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PubMed2005

Molecular characterization of a group of proteins containing ankyrin repeats in Orientia tsutsugamushi.

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The sequencing and analysis of full genomic DNA of O. tsutsugamushi has revealed at least 19 genes thus far encoding proteins with different numbers of ankyrin repeat domains. We have cloned several genes containing ankyrin repeats from the genome and produced fusion proteins to characterize their functions in host cells. It is likely that the proteins with ankyrin repeat domains expressed in O. tsutsugamushi-infected cells may control the synthesis or stability of host proteins to modulate the various cellular functions after infection. The exploitation of host factors by ankyrin repeat proteins of O. tsutsugamushi may also play a critical role in its pathogenesis.

Ankyrin Repeat↗
PubMed2001

Vertical transmission of Orientia tsutsugamushi in two lines of naturally infected Leptotrombidium deliense (Acari: Trombiculidae).

Vertical transmission of Orientia tsutsugamushi (Hayashi), the etiologic agent for scrub typhus, was studied in two lines of naturally infected Leptotrombidium deliense Walch. In one line of mites originating from a single adult (V3M), the rate of filial transmission was 100% for the first two laboratory generations, but declined to 86.6% in the third laboratory generation. The vertical infection rate in this line of mites was 100% for the parental generation, but declined to 95.6% for the F1 generation and 88.6% for F2. The transmission of O. tsutsugamushi in another line of L. deliense (V3F) was less efficient than mites originating from V3M. In the initial laboratory generation of V3F a filial transmission rate of 100% was recorded. However, none of the F2 generation of this line transmitted rickettsiae to mice (Mus musculus L.), resulting in a filial transmission rate of 0%. Transmission of O. tsutsugamushi to mice by progeny from cohort larvae originally from the same adult (V3F) was also studied in the laboratory and these were found to be relatively poor transmitters of rickettsiae. The filial infection rate of F2 larvae was 60%, F3 was 88.8%, and F4 was 55.9%. The biology of infected L. deliense was studied and compared with uninfected mites reared under the same laboratory conditions. The results showed that infected female L. deliense laid approximately the same or more eggs as uninfected adults. The rate of development of the progeny of infected L. deliense was not significantly different from uninfected mites.

Animals↗
PubMed1987

Factors influencing Rickettsia tsutsugamushi infection of cultured cells.

Several factors which influence Rickettsia tsutsugamushi infectivity of, and replication in, cultured cells have been investigated in order to establish standard laboratory conditions for reliable quantitation and maximization of rickettsial yield and to begin to explore the nature of the relationship between the rickettsia and its host cell. The extent of scrub typhus rickettsial association with host cells was dependent on the rickettsial and target cell concentrations and on the medium in which the infection took place. Brain heart infusion broth, a standard rickettsial diluent for infectivity, was markedly inhibitory. Both rickettsial and host cell replication were dependent on a component(s) supplied by serum to the tissue culture medium; rickettsial multiplication was less affected by small temperature variation (optimum 34 to 37 degrees C) than was host cell growth (optimum 37 degrees C). The antibiotic daunomycin was very useful in selectively inhibiting host cell replication and permitting attainment of somewhat greater rickettsial yields than in uninhibited cells. These findings have underscored the close relationship between parasite and infected cell and have led to more predictable, higher yields of rickettsiae in vitro.

Animals↗
PubMed1987

Improved plaque assay for Rickettsia tsutsugamushi.

The assay of Rickettsia tsutsugamushi infectivity by plaquing has been improved substantially by a number of changes which were based on our understanding of factors which enhance scrub typhus rickettsial infection of, and replication in, cultured cells. Greater numbers of plaques and/or larger plaques resulted from: use of tissue culture medium instead of brain heart infusion broth as the rickettsial diluent; plaquing in a contact-inhibited mouse embryo cell line rather than in growth-inhibited or uninhibited Vero cells; infection and incubation of monolayers at 35 degrees C instead of at lower temperatures; frequent feeding of infected cultures with medium containing ample amounts of serum; and inclusion of chicken serum in the overlay medium. Plaquing in 24-well tissue culture plates instead of in petri dishes or flasks greatly simplified the handling of large numbers of samples and was beneficial economically as well. Easily recognized rickettsial plaques were counted microscopically under x40 magnification, and maximum counts were obtained 12-14 days after infection, depending on the rickettsial strain. Slightly longer incubation yielded macroscopically visible counts. In addition to enhancing plaque number and size, the changes in standard R. tsutsugamushi plaquing methods resulted in an easier, faster, and more reliable assay, with improved reproducibility of plaque formation, maintenance of infected cell monolayers, and avoidance of microbial contamination.

Animals↗
PubMed1988

Use of avidin-biotin-glucose oxidase complex to detect antimalarial antibody in serum by light microscopy.

An immunohistochemical assay was developed combining an avidin-biotin-glucose oxidase complex procedure (ABC-GO) with light microscopy to detect specific antibody against Plasmodium falciparum. Thin blood films were prepared from culture material of P. falciparum and fixed with acetone. Antibody was detected by successive incubations with test serum, biotinylated goat antihuman antibody, avidin-biotin-glucose oxidase complex, and glucose oxidase substrate. In the presence of reactive serum, a blue precipitate formed on the parasites and could be visually observed with a 40x objective. Sera from patients with single infections for P. vivax or P. ovale were unreactive. No cross-reactivity was observed with sera from patients with rheumatoid arthritis, filariasis, amebiasis, schistosomiasis, dengue, scrub typhus, leptospirosis, or toxoplasmosis. The sensitivity of ABC-GO is comparable to that of the indirect fluorescent antibody test.

Animals↗
PubMed1988

The ELISA-U: an enzyme-linked immunosorbent assay using urease as the enzyme marker for rapid detection of Plasmodium falciparum antibody in human serum.

A visual, enzyme-linked immunosorbent assay using urease (ELISA-U) as the enzyme marker was adapted for rapid detection of antibody against Plasmodium falciparum. Flat-bottom, 96-well microtiter plates were coated with P. falciparum soluble antigen obtained by saponin and NP-40 treatment of parasite cultures. Antibody was detected by successive incubations with test sera, urease-conjugated rabbit-human antibody, and urease substrate. Reactive sera developed a definite and easily visualized purple color. Sera from patients with single infections of P. vivax or P. ovale were unreactive. No cross-reactivity was noted with sera from patients with rheumatoid arthritis, filariasis, amebiasis, schistosomiasis, dengue, scrub typhus, leptospirosis, or toxoplasmosis. The procedure can be performed at room temperature and completed within 1 hr. The sensitivity of the assay is comparable to that of the indirect fluorescent antibody test at all but the lowest dilutions tested.

Animals↗
PubMed1995

Rickettsia tsutsugamushi infection in cell culture: antibiotic susceptibility determined by flow cytometry.

Recent unpublished reports from northern Thailand of severe and sometimes fatal cases of scrub typhus, despite appropriate antibiotic therapy, suggest that resistance may occur. Current antibiotic susceptibility methods that use direct microscopic counts of Giemsa-stained cells or mouse protection assays are slow, labor-intensive, and expensive. We explored the use of flow cytometry to measure rickettsial infection in vitro in L-929 cells treated with and without doxycycline, ciprofloxacin, erythromycin, and chloramphenicol. It was possible to detect the rickettsiae down to a level of 83% of the cells infected, mean of 37 rickettsiae per cell, and 40% of cells with too many rickettsiae to count. This level of sensitivity was sufficient to determine the inhibitory effect of all four drugs at standard screening concentrations. At lower concentrations of doxycycline, flow cytometry detected inhibition of rickettsial growth at a concentration of 6.25 x 10(-2) micrograms/ml but not at 6.25 x 10(-3) micrograms/ml, suggesting that the minimum inhibitory concentration is somewhere between these two values. The data from this study show that flow cytometry permits the rapid screening of numerous rickettsial isolates for their susceptibility to a variety of antibiotics, but that visual counts of infected cells provide a more precise indication of rickettsial growth.

Animals↗
PubMed1993

Medicine in tropical Australia.

In the unique environment of Australia's tropical north there are endemic diseases inherited from Gondwana, others introduced from the north and from Europe, and a wide range of particularly venomous animals. There is continuing disparity in morbidity and mortality between Aboriginal people and other Australians in tropical areas and elsewhere. This is being addressed by the National Aboriginal Health Strategy, which emphasises social, environmental and economic issues, as well as control and coordination of services by Aboriginal and Torres Strait Islander communities. While the re-introduction of malaria remains a potential threat, together with other infections, current diseases in tropical Australia are being better elucidated; melioidosis is now recognised as the commonest cause of fatal [corrected] community-acquired pneumonia in the Top End of the Northern Territory, and a new focus of scrub typhus has been found. Sexually transmitted diseases are an urgent issue, especially for Aboriginal communities, given the potential impact of the human immunodeficiency virus.

Animals↗
PubMed1998

Current outlook of infectious diseases in Taiwan.

The "emerging" infectious diseases have received global attention. Taiwan is a country which is going through the process of becoming "developed" from being "developing". If we compare five leading causes of death in 1952 and in 1993, three were infectious diseases in 1952 and there was none in 1993. And yet today, infectious diseases remain a major problem in this country as well in every country in the world, whether developing or developed. Some of the problems Taiwan faces are old problems with old faces. They have never been adequately solved because the societal and environmental sanitary infrastructure does not ensure proper sewage disposal, safe potable water and freedom from dangerous vectors. Examples are the diarrheal diseases, parasitic diseases, scrub typhus and Japanese encephalitis. Some of the Taiwan's problems are caused by old agents which present a new face. Mortality from tuberculosis took a dramatic and gratifying plunge in the last fifty years. Yet tuberculosis is ever present and a constant public health threat. Dengue has become a problem again because of a world breakdown in the control of the mosquito, Aedes egypti, and it is partly contributed to by increased urbanization and world travel. The problem of antibiotic resistant bacteria causing hospital acquired and community acquired infections is probably the most serious "new" problem. The most important cause is excessive and indiscriminate use of antibiotics in the community and in hospitals. We propose the establishment of "Bacterial Infections Reference Laboratory" at the National Health Research Institutes to be a national facility to study the epidemiology and control of antibiotic resistance. All infectious diseases require a rigorous system of surveillance, and precise etiological diagnosis before they can be treated or prevented. This should be kept clearly in mind when one considers the changing role of the infectious disease physician in Taiwan in the face of unsolved disease problems and a new health care system. There is inadequate attention to precise microbiological definition of most infectious diseases in Taiwan. The community of infectious disease specialists may well redirect its attention to improving the competence and utilization of microbiological laboratory diagnosis.

Clinical Laboratory Techniques↗
PubMed1999

[Molecular typing of Taiwanese Orientia tsutsugamushi isolates by restriction fragment profile].

Scrub typhus, which is caused by Orientia tsutsugamushi, is one of the reportable diseases in Taiwan, ROC. Positive cases seemed to be increasing in recent years and our laboratory had confirmed a total of 247 cases in 1997. In this study, 71 strains of O. tsutsugamushi were isolated from heparinized blood of the patients by cell culture, and their DNA was isolated. Two-step polymerase chain reaction was then used to amplify a specific sequence from the DNA, which was digested with restriction enzymes, Hha I and Sfa NI, for typing. With this technique, 34 strains gave rise to the same profile as that of the Kuroki type, 6 as the Karp type and 2 as the Gilliam type. The remaining 28 strains produced fragment profiles different from those of types Karp, Kato, Kawasaki, Kuroki, Gilliam and Shimokoshi and, therefore, were classified as 6 new types designated Taiwan A to Taiwan F, respectively. These strains with profiles different from those of the standard Japanese strains may represent the local mutant strains.

Humans↗
PubMed2000

[The state of vector-borne diseases in Indonesia].

From epidemiological point of view, Indonesia is an extremely interesting area owing its insular structure and ecological, anthropological, cultural and economical diversity. As everywhere, vector-borne diseases are the result of complex and variable epidemiological systems, subject both to biogeographical rules and human activity. Two main arboviroses are present in Indonesia: dengue and Japanese encephalitis. Dengue appears as an endemoepidemic disease and is mostly circumscribed to urban areas. Haemorrhagic cases were first observed in 1968; since then, the incidence has been constantly increasing and the disease is now one of the principal causes of child lethality. Japanese encephalitis is a rural endemic disease transmitted by rice-field mosquitoes; its incidence remains relatively low since pigs, which are usual link-hosts for the virus, are uncommon in this mainly Muslem country. Human clinical cases are recorded from non-Muslem islands such as Bali or Irian Jaya which raises the question of immunisation for travellers. Recently, Japanese encephalitis was observed on east of the Wallace line which had been considered as the eastern cut-off line. Malaria is common throughout the country, Plasmodium vivax being the most frequent species. Some of the Anopheline vectors are related to brackish water as are coastal species; others have been favoured by rice growing. Several species bite and rest outdoors, rendering control measures complex. Moreover, chloroquine resistance is increasing in both P. falciparum and P. vivax. All three filaria species responsible for human lymphatic filariasis exist in Indonesia. Bancroft filariasis is present in rather limited foci on most of the islands; malayan filariasis is very prevalent on many islands, mostly in coastal areas, and Timor filariasis exist only on a few small islands. These parasitic diseases are cumulative and do not practically endanger the health of travellers. In the past, plague was common on Java island, but today, human cases are very rare. Scrub typhus is prevalent everywhere, as is murine typhus, being very frequent in harbour cities and one of the main causes of hospitalisation for febrile syndromes.. On the whole, the situation of several of these diseases has been worsening in Indonesia for about thirty years. Although epidemiological situations constantly evolve, two recent occurrences should be paid particular attention: -transmigration which is now a national priority and greatly facilitates the spread of many pathogens, arboviroses or chloroquine-resistant plasmodia, but also of rats, mosquitoes, etc. -deforestation due either to land-farming by Javanese transmigrants or to sudden climatic changes such as El Niño in 1997. Such deep ecological transformations may have considerable and unforeseeable consequences on the epidemiology of vector-borne diseases in Indonesia.

Animals↗
PubMed2002

[HIV-suppressive factors].

A number of host and microbial factors have been shown to modulate HIV-1 infection. Their inhibitory effects are either HIV-specific or non-specific, and involve many different kinds of mechanisms. Among anti-HIV host factors are natural ligands or natural antibodies to HIV coreceptors, anti-inflammatory cytokines, interferons and several body fluid components (such as lactoferrin and prostaglandins). Microbial pathogens/factors that may suppress HIV-1 infection include lipopolysaccharide, scrub-typhus rickettsia, human herpesviruses-6 or -7, and GB virus C. While simple application of these HIV-suppressive factors for HIV-infected individuals is not realistic, investigation of mechanisms involved may lead to better understanding of HIV pathogenesis and help establish novel anti-HIV strategy.

Animals↗
PubMed2003

The etiology of acute pyrexia of unknown origin in children after a flood.

Acute pyrexia of unknown origin (PUO) is a major public health problem in Thailand. We studied the etiology of 180 cases of acute PUO in children after a sudden severe flood in Hat Yai city in 2000. Dengue infection and leptospirosis accounted for more than half of the total cases. Dengue hemorrhagic fever was the most common (29.4%) followed by leptospirosis (27.2%) and scrub typhus infection (1.1%). Five serovars of leptospires were involved in this study. Leptospira interrogans bataviae was the most common (86.5%). Acute serum antibody testing could detect only 52.8% and 40.8% of dengue and leptospirosis cases, respectively. This study showed both should be included in the presumptive diagnosis of acute PUO in patients after flooding.

Adolescent↗
PubMed2004

Etiologies of acute undifferentiated febrile illness in Thailand.

BACKGROUND: Acute pyrexia of unknown origin (Acute PUO) was reported to affect approximately 200,000-400,000 patients each year reported by the national Annual Epidemiological Surveillance Report. The patients usually present with fever of less than two-week duration and non-specific symptoms such as malaise, myalgia, headache and loss of appetite. Its mortality rate is less than 0.02 percent. It would be interesting to find the etiologies and propose a management plan if the etiologies are discovered. OBJECTIVE: This prospective epidemiologic study aimed to discover the etiologies of acute undifferentiated febrile illness in a tropical region like Thailand. SUBJECTS AND METHOD: Ten community-based hospitals were chosen as representatives in each part of Thailand to enroll patients into the study. Patients aged over two years old who presented with fever at the participating hospitals during year 1991-1993 were eligible for the study. Entry criteria of acute undifferentiated febrile illnesses (AUFI) included oral temperature over 38.3 degrees C within the last 24 hours, duration of fever ranging from 3-14 days, no specific single organ involvement by history taking and physical examination, normal or non-specific results of the following investigations: complete blood count, thick film for malaria, urinalysis and chest roentgenogram. The patients were hospitalized and a preset diagnostic protocol was performed. Other diagnostic procedures deemed necessary by attending physicians were perform. Patients were followed up within one month after hospital discharge. RESULTS: 1,240 patients were enrolled but only 1,137 case records and results of the serological tests were available for analysis. Etiologies could be found in 471 cases (38.7%). Primary bacteremia was detected in 36 cases (3.2%). E. coli, streptococci, salmonella, Enterobacter spp. and S. aureus were the five most common blood isolates. Serological studies revealed positive results for scrub typhus (7.5%), influenza (6.0%), dengue fever (5.7%), murine typhus (5.3%), enteric fever (1.9%), chikunkunya infection (1.1%), leptospirosis (1.1%) and melioidosis (0.9%). Thirteen cases succumbed (1.1%) in this study. CONCLUSION: The etiologies in the majority (61.3%) of AUFI remained unknown. Rickettsial infection, influenza and dengue fever are the most common identifiable diseases in a tropical country like Thailand especially during the rainy season. A management guideline for diagnosis and treatment of the AUFI with emphasis on primary bacteremia and antimicrobial-treatable AUFI was proposed.

Acute Disease↗
PubMed2004

A study of'febrile illnesses on the Thai-Myanmar border: predictive factors of rickettsioses.

We have performed a case-control analysis to determine the significance of clinical, laboratory and epidemiological features as predictive factors of rickettsioses among patients in Sangkhla Buri, Thailand (Thai-Myanmar border). Fifteen serologically-confirmed rickettsiosis patients including Spotted Fever Group (SFG) rickettsioses, scrub typhus, and murine typhus were classified as 'cases'; one hundred and sixty-three acutely febrile patients presenting to the same hospital during the same time period, who had no serological evidence of acute rickettsiosis, were classified as 'controls'. Patients' report of rash/arthropod bite [Odds ratio (OR) 22.90, 95% CI (confidence interval) 6.23, 84.13] and history of jungle trips (OR 5.30, 95% CI 1.69-16.62) were significant risk factors. Elevated ALT (OR 3.04, 95% CI 1.04, 8.88) and depressed platelet count (OR 3.38, 95% CI 1.13, 10.10) were also useful differentiating markers of rickettsioses in this population. Definitive diagnosis of rickettsioses is difficult without specialized diagnostic capabilities that are rarely available in remote areas such as Sangkhla Buri, where other acute febrile illnesses with similar presentation are commonly found. The relative importance of predictive factors presented here may provide clinicians with some useful guidance in distinguishing rickettsioses from other acute febrile illnesses. Timely administration of empiric treatment in highly suspicious cases can deter potential morbidity from these arthropod-borne infections.

Adult↗