The use of an immunoenzyme method for the rapid diagnosis of legionellosis

An enzyme immunoassay (EIA) system for the detection of L. pneumophila antigen in clinical material (sputum, urine, bronchial washings) has been developed. The use of EIA permits the detection of L. pneumophila antigen in the urine of 75-80% of patients during the first week of the disease. The specificity and sensitivity of EIA makes it possible to recommend this method for the rapid diagnosis of L. pneumophila infection.     

Monoclonal antibodies to Legionella pneumophila cytolysin

The fusion of spleen cells, taken from BALB/c mice immunized with the purified preparation of L. pneumophila cytolysin, with cells Sp2/0 and NP has been carried out. As a result, hybridoma cells producing IgG1, IgG3 and IgM antibodies to this protein have been obtained. All monoclonal antibodies (McAb) thus obtained react with L. pneumophila strain lysates in the precipitation test, while IgG3 and IgM antibodies react with erythrocyte diagnostic agents prepared from the lysate of L. pneumophila cells in the hemagglutination test. In the Western blot assay, McAb react with the 37 KD protein (cytolysin) and a number of other proteins from L. pneumophila cultures and L. pneumophila cell lysate, but do not react with the species-specific protein with a molecular weight of 29 KD, contained in the outer membrane of L. pneumophila, as well as with other species: L. bozemanii, L. dumoffii, L. longbeachae, L. micdadei. The possibility of using these McAb conjugated with FITC and peroxidase for the rapid diagnosis of Legionella infection is shown.     

Production of monoclonal antibodies against Legionella pneumophila serogroup 1

Four monoclonal antibodies to Legionella pneumophila Philadelphia 1 were produced by the fusion of immunized BALB/c lymphocytes to a murine myeloma cell line. Two (Lp1-1 and Lp1-3) of the four monoclonal antibodies reacted with 14 L. pneumophila serogroup 1 strains, and the other (Lp1-2 and Lp1-4) reacted with only three out of 20 strains tested. These four monoclonal antibodies did not bind to any strains of L. pneumophila serogroup 2-7 and other Legionella species. In addition, it has been shown that these monoclonal antibodies may be useful not only for subserotyping of L. pneumophila but also for retrospective diagnosis using immunopathological methods.     

The biological properties of monoclonal antibodies to the cytolysin of Legionella pneumophila

The study of the biological properties of monoclonal antibodies (McAb) to L. pneumophila cytolysin has been carried out. These McAb have been shown to possess no capacity for the in vitro neutralization of cytolysin and the protection of guinea pigs from aerosol infection with L. pneumophila. Still the protective effect of the McAb under study has been observed in experiments with the intraperitoneal infection of guinea pigs, which is indicative of the possibility, in principle, of involving humoral immunity into the protection of the body from Legionella infection.     

Comparative evaluation of two commercially available antigen enzyme immunoassays (EIA) for the detection of Legionella pneumophila urinary antigen in frozen non-concentrated urine samples

We evaluated two commercial enzyme immunoassay kits, Binax EIA (for detection of soluble antigen of Legionella pneumophila serogroup 1) and Biotest EIA (for detection of antigens of Legionella pneumophila serogroups and other Legionella spp.) in order to introduce this test routinely for the diagnosis of Legionnaires' disease (LD) in our Laboratory. Frozen non-concentrated urine samples belonging to 45 patients with and without LD were tested. The sensitivity of Binax EIA and Biotest EIA was 47.4% and 42.1% respectively, the specificity was 95% by both tests. Biotest did not detect antigen from a patient with culture-proven infection of L. pneumophila serogroup 6. The detection of urinary antigen by both EIA tests is a useful tool for rapid diagnosis of LD, especially when samples are unavailable for culture; the sensitivity may be increased if the assay is performed on unfrozen and concentrated samples.     

An outbreak of Legionella infection in Armavir

In March-April 1987 an outbreak of Legionella infection was registered among 236 workers at a rubber factory in Armavir. An ARD-like syndrome and acute alveolitis were the main variants of the clinical course of the infection. Legionella infection was diagnosed on the basis of a fourfold and greater increase in the titers of antibodies to L. pneumophila, serogroup 1, as determined in the indirect immunofluorescence test and enzyme immunoassay (EIA). In urine samples obtained from patients Legionella antigen was determined with the use of EIA techniques. The culture of L. pneumophila, serogroup 1, was isolated from the recycling-type water supply system of the factory. The outbreak was stopped by a complex of sanitary, hygienic and epidemic control measures.     

Cross-reactivity and sensitivity of two Legionella urinary antigen kits, Biotest EIA and Binax NOW, to extracted antigens from various serogroups of L. pneumophila and other Legionella species

Legionella antigen detection kits for diagnosing legionellosis from urine have become widely used, but basic information about reactivity of the kits to non-serogroup (SG) 1 L. pneumophila and other Legionella species remains incomplete. We evaluated Biotest EIA and the most recently developed Binax NOW by using in-vitro extracted antigens of 22 L. pneumophila SG 1 to 15 strains and of 27 other Legionella species. Both kits showed excellent sensitivity to L pneumophila SG 1 antigens, but reacted to different sets of non-SG I L. pneumophila with different sensitivity. No cross-reactivity was observed to Legionella species other than L. pneumophila.     

A rapid coagglutination test for the detection and serogrouping of Legionellae

The applicability of coagglutination for the rapid detection and serogrouping of Legionellae has been investigated. The coagglutination reaction is carried out with the aid of self-made preparations of protein A containing staphylococci, sensitized with specific antibodies against the antigens of L. pneumophila (serogroups I to 6), L. bozemanii and L. micdadei. Preliminary heating of Legionella suspensions at 100% C for 15 min was used to prevent cross coagglutination reactions and ensure greater safety of laboratory personnel during the performance of the test. The results obtained demonstrate a high specificity of coagglutination. With the aid of the coagglutination reactions it has been shown that L. pneumophila strains isolated in Bulgaria belong to serogroup I. The coagglutination method is characterized by its rapidity, simplicity and feasibility. It is a useful and convenient means for the rapid detection and serogrouping of Legionellae.     

Immune structure of the population in some regions of the USSR with respect to Legionella pneumophila

For the first time in the USSR the structure of the immunity of different population groups to L. pneumophila was studied. Antibodies to L. pneumophila were detected in the passive hemagglutination test with the use of erythrocyte diagnosticum prepared at the Gamaleya Research Institute of Epidemiology and Microbiology (Moscow). The survey covered the total number of 2,902 persons in differ nt regions of the USSR, among them 2,430 adults and 472 children. Among the healthy adult population antibodies to L. pneumophila were detected, on the average, in 5.9% of persons. The highest number of persons with antibodies in diagnostic titers was detected among the population of the Baltic Republics (15.9%) and Kazakhstan (10.2%). The survey revealed an essential difference in the number of children aged 3-7 years, found to have antibodies to L. pneumophila, in the Brest region of the Byelorussian SSR (31.4%) and in the Turkmen SSR (1.3%). The high risk groups exposed to L. pneumophila infection were found to include narcological patients with the changed reactivity of the body and healthy adults professionally employed in earth-moving works.     

Investigation of mechanisms involved in phagocytosis of Legionella pneumophila by human cells

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive and multiply efficiently in a variety of mammalian cells. By using in vitro assays, the uptake of L. pneumophila into monocytes has shown to be mediated, at least in part, through attachment of complement-coated bacteria to complement receptors, but complement-independent phagocytosis could also be demonstrated. Since complement levels in the human lung are normally low, the role of complement-dependent phagocytosis in the pathogenesis of Legionnaires' disease is doubtful. However, the contribution of other potential phagocytosis-related host cell surface molecules to the phagocytosis of L. pneumophila has never been investigated. We therefore analyzed the role of complement receptors 1 (CD35) and 3 (CD11b/18), the lipopolysaccharide (LPS) receptor (CD14), the beta(1)-integrin chain of the fibronectin receptor (CD29), the intercellular adhesion molecule 1 (ICAM-1, CD54) and the transferrin receptor (CD71) in the complement-independent uptake of L. pneumophila. To exclude any influence of culture conditions onto phagocytosis rates, we compared a fresh clinical isolate with an agar-adapted isolate of L. pneumophila. In addition, we used three different host cell types (MM6, HeLa and Jurkat cells) expressing different rates of complement receptors. We could show that both strains of L. pneumophila were phagocytized by the three host cell lines to the same extent, but intracellular multiplication was only found in MM6 and, although to a much lesser degree, in Jurkat cells. Preincubation of MM6 cells with monoclonal antibodies directed against the above cited phagocytosis-related receptors did not result in inhibition of L. pneumophila uptake. We therefore conclude that typical phagocytosis-related cell surface receptors are not involved in the complement-independent phagocytosis of L. pneumophila.     

Immunoenzyme analysis for determining class G and M antibodies to HBsAg

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.     

Ecology of Legionella pneumophila within water distribution systems

The reservoir for hospital-acquired Legionnaires disease has been shown to be the potable water distribution system. We investigated the influence of the natural microbial population and sediment (scale and organic particulates) found in water systems as growth-promoting factors for Legionella pneumophila. Our in vitro experiments showed that: (i) water from hot-water storage tank readily supported the survival of L. pneumophila, (ii) the concentration of sediment was directly related to the survival of L. pneumophila, (iii) the presence of environmental bacteria improved the survival of L. pneumophila via nutritional symbiosis, (iv) the combination of sediment and environmental bacteria acted synergistically to improve the survival of L. pneumophila, and (v) the role of sediment in this synergistic effect was determined to be nutritional. Sediment was found to stimulate the growth of environmental microflora, which in turn stimulated the growth of L. pneumophila. These findings confirm the empiric observations of the predilection of L. pneumophila for growth in hot-water tanks and its localization to sediment. L. pneumophila occupies an ecological niche within the potable water system, with interrelationships between microflora, sediment, and temperature.     

Temperature-dependent expression of flagella in Legionella

Legionella pneumophila, the causative agent of Legionnaires' disease, was analysed by electron microscopy for production of surface structures. Crystalline surface (S-) layers and fimbriae were not detected, but monotrichous flagellation was seen. Polyclonal antibodies specific for the 47 kDa flagellin subunit of L. pneumophila Philadelphia I were used in Western blots to confirm the presence of flagella subunits in various L. pneumophila strains tested, but the antiserum also reacted with flagellin subunits of L. micdadei, L. hackelia [serogroup (SG) 1 and SG2] and L. longbeachae (SG2). Flagellation of Legionellae was shown to be temperature regulated. When the growth temperature of virulent and avirulent of strain L. pneumophila Philadelphia I was shifted from 30 degrees C to either 37 or 41 degrees C, a decrease in the percentage of flagellated bacteria within the population was observed.     

Ecology of Legionella pneumophila within water distribution systems.

The reservoir for hospital-acquired Legionnaires disease has been shown to be the potable water distribution system. We investigated the influence of the natural microbial population and sediment (scale and organic particulates) found in water systems as growth-promoting factors for Legionella pneumophila. Our in vitro experiments showed that: (i) water from hot-water storage tank readily supported the survival of L. pneumophila, (ii) the concentration of sediment was directly related to the survival of L. pneumophila, (iii) the presence of environmental bacteria improved the survival of L. pneumophila via nutritional symbiosis, (iv) the combination of sediment and environmental bacteria acted synergistically to improve the survival of L. pneumophila, and (v) the role of sediment in this synergistic effect was determined to be nutritional. Sediment was found to stimulate the growth of environmental microflora, which in turn stimulated the growth of L. pneumophila. These findings confirm the empiric observations of the predilection of L. pneumophila for growth in hot-water tanks and its localization to sediment. L. pneumophila occupies an ecological niche within the potable water system, with interrelationships between microflora, sediment, and temperature.     

Prevalence of antibodies in response to <Emphasis Type="Italic">Legionella</Emphasis> species,analysis of a healthy population from Jeollanam-do Province,Korea

Seroepidemological investigation of antibodies to Legionella species in 500 healthy individuals from a single geographical location in Korea was conducted by indirect fluorescent antibody assay (IFA). Considering an antibody titer of > or =1:128 as positive reaction, 15.2% of total sera were positive. In males and females older than 40 years old, levels of IgM and IgG were 1.2% and 14%, respectively. The sera with antibody titers of > or =1:128 to Legionella species accounted for 85 sera, and 9 sera of these were reacted to more than one Legionella species. Reactivity to L. bozemanii, L. micdadei, L. longbeachae, L. pneumophila sg 6, and L. gormanii were 32.9%, 20%, 15%, 10.6%, and 8%, respectively. However, L. pneumophila sg 1, sg 2, and sg 3 did not react to any sera. Serological analysis revealed that the level of antibody in response to L. bozemanii was more prevalent than L. pneumophila. Our results suggest that the antibodies of non-L. pneumophila species, such as L. bozemanii, may be highly prevalent in healthy population within Korea. Although conclusions based on the findings of this study must be cautiously considered given that the population sampled were sourced from a single province, we have added to the knowledge base of serodiagnosis of infections due to non-L. pneumophila species in Korea.     

Cell biology of Legionella pneumophila

Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires' disease. L. pneumophila survives and replicates inside macrophages by preventing phagosome-lysosome fusion. A large number of L. pneumophila genes, called dot or icm, have been identified that are required for intracellular growth. It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway.     

Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources

AIMS: To validate identification methods for Legionella pneumophila strains that cannot be serotyped into the known serogroups and to characterize their antigenic diversity. METHODS AND RESULTS: Fifty L. pneumophila strains that could not be serogrouped, but which had been confirmed as L. pneumophila by mip gene sequencing, were further identified phenotypically. We used (i) MONOFLUO anti-Legionella Staining Reagent (Bio-Rad) (50/50), (ii) an in-house prepared immunoblot assay for the detection of L. pneumophila- specific Mip protein epitope (50/50), (iii) fatty acid analysis using the Microbial Identifications System (MIDI) (47/50) and (iv) Oxoid agglutination tests (44/50). The serological diversity was further characterized by testing with five serogroup-cross-reactive monoclonal antibodies, resulting in nine phenons. CONCLUSIONS: The division of L. pneumophila into 15 serogroups does not reflect the serogroup heterogeneity. Results of these tests indicate that there are more serogroups. SIGNIFICANCE AND IMPACT OF THE STUDY: MONOFLUO anti-Legionella Staining Reagent is the only commercially available tool for identifying atypical strains of L. pneumophila. If necessary for epidemiological purposes, the antigenic heterogeneity of these strains can be analysed by monoclonal antibodies.     

Immunoenzyme method in the diagnosis of human brucellosis

The optimum conditions for the determination of specific antibodies in the sera of brucellosis patients by means of enzyme immunoassay (EIA) have been selected. The comparative study of the specificity and sensitivity of EIA and other serological tests has demonstrated that EIA has high diagnostic effectiveness in the diagnosis of acute and chronic brucellosis. The presence of direct correlation between the results of EIA and Coombs' test is observed, which is indicative of the capacity of EIA for detecting both complete and incomplete specific antibodies. It should be pointed out that in all cases the titer of specific antibodies in EIA has been found to be 5-16 times higher than in Coombs' test, the passive hemagglutination test, and agglutination test.     

Antipertussis immunity studied by immunoenzyme analysis

The levels of antibodies to disintegrated Bordetella pertussis and its individual fractions (protein and polysaccharide) in children immunized with different batches of adsorbed DPT vaccine have been determined with the use of EIA techniques. The background level of antibodies in the control groups has been determined, and in immunized children the levels of antibodies to disintegrated B. pertussis and its protein fraction have been shown to considerably exceed the levels of antibodies to lipopolysaccharide.     

抗嗜肺军团菌血清8型单克隆抗体的制备及双抗体夹心酶联免疫吸附试验检测法的建立

目的：制备针对嗜肺军团茵血清8型的单克隆抗体,并建立双抗体夹心酶联免疫吸附试验（ELISA）检测方法。方法：用甲醛灭活的嗜肺军团菌血清8型菌免疫BALB／c小鼠,采用杂交瘤技术制备抗嗜肺军团菌血清8型单克隆抗体,建立双抗夹心ELISA检测方法。结果：研制出8株能特异性分泌抗嗜肺军团菌血清8型单克隆抗体的杂交瘤细胞株,Ig类型分别为IgM（2株）、IgG,（1株）和IgG,（5株）;利用IgG1型单抗6G10与6C7配对,建立了双抗夹心ELISA检测方法,该方法的最低检出浓度为2．6×10^5cfu／mL,除与金黄色葡萄球菌有微弱的交叉反应外,与14株其他血清型嗜肺军团菌、17株非嗜肺军团菌及11株非军团菌均无交叉反应,具有较高的特异性。结论：制备了具有高特异性和亲和力的抗嗜肺军团菌血清8型单克隆抗体,并建立了双抗夹心EUSA检测方法。