Use of Shell-Vial Cell Culture Assay for Isolation of Bacteria from Clinical Specimens: 13 Years of Experience

Abstract

The shell-vial culture assay is performed routinely in our laboratory. Recently we revisited our experience of using the shell-vial culture assay for the isolation of microorganisms from various clinical samples. Over a 13-year period, we have isolated 580 bacterial strains (5%) from 11,083 clinical samples tested. Over the same period, 285 isolates of rickettsiae, bartonellae, or Coxiella burnetii were cultured from a total of 7,102 samples tested. These isolates include 55 Rickettsia sp. isolates, 95 Coxiella burnetii isolates, and 135 Bartonella sp. isolates. Based on our experience with the growth of fastidious microorganisms, we have used a centrifugation shell-vial technique called JNSP, for "je ne sais pas" ("I don't know [what I am growing]") for the isolation of other microorganisms. A total of 173 isolates were cultured from the 3,861 clinical samples tested using the JNSP method. Of these, 40 isolates had not been grown before on usual axenic medium. These include 2 Staphylococcus aureus isolates, 7 isolates of Streptococcus sp. and related genera, 6 Mycobacterium sp. isolates, 1 Nocardia asteroides isolate, 1 Actinomyces sp. isolate, 1 Brucella melitensis isolate, 2 Francisella tularensis isolates, 1 Mycoplasma pneumoniae isolate, and 1 Legionella pneumophila isolate. Using this protocol, we have also cultured intracellular bacteria such as Chlamydia trachomatis and we have performed the first culture and establishment of Trophyrema whipplei. Applied in our laboratory, the shell-vial culture generally exhibits a low rate of success. However, in some cases, this technique allowed microbial diagnosis when classical agar procedure and PCR were negative.The spectrum of infectious diseases is wide and changing. The reliable diagnosis of infectious disease remains a difficult problem even for specialized laboratories. Isolation of new microorganisms will allow the description of clinical features of new diseases and the characterization of new pathogens, enabling genetic descriptions, physiological analyses, improvement of diagnostic tools, and antibiotic susceptibility testing for bacteria. Therefore, the isolation of infecting bacteria is not only a mean of diagnosis but also a basis for the evaluation of much needed improved diagnostic assays and a route to enhance understanding of the diversity and the epidemiology of infections (21). The successful isolation of fastidious microorganisms is often based on cell culture (21). The success of this technique is based on two critical points. First, the ratio of microorganisms to cells should be as high as possible (21). Second, centrifugation enhances the adhesion of intracellular microorganisms to the cells (52). In 1976, a centrifugation-cell microculture system, called the shell-vial assay, was first used for the diagnosis of viral disease due to cytomegalovirus and allowed early antigen detection (22). This diagnostic tool is becoming one of the most commonly used techniques in clinical virology laboratories since it reduces turnaround time (19).For a lo...

Combining Culture Techniques for Bartonella: the Best of Both Worlds

“…The majority of studies that coculture Bartonella species with mammalian cells use 37°C; in contrast, studies that use axenic insect cell culturebased medium and agar use a culture temperature of 35°C (15,17,28). Although a 2°C difference may seem minor or trivial, it was interesting to see that there were statistically significant differences in peak growth between the two temperatures for three of the four strains tested (B. elizabethae was the exception, showing no significant difference in peak growth between the two temperatures).…”

Rickettsia sibirica mongolitimonae infection, Sri Lanka

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