Traditional and molecular techniques for the study of emerging bacterial diseases: one laboratory's perspective

Abstract

Identification of emerging bacterial pathogens generally results from a chain of events involving microscopy, serology, molecular tools, and culture. Because of the spectacular molecular techniques developed in the last decades, some authors think that these techniques will shortly supplant culture. The key steps that led to the discovery of emerging bacteria have been reviewed to determine the real contribution of each technique. Historically, microscopy has played a major role. Serology provided indirect evidence for causality. Isolation and culture were crucial, as all emerging bacteria have been grown on artificial media or cell lines or at least propagated in animals. With the use of broad-range polymerase chain reaction, some bacteria have been identified or detected in new clinical syndromes. Culture has irreplaceable advantages for studying emerging bacterial diseases, as it allows antigenic studies, antibiotic susceptibility testing, experimental models, and genetic studies to be carried out, and remains the ultimate goal of pathogen identification.

Figure 1

Diagram describing the respective places of culture-, polymerase chain reaction-, serology- and histology-based approaches for the diagnosis of acute bacterial infections, according to the natural course of the disease. Isolation and culture are possible as long as live bacteria are present in tissues, i.e., from the colonization to the treatment or to the end of the clinical manifestations (or shortly earlier). Bacterial DNA can be detected during the same period and also as far as dead microorganisms remain in tissues. Specific antibodies appear during the clinical course of the disease and persist generally for months or years. Pathologic changes can be observed soon after the contamination and, in an acute infection, will decline rapidly after elimination of the bacteria.

Figure 2

Demonstration of Bartonella henselae in cardiac valve of a patient with blood culture-negative endocarditis. The bacilli appear as black granulations (Warthin Starry, original magnification X250).

Figure 3

Demonstration of Tropheryma whipplei by immuno-histochemistry in the lamina propria of the villous tips. Bacilli are revealed in foamy macrophage cytoplasm as red-brown deposits (polyclonal rabbit.

Figure 4

Canine monocytes (DH82) cultivated in vitro and heavily infected with Ehrlichia chaffeensis, as viewed by light microscopy after Giemsa staining. Typical ehrlichial inclusions (morulae) are observed within the cytoplasm of the infected cells (Giemsa, original magnification X600).