Mimivirus in pneumonia patients.Mimivirus, the largest virus known to date, is an amebal pathogen--like Legionella Legionella /Le·gion·el·la/ (le?jah-nel´ah) a genus of gram-negative, aerobic, rod-shaped bacteria (family Legionellaceae), normal inhabitants of lakes, streams, and moist soil; they have often been isolated from cooling-tower water, sp. When Mimivirus was used as an antigen in a migration inhibition factor assay, sero-conversion was found in patients with both community- and hospital-acquired pneumonia. Mimivirus DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. was found in respiratory samples of a patient with hospital-acquired pneumonia.
The causative agent of pneumonia, the leading cause of infection-related death throughout the world, is unknown in 20% to 50% of cases (1). Therefore, identifying new causative agents of both community- and hospital-acquired pneumonia is a major public health goal. Aquatic bacteria such as Legionella spp., Pseudomonas Pseudomonas
A genus of gram-negative, nonsporeforming, rod-shaped bacteria. Motile species possess polar flagella. They are strictly aerobic, but some members do respire anaerobically in the presence of nitrate. spp., Stenotrophomonas spp., Burkholderia spp., and Acinetobacter spp. colonize col·o·nize
v. col·o·nized, col·o·niz·ing, col·o·niz·es
1. To form or establish a colony or colonies in.
2. To migrate to and settle in; occupy as a colony.
3. hospital water supplies. These bacteria, such as L. pneumophila, have been causally associated with both hospital- and community-acquired pneumonia. Legionella spp. and other bacteria are associated with free-living amebas in natural and hospital aquatic environments (2). Bacteria that resist phagocytic phag·o·cyt·ic
1. Of or relating to phagocytes.
2. Of, relating to, or characterized by phagocytosis.
emanating from or pertaining to phagocytes. destruction by amebas and are found in aerosolized water are potential agents of pneumonia (3). Ameba-associated bacteria other than L. pneumophila, including other Legionella spp., new ot-proteobacteria belonging to the Bradyrhizobiaceae (Bosea massiliensis) family, and members of the genus Parachlamydia might be implicated in hospital-acquired pneumonia (4-6).
In strict intraamebal bacteria, we found Legionella-like amebal pathogens (7), Parachlamydia acanthamebae (8), and a giant virus resembling gram-positive cocci cocci /coc·ci/ (kok´si) plural of coccus.
[L.] plural of coccus. that we named Mimivirus (9). The Mimivirus's genome is larger than that of Mycoplasma mycoplasma
Any of the bacteria that make up the genus Mycoplasma. They are among the smallest of bacterial organisms. The cell varies from a spherical or pear shape to that of a slender branched filament. and genome sequencing is finished (10). Because we found antibodies against several ameba-associated bacteria in patients with community-and hospital-acquired pneumonia (3,4) in previous studies, we tested for antibodies to Mimivirus by using a microimmunofluorescence assay on serum samples from patients with community- and hospital-acquired pneumonia. DNA of Mimivirus was also found in the bronchoalveolar lavage specimens of patients with hospital-acquired pneumonia.
We studied serum samples from 376 Canadian patients with community-acquired pneumonia (121 ambulatory and 255 hospitalized) and from 511 healthy control subjects. Extensive clinical data were available for 104 patients with community-acquired pneumonia. All of these samples were previously tested for other pneumonia agents (4). To prepare antigen for microimmunofluorescence study, Mimivirus was grown in Acanthameba polyphaga strain Linc AP-1 in 75-[cm.sup.2] cell culture flasks with peptone peptone /pep·tone/ (pep´ton) a derived protein, or a mixture of cleavage products produced by partial hydrolysis of native protein.pepton´ic
n. yeast extract glucose medium as previously described (11). After amebal lysis occurred, unlysed amebas were removed by low speed centrifugation Centrifugation
A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal at 100 g for 15 min. Mimivirus particles present in supernatant were centrifuged at 4,000 g for 30 min and washed 3 times in phosphate-buffered saline (PBS PBS
in full Public Broadcasting Service
Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural, ). The pellet obtained after the last washing was then resuspended in PBS at 2 mg/mL concentration of protein and used as antigen in microimmunofluorescence assay under previously described conditions (5). Evidence of serologic se·rol·o·gy
n. pl. se·rol·o·gies
1. The science that deals with the properties and reactions of serums, especially blood serum.
2. reaction to Mimivirus was defined as: 1) seroconversion seroconversion /se·ro·con·ver·sion/ (-con-ver´zhun) the change of a seronegative test from negative to positive, indicating the development of antibodies in response to immunization or infection. from <1:50 to [greater than or equal to] 1:100 between acute-phase and convalescent-phase serum samples or a 4-fold rise in antibody titer between acute-phase and convalescent-phase serum samples, or 2) a single or stable titer of [greater than or equal to] 1:400. The cut-off titer for single serum was chosen to have <2.5% positive rate in control subjects. We also tested paired serum samples from 26 patients with intensive care unit (ICU ICU intensive care unit.
intensive care unit
see intensive care unit.
ICU )-acquired pneumonia for a 1-year period and 50 paired serum samples from patients in our institution to determine antibodies to Rickettsia rickettsia (rĭkĕt`sēə), any of a group of very small microorganisms, many disease-causing, that live in vertebrates and are transmitted by bloodsucking parasitic arthropods such as fleas, lice (see louse), and ticks. spp. as controls.
To verify that antibodies against Mimivirus in patients with pneumonia recognize Mimivirus particles specifically, a serum sample of 1 of these patients was used to detect Mimivirus particles by immunogold technique as previously described (12). Two serum samples of patients who did not have detectable antibodies against Mimivirus were used as controls. Grids were incubated briefly twice in incubation buffer (PBS with 0.2% bovine serum albumin) for 5 min, then for 15 min in lysine lysine (lī`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. buffer (PBS with 0.05 mol lysin Lysin
A term used to describe substances that will disrupt a cell, with the release of some of its constituents. Unless the damage is minor, this action leads to the death of the cell. ). Grids were washed twice in incubation buffer for 5 min, then incubated for 3 h at 37[degrees]C in patients' diluted samples (diluted 1/1,000 in incubation buffer with 3% nonfat dry milk Noun 1. nonfat dry milk - dehydrated skimmed milk
dried milk, dry milk, milk powder, powdered milk - dehydrated milk ). Grids were washed 6 times for 5 min in incubation buffer, then incubated for 2 h at 37[degrees]C in goat antihuman immunoglobulin (IgG)-gold conjugate (Aurion Biovalley, Marne la Vallee, France) diluted 1/20 in incubation buffer with 3% nonfat dry milk. Grids were washed 6 times for 5 min in incubation buffer, then twice in PBS for 5 min. Grids were then immersed twice in gutaraldehyde (2% in PBS) for 5 min, rinsed 3 times in distilled water for 5 min, treated by R-GENT SE-EM (Aurion Biovalley) for 25 min, then rinsed 3 times in distilled water for 5 min before being stained with uranyl acetate before examination.
Genomic DNA of Mimivirus was found in bronchoalveolar lavage specimens from patients in the ICU of the Ste. Marguerite Hospital, Marseilles, France; serologic reaction was studied in serum samples of the patients for 1 year. The study was retrospective; samples were tested anonymously from 12 to 18 months after sampling. Mimivirus was found in bronchoalveolar lavage specimens from 1 of 32 patients with ICU-acquired pneumonia and none in specimens from 21 intubated control patients in ICU who did not have pneumonia (5). We designed 4 primer pairs chosen from the genome sequence of Mimivirus. To avoid any contamination, we used a nested polymerase chain reaction Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites. (PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) ), previously described as "suicide-PCR," that incorporates 2 primer pairs used only once without positive control, followed by sequencing and comparing to the targeted sequence (13). DNA from patient and control BAL (1) (Basic Assembly Language) The assembly language for the IBM 370/3000/4000 mainframe series.
(2) (Branch And Link) An instruction used to transfer control to another part of the program.
BAL - Basic Assembly Language specimens, 3 water samples, and a suspension ofA. polyphaga were extracted by using the QIAmp Tissue kit (QIAGEN GmbH, Hilden, Germany). DNA extracts from selected pathogens, including agents that are most commonly encountered in cases of hospitalacquired pneumonia (14), were tested: Enterobacter aerogenes, Proteus mirabilis, Citrobacter freundii, Escherichia coli, Citrobacter koseri, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Haemophilus influenzae, Moraxella catarrhalis, Acinetobacter baumanii, Stenotrophomonas maltophila, Pseudomonas aeruginosa, Bacteroides fragilis, Prevotella intermedia Intermedia - A hypertext system developed by a research group at IRIS (Brown University). , Streptococcus pneumoniae, Streptococcus oralis, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, CMV, HSV (Hue Saturation Value) A color space similar to HSB. See HSB.
HSV - hue, saturation, value 1, and adenovirus adenovirus
Any of a group of spheroidal viruses, made up of DNA wrapped in a protein coat, that cause sore throat and fever in humans, hepatitis in dogs, and several diseases in fowl, mice, cattle, pigs, and monkeys. . Extracted DNA was used as template with primers BCFE BCFE Boundary Committee for England
BCFE Ballyfermot College of Further Education (Dublin, Ireland)
BCFE Board Certified Forensic Examiner
Bcfe Billions of Cubic Feet Equivalent (Per Day; Gas Exploration) (5'-TTATTGGTCCCAATGCTACTC-3') and BCRE (5'TAATTACCATACGCAATTCCTG-3') as external primers and BCFI BCFI British Columbia Freshwater Institute
BCFI Business Centre Financial Industry (5'-TGTCATTCCAAATGTTAACGAAAC-3') and BCRI BCRI Birmingham Civil Rights Institute (Birmingham, Alabama)
BCRI Business Compliance Response Initiative (5'-GCCATAGCATTTAGTCCGAAAG-3') as internal primers. A minimum of 1 negative control was used for every 2 samples from patients with pneumonia; testing was conducted in a blinded manner.
In the study of 511 healthy Canadian controls, 12 (2.3%) exhibited a substantial titer of antibodies to Mimivirus. Patients with community-acquired pneumonia were positive more frequently than controls, as 36 (9.66%) were found positive (chi-square test, p < 0.01). Although the typical morphologic traits of Mimivirus make its confusion with ameba organelles unlikely (Figure), positive serum samples tested on noninfected intact amebas suspended in PBS by using the same migration inhibition factor protocol did not show any reactivity. Immunoelectron microscopic examination showed that antibodies of positive patients recognize mature Mimivirus particles specifically (Figure), whereas antibody fixation was not found in serum samples from 2 patients who were negative for Mimivirus (data not shown). We compared selected features of 14 patients with community-acquired pneumonia who had serologic evidence of infection with Mimivirus with those of 90 patients with community-acquired pneumonia who were seronegative seronegative /se·ro·neg·a·tive/ (-neg´ah-tiv) showing negative results on serological examination; showing a lack of antibody.
adj. for Mimivirus (Table). Only hospitalization from a nursing home (3/14 vs. 3/90) and rehospitalization after discharge (6/14 vs. 16/90) were significantly associated with Mimivirus antibodies (p < 0.05). Older age and diabetes mellitus were more common (both 6/14 versus 18/90) in patients with Mimivirus antibodies but not significantly so (p = 0.07). In patients with community-acquired pneumonia, more frequent rehospitalization after discharge in patients with serologic evidence of Mimivirus is likely explained by the poor efficacy of antimicrobial agents against viruses (15). Seropositive seropositive /se·ro·pos·i·tive/ (-poz´i-tiv) showing positive results on serological examination; showing a high level of antibody.
adj. patients with community-acquired pneumonia were more likely to be admitted from a nursing home; this factor suggests that Mimivirus is a particularly good candidate as an etiologic agent of pneumonia acquired in institutions, as is L. pneumophila (16). The seroprevalence seroprevalence Immunology The proportion of a population that is seropositive–ie, has been exposed to a particular pathogen or immunogen; the seropositivity of a population is calculated as the number of individuals who produce a particular antibody divided of Mimivirus was significantly higher than that of ameba-associated bacteria tested on the same samples (4).
Serologic evidence of infection was observed in 5 (19.2%) of 26 ICU patients. None of the 50 control patients was positive for Mimivirus (p < 0.01). Mimivirus DNA was detected in bronchoalveolar lavage specimen from a 60-year-old comatose co·ma·tose
1. Of, relating to, or affected with coma.
2. Marked by lethargy; torpid.
comatose (kō´m patient who had 2 episodes of hospital-acquired pneumonia during hospitalization in ICU. Mimivirus DNA was amplified from the second episode sample only. The sequenced amplified fragment was 100% homologous to the target DNA (GenBank accession no. AY026860). No serum sample was available from this patient. None of the DNA extracts from control microorganism microorganism /mi·cro·or·gan·ism/ (-or´gah-nizm) a microscopic organism; those of medical interest include bacteria, fungi, and protozoa. showed a positive PCR reaction. The finding of Mimivirus DNA in the bronchoalveolar lavage specimens from an ICU patient with nosocomial pneumonia confirms that Mimivirus may reach the respiratory tract of these patients. Because of the procedure used for suicide PCR amplification, contamination appears highly unlikely. In this patient, however, we cannot distinguish colonization and infection, but this feature is common to most microorganisms isolated from respiratory samples.
This high rate of seroconversion observed in patients with pneumonia from our seroepidemiologic study suggests that community-acquired pneumonia and hospitalacquired pneumonia patients may have contact with Mimivirus or a cross-reacting agent. As we do not report direct evidence of infection by Mimivirus, these results have to be interpreted with caution. Viruses usually have a broad range of hosts, but the extraordinary size of the Mimivirus genome (1.2 Mb), comparable to that of small bacteria such as Mycoplasma (17), suggests a possible adaptation to an extended range of hosts. We propose that Mimivirus be tested as a possible novel human pathogen among ameba-resisting microorganisms. These results are preliminary, but raise the question of the pathogenic potential of the biggest identified virus to date. Mimivirus is an agent easy to cultivate and is freely available from our laboratory on request to researchers working on pneumonia who wish to introduce Mimivirus antigen in serologic tests to confirm our results.
Table. Comparison of selected characteristics from patients with CAP who had serologic evidence of infection with Mimivirus with those of seronegative patients with CAP * Characteristic Positive Negative p (N = 14) (N = 90) Age [greater than or equal to] 80 y 6 18 0.07 Male 8 51 0.60 Length of hospital stay (d) 10.6 16.9 0.27 Days between onset and 6.9 5.2 0.77 admission Admission from nursing home 3 3 0.03 Retired 10 50 0.26 Smoked for >1 y 11 70 0.62 History of COPD 4 28 0.56 History of asthma 0 13 0.13 History of bronchiectasis 1 10 0.55 Diabetes mellitus 6 18 0.07 Hemodialysis 2 3 0.13 Rehospitalization after discharge 6 16 0.03 Death 2 2 0.08 * CAP, community-acquired pneumonia; COPD, chronic obstructive pulmonary disease.
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pertaining to or emanating from a microbe.
the breakdown of organic material, especially feedstuffs, by microbial organisms. world: protozoa and the survival of bacterial pathogens in the environment. Microbiology. 1994;140:1253 9.
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Dr. La Scola is professor of bacteriology bacteriology
Study of bacteria. Modern understanding of bacterial forms dates from Ferdinand Cohn's classifications. Other researchers, such as Louis Pasteur, established the connection between bacteria and fermentation and disease. at Universit6 de la Mdditerranee, Marseille, France. He is member of the Unite des Rickettsies (CNRS CNRS Centre National de la Recherche Scientifique (National Center for Scientific Research, France)
CNRS Centro Nacional de Referencia Para El Sida (Argentinean National Reference Center for Aids) UMR UMR Unite Mixte de Recherche (French: Mixed Unit of Research )
UMR University of Missouri - Rolla
UMR Upper Mississippi River
UMR Uniform Methods and Rules (US Department of Agriculture)
UMR Unit Manning Report 6020, World Health Organization reference center for rickettsiae and rickettsial diseases). His fields of interest are the isolation and description of fastidious bacteria, including Coxiella, Rickettsia, Bartonella, Tropheryma, and ameba-associated bacteria.
Address for correspondence: Didier Raoult, Unite des Rickettsies, Universite de la Mediterranee, Faculte de Medecine, CNRS UMR 6020, 27 Boulevard Jean Moulin, 13385 Marseille, France; fax: 33 04 91 38 77 72; email: Didier.Raoult@medecine.univ-mrs.fr
Bernard La Scola, * Thomas J. Marrie, ([dagger]) Jean-Pierre Auffray, ([double dagger]) and Didier Raoult *
* Universite de la Mediterranee, Marseille, France; ([dagger]) University of Alberta, Edmonton, Alberta, Canada; and ([double dagger]) Hopital de Ste Marguerite, Marseille, France