Rapid definitive diagnosis of Legionnaires' disease.

We describe a sporadic case of Legionnaires' disease in which the diagnosis was made by direct immunofluorescence of material obtained by percutaneous aspiration of the involved lung via a needle. Employment of this technique among selected patients with suspected Legionnaires' disease would provide for more rapid diagnosis and more prompt initiation of definitive therapy for some patients.     

Rapid diagnosis of Legionnaires' disease by latex agglutination

We developed a latex agglutination test to detect antigen(s) of serogroup 1 Legionella pneumophila in urine and evaluated its ability to diagnose legionnaires' disease. Antigen was detected in 46, or 70%, of 66 patients with serogroup 1 legionnaires' disease but in none of 51 patients with various bacteremic infections nor 60 with urinary tract infections. One of 50 patients with other pulmonary infections was antigen-positive, an immunosuppressed patient with Pneumocystis carinii infection. Seven other patients infected with P. carinii were negative. When compared with radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) using serial dilutions of a single antigen-containing urine, latex agglutination was 16-fold less sensitive. However, 82% of RIA- or ELISA-positive specimens were positive by latex agglutination. Latex agglutination is simpler and more rapid than ELISA or RIA, and it should prove valuable in situations where ELISA or RIA are unavailable or unaffordable.     

Four serogroups of Legionnaires' disease bacteria defined by direct immunofluorescence.

Thirty-five strains of Legionnaires' disease bacteria were shown to belong in four distinct serologic groups on the basis of findings obtained with direct fluorescent antibody testing. Thirty of the strains were placed in group 1, three in group 2, one in group 3, and one in group 4. Immunoelectrophoretic studies showed both unique and common antigens among the representative strains of the four serogroups.     

The laboratory diagnosis of Legionnaires' disease

Legionnaires' disease can be diagnosed by culture, by direct detection of the bacterium or its products by using immunologic means, with a DNA probe, and by serologic means. Culture diagnosis is the most specific and sensitive test. Legionellae can be isolated from sputum samples by using selective techniques. Antibody detection is more suited for epidemiologic purposes than for use in individual cases. Immunofluorescent microscopy is a useful and rapid means of diagnosis. Alternative rapid and specific tests are urinary antigen detection and the use of a DNA probe. Culture must always be performed for optimal sensitivity and for epidemiologic purposes.     

Scottish experience with the serologic diagnosis of Legionnaires' disease.

Four-hundred fifty-nine sera from 342 persons were examined at Ruchill Hospital, Glasgow, using an agar-grown heat-killed, ether-treated antigen derived from the Philadelphia 1 strain of the Legionnaires' disease bacterium. They were examined, in parallel, at the Center for Disease Control (CDC), Atlanta. Overall agreement (results agreeing +/- one twofold dilution) was reached with 90% of sera. Important differences were obtained with 10 (2.2%) sera. With three of these 10 sera the CDC alone obtained a significant titre (256) whereas with the other seven the Glasgow laboratory alone obtained a titre of 256. Altogether 28 persons (8.2%) with an agreed titre of 256 or higher were identified. However, some of these people had been ill 4 years before serum was obtained for examination. These persistent high titres make the serologic diagnosis of Legionnaires' disease difficult.     

Rapid diagnosis of pulmonary Pseudomonas aeruginosa infections by indirect immunofluorescent antibody]

167 sputum specimens collected from patients with pulmonary infection were tested by IFA (Indirect immunofluorescent antibody-staining) with serogroup-specific monoclonal antibody to Pseudomonas aeruginosa, compared with quantitative sputum culture. The results showed the minimum concentration of bacteria detectable by IFA was 10 cfu/ml. 31(86.1%) were positive in 36 specimens with more than 10 cfu/ml of Pseudomonas aeruginosa. 22(100%) were positive in 22 specimens with more than 10 cfu/ml of Pseudomonas aeruginosa. In 127 specimens with a negative culture for Pseudomonas aeruginosa, 121(95.2%) were negative, 6(4.8%) were false-positive which could be identified by Gram's-staining. The type of Pseudomonas aeruginosa could also be identified within 3 hours.     

Rapid diagnosis of Bancroftian filariasis by acridine orange staining of centrifuged parasites.

A rapid diagnostic test for detection of microfilaremia using a microhematocrit tube precoated with acridine orange (the Quantitative Buffy Coat [QBC] tube) was compared with a conventional 50 microliters-thick blood film (TF) in 119 volunteers in an area of Recife, Brazil that was endemic for Wuchereria bancrofti. Both the QBC and the TF techniques were 100% specific, and thus positive predictive values were equal at 100% for each technique in all subjects studied. Both techniques had equal negative predictive values of 100% in subjects with microfilarial counts > 100 per milliliter (mf/ml). Counts < 20 mf/ml are below a cutoff equal to the calculated limit of sensitivity of each of the two techniques. For those individuals with counts between 20 and 99 mf/ml, negative predictive values were, for practical diagnostic purposes, equivalent at 97.5% for the QBC technique and 99.0% for the TF. Because the QBC technique has predictive values as high as conventional TF, the convenience and rapidity of the technique will make the QBC technique a desirable alternative diagnostic method in those clinical settings where the equipment is available. A positive result will be available in less than six min after obtaining the specimen in individuals with counts < 100 mf/ml, and individuals with lower or no microfilaremia will have a result within 6-12 min.     

Localization of the neurofilament protein in neuroblastoma cells by immunofluorescent staining.

Neurofilament protein (54,000-56,000 daltons) has been localized in murine neuroblastoma cells by indirect immunofluorescent staining with antisera to purified calf brain neurofilament protein. In some cells with only short processes, specific staining of fibrous material was present in the perinuclear region while in other cells similar fibers, coiled to varying degrees, were present in other regions of the cytoplasm. In cells with longer processes a stained fiber extended throughout each process. The staining pattern observed followed the distribution of bundles of 100 A filaments as determined by electron microscopy. The fibers did not stain with antisera to tubulin or tropomyosin. The observations reported strongly indicate (i) that neurofilament protein isolated from calf brain is antigenically related to a component of the bundles of 100 A filaments in neuroblastoma cells, and (ii) that the neurofilament protein is an integral part of bundles of 100 A filaments in neuroblastoma cells, while neither tubulin nor tropomyosin is present in these bundles.     

Pathology of Legionnaires' disease.

The acute lesion in Legionnaires' disease pneumonia is an acute fibrinopurulent bronchopneumonia in which the alveoli are filled with many neutrophils and macrophages and abundant fibrin. There is only slight necrosis. Although characteristic, the lesion is not specific for this agent. However, the association with this lesion of myriad small pleomorphic rods, which stain well with Dieterle's silver-impregnation method but poorly or not at all with Gram-type stains, is uncommon except in Legionnaires' disease pneumonia. Final diagnosis requires isolation of the organism or immunofluorescent studies of the tissue, sera or both. The full spectrum of the pneumonia is not known, but organization has been reported once. No definite anatomic correlate for the extrathoracic manifestations of Legionnaires' disease has been identified nor has the organism been found at extrathoracic sites.     

Epidemiology of Legionnaires' disease.

Ten recorded epidemics of Legionnaires' disease are reviewed to gain a working perspective on the epidemiology of the disease. Salient features have included a summer-fall seasonality, a male predominance that may largely reflect increased exposure risk among men, and a striking absence of person-to-person spread. That the disease is spread primarily via the airborne route is well established; air-treatment and air-conditioning equipment has been implicated as the amplification and delivery system in four epidemics. Soils and excavation sites have been suggested as sources of the organism in at least one recorded epidemic. Evidence to date suggests that the Legionnaires' disease bacterium may be widespread in nature. More complete epidemiologic understanding must await development of improved microbiologic and immunologic tests.     

Lessons from Legionnaires' disease.

In July 1976 a pneumonialike epidemic from a previously unrecognized microorganism erupted among Legionnaires who had attended a meeting in Philadelphia. There were an estimated 182 cases, in which 29 patients died. Among other things the episodes shows that even in a medically sophisticated industrialized nation, a bacterical pathogen can produce a small epidemic and defy identification for almost 6 months. One historical implication of the event is the need to consider the possibility of a return of large-scale epidemic disease rivaling the sweeps of bubonic plague in fourteenth-century Europe. Such epidemics could occur through any of a variety of microorganismic mechanisms recognized as operating at the present time. It is suggested that humans would react to such a disaster much as their progenitors did centuries ago.     

Legionnaires' disease antigen detected by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay method has been developed for the rapid detection of antigen from clinical specimens such as urine and sputum. Preliminary evidence suggests that the antigen is present and can be detected in the sputum and possibly urine of patients with Legionnaires' disease. Patients not harboring the Legionnaires' disease bacterium have been uniformly negative to date.     

Radiographic features of Legionnaires' disease.

Serial chest radiographs of 35 patients with confirmed Legionnaires' disease were evaluated. A unilateral, unilobar alveolar infiltrate was, in general, the initial radiographic finding. Progression to consolidation or to new areas of involvement was typical. Every lobe was involved, but lower lobe involvement was most common. Pleural effusion was often present. Cavitation was not seen. Radiographic progression for several days following institution of therapy, and despite clinical response, was noted. In patients who survived, radiographic improvement of abnormalities was usually apparent within two weeks of therapy. Aradiographic spectrum exists and, although many features are typical, no single feature is pathognomonic.     

Microbiology of Legionnaires' disease bacterium.

Legionnaires' disease bacterium in tissue does not readily react with the Gram stain but can be seen by other stains and direct immunofluorescence. It is a slow-growing, aerobic, gram-negative rod that can be cultivated over a narrow temperature range on Mueller-Hinton agar supplemented either with complex biological mixtures or certain ferric salts and cysteine. The bacterium produces unique, branched-chain fatty acids, catalase, oxidase (weakly), and gelatinase and uses starch while ignoring other carbohydrates. Pigment production is related to tyrosine in the medium. In-vitro studies suggest susceptibility to all antibiotics except vancomycin, but a class 1 beta-lactamase has been demonstrated. Analysis of DNA confirmed the unrelatedness of this bacterium to previously recognized prokaryotes. Diagnosis of the disease has depended largely on serologic test findings and the demonstration of the bacterium in tissue and, occasionally, on isolation. Additional, simpler, and more rapid diagnostic tests should soon be available.     

Direct in-vitro isolation of the Legionnaires' disease bacterium in two fatal cases. Cultural and staining characteristics.

Two cases of Legionnaires' disease were diagnosed by direct isolation of the organism, from pleural fluid obtained before death in one case and lung tissue obtained after death in both cases. The organisms were recovered on a commercially prepared, enriched chocolate agar (Gibco, Madison, Wisconsin). Subcultures grew on commercially prepared, enriched chocolate agar (Baltimore Biological Laboratories, Cockeysville, Maryland) and on in-house enriched chocolate agar prepared with GC Medium Base (Difco, Detroit, Michigan). No growth was obtained on enriched chocolate agar prepared with trypticase soy agar. The organisms were poorly visualized in Gram-stained sections of formalin-fixed lung tissue. In Giemsa-stained sections poorly stained intracellular and extracellular slender rods were seen. However, with a silver impregnation stain, either a modified Dieterle or a modified Warthin-Starry procedure, many large, blunt-ended rods were seen. Smears prepared from minced formalin-fixed lung tissue and stained with a fluorescent antibody conjugate contained large numbers of well-stained organisms.     

Sensitivity of the virus isolation and immunofluorescent staining methods in diagnosis of infections with herpes simplex virus.

Tissues from marmoset monkeys infected with herpes simplex virus (HSV, Herpes-virus hominis) were utilized to evaluate the relative sensitivity and limitation of the virus isolation technique and the immunofluorescent (fluorescent antibody or FA) staining method for diagnosis of HSV infection. HSV encephalitis and/or disseminated infection in marmosets were established by intracerebral, intramuscular, or intravenous inoculation of the virus. Brain tissues, liver, spleen, kidney, adrenal gland, lymph node, and lung were harvested and prepared for the virus isolation procedure in tissue culture and for direct FA staining. Data from six marmosets infected with HSV type 1 and two infected with type 2 indicated that the virus isolation method was more sensitive and reliable than the FA staining technique. False-negative results by FA staining were found in two situations: (1) presence of focal lesions that were missed by the frozen sections, and (2) presence of low concentrations of virus in tissue (is less than or equal to 3.5 log10 50% tissue culture infective doses/g). FA staining provides a rapid method for detection of viral antigens, but isolation of virus in tissue culture is required for a conclusive diagnosis of active infection.