An optimized 16S–23S rRNA intergenic spacer region PCR for the detection and identification of <i>Bartonella</i> spp.

he emerging genus Bartonella comprises 60 species with unknown medical relevance (National Center for Biotechnology Information, Taxonomy Browser, 5 December 2025 [1]).Some species are human pathogenic (e.g., B. henselae: cat scratch disease, bacillary angiomatosis; B. quintana: trench fever, endocarditis; and B. bacillifor mis: Carrion's disease); other species (e.g., B. alsatica: endocarditis and B. grahamii: lymphadenopathy) are only rarely described to cause human infections (2).Accu rate diagnosis of Bartonella infections remains challenging due to unspecific clinical manifestations, difficulties in cultivation, genetic diversity within the genus, and frequent occurrence of low-level bacteremia in infected patients (3).Therefore, because of the fact that serological diagnostics have been developed only for B. henselae, B. quintana (4), and recently for B. bacilliformis (5), PCR-based assays have become indispensable for pathogen detection and differentiation.Pioneer work of Maggi and Breitschwerdt established the 16S-23S rRNA intergenic spacer (ITS) region as a valuable diagnostic target widely used in molecular diagnostics of Bartonella infections.This region is highly variable in sequence and length between Bartonella species and is embedded between conserved flanking regions, allowing the design of universal primers and enabling a reliable discrimination among closely related Bartonella species (6).Despite this great contribution to diagnostics, our results revealed that this PCR protocol with the described ITS primers fails to amplify particular species that were later fully sequenced or discovered because of numerous mismatches to the ITS target sequence (Fig. 1A).This limitation might lead to false-negative results in both clinical and epidemiological studies, ultimately impacting patient management or public health surveillance.Here, we aimed to optimize an ITS-PCR (targeting a sequence of the 16S-23S rRNA ITS) for improved diagnosis of clinically relevant Bartonella species.Genomic sequences from 16 Bartonella species (Table 1) were aligned using MAFFT v.7.490 (Geneious, Dotmatics, Auckland, New Zealand).Only 16S-23S rRNA ITS sites that were 100% conserved across all aligned sequences were considered for primer construction, covering the major pathogenic and novel emerging species (BsppITS-386s: 5-GGTTAGAGCGCGCGCTTGATAAGC-3 [3 end of the 16S rRNA gene] and BsppITS-824as: 5-CCTGCTTGCAAAGCAGGTGCTC-3 [5 end of the 23S rRNA gene]) generating PCR amplicons between 207 and 438 nucleotides.DNA was extracted from Bartonella cultures and 12 bacterial control strains (Table S1).PCR was performed under certified conditions (DIN EN ISO 15189:2024, certificate number D-ML-13102-01-00): initial denaturation (95C/3 min), followed by 35 cycles (denaturation 94C/20 s; annealing: 60C/30 s, elongation: 72C/40 s, final extension 72C/ 1 min).Products were analyzed by agarose gel electrophoresis, demonstrating a robust amplification across all tested strains, including B. bacilliformis and B. ancashensis (Fig. 1B).