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Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by the SFTS virus (SFTSV). Clinical symptoms of SFTS often involve encephalopathy and other central neurological symptoms, particularly in seriously ill patients; however, pathogenesis of encephalopathy by SFTSV is largely unknown. Herein, we present case reports of three patients with SFTS, complicated by encephalopathy, admitted to Tokushima University hospital: one patient was a 63-year-old man, while the other two were 83- and 86-year-old women. All of them developed disturbance of consciousness around the 7th day post onset of fever. After methylprednisolone pulse therapy of 500 mg/day, all of them recovered without any neurological sequelae. SFTSV genome was not detected in the cerebrospinal fluid of 2 out of the 3 patients that were available for examination. In these patients, disturbance of consciousness seemed to be an indirect effect of the cytokine storm triggered by SFTSV infection. We propose that short-term glucocorticoid therapy might be beneficial in the treatment of encephalopathy during early phase of SFTSV infection.

We performed pre-embedding electron microscopic study for visualizing the antigen and genome of severe fever with thrombocytopenia syndrome (SFTS) virus in the cytoplasm of macrophages of the human splenic red pulp, both requesting preheating treatment of sections. To pursue this, coated glass slides with unique characteristics are needed. Namely, during staining they must prevent detaching off sections, but after staining the sections must be transferred to epoxy resin. Aminopropyltriexoxysilane-coated glass slides, widely used for immunostaining, were resistant to transfer to epoxy resin. In contrast, coated glass slides designated as Thinlayer Advanced Cytology Assay System (TACAS) were suitable for this purpose. The technique is also applicable to the coated glass slide-requiring cytology practice, in which immunocytochemical evaluation is needed after cell transfer to another glass slide.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease caused by a novel Bunyavirus with a high mortality rate. We herein report a fatal case of an 86-year-old woman with SFTS complaining of a fever, fatigue, and bicytopenia. Her condition deteriorated with rapid progression of bleeding tendency, disturbance of consciousness, and multiple organ failure leading to death on Day 6 of her illness. The histopathological findings in the autopsy revealed marked infiltration of macrophages with hemophagocytosis in the bone marrow, liver, and spleen leading to a diagnosis of hemophagocytic lymphohistiocytosis (HLH). HLH might be a critical pathogenesis in fatal cases of SFTS.

We performed pre-embedding electron microscopic study for visualizing the antigen and genome of severe fever with thrombocytopenia syndrome (SFTS) virus in the cytoplasm of macrophages of the human splenic red pulp, both requesting preheating treatment of sections. To pursue this, coated glass slides with unique characteristics are needed. Namely, during staining they must prevent detaching off sections, but after staining the sections must be transferred to epoxy resin. Aminopropyltriexoxysilane-coated glass slides, widely used for immunostaining, were resistant to transfer to epoxy resin. In contrast, coated glass slides designated as Thinlayer Advanced Cytology Assay System (TACAS) were suitable for this purpose. The technique is also applicable to the coated glass slide-requiring cytology practice, in which immunocytochemical evaluation is needed after cell transfer to another glass slide.

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.

Fumihiko Mahara1,2, Kazuaki Miyamoto1,2*, Hiromi Fujita2 and Teruo Mutsuda1 1Mahara Hospital, Aratono-cho Nobusato 6-1, Anan, 779-1510, Japan 2Mahara Institute of Medical Acarology, Aratano-cho Korekuni 56-3, Anan, 779-1510, Japan *Corresponding author: Kazuaki Miyamoto, Mahara Institute of Medical Acarology, Aratano-cho Korekuni 56-3, Anan, 779-1510, Japan, Fax/Tel: +81-884-36-3601; Email: kazupitts@jtw.zaq.ne.jp

Japanese spotted fever (JSF) is caused by Rickettsia japonica, and lethal cases are reported yearly in southwest Japan. We thus established the method of diagnosing JSF by immunohistochemistry (IHC) and real-time PCR (RT-PCR) using formalin-fixed, paraffin-embedded skin biopsy specimens. Two monoclonal antibodies were used for IHC, and the 17k genus common antigen gene served as the target of RT-PCR. We collected skin biopsy (n = 61) and autopsy (n = 1) specimens from 50 patients clinically suspected of JSF. Immunohistochemically, the rickettsial antigens were localized as coarse dots in the cytoplasm of endothelial cells and macrophages. Thirty-one seropositive cases plus one autopsy case (group A) and nine seronegative cases but with positive IHC and/or RT-PCR (group B) were judged as JSF. Nine cases were regarded as non-JSF disorders based on negative serology, IHC and RT-PCR (group C). Of 50 biopsies (eschar 34, eruptions 10, and scabs 6) from groups A and B, IHC and RT-PCR positivities were 94% (32/34) and 62% (21/34) for eschar, 80% (8/10) and 30% (3/10) for eruptions, and 33% (2/6) and 50% (3/6) for scabs. For IHC, eschar was most suitable, and scabs were insufficient. Unexpectedly, 18 biopsies happened to be fixed in 100% formalin, and this lowered the detection rate by RT-PCR, but IHC was tolerant. Sequence analysis using five skin biopsy specimens confirmed a 114 bp DNA stretch homologous to that reported for the target gene of R. japonica. In 26 (84%) of the 31 seropositive patients, the diagnosis was made by IHC and/or RT-PCR earlier than serology.

Three strains of Orientia tsutsugamushi were isolated from patients in Anan City, Tokushima Prefecture. The strains were identified as Karp type by analyses of reactivities with type-specific monoclonal antibodies. One strain, Okazaki, was isolated in L cells cultivated at 31 C, but not in cells at 36 C or in mice. This strain showed better growth at 31 C than 36 C. This is the first report of an O. tsutsugamushi strain which grows preferentially at low temperatures.

Japanese spotted fever (JSF) is caused by Rickettsia japonica. Although it induces a variety of complications, acute myocarditis has never been reported as a complication of JSF. We treated a JSF patient who developed acute myocarditis. To our knowledge, this is the first case of JSF complicated with acute myocarditis.

In May 1993, we collected unfed ticks in Anan, Tokushima Prefecture, Shikoku District, Japan, a highly endemic area of Japanese spotted fever (JSF). We isolated JSF rickettsiae from these ticks by inoculating tick-emulsions into L-929 cells. Of the four genera and seven species of ticks examined, only one larva of Dermacentor taiwanensis Sugimoto yielded a rickettsial isolate. The isolate (DT-1 strain) was found to belong to the serotype common to the causative agent of JSF. This was based on its specific reactivity to mouse antisera and to a monoclonal antibody to JSF rickettsiae from the Shikoku District. This is the first tick-borne spotted fever group of rickettsia isolated in Japan. However, all stages of D. taiwanensis infrequently parasitize humans. Some ticks in the genus Haemaphysalis commonly infest humans and are frequently found to be rickettsia-positive in the hemolymph test when using the monoclonal antibody mentioned above. Therefore, the vectors of JSF rickettsiae to humans may be a complex of tick species.

We report a patient with Japanese spotted fever caused by Rickettsia japonica who developed shock associated with hypercytokinemia. Elevated levels of cytokines (macrophage colony-stimulating factor, interleukin 1 beta, interleukin 10, and gamma interferon) decreased rapidly after a combination treatment using an antibiotic (minocycline hydrochloride [MINO]) and methylprednisolone; however, tumor necrosis factor alpha levels were increased. The patient's fever relapsed and was resolved only after the addition of ciprofloxacin hydrochloride. The administration of new quinolones alone may be another useful form of treatment to eradicate R. japonica even if the symptoms of hypercytokinemia appear to improve with the administration of MINO and methylprednisolone.

In the southeast coast of Shikoku, most of the Japanese spotted fever (JSF) patients were found to be infected with the rickettsial pathogen through bamboo plantation, and also the eschar was frequently noticed in skin inspection, so that it was clinically speculated to be caused by ticks. According to out field research, the tick fauna was very rich throughout that area, and it was permissible enough to determine ticks as the vectors, based on arising of anti-SF group rickettsiae (SFGR) antibody in mice inoculated with some tick emulsions, findings of rickettsiae reactive to patient sera or a species-specific monoclonal antibody to JSFR in the hemolymph cells of some ticks, and electron microscopical observations of SFGR in various internal organs including the salivary gland of ticks. Also the transovarial and transstadial transmission of SFGR in ticks were supposed. Our results suggest that common species (adult/nymph) of the genus Haemaphysalis may be the most related vectors to man, based on their dominances, SFGR-prevalences, behavior and incidences of infestation but also indicate that various genera and species should be kept as the potential vectors, in spite of JSFR isolation from a restricted species of ticks in the future.

A gelatin particle agglutination (PA) test for Japanese spotted fever has been developed. Gelatin particles were sensitized with a sonicated causative rickettsia and used as antigens. The antibodies by PA test were detected as early as days 4-7 after the onset, whereas those by indirect immunoperoxidase (IP) test were detected after days 8-11. In addition, PA titers were higher than IP titers before days 20-23. The agglutinins detected by PA test were proven to be IgM because they were all sensitive to dithiothreitol. PA test was, however, less specific than IP test, giving a little nonspecific reaction to the sera from patients with scrub typhus and from individuals unrelated to those two rickettsioses. Nevertheless, PA test, which is simple, rapid, and easy to interpret the results, is useful for the early serodiagnosis of Japanese spotted fever.

A case of tsutsugamushi disease (scrub typhus) with atypical lymphocytes with a multilobated nucleus is reported. Although this type of atypical lymphocyte has been reported in patients with viral infections such as adult T-cell leukemia, infectious mononucleosis, human immunodeficiency virus (HIV) infection, this is the first reported case of atypical lymphocyte with a multilobated nucleus in a patient with rickettsial infection. This type of atypical lymphocyte seems to exist in a broad spectrum of infectious diseases.

The in vitro susceptibilities to five chemotherapeutic agents against a rickettsial strain which was isolated from a case of Japanese spotted fever were determined by cell culture system. Minocycline was the most effective (MIC, 0.15 micrograms/ml) followed by Tetracycline and Demethylchlor tetracycline (0.31 micrograms/ml 0.16 micrograms/ml). Chloramphenicol was less effective (5 micrograms/ml) and Aminobenzyl-penicillin was not effective with the MIC of 10 micrograms/ml or less. Thus, the earlier administration of Minocycline is recommended to clinically suspicious cases of Japanese spotted fever as well as Tsutsugamushi disease.

The acute febrile disease with characteristic rash encountered in Tokushima Prefecture was proved to be a spotted fever group rickettsiosis, which showed a significant rise in agglutinins to both Proteus OX2 and OX19 and significantly high levels of CF antibodies to Rickettsia akari and Rickettsia rickettsii.