Dried Blood Spots for qPCR Diagnosis of Acute Bartonella bacilliformis Infection

Abstract

Abstract. Bartonella bacilliformis is the etiological agent of a life-threatening illness. Thin blood smear is the most common diagnostic method for acute infection in endemic areas of Peru but remains of limited value because of low sensitivity. The aim of this study was to adapt a B. bacilliformis-specific real-time polymerase chain reaction (PCR) assay for use with dried blood spots (DBS) as a sampling method and assess its performance and use for the diagnosis and surveillance of acute Bartonella infection. Only two of 65 children (3%) that participated in this study had positive blood smears for B. bacilliformis, whereas 16 (including these two) were positive by PCR performed on DBS samples (24.6%). The use of DBS in combination with B. bacilliformis-specific PCR could be a useful tool for public health in identifying and monitoring outbreaks of infection and designing control programs to reduce the burden of this life-threatening illness.

Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples

“…It is important to remark that in the last years 2 more sensitive PCR techniques have been described in the literature: qPCR [ 13 ] in which 24.6% of DBS samples are positive, as well as a loop-mediated isothermal amplification [ 21 ] that achieves good results on analysing Lutzomyia samples. However, qPCR requires the expertise of trained personnel and is more expensive and difficult to be implemented.…”

“…Molecular diagnosis by PCR is probably the easiest way to achieve a more accurate diagnosis in endemic areas, as the equipment required is not as sophisticated or expensive, may be installed in different Health Regional Centers which may provide diagnosis to more peripheral patients, and the personnel may be easily trained in technique management. Several PCR approaches have been described in the literature in the last years [ 12 , 13 ]. However, these studies do not generally involve a large number of samples and additionally, as occurs with the remaining diagnostic tools, they are hampered by the lack of a standard case definition.…”

“…Dried blood spot (DBS) is used for the diagnosis of several infectious diseases [ 14 , 15 ], and has been proposed for use as easy method to transfer blood samples from endemic areas to reference centers in order to carry out molecular techniques for the diagnosis of Carrion’s disease [ 13 ]. Therefore, since this illness principally affects children in rural areas, DBS may be an easy solution to both the transportation of samples and for small blood volume collection in the pediatric setting.…”

Carrion's Disease: the Sound of Silence

“…Finally, recent studies have demonstrated the utility of more sensitive molecular techniques such as qPCR. Using this technique, Smit et al showed that 24.6% of 65 children were positive whereas only 3% were blood culture positive (272), and Gomes et al detected 38% of asymptomatic carriers in a postoutbreak zone and 50% in an area of endemicity (50). In another study with animal blood samples, the sequences of a region of the ssrA gene amplified by qPCR were able to discriminate Bartonella species (256).…”

“…Based on these criteria, Carrion's disease is a potentially eradicable illness, mainly due to its restricted geographic location, the lack of nonhuman reservoirs, and other factors such as the advances toward obtaining better, inexpensive, and sensitive diagnostic tools. Some of the tools proposed are based on antigenic candidates, which may also lead to the design of preventive strategies and enhance the current good activity of antibacterial agents (50,271,272,369).…”