Prevalence of Bartonella Infection among Human Immunodeficiency Virus Infected Patients with Fever

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1 MAJOR ARTICLE HIV/AIDS Prevalence of Bartonella Infection among Human Immunodeficiency Virus Infected Patients with Fever Jane E. Koehler, 1 Melissa A. Sanchez, 4 Sherilyn Tye, 4 Claudia S. Garrido-Rowland, 1 Frederick M. Chen, 4 Toby Maurer, 2 Judy L. Cooper, 5 James G. Olson, 5 Arthur L. Reingold, 4 W. Keith Hadley, 3 Russell R. Regnery, 5 and Jordan W. Tappero 6 Departments of 1 Medicine, 2 Dermatology, and 3 Laboratory Medicine, University of California at San Francisco, San Francisco, and 4 Epidemiology Program, School of Public Health, University of California at Berkeley; and 5 Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, and 6 Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia Bartonella infection can be difficult to diagnose, especially when it manifests as bacteremia, which is usually accompanied by nonspecific symptoms, such as fever. Therefore, we hypothesized that Bartonella infection represents an underrecognized cause of febrile illness. To determine the prevalence of Bartonella infection among patients presenting with fever, we evaluated 382 patients in San Francisco. Overall, 68 patients (18%) had evidence of Bartonella infection detected by culture, indirect fluorescent antibody testing, or polymerase chain reaction (PCR). Twelve patients (3%) had either Bartonella henselae or Bartonella quintana isolated from specimens of blood, tissue, or both or had DNA detected in tissue; all 12 had concomitant human immunodeficiency virus (HIV) infection. Bartonella antibodies were detected in 17% of febrile patients, including 75% of culture-positive or PCR-positive patients. In a nested, matched case-control study aimed at identifying clinical features of febrile illness associated with Bartonella infection, only bacillary angiomatosis and elevated alkaline phosphatase levels were associated with Bartonella infection ( P.03 for both). The prevalence of Bartonella infection among patients with late-stage HIV infection and unexplained fever is much greater than has previously been documented. The spectrum of Bartonella infections seen in immunocompetent and immunocompromised patients has expanded rapidly since the first HIV-infected patient Received 19 September 2002; accepted 13 March 2003; electronically published 31 July Financial support: Centers for Disease Control and Prevention (grant U64/ CCU910875); California Universitywide AIDS Research Program, National Institutes of Health (grants NIH R01 AI43703 and NIH R01 AI052813), and Pew Scholars Program in the Biomedical Sciences (to J.E.K.); and Fogarty International Center (grant D43-TW00003 to M.A.S.). Reprints or correspondence: Dr. Jane E. Koehler, Div. of Infectious Diseases, 521 Parnassus Ave., Rm. C-443, University of California at San Francisco, San Francisco, CA (jkoehler@medicine.ucsf.edu). Clinical Infectious Diseases 2003; 37: by the Infectious Diseases Society of America. All rights reserved /2003/ $15.00 with unusual, vascular proliferative lesions of bacillary angiomatosis (BA) was described in 1983 [1]. Sixteen species of Bartonella have now been identified [2]. Bartonella quintana and Bartonella henselae are the 2 Bartonella species that infect immunocompromised and immunocompetent humans most frequently in North America, and both can cause fever with vascular proliferative lesions, persistent bacteremia, or both. Despite increased recognition of the manifestations of Bartonella infection during the past 10 years, diagnosis often remains elusive because of the difficulty in culturing the organism, the difficulty in interpreting modified silver tissue stains (e.g., Warthin-Starry stain), and the lack of a rapid, readily available serologic test. Although cutaneous BA lesions and cat scratch disease HIV/AIDS CID 2003:37 (15 August) 559

2 (CSD) lymph nodes are accessible to biopsy and thus may eventually be diagnosed, the less obvious manifestations of Bartonella infection (e.g., isolated bacteremia) are rarely identified because of the nonspecific symptomatology. Undiagnosed Bartonella infections can persist for months in both immunocompetent [3 6] and immunocompromised patients [7, 8] and can cause chronic, debilitating opportunistic infection and even death [9]. We hypothesized that Bartonella infections are substantially underrecognized as a cause of fever, because focal Bartonella infection is often not initially diagnosed, and isolated Bartonella bacteremia is rarely considered in the differential diagnosis of prolonged fever. Over a 16.5-month period, we conducted a prospective study to determine the prevalence of infection due to Bartonella species among patients with unexplained febrile illness. MATERIALS AND METHODS Study Design To identify eligible study participants, we surveyed inpatients and outpatients at 4 San Francisco Bay area hospitals. Patients were eligible if they were 18 years of age and met 1 of the following 3 criteria: (1) patient had an acute febrile illness (temperature, 38 C) without an identifiable source, (2) patient had persistent or recurrent fever of 2 weeks duration without an identifiable source, and (3) patient was referred for persistent fever and subsequently noted to have lesions suspicious for BA during physical examination by a health care provider trained to recognize BA. Patients were excluded if they had received antibiotic therapy with a macrolide, tetracycline, or rifamycin in the 2 weeks before culture for Bartonella species was performed. For HIV-infected patients with fever of unknown source, patients were only eligible if they had no prior history of Mycobacterium avium complex (MAC) infection and if they had negative results of a blood culture for MAC in the 4 weeks before Bartonella culture was performed. For each patient, 1 lysis-centrifugation blood culture tube was prepared for Bartonella culture, and 1 serum separator tube was prepared for serologic testing for Bartonella species. For patients with fever who were subsequently noted to have lesions suspicious for BA during examination by a health care provider trained to recognize BA, tissue biopsy was performed, and the specimens were submitted for microbiologic and histopathologic evaluation. Blood samples were cultured simultaneously for Bartonella species and MAC at the San Francisco General Hospital Microbiology Laboratory. Serum samples were sent to the Centers for Disease Control and Prevention (CDC) for testing for Bartonella antibodies by indirect fluorescent antibody (IFA) test. Molecular and Microbiological Analysis Isolation of Bartonella species from human blood and tissues. Samples of blood, tissue, or both were obtained from patients for culture for Bartonella species. Tissue samples were homogenized [7] and plated onto both heart infusion agar (supplemented with 5% fresh defibrinated rabbit blood (HIAR) [10]) and chocolate agar. Agar plates were custom-poured with twice the standard volume (i.e., 29 ml) and were always used within 7 days. Patients blood samples were cultured using lysiscentrifugation tubes (Wampole [10]), as described elsewhere [7]. Inoculated agar plates were incubated at 34 C for 3 weeks in candle jars (to enrich for CO 2 ). Jars were opened and cultures assessed on days 7, 14, and 21 after plate inoculation, and negative cultures were blindly subcultured onto fresh agar at each time point and then held for an additional 3 weeks. Extraction of DNA from archived tissue and speciation of Bartonella isolates by sequencing. The infecting Bartonella species was determined by extraction of DNA from either Bartonella isolates (for 11 patients) or formalin-fixed, paraffinembedded tissue (for 1 patient) [7, 11]. 16S rdna was amplified under standard conditions by use of PCR with primers p24e and p12b [12], with appropriate controls, and the amplicons were cloned and sequenced [7, 8]. Speciation for each isolate was corroborated by PCR restriction fragment length polymorphism analysis of a fragment of the citrate synthase gene [13]. Serological Analysis by IFA Testing The IFA test for Bartonella antibodies was developed at the CDC [14, 15]. Serum samples were diluted 2-fold to 1:1024, and a reciprocal titer of 64 to B. henselae, B. quintana, or Bartonella elizabethae was considered to be a positive result on the basis of previous studies [14, 15]. The sensitivity of the IFA test was previously determined to be 82% 95% for patients with suspected CSD; the estimated specificity for CSD is 93% 96% [15]. With use of this IFA test, the background prevalence of Bartonella antibodies in afebrile populations was determined to be 4% 7% [14, 15]. Case-Control Study We conducted a nested, matched case-control study (ratio of case patients to control subjects, 1:2) with patients who met the eligibility requirements, to identify risk factors and to distinguish clinical features of Bartonella infection in this study population with unexplained fever. Written, informed consent was obtained from all participants, as approved by the committees for human research of the University of California at San Francisco, the University of California at Berkeley, Davies Medical Center (San Francisco, CA), and the CDC. The guidelines for human experimentation of these institutions were followed in conducting clinical research. 560 CID 2003:37 (15 August) HIV/AIDS

3 Case patient definition. Case patient was defined as a patient with a persistent or recurrent fever from whom a Bartonella species was isolated, from whose tissue Bartonella DNA was amplified, or whose anti-bartonella reciprocal antibody titer was 64, as determined by IFA test. Control subjects were matched to each case patient on the basis of the date on which blood samples were obtained for culture. Control subjects had no evidence of Bartonella infection, as determined by both culture and serologic testing performed on the date on which blood culture samples were obtained. Questionnaire. A standardized questionnaire was administered to collect information about demographic and behavioral characteristics, medical history, and environmental exposures during specific reference periods preceding the date that the culture and serum specimens were obtained. Additional clinical information was extracted from medical records with use of a structured data collection instrument. Statistical Analysis Case patients and matched control subjects were evaluated for differences using Epi Info software (CDC) [16], and the Kruskal-Wallis test was used to compare distributions of continuous variables between case patients and matched control subjects. For dichotomous variables, univariate matched ORs (MORs) with 95% CIs were calculated using the Mantel-Haenszel method. All species-stratified subanalyses were conducted on unmatched data to maintain statistical power in the setting of a diminished number of case-control sets. Although the Kruskal-Wallis test for 2 groups was again used to compare distributions of continuous variables between the unmatched case patients and control subjects, the respective P values for the univariate unmatched ORs and 95% CIs for dichotomous variables were calculated using the 2-tailed Fisher s exact test. RESULTS Blood specimens were obtained for both Bartonella culture and serologic testing from a total of 382 patients (table 1). Of these patients, 306 (80%) were culture and antibody negative for Bartonella species. The culture or serologic test specimen could not be fully evaluated for 10 patients (3%). The remaining 66 patients (17%) were found to be positive for Bartonella on the basis of 1 of the following tests: culture, PCR or IFA test. In addition, there were 2 antibody-positive patients whose cultures were contaminated or lost. Isolation and Speciation of Bartonella from Blood and Tissue Specimens A Bartonella species was isolated from the blood or tissue specimens of 11 patients, and Bartonella DNA was amplified from the tissue specimen of 1 patient (table 2). B. henselae and B. Table 1. Prevalence of Bartonella infection among patients presenting with persistent and acute febrile illness of unknown cause. Laboratory test results No. (%) of patients Culture and serologic test submitted 382 Bartonella culture and/or PCR positive 12 (3) No cutaneous BA but positive results 6 (2) IFA test positive 65 (17) IFA test and/or Bartonella culture positive 68 (18) MAC culture positive 52 (14) MAC and Bartonella culture positive 3 (1) Histoplasma culture positive 6 (2) NOTE. BA, bacillary angiomatosis; IFA, indirect fluorescent antibody; MAC, Mycobacterium avium complex. quintana were the only 2 species documented in this study (6 patients for each species). There was no difference in the ability to isolate either species from blood or tissue specimens, and no colonies were detectable until after 8 days incubation. A Bartonella species was isolated from blood and/or tissue specimens obtained from all 6 patients with cutaneous BA lesions. All 11 isolates were subsequently passaged multiple times without difficulty. During the evaluation for fever for this study, examination by physicians trained to recognize BA revealed that 6 of the 11 patients, who were ultimately found to be culture positive for Bartonella infection, had cutaneous lesions. Biopsies of the 6 patients cutaneous lesions were performed and specimens were examined, revealing histopathologic features of BA [17]. In 5 patients, no focal Bartonella infection could be identified, but Bartonella bacteremia was ultimately detected. The twelfth patient (patient 5; table 2) was hospitalized with fever of unknown origin (temperature up to 41 C) and cervical lymphadenopathy; Bartonella infection was not suspected, but examination of a lymph node biopsy specimen revealed BA. B. henselae DNA was detected in an archived tissue specimen obtained from this patient, for whom a fresh tissue sample was not available for culture. One patient with prolonged fever (patient 1; table 2), who was lost repeatedly to follow-up, provided samples for culture twice during the study period and a third time 4 months after the study period. This patient initially presented with fever, had a blood culture positive for MAC but negative for Bartonella species, and had a result of a serologic test for B. quintana that was low positive. The patient was lost to follow-up and returned with fever and cutaneous lesions 5 months later; at that time, B. quintana was isolated from a blood sample, cutaneous lesions revealed BA on histopathologic examination (the lesions were not cultured), and Bartonella antibody titers had increased. The patient again failed to return for diagnostic test results and HIV/AIDS CID 2003:37 (15 August) 561

4 Table 2. Characteristics of patients with fever and culture-documented or PCR-documented Bartonella infection. Patient, day of study Bartonella species Focal Bartonella disease Bartonella blood culture result Culture yield, cfu/10 ml of blood Tissue biopsy results Reciprocal IFA titer a Chocolate agar HIAR Culture PCR BH BQ MAC blood culture result 1 Day 0 None None 0 0 ND ND Day 152 BQ Cutaneous BA ND ND Day 583 BQ Cutaneous BA SF + b ND BH Cutaneous BA b ND BH Cutaneous BA ND ND BH None ND ND BH Lymph node BA 0 0 ND + c BQ None ND ND BQ Cutaneous BA, b ND subcutaneous nodules 8 BH Granulomatous hepatitis ND ND BH None ND ND BQ Cutaneous BA, b ND subcutaneous nodules 11 BQ None ND ND BQ Cutaneous BA ND ND NOTE. BA, bacillary angiomatosis; BH, Bartonella henselae; BQ, Bartonella quintana; HIAR, heart infusion agar supplemented with rabbit blood; IFA, indirect fluorescent antibody; MAC, Mycobacterium avium complex; ND, not done; SF, Shigella flexneri; +, positive;, negative. a Titer of 64 indicates positive IFA. b Cutaneous BA specimen. c Lymph node specimen. treatment but returned 14 months later with new cutaneous BA lesions and fever. Four of 4 blood cultures grew confluent colonies of Shigella flexneri, which obscured any Bartonella growth. However, B. quintana alone was isolated from a simultaneously obtained tissue biopsy specimen of a cutaneous BA lesion, and serologic testing revealed a further increase in the B. quintana antibody titer. The number of Bartonella colonies recovered from the semiquantitative lysis-centrifugation blood culture system was cfu/ml of blood (table 2). For 1 patient with B. henselae bacteremia (patient 4; table 2), a single colony was recovered on the primary culture plate from 10 ml of blood, and for 1 patient with B. quintana bacteremia (patient 6; table 2), 1 colony was isolated, but only on the first blind subculture plate. Of note, isolation of B. henselae occurred only on HIAR plates (never on simultaneously inoculated chocolate agar), and, except for 1 patient, isolation of B. quintana occurred only on chocolate agar plates. However, agar preference diminished with subsequent cultivation. Fifty-two (14%) of 382 patients had MAC isolated from a blood culture (table 1), despite the enrollment requirement for a negative MAC culture. Three patients (1%) were coinfected with Bartonella species and MAC. Finally, 6 (2%) of 382 patients had Histoplasma capsulatum alone isolated from blood cultures. Serologic Analysis by IFA Testing Bartonella antibodies were detected in 65 (17%) of the 382 patients tested. Fifty-six patients were seropositive and culture negative. For these 56 patients, the distribution of reciprocal titers was as follows: 64 (19 patients), 128 (16 patients), 512 (13 patients), 2048 (4 patients), 8192 (3 patients), and (1 patient). Of the 12 patients positive for Bartonella species by culture or PCR, 9 were culture positive and seropositive, 1 was PCR positive and seronegative, and 2 were culture positive and seronegative (table 2). All 3 culture-positive or PCR-positive patients who were also seronegative were infected with B. henselae. Of note, of the 9 culture-positive patients in whom antibodies were detected, all had B. quintana titers that were equal to or greater than their titers against B. henselae, even among those infected with B. henselae. Case-Control Study Among the 68 eligible patients who met the case definition, 35 were enrolled, 31 could not be located, and 2 had died (no surrogate i.e., relative or partner was available to assist with completion of the questionnaire). A total of 61 control subjects were identified; 2 control subjects were enrolled for each case 562 CID 2003:37 (15 August) HIV/AIDS

5 patient, with the exception of 9 case patients for whom only 1 control subject was enrolled each. Clinical Features, Environmental Exposures, and Socioeconomic Characteristics Matched analyses. Thirty-three (97%) of 34 case patients and 54 (93%) of 58 control subjects enrolled were infected with HIV. HIV infection status was unknown for 1 case patient and 3 control subjects. There were no statistically significant differences between case patients and control subjects with regard to median age (37.5 years overall; P p.98), race (63% overall were white; MOR, 1.8; 95% CI, ; P p.22), sex (88% overall were male; MOR, 1.2; 95% CI, ; P p.91), ethnicity (24% overall were Hispanic; MOR, 1.9; 95% CI, ; P p.32), or risk factors for HIV infection (i.e., homosexual or bisexual orientation, commercial sex work, or injection drug use; P.26 for all). There were no statistically significant differences between case patients and control subjects with regard to an AIDS diagnosis (85% overall had AIDS; MOR, 2.5; 95% CI, ; P p.34). None of the patients had received HAART, nor had virus load been determined. In addition to fever present at the time of culture and serologic testing for Bartonella species, persistent or recurrent fever was present in 30 (86%) of 35 case patients during the 3 months before Bartonella cultures were performed and in 51 (84%) of the 61 control subjects (MOR, 1.4; 95% CI, ; P p.84). With the exception of the presence of cutaneous BA lesions (in 6 case patients and no control subjects; P p.002), there was no difference between case patients and control subjects with regard to symptoms and signs at the time of presentation (e.g., chills, sweats, headache, weight loss, nausea and vomiting, abdominal pain, diarrhea, lymphadenopathy, pruritus, and bone pain; P.22 for all). With the exception of receipt of a cat scratch or bite during the 6-month period before culture and serologic tests for Bartonella species were performed (49% of case patients vs. 21% of control subjects; MOR, 2.8; 95% CI, ; P p.02) and bathing!5 times per week (46% of case patients vs. 16% of control subjects; MOR, 5.9; 95% CI, ; P p.003), there were no differences detected between case patients and control subjects with regard to a variety of other animal and insect exposures and socioeconomic factors ( P.12 for all). The median alkaline phosphatase level was higher for case patients than for control subjects (94.5 vs U/L; P p.03); however, no other laboratory values distinguished case patients from control subjects. The median CD4 + cell count for the 33 case patients with concomitant HIV and Bartonella infection was 35 cells/ mm 3, compared with 40 cells/mm 3 for the 54 HIV-infected control subjects ( P p.58). Unmatched analyses. In unmatched analyses, a number of Bartonella species specific risk factors and findings were identified (tables 3 and 4). In general, case patients with B. henselae infection were more likely than those with B. quintana infection to have more constitutional symptoms, such as weight loss, joint pain, and bone pain, and to have experienced a variety of pet cat exposures; however, not all of these associations were statistically significant. DISCUSSION Fever and persistent Bartonella bacteremia of weeks or months duration are hallmarks of Bartonella infection and occur in immunocompetent [3 6] and immunocompromised [7, 8] patients. B. henselae infection has been identified as the third most common cause of fever of unknown origin in immunocompetent children, after Epstein-Barr virus and osteomyelitis [18]. Intermittent fever and Bartonella bacteremia over a period of months have been reported in immunocompromised patients with cutaneous lesions ultimately recognized to be BA (67% of cases of B. henselae BA and 43% of cases of B. quintana BA [8]). We hypothesized that unsuspected Bartonella infection is a cause of fever in patients presenting for evaluation of persistent or undiagnosed fever, and we sought to determine the prevalence of Bartonella infection in this population. We evaluated 382 patients with acute or persistent unexplained fever and, surprisingly, found evidence of Bartonella infection in 68 (18%) by culture, IFA testing, or PCR. Either B. henselae or B. quintana was cultured from blood and/or tissue specimens obtained from 11 (3%) of the 382 patients, and B. henselae DNA was amplified from tissue from 1 patient. Although we did not select patients on the basis of HIV infection status, the majority (95%) of case patients and control subjects enrolled in the case-control study were HIV infected. For the 33 HIV-infected case patients with Bartonella infection, the median CD4 + cell count was 35 cells/mm 3. Unexplained fever occurs frequently in patients with low CD4 + cell counts [19], and disseminated MAC infection is one of the most common infections identified in this population [20]; however, in one study, more than one-half of the MAC culture positive patients also had another cause of fever identified [21]. In our study population, Bartonella infection was diagnosed more commonly (18%) than was MAC infection (14%), probably because a prior negative MAC culture result was required. Of note, 3 patients had concomitant isolation of MAC and Bartonella species. MAC grows on both HIAR and chocolate agar, with a doubling time and appearance similar to Bartonella species. This made detection of the few Bartonella colonies among the MAC colonies challenging. Finally, isolation of Histoplasma capsulatum from the blood cultures of 2% of our patients represented an unexpectedly high prevalence for this pathogen outside of a region of endemicity. Physicians trained in recognition of BA examined patients HIV/AIDS CID 2003:37 (15 August) 563

6 Table 3. Characteristics of and environmental exposures among 13 patients with Bartonella infection and 61 control subjects presenting with persistent or acute febrile illness of unknown cause, by unmatched analyses. Characteristic or exposure Case patients with Bartonella henselae infection (n p 6) a vs. control subjects Case patients with Bartonella quintana infection (n p 7) b vs. control subjects Unmatched Unmatched OR (95% CI) P c OR (95% CI) Race (percentage of patients who were white) Undefined ( ).69 HIV infection risk factor Heterosexual partner in a high-risk group 15.4 ( ) ( ) 1.00 Shared needles with an injection drug user in the past 3 months Undefined.09 Undefined 1.00 Signs and symptoms of present illness d Blood-filled skin lesions or skin nodules 9.0 ( ) ( ).05 Weight loss 0.1 ( ) ( ) 1.00 Joint pain 8.3 ( ) ( ) 1.00 Bone pain 5.0 ( ) ( ).33 Cat exposure e Household cat or significant cat exposure outside home 7.2 ( ) ( ).69 Mean no. of cats owned f NA.04 g NA.92 g Receipt of cat scratch or bite 7.4 ( ) ( ).64 Arthropod exposure h Flea bite 3.3 ( ) ( ) 1.00 Pubic lice Undefined 1.00 Undefined.10 Head or body lice Undefined.09 Undefined 1.00 NOTE. NA, not applicable. a Six case patients, including 5 culture-positive patients and 1 PCR-positive patient. b Seven case patients, including 6 culture-positive patients and 1 seropositive patient who had presumed B. quintana infection, as determined by indirect fluorescent antibody testing (B. quintana reciprocal titer, 128; B. henselae reciprocal titer, 32 [negative]). c P values were calculated by 2-tailed Fisher s exact test, unless otherwise indicated. d Symptoms during the 3-month period before performance of culture and serologic testing. e Exposure occurred during the 6-month period before performance of culture and serologic testing. f For B. henselae, the mean is 2.5 for case patients and 0.8 for control subjects; for B. quintana, the mean is 1.3 for case patients and 0.8 for control subjects. g Calculated by Kruskal-Wallis test for 2 groups. h Exposure occurred during the 12-month period before the onset of illness. P c and identified cutaneous lesions suspicious for BA in 6 (50%) of 12 culture-positive patients. Thus, the sole finding that distinguished patients with Bartonella infection from other patients with fever was the presence of cutaneous BA. As occurred for these 6 patients, even when cutaneous BA lesions develop, they can be difficult to recognize or diagnose (cases of BA remained undiagnosed for up to 8 months in one of our studies [7]). In the other 6 patients, Bartonella infection was undiagnosed until the results of special cultures or PCR for Bartonella species were positive. Of these patients, 4 had isolated Bartonella bacteremia, 1 had granulomatous hepatitis, and 1 had BA lymphadenitis. Of the 12 patients positive for Bartonella species by culture or PCR, one-half were infected with B. henselae and one-half with B. quintana. Although 3 other Bartonella species have been isolated from humans, no other Bartonella species was isolated in this study. To isolate both species, it was necessary to use 2 custom-made agar types and then to carefully read and subculture the cultures because of the low colony count from blood samples (0.1 1 cfu/ml). More sensitive techniques (e.g., cocultivation of tissue biopsy specimens with endothelial cells [7] or shell vial cultures with endothelial cells [22]) for isolating Bartonella species are not widely available and are more laborious to perform than are the lysis-centrifugation blood cultures. Our study found that 9 (75%) of our 12 HIV-infected pa- 564 CID 2003:37 (15 August) HIV/AIDS

7 Table 4. Laboratory values for 13 case patients with Bartonella infection and 61 control subjects presenting with persistent or acute febrile illness of unknown cause, by unmatched analyses. Laboratory value a Case patients b (n p 6) Bartonella henselae Control subjects Case patients d (n p 61) P c (n p 7) Bartonella quintana Control subjects (n p 61) P c Median CD4 + cell count, cells/mm Median AST level, U/L Median ALT level, U/L Median ALP level, U/L Median LDH level, U/L Median hematocrit, % NOTE. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase. a Values are those on the date closest to culture and serologic testing. b Six case patients, including 5 culture-positive patients and 1 PCR-positive patient. c P value was calculated by Kruskal-Wallis test for 2 groups. d Seven case patients, including 6 culture-positive patients and 1 seropositive patient who had presumed B. quintana infection, as determined by indirect fluorescent antibody testing (B. quintana reciprocal titer, 128; B. henselae reciprocal titer, 32 [negative]). tients with culture-positive or PCR-documented Bartonella infection had a positive antibody titer, compared with 82% 95% of immunocompetent patients [15] with suspected CSD. This difference could result from the attenuated antibody response to bacterial antigens that has been reported in HIV-infected patients [23]. The isolation of Bartonella species continues to be extremely challenging, and detection of antibodies by IFA testing remains the most practical test for diagnosis of Bartonella infection in the absence of accessible tissue (e.g., BA lesion) for biopsy; however, a more sensitive serologic test would improve diagnosis in the HIV-infected population. We performed a case-control study to identify the clinical features or risk factors that could distinguish febrile patients with Bartonella infection from other patients with fever. Except for cutaneous BA and increased alkaline phosphatase levels, there were no clinical features that were specifically associated with Bartonella infection. We were not able to stratify our matched sets of case patients and control subjects by species (i.e., B. henselae and B. quintana), because the majority of our case patients had a positive IFA result but did not have identification of the infecting species by culture or molecular methods, and because there is substantial cross-reactivity between B. henselae and B. quintana by IFA testing [15]. However, in unmatched analyses (tables 3 and 4), we found Bartonella species specific risk factors and clinical features consistent with those identified in our previous study of patients with BA [8], by comparing the 6 case patients with confirmed B. henselae infection and the 7 case patients with confirmed B. quintana infection with all 61 control subjects. Three case patients and 3 control subjects received clarithromycin in the 3-month period before onset of illness but were not receiving this antibiotic at the time that cultures were performed (patients receiving a macrolide, rifamycin, or tetracycline in the 2 weeks before culture were excluded from the study). No case patients or control subjects received azithromycin in the 3 months before illness. Of note, in our molecular epidemiologic study of 49 patients with BA [8], we found that receipt of an antibiotic from the macrolide class (for MAC prophylaxis or other reasons) was protective against development of Bartonella infection. In the present study, 13 (37%) of 35 case patients received trimethoprim-sulfamethoxazole (TMP-SMZ) in the 3 months before the onset of illness, compared with 30 (49%) of 61 control subjects. In this study and in our previous study [8], we were able to isolate Bartonella species from patients taking TMP-SMZ for prophylaxis at the time that Bartonella cultures were performed. These data corroborate our previous findings suggesting that TMP-SMZ does not have in vivo activity against Bartonella species, and they lead us to recommend that TMP-SMZ not be used to treat Bartonella infection. Our data indicate that the prevalence of Bartonella infection among HIV-infected patients is much greater than previously documented and that Bartonella infection should be considered in the differential diagnosis for patients with fever and latestage HIV disease. Although the advent of HAART has had a dramatic effect on the incidence of AIDS-associated opportunistic infections [24], we continue to diagnose Bartonella infections in HIV-infected patients, particularly in patients who lack access to HAART or for whom HAART has failed. This is especially important because Bartonella infection is one of the most treatable (with erythromycin or doxycycline [25]) causes of unexplained fever in immunocompromised patients. Because B. henselae infections are common among immunocompetent adults and children [26], future studies should determine the prevalence of Bartonella infection among immunocompetent adult patients with fever of unknown origin. HIV/AIDS CID 2003:37 (15 August) 565

8 Acknowledgments We are indebted to the study participants and the faculty, fellows, and residents in the Departments of Medicine and Dermatology at the University of California at San Francisco, for referring their patients to the study, especially from the following clinics: Ward 86 at San Francisco General Hospital (SFGH), the Emergency Department at SFGH, and the inpatient AIDS Unit at SFGH. We also thank the staff of the Clinical Microbiology Laboratory at SFGH, who provided invaluable assistance with this project. References 1. Stoler MH, Bonfiglio TA, Steigbigel RT, Pereira M. An atypical subcutaneous infection associated with acquired immune deficiency syndrome. Am J Clin Pathol 1983; 80: Cunningham ET Jr, Koehler JE. Ocular bartonellosis. Am J Ophthalmol 2000; 130: Swift HF. Trench fever. Arch Intern Med 1920; 26: Lucey D, Dolan MJ, Moss CW, et al. Relapsing illness due to Rochalimaea henselae in immunocompetent hosts: implication for therapy and new epidemiological associations. Clin Infect Dis 1992; 14: Wong MT, Dolan MJ, Lattuada CP Jr, et al. Neuroretinitis, aseptic meningitis, and lymphadenitis associated with Bartonella (Rochalimaea) henselae infection in immunocompetent patients and patients infected with human immunodeficiency virus type 1. Clin Infect Dis 1995; 21: Spach DH, Kanter AS, Dougherty MJ, et al. Bartonella (Rochalimaea) quintana bacteremia in inner-city patients with chronic alcoholism. N Engl J Med 1995; 332: Koehler JE, Quinn FD, Berger TG, LeBoit PE, Tappero JW. Isolation of Rochalimaea species from cutaneous and osseous lesions of bacillary angiomatosis. N Engl J Med 1992; 327: Koehler JE, Sanchez MA, Garrido CS, et al. Molecular epidemiology of Bartonella infections in patients with bacillary angiomatosis-peliosis. N Engl J Med 1997; 337: Cockerell CJ, LeBoit PE. Bacillary angiomatosis: a newly characterized, pseudoneoplastic, infectious, cutaneous vascular disorder. J Am Acad Dermatol 1990; 22: Slater LN, Welch DF, Hensel D, Coody DW. A newly recognized fastidious gram-negative pathogen as a cause of fever and bacteremia. N Engl J Med 1990; 323: Koehler JE, Glaser CA, Tappero JW. Rochalimaea henselae infection: a new zoonosis with the domestic cat as reservoir. JAMA 1994; 271: Relman DA, Loutit JS, Schmidt TM, Falkow S, Tompkins LS. The agent of bacillary angiomatosis: an approach to the identification of uncultured pathogens. N Engl J Med 1990; 323: Regnery RL, Anderson BE, Clarridge JE, Rodriguez-Barradas MC, Jones DC, Carr JH. Characterization of a novel Rochalimaea species, R. henselae sp nov, isolated from blood of a febrile, human immunodeficiency virus positive patient. J Clin Microbiol 1992; 30: Regnery RL, Olson JG, Perkins BA, Bibb W. Serological response to Rochalimaea henselae antigen in suspected cat-scratch disease. Lancet 1992; 339: Dalton MJ, Robinson LE, Cooper J, Regnery RL, Olson JG, Childs JE. Use of Bartonella antigens for serologic diagnosis of cat-scratch disease at a national referral center. Arch Intern Med 1995; 155: Dean AG, Dean JA, Burton AH, Dicker RC. Epi Info version 5: a word processing, database, and statistics program for epidemiology on microcomputers. Atlanta, GA: Centers for Disease Control and Prevention, LeBoit PE, Berger TG, Egbert BM, Beckstead JH, Yen TSB, Stoler MH. Bacillary angiomatosis: the histopathology and differential diagnosis of a pseudoneoplastic infection in patients with human immunodeficiency virus disease. Am J Surg Pathol 1989; 13: Jacobs RF, Schutze GE. Bartonella henselae as a cause of prolonged fever and fever of unknown origin in children. Clin Infect Dis 1998; 26: Sullivan M, Feinberg J, Bartlett JG. Fever in patients with HIV infection. Infect Dis Clin North Am 1996; 10: Sepkowitz KA, Telzak EE, Carrow M, Armstrong D. Fever among outpatients with advanced human immunodeficiency virus infection. Arch Intern Med 1993; 153: Barat LM, Gunn JE, Steger KA, Perkins CJ, Craven DE. Causes of fever in patients infected with human immunodeficiency virus who were admitted to Boston City Hospital. Clin Infect Dis 1996; 23: La Scola B, Raoult D. Culture of Bartonella quintana and Bartonella henselae from human samples: a 5-year experience ( ). J Clin Microbiol 1999; 37: Kroon FP, van Dissel JT, de Jong JC, van Furth R. Antibody response to influenza, tetanus and pneumococcal vaccines in HIV-seropositive individuals in relation to the number of CD4 + lymphocytes. AIDS 1994; 8: Centers for Disease Control and Prevention USPHS/IDSA guidelines for the prevention of opportunistic infections in persons infected with human immunodeficiency virus. MMWR Morb Mortal Wkly Rep 1999; 48(RR-10):1 59, Koehler JE, Relman DA. Bartonella species. In: Yu VL, Merigan TC Jr, Barriere SL, eds. Antimicrobial therapy and vaccines. Baltimore: Williams & Wilkins, 1999: Jackson LA, Perkins BA, Wenger JD. Cat scratch disease in the United States: an analysis of three national databases. Am J Public Health 1993; 83: CID 2003:37 (15 August) HIV/AIDS