Recently, infections with emerging zoonotic bacteria of the genus Bartonella have been reported in association with a range of CNS symptoms. Currently, it remains unknown if Bartonella spp. infection is associated with symptoms of schizophrenia/schizoaffective disorder (SCZ/SAD). The objective of this study was to determine if there is an association between Bartonella species infection and SCZ/SAD. A secondary objective was to determine if SCZ/SAD symptoms were more severe among participants with documented Bartonella spp. infection. Using a case–control study design, 17 cases and 13 controls were evaluated with a series of clinical and cognitive assessments. Blood samples were collected and tested for Bartonella spp. infection using serological, microbiological, and molecular techniques. People with SCZ/SAD were more likely than healthy volunteers to have Bartonella spp. DNA in their bloodstream, with 11 of 17 cases (65%) positive by Bartonella spp. droplet digital PCR (ddPCR). In comparison, only one healthy volunteer was Bartonella spp. ddPCR positive (8%, p = 0.0024). Based on serology, Bartonella spp. exposure was common among people with SCZ/SAD (12 of 17) as well as among healthy volunteers (12 of 13), with no significant difference between the groups ( p = 0.196). Within the case group of people with SCZ/SAD, there was no significant difference in SCZ/SAD severity scores between people with and without ddPCR evidence of Bartonella spp. infection. This pilot study provides preliminary evidence in support of future investigations that should examine a potential contribution of Bartonella spp. infection to SCZ/SAD.

Study Design and Setting

This was a prospective case-control study conducted at University of North Carolina-Chapel Hill and approved by the UNC-Chapel Hill Biomedical Institutional Review Board (#19-0114). Participants were enrolled between March 1 and October 31, 2019. Cases were recruited from the local community using fliers and newspaper advertisements. Controls were healthy volunteers recruited from the surrounding local community via targeted online and email advertising. To minimize selection bias, recruitment material did not specify the purpose of the study. Participants provided written informed consent prior to enrollment and were compensated $80 for their time. This manuscript was prepared in accordance with STROBE guidelines for case-control studies; the checklist is included as supplementary material.(Vandenbroucke et al., 2007)

Participants

Inclusion criteria for cases was a diagnosis of schizophrenia or schizoaffective disorder (confirmed by the Structured Clinical Interview for DSM-V); clinical stability as demonstrated by no psychiatric hospitalizations for the past 3 months; stable dosing of antipsychotic medications (no changes in medication or dose for 1 month prior to enrollment); ability to provide written informed consent; and at least one set of blood samples collected for microbiological and molecular testing.

Controls were considered healthy based on self-reported health status, and were excluded if reporting a previous diagnosis of schizophrenia or schizoaffective disorder. Controls were included from a local volunteer population, expected to have similar Bartonella spp. exposures.

Variables, Data Sources, and Measurement

Whole blood and serum were collected from each participant. To increase the likelihood for detection of intermittent bacteremia, blood was collected twice within a one-week period.(Pultorak et al., 2013)

As described previously,(Edward B. Breitschwerdt et al., 2019; Lantos et al., 2014; Maggi et al., 2011) each participant was tested using six indirect fluorescent antibody (IFA) assays, each representing a unique Bartonella species or subspecies. Bartonella vinsonii subsp. berkhoffii (genotypes I, II, and III), B. henselae, B. koehlerae, and B. quintana IgG antibodies were determined using cell culture-grown bacteria as antigens and following standard IFA techniques. A sample was considered Bartonella spp. seroreactive at an IFA titer of ≥1:64 for any one or more antigen.

Bartonella alpha proteobacteria growth medium (BAPGM) enrichment blood culture and qPCR was performed as previously described.(Edward B. Breitschwerdt et al., 2019) Briefly, qPCR targeting the Bartonella intergenic 16S-23S rRNA (ITS) region was performed on DNA extracted from the following: each whole blood sample, and whole blood culture-enriched in BAPGM at 7, 14, and 21 days of culture. A sample was considered BAPGM/qPCR positive if any one or more of these four qPCR tests was positive on any one or more sample.

In addition to BAPGM/qPCR, all blood and BAPGM enrichment blood culture DNA extractions were tested for Bartonella spp. DNA by droplet digital PCR (ddPCR) using the QX200 Droplet Digital PCR (Bio-Rad, Hercules, CA) system. Digital PCR amplification of the Bartonella 16S-23S ITS region, and the human hydroxymethylbilane synthase (HMBS) as house-keeping human reference gene, was conducted as previously validated and described.(Maggi et al., 2020) Bio-Rad QuantaSoft Analysis Pro software was utilized to analyze the fluorescent drop distribution and to define the positive DNA detection thresholds for each channel (FAM channel 1 for Bartonella, and HEX channel 2 for house-keeping gene amplification). A sample was considered BAPGM/ddPCR positive if any one or more of these four ddPCR tests was positive on any one or more whole blood sample.

For the primary aim, study participants were considered to have Bartonella spp. exposure if they were Bartonella spp. seroreactive at an IFA titer of ≥1:64 for any one or more antigen. Participants were considered to have Bartonella spp. infection if they were positive on any one or more BAPGM/qPCR or BAPGM/ddPCR assay. All Bartonella testing was performed by researchers blinded to participant identity and group assignment.

SCZ/SAD symptom severity was measured with a series of clinical and cognitive assessments including the Positive and Negative Syndrome Scale (PANSS),(Kay et al., 1988) Brief Assessment of Cognition in Schizophrenia (BACS),(Keefe et al., 2004) and Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES).(Ritsner et al., 2005) PANSS results were analyzed using the positive, negative, and general symptom subscores, and a total PANSS score. BACS results for each primary measure were first converted to Z scores, then the Z scores from each primary measure were averaged to create a BACS composite score. For the Q-LES, the results were normalized to the possible total score and reported as a percentage.

Data on possible confounders or effect modifiers was collected with a questionnaire (health history questionnaire, Supplementary material 2) assessing employment, geographic location, health history, and animal and insect vector contact.

See metadata tab and supplementary file #2 (Bartonella health history questionnaire).