Vol. 7, No. 1
Sonicated Diagnostic Immunoblot for Bartonellosis
´ ˜ VANIA MALLQUI,1 EMILY C. SPEELMON,1,2 MANUELA VERASTEGUI,1,3 CIRO MAGUINA-VARGAS,4 PAULA PINELL-SALLES,2 ROSA LAVARELLO,2 JOSE DELGADO,2 MARGARET KOSEK,5 SOFIAROMERO,6 YANINA ARANA,1 AND ROBERT H. GILMAN1,2,7* ´a, Departamento de Patologı 1 Departamento de Microbiologı 3 and Instituto de Medicina Tropical Alexander ´a, VonHumboldt,4 Universidad Peruana Cayetano Heredia, Asociacion Beneﬁca PRISMA,2 and United States Naval ´ ´ Medical Research Institute Detachment (NAMRID),6 Lima Peru; Department of Internal Medicine, Harbor-UCLA Medical Center, Torrance,California5; and Department of International Health, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland7
Received 26 May 1999/Returned for modiﬁcation 25 June 1999/Accepted 14 September 1999
Two simple Bartonella bacilliformis immunoblot preparation methods were developed. Antigen was prepared by two different methods: sonication of whole organisms or glycineextraction. Both methods were then tested for sensitivity and speciﬁcity. Well-deﬁned control sera were utilized in the development of these diagnostic immunoblots, and possible cross-reactions were thoroughly examined. Sera investigated for cross-reaction with these diagnostic antigens were drawn from patients with brucellosis, chlamydiosis, Q fever, and cat scratch disease, all of whom were fromregions where bartonellosis is not endemic. While both immunoblots yielded reasonable sensitivity and high speciﬁcity, we recommend the use of the sonicated immunoblot, which has a higher sensitivity when used to detect acute disease and produces fewer cross-reactions. The sonicated immunoblot reported here is 94% sensitive to chronic bartonellosis and 70% sensitive to acute bartonellosis. In ahealthy group, it is 100% speciﬁc. This immunoblot preparation requires a simple sonication protocol for the harvesting of B. bacilliformis antigens and is well suited for use in regions of endemicity. Bartonella bacilliformis is a gram-negative, facultatively intracellular bacterium that is the causative agent of bartonellosis, a biphasic illness endemic in the high-altitude river valleys of Peru,Colombia, and Ecuador. The acute febrile phase, known as Oroya fever, is characterized by severe hemolytic anemia due to B. bacilliformis invasion of as many as 90% of erythrocytes. During and brieﬂy following this febrile illness, a period of immunosuppression is associated with a high rate of secondary infections, most notably with Salmonella species (8). In the preantibiotic era, the acute phase ofillness was thought to be fatal in about 40% of patients (29), although a mortality rate of 88% in untreated cases was observed during the 1987 Shumpillan outbreak (10). Appropriate antibiotic therapy reduces mortality to 8.8% (20). Four to 8 weeks after initial infection, the patient presents with the chronic phase of bartonellosis, dubbed verruga peruana. Vascular exophytic or nodular skinlesions, resulting from the invasion of vascular endothelial cells by the bacterium and a reactive angiogenic proliferation, characterize the verruga peruana. During the chronic phase of the illness, examination of peripheral-blood smears almost always fails to reveal B. bacilliformis (12), although in a small number of cases blood cultures may be positive (11). Current methods employed for thediagnosis of bartonellosis have signiﬁcant limitations. While performing a peripheralblood smear by techniques such as Giemsa staining is rapid and simple, the sensitivity of peripheral-blood smear examination is very low in mild cases of disease and in the subclinical and chronic phases of illness. Diagnosis by blood culture is complicated by the need for prolonged incubation (1 to 6 weeks), which...