Rickettsia prowazekii and real-time polymerase chain reaction.
Rickettsia prowazekii Rickettsia pro·wa·zek·i·i
A bacterium that causes epidemic typhus fever. is the causative agent of epidemic typhus epidemic typhus
A form of typhus characterized by high fever, mental and physical depression, and macular and papular eruptions; it is caused by Rickettsia prowazekii and transmitted by body lice. and a potential bioterrorism agent. Sensitive and specific rapid assays are needed to complement existing methods of detecting this organism. We developed a real-time quantitative polymerase chain reaction Quantitative polymerase chain reaction (qPCR) is a modification of the polymerase chain reaction used to rapidly measure the quantity of DNA, complementary DNA or ribonucleic acid present in a sample. assay by using a species-specific probe targeting the gltA gene. This assay, which was rapid, specific for R. prowazekii only, and sensitive (cutoff detection of 1 to 5 copies per sample), detected and directly identified R. prowazekii in blood of 12 experimentally infected mice sampled at day 3 and 6 postinfection or in naturally or experimentally infected lice. Because our assay is highly standardized and easily adaptable, it could improve epidemic typhus surveillance in public health programs, especially for countries with underdiagnosed or unrecognized human cases.
Rickettsia prowazekii is the causative agent of epidemic for louseborne typhus typhus, any of a group of infectious diseases caused by microorganisms classified between bacteria and viruses, known as rickettsias. Typhus diseases are characterized by high fever and an early onset of rash and headache. , which is transmitted by the human body louse body louse
A parasitic louse that infests the body and clothes of humans. . This disease can be fatal and, without treatment with doxycycline doxycycline /doxy·cy·cline/ (dok?se-si´klen) a semisynthetic broad-spectrum tetracycline antibiotic, active against a wide range of gram-positive and gram-negative organisms; used also as d. calcium and d. hyclate. , will cause death in up to 30% of cases (1-3). More than 30 million cases of epidemic typhus occurred during and immediately after World War I, resulting in an estimated 3 million deaths (1). Although the incidence of epidemic typhus is low today, the infection could reemerge in epidemic form in human populations, as recently reported in Burundi (4), Russia (5), and Algeria (6). Infections with R. prowazekii have been rarely reported in the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. : only 39 cases were reported from 1976 to 2001, all in persons who had no reported contact with body lice body lice Vox populi Pediculosis humanis corporis. See Louse. but did have contact with flying squirrels (7,8).
The ability to be acquired by the aerosol route, efficient arthropod arthropod
Any member of the largest phylum, Arthropoda, in the animal kingdom. Arthropoda consists of more than one million known invertebrate species in four subphyla: Uniramia (five classes, including insects), Chelicerata (three classes, including arachnids and horseshoe transmission, and severe clinical outcome and death in untreated cases make R. prowazekii a category B bioterrorism agent. The former Soviet Union's Red Army developed R. prowazekii as a battlefield weapon, and the Japanese army Japanese Army can refer to:
Since clinical signs of epidemic typhus are usually nonspecific nonspecific /non·spe·cif·ic/ (non?spi-sif´ik)
1. not due to any single known cause.
2. not directed against a particular agent, but rather having a general effect.
1. , including fever, headaches, and severe myalgia myalgia /my·al·gia/ (mi-al´jah) muscular pain.myal´gic
epidemic myalgia see under pleurodynia.
n. , appropriate diagnostic methods are important (10). Despite recent developments in cell culture and molecular detection methods for the diagnosis of rickettsial diseases (11), 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. assays remain the simplest diagnostic tests to perform, even if serum samples are sent on filter paper (12). Nevertheless, serologic tests lack specificity because most also detect cross-reactive antibodies among the typhus-group rickettsioses Rickettsioses
Often severe infectious diseases caused by several diverse and specialized bacteria, the rickettsiae and rickettsia-like organisms. The best-known rickettsial diseases infect humans and are usually transmitted by parasitic arthropod vectors. . Moreover, a definite diagnosis of epidemic typhus is often delayed because the sensitivity of cell culture and polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) ) methods is low (13), and serologic diagnosis can be obtained only by using advanced serologic methods such as Western blot analysis West·ern blot analysis
An electrophoretic procedure for separating proteins. after cross-adsorptions. These methods are restricted to laboratories with biosafety level biosafety level Epidemiology A classification for the degree of caution required when working with specific groups of pathogens. See Maximum containment facility. 3 (BSL-3) facilities and trained technicians (14). Recent studies have demonstrated the usefulness of PCR of body lice in ongoing surveillance of louse-associated infections, especially in outbreaks of epidemic typhus (15). The aim of our study was to develop a real-time quantitative PCR assay by using a species-specific probe that is rapid, sensitive, and specific for detecting R. prowazekii in clinical samples or in body lice in outbreaks of epidemic typhus.
Materials and Methods
The gltA sequences of 22 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. species were aligned by using the multiple-sequence alignment program ClustalW supported by the Infobiogen website (www.infobiogen.fr). Within the alignments, primers and probe were selected that were specific for R. prowazekii.
R. prowazekii strain Breinl (ATCC ATCC American Type Culture Collection, see there VR-142) was grown in Vero cell Vero cells are lineages of cells used in cell cultures.
The Vero lineage was isolated from kidney epithelial cells extracted from African green monkey (Cercopithecus aethiops). monolayers cultured in minimal essential medium supplemented with 4% fetal calf serum and 2 mmol/L L-glutamine as previously described (16). Infected cells were harvested by using sterile glass beads and sonicated. Cell fragments were removed by 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 , and the supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material.
the liquid lying above a layer of precipitated insoluble material. was centrifuged for 10 min at 7,500 x g. The resulting pellet was resuspended in 20 mL phosphate-buffered saline, pH 7.5. R. prowazekii inoculum inoculum /in·oc·u·lum/ (-ok´u-lum) pl. inoc´ula material used in inoculation.
n. pl. was quantified by using either the plaque assay method (17) or comparatively by 10-fold serial dilutions of a known plasmid standard of R. prowazekii containing 2.0 x [10.sup.7] copies per sample in an independent real-time PCR as previously described (18).
In this assay 4 R. prowazekii strains, 21 strains of Rickettsia spp., and 14 strains of bacteria from genera other than Rickettsia were evaluated (Table). We also included 31 lice from an outbreak of epidemic typhus in Rwanda in 2004 and 10 R. prowazekii laboratory-infected lice (19), 10 R. typhi laboratory-infected lice (20), and 10 Borrelia Borrelia
A genus of spirochetes that have a unique genome composed of a linear chromosome and numerous linear and circular plasmids. Borreliae are motile, helical organisms with 4–30 uneven, irregular coils, and are 5–25 micrometers long and 0. recurrentis laboratory-infected lice (21). Finally, we also included 30 lice received in June 2005 from Rwanda, which were tested only with the quantitative PCR (qPCR) assay (Table). Negative controls included 10 pathogen-free lice, distilled sterile water, and PCR mixture. All experiments were repeated 4 times. For mice samples, 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. samples extracted from blood of uninfected mice were used as negative controls.
We have also tested R. prowazekii-infected mice by using a currently available experimental model similar to the previous model described for R. typhi (22). We used 7-week-old female BALB/C mice (Charles River Charles River
River, eastern Massachusetts, U.S. The longest river wholly in the state, it flows into Boston Bay after a course of about 80 mi (130 km). Navigable for about 7 mi (11 km), its estuary separates the cities of Boston and Cambridge. Laboratories, Arbresle, France) that were maintained in cages with sterile food and water. All experiments were performed in a BSL-3 laboratory. Twelve mice were injected with 1.8 x [10.sup.5] PFU/mL R. prowazekii strain Breinl (ATCC VR-142), and 6 mice were injected with uninfected cells. The solution containing bacteria was injected into the retroorbital venous plexus Venous plexus can refer to:
Total genomic DNA genomic DNA
The full complement of DNA contained in the genome of a cell or organism. from bacterial strains was extracted with the Qiagen QIAamp Blood Kit (Qiagen, Hilden, Germany), and lice DNA and blood and biopsy samples from infected mice were extracted by using the Qiagen QIAamp Tissue Protocol (Qiagen). PCR was performed by using a LightCycler instrument (Roche Biochemicals, Mannheim, Germany).The PCR mixture included a final volume of 20 [micro]L with 10 [micro]L of the Probe Master kit (Qiagen), 0.5 [micro]L (10 pmol/[micro]L) of each primer, 2 [micro]L (2 [micro]mol/[micro]L) probe, 5 [micro]L distilled water Noun 1. distilled water - water that has been purified by distillation
H2O, water - binary compound that occurs at room temperature as a clear colorless odorless tasteless liquid; freezes into ice below 0 degrees centigrade and boils above 100 degrees centigrade; , and 2 [micro]L extracted DNA. The amplification conditions were as follows: an initial denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. step at 95[degrees]C for 15 min, followed by 40 cycles of denaturation at 95[degrees]C, annealing annealing (ənēl`ĭng), process in which glass, metals, and other materials are treated to render them less brittle and more workable. and elongation at 60[degrees]C for 120 s, with fluorescence acquisition in single mode. Each sample was also tested with a standard PCR that was performed on a PCR instrument (Eppendorf, Mastercycler, Hamburg, Germany) using primers of the gltA gene (23).
The R. prowazekii inoculum used in this study was of 1.8 x [10.sup.5] PFU/mL using the plaque assay quantification method (17) and contained 1.16 x [10.sup.6] copies per sample when quantified with our plasmid standard (18). The selected primers and probe of the gltA gene specific only for R. prowazekii were as follows: RproF (5'-TCGGTAAAGATGTAATCGATATAAG-3'), RproR (5'-CATATCCTCGATACCATAATATGC-3') and Rp.probe (FAM-ACTTTACTTATGATCCGGGTTTTATGTAMRA), leading to a PCR product size of 154 bp. When qPCR assay was used, only R. prowazekii strains were positive, whereas the standard PCR assay detected all rickettsial rickettsial /rick·ett·si·al/ (ri-ket´se-al) pertaining to or caused by rickettsiae.
Relating to, or caused by a member of the genus Rickettsia. species (Table). The standard PCR assay was positive for all 20 laboratory-infected lice (R. typhi or R. prowazekii), while qPCR assay was positive only for the 10 R. prowazekii laboratory-infected lice. Finally, blood samples obtained from our experimental model of R. prowazekii-infected mice at days 3 and 6 PI were also positive by using the protocol described above. The mean number of cycle thresholds (Ct value) for mice sampled at day 3 PI was 32.47 [+ or -] 2.11; at day 6 PI, the Ct value was of 35.52 [+ or -] 2.01 (p = 0.001). All uninfected lice, B. recurrentis--infected lice, and mice samples were negative with both assays.
The sensitivity of qPCR and the standard PCR was determined by using 10-fold serial dilutions of our known R. prowazekii inoculum (1.16 x [10.sup.6] DNA copies per sample). The sensitivity of the qPCR was increased 10-fold over that of the standard PCR. Compared to our plasmid standard, the cutoff detection of the qPCR was 1-5 copies per sample, whereas the cutoff detection was >10 copies for the standard PCR.
Among the 31 lice from Rwanda sampled in 2004, 17 were positive by real-time PCR, whereas only 10 of these 17 lice were positive by standard PCR. The latter 10 samples hada mean number of 1,300 DNA copies (Ct value 26.82-35.22). The 7 samples positive only by real-time PCR had a mean number of 8.5 DNA copies (Ct value 33.72-38.73). The real-time PCR therefore appears to be more sensitive. However, this difference was not significant (p = 0.07) perhaps because of the small number of tested lice. Finally, 5 of the 30 lice received from Rwanda in June 2005 were positive when the qPCR was used (Table).
We developed a real-time quantitative PCR for specific detection of R. prowazekii. The selected primers and probe were 100% complementary to R. prowazekii only and to no other rickettsial strains. We confirmed the specificity of these primers and probe on rickettsial isolates and other common bacteria and repeated the experiments 4 times without discrepancies. Real-time quantitative PCR for rickettsiae was first developed to test antimicrobial drug susceptibility (24) and then was used to detect R. rickettsii and closely related spotted fever spot·ted fever
A tick typhus caused by Rickettsia rickettsii, such as Rocky Mountain spotted fever.
spotted fever Rocky Mountain spotted fever, see there group rickettsiae (17) or R. prowazekii strains (25).
Our assay has a greater sensitivity than the standard assay, with a cut-off detection of only 1 to 5 DNA copies per sample, as measured comparatively to plasmid DNA Noun 1. plasmid DNA - a small cellular inclusion consisting of a ring of DNA that is not in a chromosome but is capable of autonomous replication
plasmid quantification. The sensitivity found with our standard PCR has been previously estimated at 1-10 DNA copies of the gene (23). The first use of standard PCR for detecting R. prowazekii using primers derived from the 17-kDa antigen sequence hada cut-off detection of as few as 30 rickettsiae (26). Cutoff detection of rickettsiae with real-time quantitative PCR ranges from 5 copies (17) to 10 copies (25). Using our LightCycler assay, we detected an extra 7 samples in lice from Rwanda as compared to standard PCR. Only 1 report exists of real-time detection of R. prowazekii using molecular beacon Molecular beacons are oligonucleotide probes that can report the presence of specific nucleic acids in homogenous solutions. The terms more often used is molecular beacon probes. probes targeting the ompB gene (25). In this report, only 2 R. prowazekii strains were tested (25). Moreover, we showed that R. prowazekii can be amplified from blood of experimentally infected mice. This experimental model of R. prowazekii infection and the ability to quantify the bacteria with the real-time PCR could be used to better study the pathogenesis of the organism. We found in this mouse model that the number of bacteria in blood was lower at day 6 PI than that at day 3 PI, which suggests that mice can eradicate infection at this dose.
The assay we describe can be performed wherever a real-time quantitative PCR machine is available. The reagents and the machine are standardized; this method gives rapid results (sequencing is not necessary) and decreases the likelihood of error. This assay was applied successfully in lice received from Rwanda in June 2005. Indeed, using our assay we were able to alert the World Health Organization of the presence of R. prowazekii-positive lice within 1 working day.
Because body lice and their associated diseases are generally encountered in areas where medical and biologic assistance is limited, local assessment of their roles as sources of infection is difficult. Lice are easy to collect and to transport to reference laboratories, where suitable molecular biologic approaches can be used (23). Although sucking lice die within 24 h of their final blood meal, the infecting bacterial DNA will remain intact for extraction for several weeks if the samples are kept dry (15). Upon arrival in the laboratory, the lice can be processed very quickly, and a diagnosis can be established rapidly (DNA extraction DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis. Outline of a DNA extraction
There are three basic steps in a DNA extraction, the details of which may vary depending on the type of sample and any substances that may and LightCycler PCR take [approximately equal to] 5 h). Several weeks are necessary to obtain bacterial culture and serologic results, and those procedures do not always highlight the presence of bacteria. The usefulness of bacterial DNA detection in lice by PCR has been demonstrated by recent investigations. In central Africa, large outbreaks of lice infections occurred during civil wars in Burundi, Rwanda, and Zaire and preceded the outbreak of epidemic typhus by 2 years (4). Finally, our data obtained in experimentally infected mice suggest that real-time PCR could also be useful for detecting R. prowazekii directly from blood specimens. Because our assay is highly standardized and easily adaptable anywhere and anytime, it could improve epidemic typhus surveillance in public health programs, especially for countries with underdiagnosed or unrecognized human cases (4).
We thank Esther Platt for reviewing the manuscript.
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The complete set of proteins that are produced by the genes of an organism.
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pertaining to or emanating from serology.
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Sanela Svraka, * Jean-Marc Rolain, * Yassina Bechah, * John Gatabazi, ([dagger]) and Didier Raoult *
* Universite de la Mediterranee, Marseilles, France; and ([dagger]) National Reference Laboratory, Kigali, Rwanda
Ms. Svraka is completing her PhD fellowship at National Institute for Public Health and Environment in Bilthoven, the Netherlands.
Address for correspondence: Didier Raoult, 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, IFR IFR
instrument flight rules 48, Faculte de Medecine, Universite de la Mediterranee, 27 Boulevard Jean Moulin Jean Moulin (June 20, 1899–July 8, 1943) was a high-profile member of the French Resistance during World War II. He is remembered today as an emblem of the Resistance primarily due to his courage and death at the hands of the Germans. , 13385 Marseilles Cedex 05, France; fax: 33491-83-03-90; email: Didier.Raoult@medecine.univ-mrs.fr
Table. Strains used in real-time PCR * Strains Source Rickettsia prowazekii Breinl ATCC R. prowazekii Evir UR R. prowazekii BatnaRp22 UR R. prowazekii Russian sample UR R. typhi Wilmington ATCC R. massiliae Mtul ATCC R. montanensis ATCC "R. aeschlimanii" UR R. massiliae strain Bar 29 UR R. helvetica C6P9 ATCC R. felis UR "R. sibirica mongolitimonae" UR R. rickettssii ATCC R. conorii moroccan ATCC R. sibirica sibirica 246 ATCC R. conorii subsp. israelensis CDC1 G.A. Dasch R. africae ESF-5 UR R. japonica YM ATCC Thai tick typhus rickettsia G.A. Dasch R. slovaca UR R. conorii subsp. caspia A-167 UR R. australis Phillips G.A. Dasch R. honei RB GRIC Rickettsia sp. AT1 UR R. bellii 369L42-1 D.H. Walker 31 lice from Rwanda (2004) J. Bosco 30 lice from Rwanda (2005) J. Gatabazi R. prowazekii laboratory-infected lice UR R. typhi laboratory-infected lice UR R. prowazekii-infected BALB/C mice UR B. recurrentis laboratory-infected lice UR Borrelia recurrentis ATCC Escherichia coli CIP Proteus mirabilis CIP Staphylococcus aureus CIP Streptococcus salivarius CIP Orientia tsutsugamushi CIP Streptococcus pyogenes CIP Mycobacterium xenopi CIP Chlamydia trachomatis Human isolate Propionibacterium acnes UR HGE agent ATCC Bartonella quintana Oklahoma ATCC Tropheryma whipplei Twist UR M. tuberculosis CIP Strains Standard PCR Rickettsia prowazekii Breinl + R. prowazekii Evir + R. prowazekii BatnaRp22 + R. prowazekii Russian sample + R. typhi Wilmington + R. massiliae Mtul + R. montanensis + "R. aeschlimanii" + R. massiliae strain Bar 29 + R. helvetica C6P9 + R. felis + "R. sibirica mongolitimonae" + R. rickettssii + R. conorii moroccan + R. sibirica sibirica 246 + R. conorii subsp. israelensis CDC1 + R. africae ESF-5 + R. japonica YM + Thai tick typhus rickettsia + R. slovaca + R. conorii subsp. caspia A-167 + R. australis Phillips + R. honei RB + Rickettsia sp. AT1 + R. bellii 369L42-1 + 31 lice from Rwanda (2004) + (10/31) ([dagger]) 30 lice from Rwanda (2005) ND R. prowazekii laboratory-infected lice +(10/10) R. typhi laboratory-infected lice +(10/10) R. prowazekii-infected BALB/C mice +(12/12) ([double dagger]) B. recurrentis laboratory-infected lice -(0/10) Borrelia recurrentis - Escherichia coli - Proteus mirabilis - Staphylococcus aureus - Streptococcus salivarius - Orientia tsutsugamushi + Streptococcus pyogenes - Mycobacterium xenopi - Chlamydia trachomatis - Propionibacterium acnes - HGE agent - Bartonella quintana Oklahoma - Tropheryma whipplei Twist - M. tuberculosis - Strains LC PCR assay Rickettsia prowazekii Breinl + R. prowazekii Evir + R. prowazekii BatnaRp22 + R. prowazekii Russian sample + R. typhi Wilmington - R. massiliae Mtul - R. montanensis - "R. aeschlimanii" - R. massiliae strain Bar 29 - R. helvetica C6P9 - R. felis - "R. sibirica mongolitimonae" - R. rickettssii - R. conorii moroccan - R. sibirica sibirica 246 - R. conorii subsp. israelensis CDC1 - R. africae ESF-5 - R. japonica YM - Thai tick typhus rickettsia - R. slovaca - R. conorii subsp. caspia A-167 - R. australis Phillips - R. honei RB - Rickettsia sp. AT1 - R. bellii 369L42-1 - 31 lice from Rwanda (2004) +(17/31) ([dagger]) 30 lice from Rwanda (2005) +(5/30) R. prowazekii laboratory-infected lice +(10/10) R. typhi laboratory-infected lice - R. prowazekii-infected BALB/C mice +(12/12) ([double dagger]) B. recurrentis laboratory-infected lice -(0/10) Borrelia recurrentis - Escherichia coli - Proteus mirabilis - Staphylococcus aureus - Streptococcus salivarius - Orientia tsutsugamushi - Streptococcus pyogenes - Mycobacterium xenopi - Chlamydia trachomatis - Propionibacterium acnes - HGE agent - Bartonella quintana Oklahoma - Tropheryma whipplei Twist - M. tuberculosis - * PCR, polymerase chain reaction; LC, LightCycler; ATCC, American Type Culture Collection, Rockville, MD, USA; GRIC, Gamaleya Research Institute Collection; G.A. Dasch, Naval Medical Research Institute, Bethesda, MD, USA; UR, Unite des Rickettsias, CNRS UPRES A, Marseille, France; D.H. Walker, University of Texas, Galveston; CIP, Collection Institute Pasteur, Paris, France; HGE, human granulocytic ehrlichiosis. ([dagger]) Number of positive lice/total number of tested lice. ([double dagger]) PCR for each mouse was positive in blood at days 3 and 6 postinfection (for cycle thresholds see results in text).