Legionella-Like and Other Amoebal Pathogens as Agents of Community-Acquired Pneumonia

Thomas J. Marrie, Department of Medicine University of Alberta, Edmonton, Alberta, Canada; Didier Raoult, Bernard La Scola, Richard J. Birtles, Faculté de Médecine de Marseille, Marseille, France; Emidio de Carolis, Pfizer Canada Inc., Montreal, Quebec, Canada; the Canadian Community-Acquired Pneumonia Study Group(1)


Emerging Infectious Diseases. 2001;7(6) 

In This Article

The Study

We used 511 specimens from a 1985 study of a random sample of the Nova Scotia population[6]; 121 acute- and convalescent-phase serum specimens from a study (Nova Scotia, 1991-1994) of 149 ambulatory patients with community-acquired pneumonia[7]; and specimens from a prospective study of community-acquired pneumonia requiring hospitalization conducted at 15 teaching hospitals in eight Canadian provinces (1996-1997).

All serum specimens from both groups of patients with pneumonia were tested for antibodies to Mycoplasma pneumoniae; influenza viruses A and B; parainfluenza viruses 1,2,3; adenovirus; and Respiratory syncytial virus (RSV) by a standard complement fixation technique in microtiter plates. Serum specimens from 60% of the patients (randomly selected from the group of patients with community-acquired pneumonia requiring hospitalization) were tested by the microimmunofluorescence test[8,9,10] for immunoglobulin (Ig) G and IgM antibodies to Chlamydia pneumoniae (AR 39 strain); C. psittaci (avian strain 6BC, feline pneumonitis strain FP, turkey strain TT 3, and pigeon strain CP 3); C. pecorum (ovine polyarthritis strain); and C. trachomatis (pooled antigens of serovars BED, CJHI, and FGK). Serum specimens from hospitalized patients with pneumonia were tested for antibodies to Streptococcus pneumoniae pneumolysin, pneumolysin immune complexes, C polysaccharide, surface protein A, Haemophilus influenzae, and Branhamella catarrhalis by Dr. M. Leinonen, National Public Health Institute, Oulu, Finland, as reported previously[11,12,13].

Acute- and convalescent-phase serum specimens from 150 patients also had been previously tested by enzyme-linked immunosorbent assay (ELISA) for antibodies to L. pneumophila serogroups 1-6 by Yu[14]. A urine sample collected from each patient within 24 hours of hospitalization was tested for L. pneumophila serogroup 1 antigen by ELISA[15] (Binax, Inc., Portland, ME). Antibodies to Coxiella burnetii phase 1 and 11 antigens and to Chlamydia pneumoniae were determined by a microimmunofluorescence test, as described[16,17].

Antibody titers to LLAPs also were determined by the indirect fluorescent antibody technique. These included Acanthamoeba polyphaga strain Linc AP 1 and LLAP strains 1, 2, 4, 6, 7, 9, 10, 12; L. lytica (strains LLAP 3 and L 2, formerly Sarcobium lyticum); and Parachlamydia acanthamoeba (strain BN 9 and Hall's coccus). A. felis ATCC 53690 was from the American Type Culture Collection. LLAPs were cultured in A. polyphaga in 150 peptone-yeast extract-glucose broth[18] at 30°C. When maximally infected, amoebae were lysed through three cycles of freeze-thawing in liquid nitrogen. This suspension was then resuspended in 30 mL of phosphate-buffered saline and centrifuged at 10,000 rpm for 10 minutes. Supernatant fluid was removed, and pellets containing respective LLAPs were resuspended in the smallest possible volume of sterile distilled water and were adjusted to a concentration of 2 mg/mL, as determined spectrophotometrically. Antigen prepared in this manner was frozen at -20°C until required.

Specimens with an IgM titer of ≥1:100, seroconversion from 0 to 100, a fourfold rise in antibody titer between acute- and convalescent-phase serum, and a single or stable titer of ≥400 were considered indicative of recent infection. An antibody titer of ≥50 was considered seropositive, i.e., evidence of prior but not recent infection.

The seropositivity rate to various antigens is shown in Table 1. The background rate of such infection is low. Only one healthy Nova Scotian had serologic evidence of recent infection with an LLAP, LLAP 4. None of the patients with ambulatory pneumonia had such an infection. LLAP 4 was the most common LLAP-causing pneumonia: four such infections among patients with community-acquired pneumonia required hospitalization. Two of the 58 patients from the Nova Scotia site had infection with LLAP 4, versus one of 511 healthy Nova Scotians (p<0.029, Fisher exact test). BN 9 caused two infections, and LLAP 1 and 12 caused one each among patients with community-acquired pneumonia requiring hospitalization.

LLAP 1 and 12 infections. A 40-year-old floral designer was hospitalized in Edmonton, Alberta, with a 21-day history of diarrhea, myalgias, headache, chills, and shortness of breath. She had traveled to Los Angeles and Palm Springs in the previous 10 days. On admission, her oral temperature was 36.9°C, and fine bilateral interstitial infiltrates were present on a chest radiograph. She was treated with erythromycin and doxycycline, was discharged on day 7, and was readmitted 2 weeks later, at which time a transbronchial biopsy specimen yielded Mycobacterium avium intracellulare on culture. No evidence of HIV infection was found. The acute- and convalescent-phase antibody titers to LLAP 1 were 1:400 in the IgG fraction and 1:25 and 0 in the IgM fraction.

A 34-year-old clerical worker in a hospital radiology department was hospitalized on April 11, 1996, with pleuritic chest pain and shortness of breath of 10 days' duration. His oral temperature was 38.7°C. The leukocyte count was 9.2 x 109/L. A chest radiograph showed multilobar patchy opacities on the left and a 3-cm nodular opacity on the right. The patient was treated with erythromycin and cefuroxime intravenously for 36 hours, followed by oral clarithromycin. The nodule did not resolve over the next 6 weeks, and an open lung biopsy was performed. All cultures were negative. Histologic examination revealed acute and chronic inflammation. The acute-phase serum sample had an IgM antibody titer of 1:200 to LLAP 12, and the convalescent-phase titer was 1:100; the corresponding values for IgG were 0 and 1:50.

BN 9 infection. A 21-year-old university student was hospitalized with fever, abdominal pain, nausea, vomiting, diarrhea, pleuritic chest pain, and nonproductive cough. He also complained of a sore throat and shortness of breath. On examination, he looked acutely ill and had a diffuse erythematous rash. His oral temperature was 38.3°C. A chest radiograph showed diffuse opacities involving both lower lobes. He was treated with erythromycin. The next day desquamation of the lips and the skin of the digits was noted, and a diagnosis of adult Kawasaki disease was entertained. Treatment with aspirin and gamma globulin was instituted, and the patient made an uneventful recovery. There was no evidence of cardiac involvement as indicated by normal serial electrocardiograms and a normal echocardiogram. The BN 9 antibody titer was 1:50 and 1:6,400 in the acute- and convalescent-phase serum specimens. There was a stable antibody titer to Hall's coccus of 1:400 in both. Blood and urine cultures, as well as other microbiologic tests were negative.

A 68-year-old man was hospitalized on October 15, 1996, with nausea, vomiting, diarrhea, a nonproductive cough, shortness of breath, chills, and pleuritic chest pain. The year before, he had received a cadaveric renal transplant and was maintained on corticosteroid and cyclosporin therapy. His oral temperature was 39.2°C, and consolidation was found on examination of the right lung. A chest radiograph showed a single lobar opacity on the right. The leukocyte count was 17 x 109/L. S. pneumoniae was isolated from the sputum. The patient was treated with cefuroxime intravenously for 4 days and was discharged on oral cefaclor. He made an uneventful recovery. The acute- and convalescent-phase titers to BN 9 were stable at 1:400.

LLAP 4 infection. Four patients met our definition for infection with LLAP 4 (Table 2). Appearance of pneumonia was similar in all four chest radiographs. Patient ML 13 had diffuse interstitial infiltrates, but this patient, who had had a bone marrow transplant, also had RSV infection. All patients with LLAP 4 recovered from pneumonia.


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