Improvement of Bartonella henselae DNA Detection in Cat Blood Samples by Combining Molecular and Culture Methods

Abstract

spp. are bacteria of worldwide distribution that cause asymptomatic to fatal infections in animals and humans. The most common zoonotic species is , for which cats are the major natural reservoir host. To better understand sp. diagnostic limitations, we determined the frequency of bloodstream infection in 112 cats by comparing and combining the results of multiple conventional and nested PCRs from blood and liquid culture samples. Using liquid culture conventional PCR, sp. DNA was amplified from 27.7% of samples (31/112) compared to 90.2% of samples (101/112) by combining nested PCR from blood and liquid culture, indicating that PCR testing of more than one type of sample provides better sensitivity than a standalone PCR and that bloodstream infection is very frequent among cats in southeastern Brazil. This study reinforces the need for multistep testing for sp. infection to prevent false-negative diagnostic results, even in reservoir hosts such as cats that typically maintain higher bacteremia levels.

Aortic valve endocarditis due to Bartonella clarridgeiae in a dog in Brazil

“…While in the present study we used chocolate agar under 37°C and 5% CO 2 atmosphere for culturing, Chomel et al (2001Chomel et al ( , 2003 successfully isolated B. clarridgeiae and B. washoensis from dogs with aortic and mitral valve endocarditis, respectively, by culturing dogs' blood samples on heart infusion agar containing 5% rabbit blood and incubated at 5% CO 2 in a 35°C atmosphere for up to 4 weeks. Recently, B. henselae was isolated from cats blood samples in southeastern Brazil using a platform combining BAPGM liquid culture for 10 days followed by inoculation on agar containing 30% sheep blood, under incubation at 5% CO 2 and 37°C for 45 days (DRUMMOND et al, 2018). Nonetheless, the absence of colonies in solid medium has already been reported for blood samples from dogs showing positive results in qPCR assays based on BAPGM liquid cultures (DUNCAN et al, 2007).…”

“…The tissue and biological samples were subjected to pre-enrichment liquid culturing using an insect-based medium, as previously described, but with modifications (MAGGI et al, 2005;DUNCAN et al, 2007;DRUMMOND et al, 2018). The fragments were macerated in 2 mL of Bartonella-alpha proteobacteria growth medium (BAPGM) and were kept under agitation at 37 °C with 5% CO 2 for 10 days.…”

False Negative Results in Bartonellosis Diagnosis

“…These divergent serological, molecular, and microbiological findings from a long-term antibiotic treatment failure case exemplify the complexity of bartonellosis diagnosis. The low and cyclic Gram-negative bacteremia and the stochastic and dilution phenomena are already discussed in the literature (Wilson 1997, Drummond et al 2018. These factors can possibly explain false negative results related to this fatal bartonellosis case.…”

“…growth BAPGM (Galaxy Diagnostics) that was incubated at 37°C in 5% CO 2 in a water-saturated atmosphere and maintained with a constant shaking motion for 14 days. After this incubation, a 1 mL aliquot was seeded over solid medium slant tubes incubated at 37°C in 5% CO 2 in a water-saturated atmosphere for up to 45 days (Drummond et al 2018). To extract the DNA from all samples, we used QIAamp DNA minikit (Qiagen, Inc.), according to the manufacturer's instructions.…”

“…DNA from the patient's blood or liquid blood culture specimens (Diniz et al 2007). Bartonella henselae DNA was successfully amplified from the liquid blood culture using a specie-specific nested PCR targeting the ftsZ gene (Drummond et al 2018). Subsequently, B. henselae colonies were isolated by subculture from the liquid medium and, due to the large quantity of bacterial DNA, both ITS and ftsZ PCRs were positive ( Table 1).…”