Bartonella spp.

Antibodies in Forensic Samples from Swedish Heroin Addicts

SVENA MCGILL,a EVA HJELM,b JOVAN RAJS,c OLLE LINDQUIST,d AND GRAN FRIMANa
aInfectious

Diseases and bClinical Microbiology, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden

Units of Forensic Medicine, cKarolinska Institute, Stockholm and dUppsala University, Uppsala, Sweden

ABSTRACT: A high frequency of Bartonella elizabethae seropositivity (39%) was recorded among intravenous heroin addicts in Stockholm, Sweden, who died from a lethal injection. Some of the B. elizabethaeseropositive 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 frequent finding but no case had peliosis hepatitis. There was no case of endocarditis, but in three persons active subacute-to-chronic myocarditis was found; two of these cases were Bartonella-positive and HIV-negative. KEYWORDS: Bartonella spp.; Stockholm; heroin addicts; IV drug users; myocarditis

INTRODUCTION Bartonella infections have been found in inner city homeless, alcoholic men as a cause of endocarditis in southern France and the U.S.,1,2 and high Bartonella seroprevalences have been reported in other socially underprivileged groups, including intravenous drug (IVD) users.35 High prevalences of antibodies to B. elizabethae (33%46%) were found among inner-city IVD users in the USA.4,5 In Sweden, a high seropositivity rate to B. elizabethae has been reported in domestic cats,6 clinical samples,7 and orienteer sportsmen, many of whom have been struck by sudden unexpected death due to subacute myocarditis.8,9 In this retrospective study, the seroreactivities to various Bartonella spp. were tested in forensic serum samples from IVD users of Stockholm, who died in connection with heroin injection. MATERIALS AND METHODS Cases The study was based on death cases investigated at the Government Institute of Forensic Medicine in Stockholm, Sweden, from 1987 to 1992. A total of 17,699
Address for correspondence: Gran Friman, Infectious Diseases, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden. Goran.friman@medsci.uu.se
Ann. N.Y. Acad. Sci. 990: 409413 (2003). 2003 New York Academy of Sciences.

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bodies were examined, and of these, 149 deaths occurred in direct association with heroin injection. Blood or serum specimens from 59 of these 149 addicts were obtained from the National Institute of Infectious Disease Control, Stockholm. The majority of the cases were from the inner city, and direct IV heroin injection was implicated as the primary cause of death. The subjects were all European (Caucasian), age range 2243 years, mean 32.1; 85% were male. All subjects were tested for human immunodeciency virus (HIV). Controls Sera from 44 forensic autopsy cases from Stockholm with causes of death unrelated to any type of substance abuse were used. The age range was 2055 years, mean 40.9; 84% were male. Forensic Analysis Bodies were kept in refrigerated rooms of the mortuary at +48C until autopsied (06 days after death; mean 3.6 days). Prior to autopsy, blood samples were analyzed for HIV antibodies. Heart sections for histopathological analysis were taken from the anterior wall, interatrial and interventricular septum, the left anterior and posterior ventricular wall, and papillary muscles. Inammation was dened by the presence of inammatory cells (n 15/focus) adjacent to necrotic or degenerated myocytes with or without brosis according to the Dallas classication.10 Myocardial brosis was dened as focal presence of noninammatory brous tissue in the ventricular myocardium, except 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, and bladder urine was collected. Serological Testing The serology was performed by indirect immunouorescence, as described by Regnery et al.11 to the following antigens: B. elizabethae (F9251; ATCC 49927), B. grahamii (ATCC 700132), B. henselae (Houston-1, ATCC 49882), B. henselae (Marseille, URLLY8), B. quintana (OK 90-268), and B. vinsonii subsp. vinsonii (ATCC VR-152).

RESULTS In serological testing, 39% (23/59) of the IVD users had antibodies to at least one of the 6 Bartonella antigens tested (see FIGURE 1). All 23 seropositive sera reacted to B. elizabethae, ranging up to 1:512 in four cases. Geometric mean titers (GMT) for B. elizabethae, B. grahamii, B. henselae (Houston-1) and B. quintana among the Bartonella-positive sera were 144, 181, 117, and 64, respectively. The autopsy ndings are described in TABLE 1. Nearly all of the completely autopsied IVD users had hepatitis but none had peliosis hepatis. Myocarditis and

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FIGURE 1. Seroreactivity to Bartonella spp. in 59 Swedish heroin addicts.

myocardial brosis were found in a few cases, but no case with endocarditis was recorded. The average concentration of morphine in the femoral vein blood or heart at autopsy in the seropositive IVD users was 0.37 g morphine/g blood. Cocaine or alcohol was not present.

TABLE 1. Age and sex distribution of heroin addicts and controls. HIV-positivity rate and autopsy ndings in addicts Heroin addicts ( n = 59) Seropositive (n = 23) Mean age (years) Female/male HIV-positive Hepatitisa Myocarditisa Myocardial
aComplete

Control patients ( n = 44) Seropositive (n = 9) 42.0 2/9 (22%) Seronegative (n = 35) 40.5 5/35 (14%)

Seronegative (n = 36) 31.8 7/36 (19%) 13/36 (36%) 14/14 (100%) 1/14 (7%) 3/14 (21%)

32.6 2/23 (9%) 11/23 (48%) 10/11 (91%) 2/11 (18%) 3/11 (27%)

fibrosis a

autopsy, including microscopical investigation, was performed in 11 of the Bartonella seropositive cases and in 14 of the Bartonella seronegative cases. In consideration of the autopsy personnel, complete autopsy was not carried out in such HIV-positive cases where the cause of death was obvious.

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The control sera manifested a 20% (9/44) Bartonella seropositivity rate. This is a statistically signicant difference (P < 0.01) compared to that of the IVD users. All positive sera reacted to 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.

DISCUSSION A considerable proportion, 39%, of the Swedish intravenous drug (IVD) users were seropositive to Bartonella spp. In all of the Bartonella-positive sera (both in cases and controls) antibodies to B. elizabethae were detected. The seroprevalence in Swedish blood donors to the same antigen was earlier found to be 14%.12 Nine of the 23 (39%) B. elizabethae-positive sera had antibodies to other Bartonella spp. as well, but cross-reactions between strains cannot be excluded. The 14% seroprevalence to B. henselae H-1 is comparable to the results in the IVD users in the U.S.,4,5 but much higher than the prevalence in healthy blood donors in Sweden (1.2%).12 B. grahamii seropositivity was found at the same rate in the addicts as in Swedish blood donors (3 and 2.6%, respectively).12 B. grahamii has been isolated from several rodent species, including specimens collected in Sweden.13,14 The 3% seroprevalence to B. quintana among our IVD users is comparable to the 2% among IVD users from New York City,5 whereas 10% was reported among Baltimore inner-city IVD users.4 Studies of healthy blood donors in Seattle and Sweden reported 2% and 0.2%, respectively.3,12 HIV-positivity was more frequent among the IVD users with antibodies to Bartonella spp. (48%) than among the Bartonella seronegative addicts (36%). In the Baltimore study, an opposite result was found for B. henselae and B. quintana, where HIV-negativity was associated with having antibody to these agents, however not to B. elizabethae.4 Furthermore, among the HIV-seropositive individuals of that study, a signicant inverse association was found of antibody prevalence to each of B. henselae and B. quintana and CD4 cell count. In three cases myocarditis was found, two of whom were Bartonella seropositive and HIV seronegative. Subacute myocardits was recently reported as a manifestation of Bartonella infection in Swedish elite orienteers succumbing to sudden unexpected death,9 and chronic active myocarditis has subsequently been reported in a case of cat scratch disease in the USA.15 However, myocarditis may also result from heroin abuse.16 No case of endocarditis was found.

ACKNOWLEDGMENTS Our appreciation is extended to Dr. Russell Regnery, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA for his assistance with antigen production. A more detailed article has been submitted for subsequent publication in Acta Patho-

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logica et Microbiologica Scandinavica (the present condensed report was published with permission).
REFERENCES 1. DRANCOURT, M., J.L. M AINARDI, P. BROUQUI, et al. 1995. Bartonella (Rochalimaea) quintana endocarditis in three homeless men. N. Engl. J. Med. 332(7): 419423. 2. SPACH, D.H., A.S. K ANDER, M.J. DOUGHERTY, et al. 1995. Bartonella (Rochalimaea) quintana bacteremia in inner-city patients with chronic alcoholism. New. Engl. J. Med. 332(7): 424428. 3. JACKSON, L.A., D.H. S PACH, D.A. KIPPEN, et al. 1996. Seroprevalence to Bartonella quintana among patients at a community clinic in downtown Seattle. J. Infect. Dis. 173(4):10231026. 4. COMER, J.A., C. FLYNN, R.L. REGNERY, et al. 1996. Antibodies to Bartonella species in inner-city intravenous drug users in Baltimore, MD. Arch. Intern. Med. 156: 2491 2495. 5. COMER, J.A., T. DIAZ, D. VLAHOV, et al. 2001. 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. 65(6): 855860. 6. HJELM, E., G. BLOMQVIST, S. MCGILL. 2002. Seroprevalence of Bartonella antibodies in Swedish domestic cats. Scand. J. Infect. Dis. 34: 192196. 7. HOLMBERG, M., S. MCGILL, C. EHRENBORG, et al. 1999. Evaluation of human seroreactivity to Bartonella species in Sweden. J. Clin. Microbiol. 37: 13811384. 8. MCGILL, S., L. WESSLN, E. HJELM, et al. 2001. Serological and epidemiological analysis of the prevalence of Bartonella spp. antibodies in Swedish elite orienteers 19921993. Scand. J. Infect. Dis. 33(6): 423428. 9. WESSLN, L., C. EHRENBORG, M. HOLMBERG, et al. 2001. Subacute Bartonella infection in Swedish orienteers succumbing to sudden unexpected cardiac death or having malignant arrhythmias. Scand. J. Infect. Dis. 33(6):429438. 10. ARETZ, H.J., M.E. B ILLINGHAM & W.D. EDWARDS. 1987. Myocarditis: a histopathologic definition and classification. Am. J. Cardiovasc. Pathol. 1: 314. 11. REGNERY, R.L., J.G. O LSON, B.A. PERKINS & W. BIBB. 1992. Serological response to Rochalimaea henselae antigen in suspected cat-scratch disease. Lancet 339: 1443 1445. 12. MCGILL, S., L. WESSLN, E. HJELM, et al. 2002. Bartonella spp. seroprevalence in healthy Swedish blood donors. Scand. J. Infect. Dis. Submitted. 13. BIRTLES, R.J., T.G. H ARRISON & D.H. MOLYNEUX. 1994. Grahamella in small woodland mammals in the U.K.: isolation, prevalence and host specificity. Ann. Trop. Med. Parasitol. 88(3): 317327. 14. HOLMBERG, M., J.N. M ILLS, S. MCGILL, et al. 2003. Bartonella infection in sylvatic small mammals of central Sweden. Epidemiol. Infect. 130(1): 149157. 15. MEININGER, G.R., T. N ADASDY, R.H. HRUBAN, et al. 2001. Chronic active myocarditis following acute Bartonella henselae infection (cat scratch disease). Am. J. Surg. Pathol. 25(9): 12111214. 16. RAJS, J. & B. FALCONER. 1979. Cardiac lesions in intravenous drug addicts. Forensic Sci. Int. 13(3): 193209.