Case 5 -

Legionella Pneumonia Ricardo V. Lloyd and Arie Perry

Clinical History A 60-year-old man presented with high fever and diarrhea. Chest x-ray showed areas of consolidation. He had an open lung biopsy. Case 5 - Figure 1 Case 5 - Figure 2 Case 5 - Figure 3 Microscopic Description The lung had a mixed inflammatory infiltrate in the alveolar spaces consisting of neutrophils and mononuclear phagocytes. A Warthin-Starry stain was suspicious for Legionella. In situ hybridization with an oligonucleotide probe cocktail was positive for Legionella. Discussion Legionnaires disease was first described in 1976 when a lethal pneumonia struck a group of conventioners at the American Legion in Philadelphia. The causative agent was subsequently characterized as a gram-negative bacterial pathogen, Legionella pneumophilia. Although it was probably associated with an outbreak of fever in Pontiac, Michigan, in 1968, the causative agent was not detected because of the special culture requirements on special media and the need for silver or other special stains. The bacterium is resistant to chlorine, and epidemics have been associated with air conditioners in building. The infectivity can range from a self-limited fever in healthy individuals to severe pneumonia in the elderly, compromised individuals such as smokers, patients with chronic lung disease, or who are immunosuppressed. The bacterium is a facultative intracellular parasite of macrophages and of aquatic amebae (Hartmanella and Tetrahymena). After it enters the macrophages via the Fc receptors or complement receptors on macrophages, it blocks phagocyte function with the lysosomes, replicates and eventually lyses the macrophages. A macrophage infectivity potentiator a 24 Kda surface protein on the bacteria is needed for growth in the macrophages and amebae and for infectivity. Erythromycin is the antibiotic of choice for treatment. The genus Legionella causes human disease, most commonly opportunistic pneumonia in immunocompromised patients. The vast majority of such cases (approximately 85%) are due to L. pneumophila, with a substantial minority due to other species, most commonly L. micdadei, L. bozemanii, L. dumoffii, and L. longbeachae. A number of methods have been used to identify these organisms in paraffin-embedded tissue sections, including various histochemical and immunohistochemical techniques. The environmental pathogen Legionella pneumophila, which is the etiologic agent of Legionnaires' disease, usually survives in nature as an intracellular parasite of amoebae and ciliates. Intracellular growth of L. pneumophila in trophozoites of a variety of amoebae has been demonstrated under laboratory conditions. Development of legionellosis has been attributed to the inhalation of viable organisms in fine aerosols into the lung. The organism subsequently invades the alveolar macrophages and other phagocytic cells. Due to slow growth and lack of suitable phenotypic tests, identification of Legionella spp. remains difficult. Direct antibody techniques and DNA hybridization assays may be used to confirm Legionella spp. or colonies isolated on selective medium but are hampered by nonspecific binding of antibodies and probes to other bacteria or by phenotypic variation. Direct fluorescent antibody techniques may also be applied to tissue, however, the sensitivity of these assays is poor. L. pneumophila infection may also be diagnosed by noninvasive means including detection of L. pneumophila antigen in the urine and Legionella antibodies in the serum. Detection of a four-fold rise in serum antibodies is frequently required to make the diagnosis; but this assessment may require 4-6 weeks from the time of acute infection. Nonradioactive in situ hybridization of whole cells with rRNA-targeted oligonucleotide probes has become a highly valuable tool for the specific detection of individual microbial cells without cultivation. Many of Legionella species have been associated with illness, but Legionella pneumophila accounts for the majority of reported pneumonic illness caused by Legionella spp., which is a facultative intracellular parasite within human mononuclear phagocytes. Because Legionella pneumonia cannot be reliably distinguished from other pneumonias clinically, diagnosis depends upon the laboratory. Culture is the current standard for diagnosis, with a sensitivity of 50-80% and a specificity of 100%. Legionella does not grow on routine media, and at least 2-3 days are required for cultures to yield positive results. Direct fluorescent antibody (DFA) staining is rapid and highly specific, but its sensitivity is only 25-50%. The nonradioactive in situ hybridization technique is both sensitive and highly specific for detection of Legionella in formalin-fixed paraffin embedded lung sections. We used two oligonucleotide probes directed against the 16s rRNA sequence of L. pneumophila for in situ hybridization. One probe was from the literature (4). The other probe was designed in house based on the analysis of sequence matches and mismatches (Gen Bank). The specificity of the probe was checked against the sequence of other bacteria and fungi, using Genetics Computer software (Madison, WI). Probes were labeled at the 31 end with digoxigenin-11 dUTP and used for in situ hybridization. References Stout JE, Yu VL. Legionellosis. [Review] New England Journal of Medicine. 337(10):682-7, 1997 Levy I, Rubin LG. Legionella pneumonia in neonates: a literature review. Journal of Perinatology. 18(4):287-90, 1998 Desai R, Welsh C, Summy M, Farone M, Newsome AL. The potential of in situ hybridization and an immunogold assay to identify Legionella associations with other microorganisms. Journal of Microbiological Methods. 37(2):155-64, 1999 Grimm D, Merkert H, Ludwig W, Schleifer KH, Hacker J, Brand BC. Specific detection of Legionella pneumophilia: construction of a new 16S rRNA-targeted oligonucleotide probe. Applied & Environmental Microbiology. 64(7):2686-90, 1998 Manz W, Amann R, Szewzyk R, Szewzyk U, Stenstrom TA, Hutzler P, Schleifer KH. In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Microbiology. 141 (Pt1):29-39, 1995 Fain JS, Bryan RN, Cheng L, Lewin KJ, Porter DD, Grody WW. Rapid diagnosis of Legionella infection by a nonisotopic in situ hybridization method. American Journal of Clinical Pathology. 95(5):719-24, 1991 Hayden RT, Qian X, Uhl JR, Hopkins MK, Aubry MC, Limper AH, Lloyd RV, Cockerill FR. Direct detection of Legionella species from bronchoalveolar lavage and open lung biopsy specimens: A comparison of rapid-cycle real-time PCR, in situ hybridization, direct fluorescence antigen detection and culture. Journal of Clinical Microbiology. 39:2618-2626, 2001 Table 1. Comparison of Results for Open-Lung Biopsies Compared to Culture-Positive Cases in the detection of Legionella [7] Method Positive Cases (%) DFA 7/16 (44%) Warthin-Starry 10/16 (62.5%) ISH 13/14 (92.8%) PCR 11/16 (68.8%) DFA — Direct fluorescent antibody ISH — In situ hybridization PCR — Polymerase chain reactions