Serological testing for Bartonella henselae infections in The Netherlands: clinical evaluation of immunofluorescence assay and ELISA

Abstract

Cat-scratch disease (CSD), caused by Bartonella henselae infection, can mimic malignancy and can manifest atypically. Reliable serological testing is therefore of great clinical importance. The diagnostic performance of immunofluorescence assay (IFA) and ELISA was evaluated in a group of Dutch patients with proven CSD (clinical diagnosis confirmed by PCR). Sera of 51 CSD patients and 56 controls (patients with similar symptoms, but who were B. henselae PCR-negative and had an alternative confirmed diagnosis) were tested for anti-B. henselae IgM and IgG by IFA and ELISA. A commercially available IFA test for IgM had a sensitivity of 6%. In-house assays for IgM showed specificities of 93% (IFA) and 91% (ELISA), but with low sensitivities (53% and 65%, respectively). With a specificity of 82% (IFA) and 91% (ELISA), in-house IgG testing showed a significantly higher sensitivity in IFA (67%) than in ELISA (28%, p <0.01). Sensitivity was higher for genotype I (38-75%) than for genotype II (7-67%) infections, but this was only statistically significant for IgG ELISA (p <0.05). In conclusion, detection of IgM against B. henselae by in-house ELISA and IFA was highly specific for the diagnosis of CSD. The high seroprevalence in healthy individuals limits the clinical value of IgG detection for diagnosing CSD. Given the low sensitivity of the serological assays, negative serology does not rule out CSD and warrants further investigation, including PCR. Adding locally isolated (e.g., genotype II) B. henselae strains to future tests might improve the sensitivity.

Low sensitivity of Bartonella henselae PCR in serum samples of patients with cat-scratch disease lymphadenitis

“…The collection of serum occurred 0-17 days (median 5 days) before tissue sampling. A 'control' group consisted of 50 patients (mean age 35 years) who were clinically suspect for CSD but tested negative for B. henselae IgM antibodies using an in-house-prepared immunofluorescence assay as described previously (Bergmans et al, 1997;Vermeulen et al, 2007). A real-time PCR assay targeted at the heat-shock protein groEL was used to detect B. henselae DNA in all serum samples (Diederen et al, 2007a).…”

Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N -Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease

“…The presence of IgM in patients is clear evidence of recent contact with B. henselae; however, the sensitivity of assays in the detection of the IgM is low (5)(6)(7)(8)(9)(10)12). IgM remains in the serum for approximately 3 months (7,13). The development of a more sensitive as well as more specific IgM assay for B. henselae is crucial for improving the clinical diagnosis of CSD.…”

“…We proved that the ELISAs using antigen II, IV, and V could clearly distinguish between the two sets of sera, whereas the ELISAs with antigen I and III showed overlapping results for the two sets, indicating that these two antigens were not suitable for use in the IgM-ELISA. There have been many previous reports on the development of IgM-ELISAs using whole-cell protein (antigen I) (6)(7)(8)(9)(10), which have generally shown poor sensitivity. We confirmed that whole-cell protein was inappropriate for use in the IgM-ELISA.…”

“…Although the detection of IgM antibodies is crucial for the diagnosis of active B. henselae infection, the sensitivity of IFA for anti-B. henselae IgM (IgM-IFA) is reported to be especially low (5)(6)(7)(8).…”

“…henselae IgM by various enzymelinked immunosorbent assays (IgM-ELISA) has been proposed as an alternative to IgM-IFA. Several IgM-ELISAs use sonically disrupted B. henselae antigens (whole-cell proteins) (6,7,9,10) or the putative outer membrane proteins which correspond to antigens fractionated from surfactant (i.e., N-lauroyl-sarcosine)-disrupted B. henselae (sarcosine-insoluble proteins) (11)(12)(13). However, the constituents of the crude preparations vary widely depending on the methods used for the isolation of the antigens.…”