) in the seropositive IVD users with such recordings was 26.7 (Table 1). Controls The Bartonella seropositivity rate in the IVD user case sera was signicantly higher (p0.01) than that in the control group of autopsy sera. The latter manifested an overall 21% (9/44) seropositivity rate when tested against Bartonella spp., 5/9 cases belonging to the non-violent death group and 4/9 cases to the violent death group. All of these positive reactions included seroreactivity against B. elizabethae, with one serum cross-reacting with both B. henselae (Houston-1) and B. quintana. There were no positive reactions against the other Bartonella antigens tested. GMT values for B. elizabethae, B. henselae (Houston-1), and B. quintana were 102, 64, and 64, respectively. In the 9 seropositive controls, 22% (2/9) were female; in the 35 seronegative controls, 14% (5/35) were female. 509

Fig. 1. Seroprevalence of Bartonella spp. in 59 Swedish heroin addicts. Hatched column denotes single reactivity to B. elizabethae.

510
TABLE 1. Postmortem characteristics and serology results of 23 Bartonella spp. seropositive heroin addicts ID Sex Age BMI Morphine HIV Hepatitis Endocarditis Active Myocardial Bea Bg Bh Bh Bq Bv 2 no. [kg/m ] conc. (mg/g myocarditis brosis (H-1) (M) blood) 1 M 28 52.01 0.30 (F)b N/Ac N/A N/A N/A 64 32 32 32 32 32 2 M 28 23.51 0.40 (F) 128 32 32 32 32 32 3 M 28 N/A 0.30 (F) N/A N/A N/A N/A 128 32 32 32 32 32 4 F 30 22.10 0.50 (F) 128 32 32 32 32 32 5 M 30 N/A 0.65 (F) N/A N/A N/A 128 32 64 32 32 32 6 M 30 N/A 0.18 (F) N/A N/A N/A N/A 256 256 128 32 64 32 7 M 30 N/A 0.10 (F) N/A N/A N/A N/A 64 32 32 32 32 32 8 M 31 20.72 0.30 (F) 256 256 128 32 32 32 9 M 31 26.99 0.10 (F) 128 32 32 32 32 32 10 M 32 N/A 0.14 (F) N/A N/A N/A N/A 64 32 32 32 32 32 11 M 32 N/A 0.10 (F) N/A N/A N/A N/A 64 32 32 32 32 32 12 M 33 20.60 0.40 (F) 128 32 64 32 32 32 13 M 34 N/A 0.40 (F) N/A N/A N/A N/A 128 32 32 32 32 32 14 M 34 N/A 0.30 (F) 512 128 512 32 32 32 15 F 35 N/A 0.30 (F) N/A N/A N/A N/A 64 32 32 32 32 32 16 M 35 29.07 0.42 (F) 128 32 64 32 32 32 17 M 36 26.30 0.90 (F) 64 32 32 32 64 32 18 M 38 21.88 0.18 (F) 64 32 32 32 32 32 19 M 39 27.46 0.40 (F) N/A N/A N/A 128 32 32 32 32 32 20 M 39 28.36 0.20 (F) N/A N/A N/A N/A 512 128 128 32 32 32 21 M 40 25.88 0.40 (F) 512 32 32 32 32 32 22 M 41 24.30 1.40 (F) 512 32 32 32 32 32 23 M 42 24.21 0.20 (F) N/A N/A N/A 256 32 128 32 32 32 a 3 Titers reported as reciprocal values of serum IFA endpoint dilutions. Be, B. elizabethae; Bg, B. grahamii; Bh(H-1) , B. henselae (Houston-1); Bh(M), B. henselase (Marseille); Bq, B. quintana; Bv, B. vinsonii. b (F) represents postmortem morphine concentration in femoral vein blood taken at autopsy. c N/Adata not available from autopsy reports.
McGILL et al.

BARTONELLA IN SWEDISH INTRAVENOUS DRUG ADDICTS

DISCUSSION A considerable proportion (39%) of the forensic samples from the Stockholm city intravenous drug (IVD) users in this study were seropositive for Bartonella spp. by IFA in their postmortem blood or serum. This percentage is twice that found in non-IVD user forensic cases that served as controls, of whom 21% were seropositive. In Swedish blood donors, a seropositivity rate of 14% has been reported (23). In all of the Bartonella-seropositive IVD user samples, antibodies to B. elizabethae were detected. Comparably high overall seropositivities for B. elizabethae were found in inner-city IVD users in Baltimore, MD (33%) and Central and East Harlem, New York City (46%) (7, 8). Because B. elizabethae has been isolated as the causative agent in only one case of endocarditis (13), it is regarded as one of the lesser pathogenic Bartonella species. In addition to B. elizabethae, some of our Stockholm IVD user forensic samples reacted to B. grahamii, B. henselae (Houston-1 strain), and B. quintana. Because cross-reactivity is known to exist in Bartonella species in the IFA test (7), it cannot be determined which species gave rise to the serologic responses in these cases. B. grahamii seropositivity was found in 2.6% of Swedish blood donors (23), a percentage comparable to the 3% seropositivity in the present IVD abusers. The Baltimore and New York City addicts were not tested for B. grahamii antibodies. The Houston-1 strain of B. henselae, the etiologic agent of cat scratch disease (1), has a background seroprevalence of 1% in Sweden (23). Our IVD users had a 14% seroprevalence for this agent. Comparably high seroprevalences for B. henselae Houston-1 were detected in the Baltimore (11%) and New York City (10%) IVD users (7, 8). B. quintana, the etiologic agent of trench fever during the two world wars, has fairly recently emerged as a cause of endocarditis in alcoholics and homeless men (3, 4). Our IVD users had a 3% seroprevalence for B. quintana, similar to the 3% seropositivity found in IVD users from New York City (8), whereas the seroprevalence in Swedish blood donors was 0.2% (23). A higher seroprevalence of 10% was reported in the Baltimore IVD users, with no correlation with homelessness or alcoholism (7).

Bartonella species have been found to be opportunistic pathogens in immunocompromised individuals (1, 2, 5). Our sample population of city drug addicts consisted of 41% HIV-positive individuals. There was no statistical difference in HIV-positivity rate in the IVD users with positive serology for Bartonella spp. as compared to the Bartonella-seronegative addicts. In the Baltimore study (7), HIV seronegative status was associated with Bartonella seropositivity. Invasive bacterial infections, including endocarditis and recurrent bacteremia, have been found to be common complications of IVD abuse affecting multiple systems, including cardiac valves, larger arteries, bones, joints, the central nervous system, etc. These complications are due to repeated injections of nonsterile materials (24). Bartonella should be no exception. The intravenous route of administration could represent the source of entry of Bartonella from the environment into the human system, although potential exposure to arthropod vectors and perhaps vertebrate reservoirs is also possible (7). Although several species of Bartonella have been implicated as agents of endocarditis, namely B. quintana, B. henselae (both Houston-1 and Marseille strains), and B. elizabethae (3, 4, 1315), no histopathologic evidence of endocarditis was found in any of our cases. Likewise, no association between endocarditis and Bartonella infection could be detected in the Baltimore IVD study (7). However, myocarditis was found in three of our cases, two of which were Bartonella seropositive and HIV seronegative. Only recently myocarditis was reported as a manifestation of Bartonella infection, as found in Swedish elite orienteers who succumbed to sudden unexpected death caused by subacute myocarditis (11). Myocarditis may also occur in HIV infection (25) and result from intravenous drug use (21). The high incidence of chronic hepatitis in this series is in good agreement with earlier IVD user studies (26). Neither peliosis hepatis, often found in B. henselae infection (19), nor liver cirrhosis was observed in our study. All of the 59 IVD users died in direct connection with administration of heroin, as determined by autopsy and toxicological examination. The mean blood morphine concentration was high, 0.37 mg/g blood, i.e. exceeding 0.30 mg/g, which is considered critical for causing 511

McGILL et al.

death (27). Although some of the addicts had a morphine concentration lower than 0.3 mg/g at time of autopsy, it can safely be argued that heroin administration caused all these deaths (cocaine or alcohol was not present). In sharp contrast, in a longitudinal study on IVD users in Baltimore, MD, merely 8% of the addicts used heroin only, whereas 65% used both heroine and cocaine (28). It is noteworthy that the BMI values of Swedish IVD users, including the present heroin addicts, are above average for the normal population, reecting a certain level of obesity (Rajs et al., unpublished observations). This is at variance with most studies in other countries, showing decreased BMI in IVD users. In conclusion, a high frequency of B. elizabethae seropositivity (39%) was recorded in heroin addicts from Stockholm, Sweden, all of whom died from a lethal injection. Some of the B. elizabethae-seropositive individuals also had antibodies to B. henselae Houston-1, B. grahamii, and B. quintana, but none had antibodies to B. henselae Marseille or B. vinsonii subsp. vinsonii. Hepatitis was a common nding, evidently ascribable to frequent drug abuse; no case had peliosis hepatis. There was no case of endocarditis, but active subacute to chronic myocarditis was found in three cases, two of which were Bartonella-positive and HIV-negative. Myocarditis may have various underlying causes, including heroin abuse, Bartonella, and HIV.
Our appreciation is extended to: Rigmor Thorstensson at the National Institute of Infectious Disease Control, Stockholm, for supplying the forensic blood and serum samples of the drug abuser cases; Anna Fugelstad, for assisting in the forensic work; and Russell Regnery, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, for assistance with antigen production. This study was supported nancially by a grant from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) and from the Faculty of Medicine, Uppsala University.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

REFERENCES
1. Regnery RL, Anderson BE, Clarridge JE, Rodriguez-Barradas MC, Jones DC, Carr JH. Characterization of a novel Rochalimea species, R. 13.

henselae sp. nov., isolated from blood of a febrile, human immunodeciency virus-positive patient. J Clin Microbiol 1992;30:26574. Regnery RL, Childs JE, Koehler JE. Infections associated with Bartonella species in persons infected with human immunodeciency virus. Clin Infect Dis 1995;21 Suppl 1:S948. Drancourt M, Mainardi JL, Brouqui P, Vandenesch F, Carta A, Lehnert F, et al. Bartonella (Rochalimaea) quintana endocarditis in three homeless men. N Engl J Med 1995;332:41923. Spach DH, Kanter AS, Dougherty MJ, Larson AM, Coyle MB, Brenner DJ, et al. Bartonella (Rochalimaea) quintana bacteremia in inner-city patients with chronic alcoholism. New Engl J Med 1995;332:4248. Wong MT, Dolan MJ, Lattuada CP, Jr., Regnery RL, Garcia ML, Mokulis EC, et al. Neuroretinitis, aseptic meningitis, and lymphadenitis associated with Bartonella (Rochalimaea) henselae infection in immunocompetent patients and patients infected with human immunodeciency virus type 1. Clin Infect Dis 1995;21:35260. Jackson LA, Spach DH, Kippen DA, Sugg NK, Regnery RL, Sayers MH, et al. Seroprevalence to Bartonella quintana among patients at a community clinic in downtown Seattle. J Infect Dis 1996;173:10236. Comer JA, Flynn C, Regnery RL, Vlahov D, Childs JE. Antibodies to Bartonella species in inner-city intravenous drug users in Baltimore, Md. Arch Intern Med 1996;156:24915. Comer JA, Diaz T, Vlahov D, Monterroso E, Childs JE. Evidence of rodent-associated Bartonella and Rickettsia infections among intravenous drug users from Central and East Harlem, New York City. Am J Trop Med Hyg 2001;65:85560. Hjelm E, McGill S, Blomqvist G. Prevalence of antibodies to Bartonella henselae, B. elizabethae and B. quintana in Swedish domestic cats. Scand J Infect Dis 2002;34:1926. Holmberg M, McGill S, Ehrenborg C, Wessle n L, Hjelm E, Darelid J, et al. Evaluation of human seroreactivity to Bartonella species in Sweden. J Clin Microbiol 1999;37:13814. Wesslen L, Ehrenborg C, Holmberg M, McGill S, Hjelm E, Lindquist O, et al. Subacute Bartonella infection in Swedish orienteers succumbing to sudden unexpected cardiac death or having malignant arrhythmias. Scand J Infect Dis 2001;33:42938. McGill S, Wesslen L, Hjelm E, Holmberg M, Rolf C, Friman G. Serological and epidemiological analysis of the prevalence of Bartonella spp. antibodies in Swedish elite orienteers 1992 93. Scand J Infect Dis 2001;33:4238. Daly JS, Worthington MG, Brenner DJ, Moss CW, Hollis DG, Weyant RS, et al. Rochalimea

512

BARTONELLA IN SWEDISH INTRAVENOUS DRUG ADDICTS

14.

15. 16.

17.

18.

19.

20. 21.

elizabethae sp. nov. isolated from a patient with endocarditis. J Clin Microbiol 1993;31:87281. Drancourt M, Moal V, Brunet P, Dussol B, Berland Y, Raoult D. Bartonella (Rochalimea) quintana infection in a seronegative hemodialyzed patient. J Clin Microbiology 1996;34:115860. Hadeld T, Warren R, Kass M, Brun E, Levy C. Endocarditis caused by Rochalimaea henselae. Hum Pathol 1993;24:11401. Kerkhoff FT, Bergmans AM, van Der Zee A, Rothova A. Demonstration of Bartonella grahamii DNA in ocular uids of a patient with neuroretinitis. J Clin Microbiol 1999;37:40348. OHalloran HS, Draud K, Minix M, Rivard AK, Pearson PA. Lebers neuroretinitis in a patient with serologic evidence of Bartonella elizabethae. Retina 1998;18:2768. Roux V, Eykyn SJ, Wyllie S, Raoult D. Bartonella vinsonii subsp. berkhofi as an agent of afebrile blood culture-negative endocarditis in a human. J Clin Microbiol 2000;38:1698700. Welch DF, Pickett DA, Slater LN, Steigerwalt AG, Brenner DJ. Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis. J Clin Microbiol 1992;30:27580. Rajs J, Fugelstad A. Suicide related to human immunodeciency virus infection in Stockholm. Acta Psychiatr Scand 1992;85:2349. Rajs J, Falconer B. Cardiac lesions in intra-

22. 23.

24.

25.

26. 27. 28.

venous drug addicts. Forensic Sci Int 1979;13: 193209. Aretz HJ, Billingham ME, Edwards WD. Myocarditis: a histopathologic denition and classication. Am J Cardiovasc Pathol 1987;1:314. McGill S, Wessle n L, Hjelm E, Holmberg M, Auvinen MK, Berggren B, et al. Bartonella spp. seroprevalence in healthy Swedish blood donors. Submitted to Scand J Infect Dis 2003. Burke AP, Kalra P, Li L, Smialek J, Virmani R. Infectious endocarditis and sudden unexpected death: incidence and morphology of lesions in intravenous addicts and non-drug abusers. J Heart Valve Dis 1997;6:198203. Barbaro G, Di Lorenzo G, Grisorio G, Barbarini G. Incidence of dilated cardiomyopathy and detection of HIV in myocardial cells of HIV-positive patients. N Engl J Med 1998;339:10939. Paties C, Peveri V, Falzi G. Liver histopathology in autopsied drug-addicts. Forensic Sci Int 1987;35:1126. Monforte JR. Some observations concerning blood morphine concentrations in narcotic addicts. J Forensic Sci 1977;22:71824. Vlahov D, Anthony JC, Munoz A, Margolick J, Nelson KE, Celentano DD, et al. The ALIVE study, a longitudinal study of HIV-1 infection in intravenous drug users: description of methods and characteristics of participants. NIDA Res Monogr 1991;109:75100.

513