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Decrease in human immunodeficiency virus type 1 load during acute dengue fever.

Rather than the expected increase in human immunodeficiency virus type 1 (HIV-1) load, there was transient suppression of HIV-1 replication during acute dengue infection in a 29-year-old Thai woman. Acute-phase (but not convalescent-phase) serum samples obtained from an HIV-1-uninfected patient with dengue fever reduced HIV-1 infectivity, as determined by a peripheral blood mononuclear cell assay, suggesting the possibility that HIV-1 replication is suppressed during acute dengue fever, as occurs during some cases of scrub typhus infection and measles.

Acute Disease↗

Bacteriostatic and bactericidal activity of levofloxacin against Rickettsia rickettsii, Rickettsia conorii, 'Israeli spotted fever group rickettsia' and Coxiella burnetii.

Levofloxacin, the L-isomer of ofloxacin, is approximately twice as active as ofloxacin against most Gram-positive and Gram-negative bacteria, and has improved intracellular pharmacokinetic and pharmacodynamic properties. The present work deals with the in-vitro activity of levofloxacin against the obligate intracellular bacteria Rickettsia rickettsii, Rickettsia conorii, 'Israeli spotted fever group rickettsia' (Israeli SFGR) and Coxiella burnetii. Fluoroquinolones, including ofloxacin, have previously been shown to be bacteriostatic against Rickettsia spp. and C. burnetii in vitro. They are reliable alternatives to tetracycline therapy for Mediterranean spotted fever, scrub typhus and acute Q fever. Levofloxacin was bacteriostatic against R. rickettsii, R. conorii and the Israeli SFGR at concentrations of 0.5-1 mg/L, as determined by both a plaque assay and a dye uptake assay. It was also bacteriostatic against C. burnetii isolates, including the Nine Mile, Priscilla and Q212 strains, at concentrations of 0.5-2 mg/L, as determined using the shell vial assay. Overall, levofloxacin could inhibit rickettsial growth at concentrations equal to or half of those necessary for growth inhibition by ofloxacin. Levofloxacin was not bactericidal against C. burnetii at concentrations up to 4 mg/L.

Anti-Infective Agents↗

Seasonal development of Leptotrombidium pallidum (Acari: Trombiculidae) observed by experimental rearing in the natural environment.

Engorged larvae of Leptotrombidium pallidum Nagayo, Miyagawa, Mitamura & Tamiya, the vector mite of scrub typhus in Japan, were reared by feeding them with fresh eggs of the collembolan Sinella curviseta Brook while confined in small plastic containers under natural conditions in a copse. The larvae were collected from wild rodents (Apodemus speciosus) in autumn 1985 and spring 1986. Adults were kept alive for 2 yr or longer. The larvae obtained in autumn became dormant in the cold winter season, and growth recommenced in the spring. Thus, the development of mites collected in April became synchronized with that of larvae obtained in the autumn. Most larvae developed into protonymphs in May, deutonymphs in June, tritonymphs in July, and adults in August. The females laid eggs in two consecutive summers. Some larvae collected in autumn were kept in a refrigerator until the following summer. They developed into deutonymphs, tritonymphs, or adults and then became dormant in the winter. Development restarted the next spring and all became adult by summer, when the females laid eggs. Under experimental conditions, all larvae are hatched in the autumn, unlike the natural situation in which two peaks of larval occurrence on wild rodents are observed in autumn and spring.

Animals↗

Rickettsia tsutsugamushi in chiggers (Acari: Trombiculidae) associated with rodents in central Thailand.

Chiggers were collected from rodents trapped at two military bases located 10 km apart in central Thailand. One site was swampy and nearly treeless and the other site was well-drained and partially wooded. Although 13 species of chiggers were collected, only three species were found to be positive for Rickettsia tsutsugamushi (Hayashi) Ogata using a direct fluorescent antibody test: Blankaartia acuscutellaris Walch (7.3% infected), Leptotrombidium delinese Walch (3.1% infected), and an undescribed species of Ascoschoengastia near A. indica Hirst (1.2% infected). This was the first record of R. tsutsugamushi in B. acuscutellaris. The Ascoschoengastia species occurred with equal frequency at the two study sites, L. deliense occurred more frequently at the well-drained site, and B. acuscutellaris occurred more frequently at the swampy site. Results suggest that there are important foci of scrub typhus in central Thailand and that B. acuscutellaris may be a vector in this area.

Animals↗

Detection and characterization of Rickettsia tsutsugamushi (Rickettsiales: Rickettsiaceae) in infected Leptotrombidium (Leptotrombidium) fletcheri chiggers (Acari: Trombiculidae) with the polymerase chain reaction.

We developed a method for detecting and characterizing the DNA of Rickettsia tsutsugamushi in chiggers (larval trombiculid mites) by polymerase chain reaction (PCR). Three procedures for extracting DNA from frozen chiggers were compared by evaluating the yield of PCR amplicand obtained with nine oligonucleotide primer pairs derived from the rickettsial 22 kD, 47 kD, groESL, 56 kD, and 110 kD antigen genes. Although extracts and primer pairs differed in amplification efficiency, R. tsutsugamushi DNA was successfully detected in extracts of colonized infected Leptotrombidium (Leptotrombidium) fletcheri (Wormersley & Heaslip) chiggers and in uninfected chigger extracts seeded with known amounts of Karp-strain rickettsiae. The 22 kD gene restriction fragment length polymorphisms (RFLP) observed in PCR amplicands from five rickettsial isolates obtained from the infected chigger colony over a 26-yr period were identical to those of PCR amplicands derived directly from infected chiggers taken from the same colony. This suggests that stable transmission of R. tsutsugamushi occurs in mites (62 generations), and isolates encompass the full genetic heterogeneity found in the chigger. PCR/RFLP analysis is an important new tool for investigating the complex epidemiology of scrub typhus rickettsiae in their mite vectors.

Animals↗

Seasonal development of Leptotrombidium akamushi (Acari: Trombiculidae) under field temperatures.

Engorged larvae of Leptotrombidium akamushi (Brumpt), a vector of scrub typhus, were reared in small plastic containers placed on the ground and fed fresh eggs of the collembolan Sinella curviseta Brook. Engorged larvae obtained in October developed into deutonymphs through protonymphs approximately 1 mo before winter and became dormant in the cold winter season (approximately 3 mo). Most deutonymphs developed into tritonymphs in April and adults in May. Females began laying eggs in mid-June and the numbers of unfed larvae showed a peak in August. The mites reared from July rapidly developed into adults by August, and laid eggs in September. Larvae were most abundant in October, and adults became dormant in the winter. The same adults laid eggs from early May to late June and, upon hatching, the larval population peaked in early July of the 2nd summer. Most larvae died before the 2nd winter. Eggs hatched approximately 3 wk after oviposition and longevity of unfed larvae was 2 mo. Because of this very short incubation period, L. akamushi larvae occur in the summer, whereas L. pallidum Nagayo, Miyagawa, Mitamura & Tamiya, and L. scutellare Nagayo, Miyagawa, Mitamura, Tamiya & Tenjin occur in the autumn, although 3 species lay eggs from May to August.

Animals↗

A new species of Leptotrombidium (Acari:Trombiculidae) collected in active rice fields in northern Thailand.

Leptotrombidium (Leptotrombidium) chiangraiensis Tanskul & Linthicum is described and illustrated as new from specimens collected from the rodents Rattus rattus (L., 1758), Rattus argentiventer (Robinson & Kloss, 1916), Rattus losea (Swinhoe, 1870), and Bandicota indica (Bechstein, 1800) in Chiangrai Province northern Thailand. The new species was collected in active rice fields and adjacent fruit plantation areas. The etiological agent of scrub typhus, Orientia (formerly Rickettsia) tsutsugamushi (Hayashi), has been isolated from patients who live and work in the same habitat where L. chiangraiensis is the predominant Leptotrombidium species.

Animals↗

Redescription of Leptotrombidium (Leptotrombidium) imphalum (Acari: Trombiculidae), with observations on bionomics and medical importance in northern Thailand.

Leptotrombidium (Leptotrombidium) imphalum Vercammen-Grandjean & Langston is redescribed and illustrated. Specimens were collected from the rodents Rattus rattus, Rattus losea, and Bandicota indica in Chiangrai Province, northern Thailand. The species was found on hosts collected on dikes at the margins of rice fields and in adjacent fruit plantations and along irrigation canals, especially in areas covered with the grasses Imperata cylindrica (lalang grass) and Saccharum arudinaceum. The etiological agent of scrub typhus, Orientia (formerly Rickettsia) tsutsugamushi has been isolated from L. (L.) imphalum, rodent hosts, and patients who live and work in the same habitats.

Animals↗

Seasonal occurrence of Leptotrombidium deliense (Acari: Trombiculidae) attached to sentinel rodents in an orchard near Bangkok, Thailand.

Leptotrombidium deliense Walch that attached to sentinel laboratory mice and the roof rat, Rattus rattus (L.), placed in an orchard habitat near Bangkok, Thailand, were studied between April 1993 and April 1995. A single L. deliense larva was attached to only 1 of 51 laboratory mice placed in the study area between April and September 1993. Overall, 89/202 (44.1%) R. rattus had 1 or more L. deliense larvae attached, and Orientia tsutsugamushi (Hayashi), the etiologic agent for scrub typhus, was isolated from liver/spleen samples of 2/202 (1.0%) rats placed in an endemic area for a single night. A total of 474 L. deliense attached to sentinel R. rattus, of which 314 larvae successfully fed to repletion and were recovered, and 2 (0.6%) of these were naturally infected with O. tsutsugamushi. The occurrence of L. deliense was influenced by rainfall, with more chiggers attached to rodents in the wetter months of the year. The study showed that the risk of exposure to infection with O. tsutsugamushi is greater during the wetter months of the year, and that only a relatively small number of chigger attachments are needed to infect potential hosts.

Animals↗

Comparison of PanBio dengue duo enzyme-linked immunosorbent assay (ELISA) and MRL dengue fever virus immunoglobulin M capture ELISA for diagnosis of dengue virus infections in Southeast Asia.

The performances of the MRL dengue fever virus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the PanBio Dengue Duo IgM capture and IgG capture ELISA were compared. Eighty sera from patients with dengue virus infections, 24 sera from patients with Japanese encephalitis (JE), and 78 sera from patients with nonflavivirus infections, such as malaria, typhoid, leptospirosis, and scrub typhus, were used. The MRL test showed superior sensitivity for dengue virus infections (94 versus 89%), while the PanBio test showed superior specificity for JE (79 versus 25%) and other infections (100 versus 91%). The PanBio ELISA showed better overall performance, as assessed by the sum of sensitivity and specificity (F value). When dengue virus and nonflavivirus infections were compared, F values of 189 and 185 were obtained for the PanBio and MRL tests, respectively, while when dengue virus infections and JE were compared, F values of 168 and 119 were obtained. The results obtained with individual sera in the PanBio and MRL IgM ELISAs showed good correlation, but this analysis revealed that the cutoff value of the MRL test was set well below that of the PanBio test. Comparing the sensitivity and specificity of the tests at different cutoff values (receiver-operator analysis) revealed that the MRL and PanBio IgM ELISAs performed similarly in distinguishing dengue virus from nonflavivirus infections, although the PanBio IgM ELISA showed significantly better distinction between dengue virus infections and JE. The implications of these findings for the laboratory diagnosis of dengue are discussed.

Adolescent↗

Exploitation of the endocytic pathway by Orientia tsutsugamushi in nonprofessional phagocytes.

Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular bacterium that requires the exploitation of the endocytic pathway in the host cell. We observed the localization of O. tsutsugamushi with clathrin or adaptor protein 2 within 30 min after the infection of nonprofessional phagocytes. We have further confirmed that the infectivity of O. tsutsugamushi is significantly reduced by drugs that block clathrin-mediated endocytosis but not by filipin III, an inhibitor that blocks caveola-mediated endocytosis. In the present study, with a confocal microscope, O. tsutsugamushi was sequentially colocalized with the early and late endosomal markers EEA1 and LAMP2, respectively, within 1 h after infection. The colocalization of O. tsutsugamushi organisms with EEA1 and LAMP2 gradually disappeared until 2 h postinfection, and then free O. tsutsugamushi organisms were found in the cytoplasm. When the acidification of endocytic vesicles was blocked by treating the cells with NH(4)Cl or bafilomycin A, the escape of O. tsutsugamushi organisms from the endocytic pathway was severely impaired, and the infectivity of O. tsutsugamushi was drastically reduced. To our knowledge, this is the first report that the invasion of O. tsutsugamushi is dependent on the clathrin-dependent endocytic pathway and the acidification process of the endocytic vesicles in nonprofessional phagocytes.

Animals↗

Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens.

The genetic and antigenic relatedness of eight antigens in three strains of Rickettsia tsutsugamushi has been studied by using recombinant organisms expressing epitopes of the 150-, 110-, 72-, 58-, 56-, 49-, 47-, and 20-kilodalton (kDa) polypeptide antigens of the Karp strain. Southern blot analysis of Karp, Kato, and Gilliam strain genomic DNA by using probes specific for each antigen class indicated that while strong homology exists between each of the corresponding antigen genes in these three strains, some restriction fragment length polymorphism exists. Antibodies affinity purified against each recombinant antigen class reacted with a comparably sized polypeptide in the Karp, Kato, and Gilliam strains in Western blots (immunoblots). Against more recent human isolates of R. tsutsugamushi, the affinity-purified antibodies against the 58-kDa recombinant antigen (anti-58-kDa) reacted with all nine isolates, anti-56-kDa reacted with eight of nine isolates, anti-47-kDa reacted with eight of nine isolates, anti-72-kDa reacted with eight of nine isolates, and anti-110-kDa reacted with four of nine isolates. Additional analysis indicated that the 110-kDa antigen may contain strain-specific epitopes similar to those previously reported for the 56-kDa polypeptide. Evidently, the strain heterogeneity among scrub typhus rickettsiae is a result of multiple components that exhibit variability in a background of strong homology.

Antigens, Bacterial↗

Growth characteristics and proteins of plaque-purified strains of Rickettsia tsutsugamushi.

Six plaque-purified strains of Rickettsia tsutsugamushi (Karp, Gilliam, Kato, JC472B, TA716, and TA763) that fall into three categories of virulence for mice were compared by several parameters. Five of the six strains formed plaques of identical size in mouse cells, but each of three strains tested (representing three mouse virulence types) had a different doubling time in mouse cell cultures. Neither of these properties correlated strictly with virulence in mice, although the avirulent TA716 strain replicated much more slowly than the more virulent Karp and Gilliam strains. R. tsutsugamushi strain heterogeneity was also manifested at the polypeptide level by migration rates in sodium dodecyl sulfate-polyacrylamide gels of three of the major scrub typhus antigens (Sta110, Sta56, and Sta47), with those of Sta110 differing most widely. As expected, immunoblotting with polyclonal mouse sera showed substantial cross-reactivity among the major antigens of the six strains. Similar tests with Karp-induced monoclonal antibodies (MAb) demonstrated that some epitopes on Sta110 and Sta56 were shared by fewer than the six strains, but they identified no epitope unique to Karp. In contrast to the ready demonstration of antigenic heterogeneity in Sta110 and Sta56, four of the five Sta47-specific MAb reacted well with Sta47 from each of the six strains; the remaining MAb bound Sta47 from Karp and the Karp-like JC472B strain more strongly than Sta47 from the other four strains. The MAb also were useful in indicating the possible occurrence of Sta47 as dimers and trimers, the presence of Sta110 (as well as Sta56 and Sta47) in the rickettsial membrane, and the apparent interaction of the putative heat shock protein Sta58 with Sta47 or Sta47-Sta56 complexes.

Animals↗

Mapping of antigenic determinant regions of the Bor56 protein of Orientia tsutsugamushi.

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.

Animals↗

Expression of chemokine genes in murine macrophages infected with Orientia tsutsugamushi.

Scrub typhus, caused by Orientia tsutsugamushi infection, is characterized by local as well as systemic inflammatory manifestations. Inflammation is initiated by O. tsutsugamushi-infected macrophages and endothelial cells in the dermis. We investigated the regulation of chemokine induction in macrophage cell line J774A.1 in response to O. tsutsugamushi infection. The mRNAs for macrophage inflammatory proteins 1alpha/beta (MIP-1alpha/beta), MIP-2, and macrophage chemoattractant protein 1 were induced within 30 min, and their levels showed a transitory peak for 3 to 12 h. However, the lymphotactin, eotaxin, gamma interferon-inducible protein 10, and T-cell activation gene 3 mRNAs were not detected by RNase protection assays. Heat-killed O. tsutsugamushi induced a similar extent of chemokine responses. Induction of the chemokine genes was not blocked by the eukaryotic protein synthesis inhibitor cycloheximide, suggesting that de novo synthesis of host cell protein is not required for these transcriptional responses. The induction of chemokine mRNAs by O. tsutsugamushi was blocked by the inhibitors of NF-kappaB activation. Furthermore, O. tsutsugamushi induced the nuclear translocation and activation of NF-kappaB. These results demonstrate that heat-stable molecules of O. tsutsugamushi induce a subset of chemokine genes and that induction involves activation of the transcription factor NF-kappaB.

Animals↗

Expression of chemokine genes in human dermal microvascular endothelial cell lines infected with Orientia tsutsugamushi.

Scrub typhus, caused by Orientia tsutsugamushi, is characterized by local as well as systemic inflammatory manifestations. The main pathologic change is focal or disseminated multiorgan vasculitis, which is caused by the destruction of endothelial cells and perivascular infiltration of leukocytes. We investigated the regulation of chemokine induction in transformed human dermal microvascular endothelial cells (HMEC-1) in response to O. tsutsugamushi infection. The monocyte chemoattractant protein-1 (MCP-1) and interleukin 8 (IL-8) mRNAs were induced, and their levels showed a transitory peak at 3 and 6 h, respectively. The RANTES transcript was detected at 6 h after infection, with increased levels evident by 48 h. The induction of the MCP-1 and IL-8 genes was not blocked by cycloheximide, suggesting that de novo protein synthesis of host cell proteins is not required for their transcriptional activation. Heat- or UV-inactivated O. tsutsugamushi induced a similar extent of MCP-1 and IL-8 responses. The induction of MCP-1 and IL-8 transcripts in the endothelial cells by O. tsutsugamushi was not blocked by the inhibitors of NF-kappaB. Furthermore, the activation of NF-kappaB was not detected in HMEC-1 stimulated with O. tsutsugamushi. These results demonstrate that heat-stable molecules of O. tsutsugamushi induce the MCP-1 and IL-8 genes and the induction of the chemokine genes may be mediated by an NF-kappaB independent mechanism. We also showed that another major transcription factor, activator protein-1 (AP-1), was up-regulated in HMEC-1 after O. tsutsugamushi infection. This suggests the possible involvement of AP-1 in the chemokine gene expression.

Cells, Cultured↗

Evaluation of immunoglobulin M (IgM) and IgG rapid cassette test kits for diagnosis of melioidosis in an area of endemicity.

An enzyme-linked immunosorbent assay-based rapid cassette immunoglobulin G (IgG) and IgM immunochromogenic test kit was compared to the indirect hemagglutination test (IHA) for the diagnosis of acute melioidosis in northeastern Thailand. Admission sera from 70 culture-confirmed septicemic melioidosis patients and 30 patients with localized infections were tested. As a control group, 80 patients with other acute febrile illnesses (other bacterial infections, leptospirosis, or scrub typhus) and 119 healthy individuals were tested. The diagnostic sensitivity of the IgG and IgM tests and the IHA test were 79, 67, and 72%, respectively, with corresponding specificities of 90, 80, and 68%. This kit represents an improvement over IHA for the diagnosis of melioidosis an area of endemicity although, as with other serological tests, it has reduced diagnostic utility in a population with high background seropositivity.

Bacterial Infections↗

Failure of cyclophosphamide to significantly enhance isolation of Rickettsia tsutsugamushi from wild Philippine rats.

Treatment of wild rats from a known scrub typhus focus in the Philippines with cyclophosphamide did not significantly enhance the isolation rate of Rickettsia tsutsugamushi using mouse inoculation techniques. Similarly, cyclophosphamide treatment of mice inoculated with organs of wild rats did not increase the recovery of rickettsiae, and isolation of rickettsiae was about equal from biopsies obtained before and after immunosuppression of wild rats.

Animals↗