Succinyl-CoA Synthetase: New Antigen Candidate of Bartonella bacilliformis

Abstract

Background: Bartonella bacilliformis is the causative agent of Carrion's disease, a neglected illness with mortality rates of 40-85% in the absence of treatment. The lack of a diagnostic technique to overcome misdiagnosis and treat asymptomatic carriers is of note. This study aimed to identify new B. bacilliformis antigenic candidates that could lead to a new diagnostic tool able to be implemented in endemic rural areas.

Methodology/principal findings: Blood (n = 198) and serum (n = 177) samples were collected in northern Peru. Clinical data were recorded. Specific 16S rRNA amplification by RT-PCR, IFA and ELISA for IgM/IgG with whole cells as antigens was done. Western blot analysis and N-terminal amino acid sequencing detected seroreactive proteins. ELISAs for IgM/IgG for the antigenic candidates were performed. Of the population 33.3% reported at least one symptom compatible with Carrion's disease; 25.4% (IFA), 27.1% (ELISA-IgG), 33.9% (ELISA-IgM) and 38.9% (RT-PCR) of samples were positive. Four proteins were considered potential antigenic candidates, including two new antigenic candidates, succinyl-CoA synthetase subunit α (SCS-α) and succinyl-CoA synthetase subunit β (SCS-β). On Western blot both Pap31 and SCS-α interacted with IgM, while GroEL and SCS-β interacted with IgG. The presence of specific antibodies against the antigenic candidates varied from 34.5% (IgG against SCS-α) to 97.2% (IgM against Pap31).

Conclusions/significance: RT-PCR and the high levels of positivity for specific ELISAs demonstrate high levels of B. bacilliformis exposure and asymptomatic carriers among inhabitants. The new antigens identified might be used as a new rapid diagnostic tool to diagnose acute Carrion's disease and identify asymptomatic carriers.

Fig 1. Distribution of B. bacilliformis antibodies by IFA assay among the study population (n = 177).

Fig 2. Example of a positive IFA test.

A) 20X objective and 10X oculars. B) 100X objective and 10X oculars.

Fig 3. IgM and IgG antibodies in the study population.

A) Overall data. Data are presented as boxplots that illustrate the medians and the maximum and minimum. B) IgM and IgG distribution by sex; C) IgM and IgG distribution by locality. P-values obtained on comparing the 4 post-outbreak areas with the endemic area. When comparing pairs of localities we obtained significance for IgG with the following analysis: Guayaquiles and Huancabamba (P = 0.0001); Los Ranchos and Huancabamba (P = 0.0002); Mayland and Huancabamba (P = 0.0201); Tunal and Huancabamba (P<0.0001). D) IgM and IgG distribution by age groups. P-values obtained comparing the antibody levels of subjects between ≤ 25 years with ≥ 26 years. In panels B, C and D the data are presented as boxplots that illustrate the medians and the 25th and 75th quartiles, and the whiskers represent the 10% and 90% percentiles. Outliers are marked with circles. P-values were calculated using the Mann-Whitney test.

Fig 4. Antigenic candidates of B. bacilliformis selected.

A) SDS gel with sonicated whole cell B. bacilliformis. B) Example of a Western blot with a positive serum by ELISA. The left panel corresponds to IgG and the right panel to IgM.

Fig 5. IgM and IgG levels for antigenic candidate ELISAs.

A) Overall IgM and IgG data for the study population. Data are presented as boxplots that illustrate the medians and the 1 and 99% percentiles. Outliers are marked with circles. B) IgM ELISA results of post-outbreak localities for SCS-α, Pap31, SCS-β and GroEL according to IFA results. C) IgM ELISA results of post-outbreak localities for SCS-α, Pap31, SCS-β and GroEL according to RT-PCR results. D) IgM and IgG ELISA result of post-outbreak population for SCS-α, Pap31, SCS-β and GroEL taking into account the whole cell B. bacilliformis ELISA assay. E) IgM and IgG ELISA result for the endemic area (Huancabamba) for SCS-α, Pap31, SCS-β and GroEL according to the whole cell B. bacilliformis ELISA assay. For B, C, D and E data are presented as boxplots that illustrate the medians and the 25th and 75th quartiles, and the whiskers represent the 1% and 99% percentiles. Outliers are marked with circles. P-values were calculated using the Mann-Whitney test.