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Comparative evaluation of a commercial enzyme immunoassay for the detection of human antibody to Rickettsia typhi.

A commercial enzyme immunoassay kit called the Dip-S-Ticks (DS) for the detection of total immunoglobulin (Ig) G and IgM human antibodies to Rickettsia typhi was evaluated. In tests with 340 serum samples from patients with diagnosed cases of rickettsial diseases, patients suffering from other febrile illnesses, and normal subjects, the DS compared favorably with the standard indirect fluorescent-antibody (IFA) test. At IFA cutoff titers of > or = 1.64 and > or = 1:128, the DS showed sensitivities of 88.2 and 91.4% and specificities of 91.8 and 87.7%, respectively. The DS test correlated significantly with both the IFA IgG (r = 0.84, P < 0.0005) and IgM (r = 0.63, P < 0.0005) titers. Only 80% of IgG and 82% of IgM IFA readings determined by two technicians were within one dilution, while the DS was more reliable, with 100% within one dot. The rapidity, reliability, and simplicity of the DS suggest that it is a suitable test for use in clinical laboratories unable to perform the IFA test.

Antibodies, Bacterial↗

Evaluation ofprimary blood monocyte and bone marrow cell culture for the isolation of Rickettsia rickettsii.

Rickettsia rickettsii was isolated from experimentally infected guinea pigs by culture of blood monocytes and bone marrow cells, and from experimentally infected rhesus monkeys by blood monocyte culture. Rickettsiae were identified in monocyte-macrophage monolayers stained by Giménez or flourescent antibody techniques. A total of 78 culture attempts were made from 20 guinea pigs and 16 monkeys. The success of isolation of R. rickettsii in culture was positively correlated with the numbers of rickettsiae present in the blood and bone marrow. in cultures derived from infected guinea pigs, rickettsiae were usually observed after 5 to 7 days of culture, and in monkeys monocyte cultures they were usually observed within 3 to 5 days. Positive cultures were derived from guinea pigs and monkeys as early as the first day of fever and 1 to 3 days before the appearance of other clinical signs. Monocyte cultures became negative with the resolution of rickettsemia and concomitantly with the appearance of serum antibody. Monocyte culture isolation of R. rickettsii may be as sensitive for the detection of rickettsiae in blood and marrow as the intraperitoneal inoculation of guinea pigs or the plaque assay technique. Because of the simplicity of the method and because rickettsiae were often identified within 3 to 5 days after initiation, the monocyte culture technique may be useful in the early diagnois of human rickettsial disease.

Animals↗

von Willebrand factor release and thrombomodulin and tissue factor expression in Rickettsia conorii-infected endothelial cells.

Mediterranean spotted fever, a tick-borne rickettsiosis caused by Rickettsia conorii, may lead to small-vessel or deep-vein thrombosis. In order to evaluate the role of endothelial cell alteration in this lesion, we infected human endothelial cells derived from umbilical veins with R. conorii. We report the induction of two previously unreported prothrombotic mechanisms in rickettsial disease: (i) a progressive decline in thrombomodulin antigen and (ii) early expression of tissue factor, and, as described for R. rickettsii infection, later release of von Willebrand factor from Weibel-Palade bodies. Thrombomodulin expression in infected endothelial cells, measured by the thrombin-dependent activation of protein C or flow cytometric analysis, decreased steadily between 4 and 24 h after inoculation with rickettsiae. R. conorii infection induced tissue factor expression, measured by clotting assay and flow cytometric analysis, which was detectable 2 h postinoculation, reached its maximum 4 h postinoculation, and progressively decreased thereafter. Infection resulted in a relatively late release of von Willebrand factor antigen into the culture medium. A double-label immunofluorescence assay for the simultaneous evaluation of von Willebrand factor and R. conorii showed that the depletion of cytoplasmic von Willebrand factor stored in Weibel-Palade bodies was due to a direct effect of the intracellular R. conorii. These disturbances of endothelial function observed with R. conorii-infected cells may provide a paradigm for the elucidation of thrombotic pathobiology with Mediterranean spotted fever.

Cells, Cultured↗

Alternatives to the standardized laboratory branch complement fixation test for detection of antibodies to Coccidioides immitis.

Antibody titers to Coccidioides immitis, using coccidioidin antigen, were determined by three methods: the standardized Laboratory Branch complement fixation method (LBCF), a modified version of the Viral and Rickettsial Disease Laboratory complement fixation test (VRDL-CF), and a quantitative immunodiffusion test (QID). Of the 133 samples evaluated, 72 were negative by each method and 57 (42 serum samples, 15 cerebrospinal fluid samples) were positive by all three methods. Four additional specimens (1 serum sample, 3 cerebrospinal fluid samples) were positive by QID alone. All positive patients were diagnosed clinically as having pulmonary or extrapulmonary coccidioidomycosis or both. When titers from two methods were compared, the agreement within +/- 1 dilution was VRDL-CF/QID, 88.5%; VRDL-CF/LBCF, 85.2%; and LBCF/QID, 82.0%. The agreement of these methods within +/- 2 dilutions was VRDL-CF/QID, 98.4%; VRDL-CF/LBCF, 96.7%; and LBCF/QID, 93.4%. The VRDL-CF and QID methods are simpler to perform; however, they are yet unrecognized as suitable alternatives to the more cumbersome LBCF. Our data show that they should be considered as options for C. immitis serology.

Antibodies, Fungal↗

Addition of monoclonal antibodies specific for Rickettsia akari to the rickettsial diagnostic panel.

Monoclonal antibodies were produced from mice infected with Rickettsia akari (the etiologic agent of rickettsialpox) and evaluated for specificity in indirect fluorescent-antibody tests with 23 different rickettsial antigens. Of the nine antibodies that were evaluated, two were specific for R. akari and four reacted with R. akari and all other spotted fever group rickettsiae. The remaining three antibodies reacted with some, but not all, members of the spotted fever group. None of the antibodies reacted with typhus, scrub typhus, trench fever, or Q fever rickettsiae. Adding these antibodies to the list of available diagnostic reagents will facilitate identification of rickettsial diseases, particularly those caused by members of the spotted fever group, where the clinical presentations are similar and the etiologic agents are closely related antigenically.

Animals↗

A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks.

Heartwater, caused by Cowdria ruminantium and transmitted by ticks of the genus Amblyomma, is a constraint to ruminant animal production in sub-Saharan Africa. This rickettsial disease could spread from endemically infected areas of sub-Saharan Africa and certain Caribbean islands to other countries, including the United States, in which Amblyomma ticks exist. To detect C. ruminantium in tick vectors and animals, we made DNA probes from C. ruminantium DNA isolated from endothelial cell cultures. Two clones were evaluated; pCS20 from Crystal Springs (Zimbabwe) strain DNA had a 1,306-bp insert, and pCR9 from Kiswani (Kenya) strain DNA had a 754-bp insert. Both DNA probes detected 1 ng of Crystal Springs DNA; however, the pCS20 probe had a 10-fold-greater ability to discriminate between C. ruminantium DNA and DNA from other organisms. Also, the pCS20 probe did not hybridize to 400 ng (highest amount tested) of DNA from bovine cells, 3 protozoa, 3 rickettsiae, and 12 bacteria. In all experiments, C. ruminantium DNA was detected in midguts from 99 of 160 Amblyomma variegatum nymphs infected as larvae and in midguts from 38 of 80 adult ticks infected as nymphs but not in midguts from control nymphs and adults. The presence of C. ruminantium in nymphs and adults was confirmed by transmission of heartwater to goats. The DNA sequences of both probes were determined; synthetic oligonucleotides from pCS20 are recommended as DNA probes for C. ruminantium.

Animals↗

Human and tick spotted fever group Rickettsia isolates from Israel: a genotypic analysis.

The genomes of spotted fever group rickettsiae isolated in different geographical areas of Israel (two from ticks and four from humans, obtained over a span of 20 years) were studied by polymerase chain reaction (PCR) and restriction endonuclease fragment length polymorphism (RFLP) analysis. The human isolates were obtained from patients suffering from rickettsial disease of different degrees of severity. The PCR products obtained with five pairs of oligonucleotide primers (two primer sets derived from the 190-kDa polypeptide gene and three from the 120-kDa polypeptide gene) and cleaved with restriction endonucleases were used to study the Israeli isolates and reference Rickettsia conorii isolates. Subtle differences between the PCR-RFLP patterns of Israeli isolates and the two R. conorii reference strains (Moroccan and no. 7) were seen when the PCR products derived from the 190-kDa gene-derived primer sets were digested. All of the Israeli isolates were identical by RFLP analysis using all of the primer sets. This study showed that the Israeli spotted fever group isolates (from both ticks and humans) were genetically homogeneous by the criteria used in this study, despite the time and location differences in their original isolation, and different as a group from R. conorii.

Animals↗

Enzyme-linked immunosorbent assay for detection of human immunoglobulin G to lipopolysaccharide of spotted fever group rickettsiae.

An enzyme-linked immunosorbent assay for detecting human immunoglobulin G to spotted fever group rickettsiae was developed and tested. The assay uses proteinase K-resistant material, characteristic of the rickettsial lipopolysaccharides shown to be group specific by immunoblots, as the antigen. The results indicate that the assay provides a sensitive, yet specific, alternative method for diagnosing rickettsial diseases.

Endopeptidase K↗

Immunodiagnosis of Ehrlichia canis infection with recombinant proteins.

Ehrlichia canis causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive E. canis proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive E. canis surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in E. chaffeensis DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with E. chaffeensis as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for E. canis, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for E. canis infections.

Amino Acid Sequence↗

Gastrointestinal and hepatic manifestations of human ehrlichiosis: 8 cases and a review of the literature.

BACKGROUND: Ehrlichiosis has emerged as an increasingly recognized tick-borne rickettsial disease. It can affect multiple organs including the gastrointestinal tract and liver. Signs and symptoms include abdominal pain, nausea, vomiting, diarrhea, jaundice, and hepatosplenomegaly. Patients commonly have elevated liver transaminases early in the course of illness as well as leukopenia and thrombocytopenia. If not diagnosed and treated in a timely fashion, ehrlichiosis can progress to multiorgan failure. METHODS: Between 1992 and 1998, 8 patients meeting the CDC criteria for ehrlichiosis were diagnosed and treated at the John L. McClellan Memorial Veterans Hospital and University Hospital in Little Rock, Ark. These cases and English-language articles relating to gastrointestinal and hepatic manifestations of ehrlichiosis, identified by searching Medline and manually reviewing bibliographies of retrieved articles, are reviewed. RESULTS: Seven of the eight patients had elevated transaminases on presentation which increased rapidly initially and slowly returned to normal. Three of our patients had hyperbilirubinemia. One of the eight patients presented with hepatomegaly. Three had hyperbilirubinemia with a peak bilirubin of 13.8 mg/dl. Two patients (25%) had a rash on presentation. All 8 patients were treated with and responded to doxycycline. One developed multiorgan failure but eventually recovered with intensive medical care and doxycycline. CONCLUSION: To avoid treatment delay and serious complications, in the appropriate clinical setting ehrlichiosis should be considered as a cause of elevated liver transaminases.

Adult↗

Two new candidate adenovirus serotypes.

Two new adenovirus (AV) subgenus D serotypes, which we propose as AV candidates 48 and 49, are described. Both AV 48 and 49 were unique by serum neutralization and the pattern of restriction endonuclease fragments. By the hemagglutination inhibition assay there was a significant reciprocal cross between AV 48 and 44 and one-way crosses between AV 48 and AV 37 sera and between AV 30 and AV 49 sera. Eleven AV 48 and 5 AV 49 isolates, mainly from AIDS patients, have been identified at the Viral and Rickettsial Disease Laboratory.

Acquired Immunodeficiency Syndrome↗

Visual loss in a returning traveller with tick typhus.

Rickettsial diseases are increasingly found world-wide and should be considered in febrile patients returning from abroad. This case report describes the vasculitic complications of a patient returning from the Republic of South Africa with tick typhus.

Adult↗

Editorial

As dermatologists, we have all been active in educating patients about sun awareness and sun protection. This is even more important for children, as childhood exposure to ultraviolet light is a significant risk for both melanoma and nonmelanoma skin cancers. The importance of an educational approach in appropriate sun awareness in childhood is further underscored by the recent findings by Rivers et al., in the Vancouver Moles Cohort study, presented at the 1999 American Academy of Dermatology meeting. In a placebo-controlled trial, the findings of Rivers et al. clearly demonstrated that the use of sunscreens can significantly decrease the formation of nevae in children, providing further evidence to support sun awareness education initiatives. The lead article by Gooderham and Guenther in the Basic and Clinical Sciences section evaluates the effectiveness of a particular sun awareness program, and gives valuable insights into how more effective approaches may be used in the future. In addition to ultraviolet light playing a causal role in cutaneous malignancies, it is known to induce a number of other skin problems. One particularly difficult group of disorders is the photosensitive dermatoses, including solar urticaria. Bissonnette et al. describe an innovative approach to the management of refractory solar urticaria with plasma exchange. In the Grand Rounds section, Strauss et al. review the case of an acute SLE and give an insightful discussion related to bullous eruptions in acutely ill children. The mechanism of ultraviolet-light-induced carcinogenesis involves UV-induced DNA damage. Over the past decade, it has become clear that tumour suppressor genes can regulate these processes. In the Review section, Tron et al. discuss the role of the suppressor gene p53, which is mutated or lost in nonmelanoma skin cancer. P53 is crucial in protecting keratinocytes from the harmful effects of ultraviolet radiation, and in their instructive article, these authors use gene-targeted mutant mice lacking p53 to further evaluate the role in UV-induced DNA damage. With the warm weather upon us, we are spending more time in the outdoors and, as a result, are exposed to a vast number of environmental onslaughts. These include such things as Rickettsial disease, summarized in our CME section Summary Notes. Furthermore, in a comprehensive review, Dr. Sasseville examines another outdoor threat as he delineates the wide spectrum of plant contact dermatitis. This represents an important and in-depth reference on phytodermatitis. Our specialty, and indeed all of medicine, is being dramatically altered by recent advances in our understanding of disease at a molecular level. This new understanding of disease has led to the potential of modifying gene expression through the use of gene therapy. This is particularly attractive in skin disease, where gene therapy can be delivered quite readily through the skin. This advancement is insightfully discussed in the article by Somani et al., "Gene Therapy and Dermatology," which is both valuable for the cognoscenti and noncognoscenti alike, and serves as an important reference work in this area.

Journal Article↗

Potential roles for regulatory oxygenases in rickettsial pathogenesis.

Clinical and experimental evidence suggests an important role for oxidative stress and associated cellular defense mechanisms in the pathogenesis of vasculopathic rickettsioses. Our laboratory has reported that R. rickettsii infection of endothelial cells in vitro induces the expression of HO-1, the inducible isoform of the antioxidant defense enzyme heme oxygenase. HO-1 plays a critical role in maintaining the integrity of the vasculature and controls the functioning of the cyclooxygenase (COX) system. This study was undertaken to investigate the expression of COX and HO isozymes during in vitro infection of EC with two major representatives of spotted fever group Rickettsia species. The mRNA expression of COX-2 was significantly increased in endothelial cells infected with R. rickettsii and R. conorii, while that of COX-1 remained unaffected. Western blot analysis using total protein lysates from infected endothelial cells and corresponding uninfected controls further confirmed specific induction of COX-2 in response to infection. ELISA measurements on culture supernatants also suggested enhanced secretion of 6-keto PGF(1alpha) (stable hydrolysis product of PGI(2) and PGE(2). As a functional consequence of HO-1 upregulation, increased expression of the iron storage protein ferritin following R. rickettsii and R. conorii infection was also evident. Since products of HO-1 and COX-2 reactions govern a variety of physiologically important functions in the vasculature, further studies to define their regulation in the host cell should provide useful insights into the pathogenesis of rickettsial diseases.

Cells, Cultured↗

Rickettsioses in Japan and the far East.

Three rickettsial diseases are known to exist in Japan currently: Japanese spotted fever (JSF), Tsutsugamushi disease (TD; scrub typhus), and Q fever. Since April 1999, the system for infection control and prevention in Japan has changed drastically. JSF, Q fever, and TD, as emerging infectious diseases, are designated as national notifiable diseases.The geographic distribution of JSF patients is along the coast of central and southwestern Japan, whereas TD and Q fever occur almost all over the country. The number of JSF patients reported was 216 cases during 1984-1998 and 268 cases, under the revised law, in 1999-2004. About 300-1000 cases of TD occur every year, and 7-46 cases of Q fever in 1999-2004. The number of cases of JSF and its endemic area are gradually increasing. There was only one fatality due to JSF until 2003, whereas two patients died of JSF in 2004, so JSF is still a life-threatening disease in Japan. Treatment of fulminant JSF consists of prompt administration of a combination of tetracycline and quinolone. Recent tick surveys revealed that the most probable vectors of JSF are Haemophysalis flava and Haemophysalis hystericis. In addition to R. japonica, two serotypes or species of spotted fever group rickettsiae have been isolated from ticks in Japan; one is closely related to R. helvetica and the other is a new genotype of unknown genotype AT, which is closely related to a Slovakian genotype. These serotypes are of uncertain clinical significance. Epidemiology of rickettsioses in the Far East is mentioned briefly.

Anti-Bacterial Agents↗

Rickettsioses in Australia.

Australia, an island continent in the southern hemisphere, has a range of rickettsial diseases that include typhus group rickettsiae (Rickettsia typhi), spotted fever group rickettsiae (R. australis, R. honei), scrub typhus group rickettsiae (R. tsutsugamushi), and Q fever (C. burnetii). Our knowledge of Australian rickettsiae is expanding with the recognition of an expanded range of R. honei (Flinders Island spotted fever) to Tasmania and southeastern mainland Australia (not just on Flinders Island), and the detection of a new SFG species (or subspecies), tentatively named "R. marmionii" in the eastern half of Australia. This rickettsia causes both acute disease (7 cases, recognized so far) and is also associated (as a "R. marmionii" bacteriaemia) with patients having a chronic illness. The significance of the latter is under investigation. It may be a marker of autoimmune disease or chronic fatigue in some patients.

Animals↗

Ticks and tick-borne Rickettsiae surveillance in Montesinho Natural Park, Portugal.

This study constitutes the first contribution to the knowledge of tick dynamics and its implication in the epidemiology of rickettsial diseases in Montesinho Natural Park (MNP), Bragança district of Portugal. Of 76 ticks collected, 12 (15.8%) were Dermacentor (D.) marginatus, 36 (47.4%) D. reticulatus, and 28 (36.8%) Rhipicephalus (R.) sanguineus. Isolation assays were performed by shell-vial technique on 41 ticks. Israeli spotted fever strain was an isolate from R. sanguineus, and three isolates of Rickettsia slovaca were obtained from D. reticulatus. All 76 ticks were screened by PCR for Rickettsia sp., Ehrlichia (E.) chaffeensis, and Anaplasma (A.) phagocytophilum. Rickettsia RpA4 strain DNA was detected in 10 D. marginatus and 2 D. reticulatus, and Israeli spotted fever strain in 1 R. sanguineus. No E. chaffeensis or A. phagocytophilum infection was detected. New host records are provided for D. reticulatus. Also described for the first time in Portugal is the isolation of R. slovaca from D. reticulatus and the isolation of Israeli spotted fever strain from R. sanguineus. This confirms the association of the last rickettsiae strain with the same vector tick as previously described in Israel and Sicily.

Animals↗