Specimen Collection, Transport, and Processing

Clinical specimens submitted to the laboratory for direct examination and culture include blood, which has been collected in a lysis-centrifugation blood culture tube (Isolator; Wampole Laboratories, Cranbury, New Jersey), as well as aspirates and tissue specimens (e.g., lymph node, spleen, or cutaneous biopsies). There are no special requirements for specimen collection, transport, or processing that enhances organism recovery.

Direct Detection Methods

Detection of Bartonella spp. during the histopathologic examination of tissue biopsies is enhanced with staining using the Warthin-Starry silver stain orimmunofluorescence and immunohistochemical techniques. Because of the fastidious nature of the organisms and slow growth, molecular methods to identify Bartonella spp. directly in clinical specimens allows earlier detection. Polymerase chain reaction (PCR) targeting the 16S-23S rRNA gene intergenic transcribed spacer region has been proposed as a reliable method for the detection of Bartonella DNA in clinical samples. However, a recent study revealed some potential limitations based on insufficient primer specificity. As the number of species included in the genus expand, PCR and restriction fragment length poly morphism (RFLP) as well as sequencing may require targeting several genes and subsequent sequencing for accurate species identification.

Cultivation

The optimum conditions required for recovery of bartonellae from clinical specimens has yet to be fully defined. Currently, two methods are recommended including direct inoculation onto fresh chocolate agar plates (less than 2 weeks old) and co-cultivation in cell culture. Fresh agar helps supply moisture necessary for growth. Lysed, centrifuged sediment of blood collected in an isolator tube or minced tissue is directly inoculated onto fresh chocolate agar plates and incubated at 35° C in a very humid atmosphere containing 5% to 10% carbon dioxide (CO2), examined daily for 3 days, and examined again after 2 weeks of incubation. One study indicated that collection of blood in EDTA and subsequent freezing may improve the sensitivity of recovering B. henselae. Biopsy material is co-cultivated with an endothelial cell culture system; co-cultures are incubated at 35° C in 5% to 10% CO2 for 15 to 20 days. Blood-enriched agar, such as Columbia or heart infusion agar base with 5% sheep blood, has been used, but horse or rabbit blood has been reported to be a more effective supplement for recovery of organisms. Lymph node tissue, aspirates, or swabs can be inoculated onto laked horse blood agar slopes supplemented with hemin; plates are sealed and incubated in 5% CO2 up to 6 weeks at 37° C with 85% humidity.

Approach to Identification

 Bartonella spp. should be suspected when colonies of small, gram-negative bacilli are recovered after pro longed incubation (Figure 1). Organisms are all oxidase, urease, nitrate reductase, and catalase negative.

Fig1. A, Colonies of Bartonella henselae on blood agar. B, Gram stain of a colony of B. henselae from blood agar. 

Various methods may be used for confirmation and identification of Bartonella spp. Species identification is possible by adding 100 μg/mL of hemin to the test medium, as well as biochemical profiling using the MicroScan rapid or Rapid ANAII system (Innovative Diagnostic Systems, Norcross, Georgia) anaerobe panels, polyvalent antisera, or a variety of molecular methods.

Serodiagnosis

 Several serologic methods for detecting antibodies to Bartonella spp. have been developed. An indirect fluorescent antibody has been developed using antigen pre pared from Bartonella spp. co-cultivated with Vero cells and enzyme-linked immunoassays. However, the sensitivity and specificity of these assays have been questioned. Cross-reactivity between Bartonella, Chlamydia spp. and Coxiella burnettii has been reported. Serology testing is not recommended in HIV-positive or immunocompromised patients because of a decreased antibody response to infection.

A 5-year study by LaScola and colleagues of various samples obtained for culture for Bartonella species demonstrated that successful recovery or detection of B. henselae or B. quintana was dependent on several factors. These factors include the clinical form of the disease (i.e., endocarditis, bacteremia, bacillary angiomatosis, or CSD), previous antibiotic therapy, the type of clinical specimen (e.g., blood, heart valve, skin, or lymph node), and the type of laboratory diagnostic method employed (serology, PCR, shell vial cultures with human endothelial cell monolayers, direct plating of blood onto agar or broth blood cultures). In other words, the organisms are not unlike other microorganisms cultured and identified in the microbiology laboratory. The knowledge required for sufficient recovery and appropriate methods is yet to be established.

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مواضيع ذات صلة

Laboratory Diagnosis of Brucella

Epidemiology and Pathogenesis of Helicobacter

Clostridium botulinum

Bacillus Cereus

Bacillus anthracis

Epidemiology and Pathogenesis of Bartonella

Laboratory Diagnosis of Haemophilus

Pathogenesis and Spectrum of Disease of Haemophilus

Epidemiology of Haemophilus

COXIELLA

Ehrlichia and Anaplasma

Rickettsia