Comparison of Immunofluorescence, Western Blotting, and Cross-Adsorption Assays for Diagnosis of African Tick Bite Fever

Abstract

In testing paired serum samples from 40 consecutive cases of African tick bite fever, we detected diagnostic antibodies against spotted fever group rickettsiae in 45% of the patients by immunofluorescence assay (IFA) and in 100% of the patients by Western blotting (WB) (P < 0.01). A specific diagnosis of Rickettsia africae infection could be established in 15% of the patients by IFA and in 73% of the patients by a combination of WB and cross-adsorption assays (P < 0.01).African tick bite fever (ATBF) is a flu-like illness frequently accompanied by inoculation eschars, headache, and neck myalgia (7). The disease is caused by Rickettsia africae, a recently identified spotted fever group (SFG) rickettsia, and is transmitted by cattle ticks in large parts of rural sub-Saharan Africa (9). ATBF typically occurs in clusters and has recently emerged as a common cause of acute febrile illness in international visitors to the region (2,4,8,13,14). Serology is the most widely used microbiological method for diagnosing rickettsial infections. Several tests are available, but with the exception of immunofluorescence assay (IFA), Western blotting (WB), and cross-adsorption assay, most are not recommended (10, 11). IFA, which can detect immunoglobulin G (IgG) and IgM antibodies, has a sensitivity rate ranging from 84 to 100% (1,6,13,15). Poor species specificity is a major drawback, but this may be circumvented by using a multiple-antigen IFA where reactions to several species can be compared directly (4, 13). WB detects both early occurring antibodies against nonspecific lipopolysaccharide (LPS) antigens and late occurring antibodies against specific protein antigens (SPAs) located in the rickettsial outer membrane (15). In cases where WB is unable to identify the causative species, a cross-adsorption assay followed by a second WB on the resulting supernatant may be conclusive (11).(Part of this paper was presented at the 14th European Congress of Clinical Microbiology and Infectious Diseases, Prague, Czech Republic, 1 to 4 May 2004.) Eighty serum samples collected between days 1 and 45 after the onset of symptoms from 40 consecutive patients with ATBF were obtained from a prospective cohort study of tropical fevers in Norwegian safari travelers to rural sub-Saharan Africa in 1999 to 2001 (8). All cases met the epidemiological and clinical criteria for ATBF, including myalgia and fever commencing no later than 10 days after a safari, and 26 (65%) cases occurred in clusters. Twenty-nine (73%) patients were males, and the mean age was 38.3 years (range, 14 to 57 years). Nineteen (48%) patients were treated with doxycycline, one (3%) had a complicated course (reactive arthritis), and one (3%) was hospitalized.A multiple-antigen IFA was performed as previously reported (4), using seven SFG rickettsial antigens: R. africae strain ESF-5, R. conorii strain seven (Malish) ATCC VR-613T, Rickettsia sibirica mongolotimonae strain HA-91T, Rickettsia aeschlimannii strain MC16T, Rickettsia massiliae strain Mtu1T, Rickettsia akari strain MK...

A Focus of Dogs and Rickettsia massiliae–Infected Rhipicephalus sanguineus in California

“…Protein concentration was measured by using the bicinchoninic acid protein assay (Pierce, Rockford, IL) and normalized to a concentration of 1 mg/mL of the estimated rickettsial protein content. Serum absorptions were performed overnight at room temperature 31 by using the solubilized nuclear fractions. Unabsorbed and absorbed serum specimens were incubated for 1.5 hours with membranes at final dilutions of 1:200-1:10,000 in 2% nonfat dry milk in TTBS buffer.…”

Human Infections by Multiple Spotted Fever Group Rickettsiae in Tennessee

“…Cross-absorption has been previously used to determine the causative agent of various rickettsial diseases. [25][26][27] However, the high number of indeterminate samples even after crossabsorption was unexpected. Of 91 samples that were crossabsorbed, 31 had less than 4-fold difference in titers even after cross-absorbing with all four species and in 20 samples, all of the titers disappeared after cross-absorption.…”

Seroepidemiologic Study of Human Infections with Spotted Fever Group Rickettsiae in North Carolina

“…Membranes were washed twice in TBS for 10 min, and bands were visualized using the BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium) phosphatase substrate (KPL) and washed in distilled water to stop the reaction. Membranes were examined for the presence of the typical rickettsial lipopolysaccharide (LPS) antigen washboard pattern at 20 to 60 kDa and specific protein antigens (SPAs) in the 110-to 140-kDa region (20,21).…”